ATSDR Nitrate and Nitrite Tox Profile

TOXICOLOGICAL PROFILE FOR

NITRATE AND NITRITE

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

Agency for Toxic Substances and Disease Registry

July 2017

ii NITRATE AND NITRITE

DISCLAIMER

Use of trade names is for identification only and does not imply endorsement by the Agency for Toxic

Substances and Disease Registry, the Public Health Service, or the U.S. Department of Health and Human

Services.

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UPDATE STATEMENT

A Toxicological Profile for Nitrate and Nitrite, Draft for Public Comment was released in September

2015. This edition supersedes any previously released draft or final profile.

Toxicological profiles are revised and republished as necessary. For information regarding the update

status of previously released profiles, contact ATSDR at:

Agency for Toxic Substances and Disease Registry

Division of Toxicology and Human Health Sciences

Environmental Toxicology Branch

1600 Clifton Road NE

Mailstop F-57

Atlanta, Georgia 30329-4027

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FOREWORD

This toxicological profile is prepared in accordance with guidelines* developed by the Agency for Toxic

Substances and Disease Registry (ATSDR) and the Environmental Protection Agency (EPA). The

original guidelines were published in the Federal Register on April 17, 1987. Each profile will be revised

and republished as necessary.

The ATSDR toxicological profile succinctly characterizes the toxicologic and adverse health effects

information for these toxic substances described therein. Each peer-reviewed profile identifies and

reviews the key literature that describes a substance's toxicologic properties. Other pertinent literature is

also presented, but is described in less detail than the key studies. The profile is not intended to be an

exhaustive document; however, more comprehensive sources of specialty information are referenced.

The focus of the profiles is on health and toxicologic information; therefore, each toxicological profile

begins with a public health statement that describes, in nontechnical language, a substance's relevant

toxicological properties. Following the public health statement is information concerning levels of

significant human exposure and, where known, significant health effects. The adequacy of information to

determine a substance's health effects is described in a health effects summary. Data needs that are of

significance to the protection of public health are identified by ATSDR.

Each profile includes the following:

(A) The examination, summary, and interpretation of available toxicologic information and

epidemiologic evaluations on a toxic substance to ascertain the levels of significant human

exposure for the substance and the associated acute, subacute, and chronic health effects;

(B) A determination of whether adequate information on the health effects of each substance

is available or in the process of development to determine levels of exposure that present a

significant risk to human health of acute, subacute, and chronic health effects; and

(C) Where appropriate, identification of toxicologic testing needed to identify the types or

levels of exposure that may present significant risk of adverse health effects in humans.

The principal audiences for the toxicological profiles are health professionals at the Federal, State, and

local levels; interested private sector organizations and groups; and members of the public.

This profile reflects ATSDR’s assessment of all relevant toxicologic testing and information that has been

peer-reviewed. Staffs of the Centers for Disease Control and Prevention and other Federal scientists have

also reviewed the profile. In addition, this profile has been peer-reviewed by a nongovernmental panel

and was made available for public review. Final responsibility for the contents and views expressed in

this toxicological profile resides with ATSDR.

Patrick N. Breysse, Ph.D., CIH

Director, National Center for Environmental Health and

Agency for Toxic Substances and Disease Registry

Centers for Disease Control and Prevention

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*Legislative Background

The toxicological profiles are developed under the Comprehensive Environmental Response,

Compensation, and Liability Act of 1980, as amended (CERCLA or Superfund). CERCLA section

104(i)(1) directs the Administrator of ATSDR to “…effectuate and implement the health related

authorities” of the statute. This includes the preparation of toxicological profiles for hazardous

substances most commonly found at facilities on the CERCLA National Priorities List and that pose the

most significant potential threat to human health, as determined by ATSDR and the EPA. Section

104(i)(3) of CERCLA, as amended, directs the Administrator of ATSDR to prepare a toxicological profile

for each substance on the list. In addition, ATSDR has the authority to prepare toxicological profiles for

substances not found at sites on the National Priorities List, in an effort to “…establish and maintain

inventory of literature, research, and studies on the health effects of toxic substances” under CERCLA

Section 104(i)(1)(B), to respond to requests for consultation under section 104(i)(4), and as otherwise

necessary to support the site-specific response actions conducted by ATSDR.

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QUICK REFERENCE FOR HEALTH CARE PROVIDERS

Toxicological Profiles are a unique compilation of toxicological information on a given hazardous

substance. Each profile reflects a comprehensive and extensive evaluation, summary, and interpretation

of available toxicologic and epidemiologic information on a substance. Health care providers treating

patients potentially exposed to hazardous substances may find the following information helpful for fast

answers to often-asked questions.

Primary Chapters/Sections of Interest

Chapter 1: Public Health Statement: The Public Health Statement can be a useful tool for educating

patients about possible exposure to a hazardous substance. It explains a substance’s relevant

toxicologic properties in a nontechnical, question-and-answer format, and it includes a review of

the general health effects observed following exposure.

Chapter 2: Relevance to Public Health: The Relevance to Public Health Section evaluates, interprets,

and assesses the significance of toxicity data to human health.

Chapter 3: Health Effects: Specific health effects of a given hazardous compound are reported by type

of health effect (e.g.,death, systemic, immunologic, reproductive), by route of exposure, and by

length of exposure (acute, intermediate, and chronic). In addition, both human and animal studies

are reported in this section.

NOTE: Not all health effects reported in this section are necessarily observed in the clinical

setting. Please refer to the Public Health Statement to identify general health effects observed

following exposure.

Pediatrics: Four new sections have been added to each Toxicological Profile to address child health

issues:

Chapter 1 How Can (Chemical X) Affect Children?

Chapter 1 How Can Families Reduce the Risk of Exposure to (Chemical X)?

Section 3.7 Children’s Susceptibility

Section 6.6 Exposures of Children

Other Sections of Interest:

Section 3.8 Biomarkers of Exposure and Effect

Section 3.11 Methods for Reducing Toxic Effects

ATSDR Information Center

Phone: 1-800-CDC-INFO (800-232-4636) or 1-888-232-6348 (TTY)

Internet: http://www.atsdr.cdc.gov

The following additional materials are available online:

Case Studies in Environmental Medicine are self-instructional publications designed to increase primary

health care providers’ knowledge of a hazardous substance in the environment and to aid in the

evaluation of potentially exposed patients (see https://www.atsdr.cdc.gov/csem/csem.html).

viii NITRATE AND NITRITE

Managing Hazardous Materials Incidents is a three-volume set of recommendations for on-scene

(prehospital) and hospital medical management of patients exposed during a hazardous materials

incident (see https://www.atsdr.cdc.gov/MHMI/index.asp). Volumes I and II are planning guides

to assist first responders and hospital emergency department personnel in planning for incidents

that involve hazardous materials. Volume III—Medical Management Guidelines for Acute

Chemical Exposures—is a guide for health care professionals treating patients exposed to

hazardous materials.

Fact Sheets (ToxFAQs™) provide answers to frequently asked questions about toxic substances (see

https://www.atsdr.cdc.gov/toxfaqs/Index.asp).

Other Agencies and Organizations

The National Center for Environmental Health (NCEH) focuses on preventing or controlling disease,

injury, and disability related to the interactions between people and their environment outside the

workplace. Contact: NCEH, Mailstop F-29, 4770 Buford Highway, NE, Atlanta, GA

30341-3724 • Phone: 770-488-7000 • FAX: 770-488-7015 • Web Page:

https://www.cdc.gov/nceh/.

The National Institute for Occupational Safety and Health (NIOSH) conducts research on occupational

diseases and injuries, responds to requests for assistance by investigating problems of health and

safety in the workplace, recommends standards to the Occupational Safety and Health

Administration (OSHA) and the Mine Safety and Health Administration (MSHA), and trains

professionals in occupational safety and health. Contact: NIOSH, 395 E Street, S.W., Suite 9200,

Patriots Plaza Building, Washington, DC 20201 • Phone: 202-245-0625 or 1-800-CDC-INFO

(800-232-4636) • Web Page: https://www.cdc.gov/niosh/.

The National Institute of Environmental Health Sciences (NIEHS) is the principal federal agency for

biomedical research on the effects of chemical, physical, and biologic environmental agents on

human health and well-being. Contact: NIEHS, PO Box 12233, 104 T.W. Alexander Drive,

Research Triangle Park, NC 27709 • Phone: 919-541-3212 • Web Page:

https://www.niehs.nih.gov/.

Clinical Resources (Publicly Available Information)

The Association of Occupational and Environmental Clinics (AOEC) has developed a network of clinics

in the United States to provide expertise in occupational and environmental issues. Contact:

AOEC, 1010 Vermont Avenue, NW, #513, Washington, DC 20005 • Phone: 202-347-4976

• FAX: 202-347-4950 • e-mail: AOEC@AOEC.ORG • Web Page: http://www.aoec.org/.

The American College of Occupational and Environmental Medicine (ACOEM) is an association of

physicians and other health care providers specializing in the field of occupational and

environmental medicine. Contact: ACOEM, 25 Northwest Point Boulevard, Suite 700, Elk

Grove Village, IL 60007-1030 • Phone: 847-818-1800 • FAX: 847-818-9266 • Web Page:

http://www.acoem.org/.

The American College of Medical Toxicology (ACMT) is a nonprofit association of physicians with

recognized expertise in medical toxicology. Contact: ACMT, 10645 North Tatum Boulevard,

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Suite 200-111, Phoenix AZ 85028 • Phone: 844-226-8333 • FAX: 844-226-8333 • Web Page:

http://www.acmt.net.

The Pediatric Environmental Health Specialty Units (PEHSUs) is an interconnected system of specialists

who respond to questions from public health professionals, clinicians, policy makers, and the

public about the impact of environmental factors on the health of children and reproductive-aged

adults. Contact information for regional centers can be found at http://pehsu.net/findhelp.html.

The American Association of Poison Control Centers (AAPCC) provide support on the prevention and

treatment of poison exposures. Contact: AAPCC, 515 King Street, Suite 510, Alexandria VA

22314 • Phone: 701-894-1858 • Poison Help Line: 1-800-222-1222 • Web Page:

http://www.aapcc.org/.

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CONTRIBUTORS

CHEMICAL MANAGER(S)/AUTHOR(S):

Carolyn Harper, Ph.D.

Sam Keith, M.S., C.H.P.

G. Daniel Todd, Ph.D.

Malcolm Williams, D.V.M., Ph.D.

ATSDR, Division of Toxicology and Human Health Sciences, Atlanta, GA

David W. Wohlers, Ph.D.

Gary L. Diamond, Ph.D.

Fernando Llados, Ph.D.

Christina Coley, B.S.

Mario Citra, Ph.D.

SRC, Inc., North Syracuse, NY

THE PROFILE HAS UNDERGONE THE FOLLOWING ATSDR INTERNAL REVIEWS:

1. Health Effects Review. The Health Effects Review Committee examines the health effects

chapter of each profile for consistency and accuracy in interpreting health effects and classifying

end points.

2. Minimal Risk Level Review. The Minimal Risk Level Workgroup considers issues relevant to

substance-specific Minimal Risk Levels (MRLs), reviews the health effects database of each

profile, and makes recommendations for derivation of MRLs.

3. Data Needs Review. The Environmental Toxicology Branch reviews data needs sections to

assure consistency across profiles and adherence to instructions in the Guidance.

4. Green Border Review. Green Border review assures the consistency with ATSDR policy.

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PEER REVIEW

A peer review panel was assembled for nitrate and nitrite. The panel consisted of the following members:

1. Dr. John Fawell, Visiting Professor, School of Applied Sciences, Cranfield University, Cranfield,

Bedfordshire MK43 OAL, United Kingdom;

2. Dr. Richard B. Ferguson, Professor of Soil Science, Associate Head of the Department of

Agronomy & Horticulture, University of Nebraska-Lincoln, Lincoln, Nebraska; and

3. Dr. Stephen M. Roberts, Director, Center for Environmental & Human Toxicology; Professor,

College of Veterinary Medicine, College of Medicine, College of Public Health and Health

Professions, University of Florida, Gainesville, Florida.

These experts collectively have knowledge of nitrate’s and nitrite’s physical and chemical properties,

toxicokinetics, key health end points, mechanisms of action, human and animal exposure, and

quantification of risk to humans. All reviewers were selected in conformity with the conditions for peer

review specified in Section 104(I)(13) of the Comprehensive Environmental Response, Compensation,

and Liability Act, as amended.

Scientists from the Agency for Toxic Substances and Disease Registry (ATSDR) have reviewed the peer

reviewers' comments and determined which comments will be included in the profile. A listing of the

peer reviewers' comments not incorporated in the profile, with a brief explanation of the rationale for their

exclusion, exists as part of the administrative record for this compound.

The citation of the peer review panel should not be understood to imply its approval of the profile's final

content. The responsibility for the content of this profile lies with the ATSDR.

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CONTENTS

DISCLAIMER .............................................................................................................................................. ii

UPDATE STATEMENT ............................................................................................................................. iii

FOREWORD ................................................................................................................................................ v

QUICK REFERENCE FOR HEALTH CARE PROVIDERS .................................................................... vii

CONTRIBUTORS ....................................................................................................................................... xi

PEER REVIEW ......................................................................................................................................... xiii

CONTENTS ................................................................................................................................................ xv

LIST OF FIGURES ................................................................................................................................... xix

LIST OF TABLES ..................................................................................................................................... xxi

1. PUBLIC HEALTH STATEMENT FOR NITRATE AND NITRITE ..................................................... 1

2. RELEVANCE TO PUBLIC HEALTH ................................................................................................... 9

2.1 BACKGROUND AND ENVIRONMENTAL EXPOSURES TO NITRATE AND NITRITE

IN THE UNITED STATES ........................................................................................................... 9

2.2 SUMMARY OF HEALTH EFFECTS ........................................................................................... 9

2.3 MINIMAL RISK LEVELS (MRLs) ............................................................................................ 21

3. HEALTH EFFECTS .............................................................................................................................. 29

3.1 INTRODUCTION ........................................................................................................................ 29

3.2 DISCUSSION OF HEALTH EFFECTS BY ROUTE OF EXPOSURE ..................................... 29

3.2.1 Inhalation Exposure .............................................................................................................. 30

3.2.1.1 Death .............................................................................................................................. 30

3.2.1.2 Systemic Effects............................................................................................................. 31

3.2.1.3 Immunological and Lymphoreticular Effects ................................................................ 32

3.2.1.4 Neurological Effects ...................................................................................................... 32

3.2.1.5 Reproductive Effects ......................................................................................................

32

3.2.1.6 Developmental Effects ................................................................................................... 32

3.2.1.7 Cancer ............................................................................................................................ 32

3.2.2 Oral Exposure ........................................................................................................................ 34

3.2.2.1 Death .............................................................................................................................. 34

3.2.2.2 Systemic Effects............................................................................................................. 34

3.2.2.3 Immunological and Lymphoreticular Effects ................................................................ 71

3.2.2.4 Neurological Effects ...................................................................................................... 71

3.2.2.5 Reproductive Effects ...................................................................................................... 72

3.2.2.6 Developmental Effects ................................................................................................... 74

3.2.2.7 Cancer ............................................................................................................................ 80

3.2.3 Dermal Exposure ................................................................................................................. 105

3.2.3.1 Death ............................................................................................................................ 105

3.2.3.2 Systemic Effects........................................................................................................... 105

3.2.3.3 Immunological and Lymphoreticular Effects .............................................................. 105

3.2.3.4 Neurological Effects .................................................................................................... 105

3.2.3.5 Reproductive Effects .................................................................................................... 105

3.2.3.6 Developmental Effects ................................................................................................. 105

3.2.3.7 Cancer .......................................................................................................................... 105

3.3 GENOTOXICITY ...................................................................................................................... 105

3.4 TOXICOKINETICS ................................................................................................................... 109

3.4.1 Physiologically Based Pharmacokinetic (PBPK)/Pharmacodynamic (PD) Models ........... 115

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3.5 MECHANISMS OF ACTION ................................................................................................... 122

3.5.1 Pharmacokinetic Mechanisms ............................................................................................. 122

3.5.2 Mechanisms of Toxicity ...................................................................................................... 123

3.5.3 Animal-to-Human Extrapolations ....................................................................................... 123

3.6 TOXICITIES MEDIATED THROUGH THE NEUROENDOCRINE AXIS ........................... 124

3.7 CHILDREN’S SUSCEPTIBILITY ............................................................................................ 125

3.8 BIOMARKERS OF EXPOSURE AND EFFECT ..................................................................... 128

3.8.1 Biomarkers Used to Identify or Quantify Exposure to Nitrate and Nitrite ......................... 129

3.8.2 Biomarkers Used to Characterize Effects Caused by Nitrate and Nitrite ........................... 130

3.9 INTERACTIONS WITH OTHER CHEMICALS ..................................................................... 130

3.10 POPULATIONS THAT ARE UNUSUALLY SUSCEPTIBLE ................................................ 131

3.11 METHODS FOR REDUCING TOXIC EFFECTS .................................................................... 132

3.11.1 Reducing Peak Absorption Following Exposure ............................................................. 133

3.11.2 Reducing Body Burden ................................................................................................... 133

3.11.3 Interfering with the Mechanism of Action for Toxic Effects .......................................... 133

3.12 ADEQUACY OF THE DATABASE ........................................................................................ 134

3.12.1 Existing Information on Health Effects of Nitrate and Nitrite ........................................ 134

3.12.2 Identification of Data Needs ............................................................................................ 137

3.12.3 Ongoing Studies .............................................................................................................. 150

4. CHEMICAL AND PHYSICAL INFORMATION .............................................................................. 151

4.1 CHEMICAL IDENTITY ............................................................................................................ 151

4.2 PHYSICAL AND CHEMICAL PROPERTIES ......................................................................... 152

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL .......................................................... 161

5.1 PRODUCTION ..........................................................................................................................

161

5.2 IMPORT/EXPORT .................................................................................................................... 170

5.3 USE ............................................................................................................................................ 173

5.4 DISPOSAL ................................................................................................................................. 173

6. POTENTIAL FOR HUMAN EXPOSURE ......................................................................................... 177

6.1 OVERVIEW ............................................................................................................................... 177

6.2 RELEASES TO THE ENVIRONMENT ................................................................................... 181

6.2.1 Air ....................................................................................................................................... 181

6.2.2 Water ................................................................................................................................... 184

6.2.3 Soil ...................................................................................................................................... 190

6.3 ENVIRONMENTAL FATE ...................................................................................................... 191

6.3.1 Transport and Partitioning ................................................................................................... 192

6.3.2 Transformation and Degradation ........................................................................................ 193

6.3.2.1 Air ................................................................................................................................ 193

6.3.2.2 Water ............................................................................................................................ 194

6.3.2.3 Sediment and Soil ........................................................................................................ 194

6.4 LEVELS MONITORED OR ESTIMATED IN THE ENVIRONMENT .................................. 195

6.4.1 Air ....................................................................................................................................... 195

6.4.2 Water ................................................................................................................................... 196

6.4.3 Sediment and Soil ............................................................................................................... 199

6.4.4 Other Environmental Media ................................................................................................ 200

6.5 GENERAL POPULATION AND OCCUPATIONAL EXPOSURE ........................................ 204

6.6 EXPOSURES OF CHILDREN .................................................................................................. 211

6.7 POPULATIONS WITH POTENTIALLY HIGH EXPOSURES .............................................. 212

6.8 ADEQUACY OF THE DATABASE ........................................................................................

212

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6.8.1 Identification of Data Needs ............................................................................................... 212

6.8.2 Ongoing Studies .................................................................................................................. 215

7. ANALYTICAL METHODS................................................................................................................ 217

7.1 BIOLOGICAL MATERIALS .................................................................................................... 217

7.2 ENVIRONMENTAL SAMPLES .............................................................................................. 220

7.3 ADEQUACY OF THE DATABASE ........................................................................................ 225

7.3.1 Identification of Data Needs ............................................................................................... 225

7.3.2 Ongoing Studies .................................................................................................................. 226

8. REGULATIONS, ADVISORIES, AND GUIDELINES ..................................................................... 227

9. REFERENCES .................................................................................................................................... 237

10. GLOSSARY ...................................................................................................................................... 273

APPENDICES

A. ATSDR MINIMAL RISK LEVELS AND WORKSHEETS ............................................................. A-1

B. USER’S GUIDE .................................................................................................................................. B-1

C. ACRONYMS, ABBREVIATIONS, AND SYMBOLS ...................................................................... C-1

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LIST OF FIGURES

3-1. Levels of Significant Exposure to Nitrate/Nitrite - Oral ..................................................................... 52

3-2. The Nitrate-Nitrite-Nitric Oxide Cycle in Humans .......................................................................... 113

3-3. Conceptual Representation of a Physiologically Based Pharmacokinetic (PBPK) Model for a

Hypothetical Chemical Substance .................................................................................................... 117

3-4. Structure of the Zeilmaker et al. (1996, 2010b) Model .................................................................... 119

3-5. Existing Information on Health Effects of Nitrate ............................................................................ 135

3-6. Existing Information on Health Effects of Nitrite ............................................................................ 136

5-1. Simplified Schematic of the Nitrogen Cycle .................................................................................... 168

6-1. Frequency of NPL Sites with Ammonium Nitrate Contamination ................................................... 178

6-2. Frequency of NPL Sites with Sodium Nitrate Contamination ......................................................... 179

6-3. Frequency of NPL Sites with Sodium Nitrite Contamination .......................................................... 180

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LIST OF TABLES

3-1. Levels of Significant Exposure to Nitrate/Nitrite - Oral ..................................................................... 36

3-2. Selected Cohort and Case-Control Studies Published Since 2006 Examining Possible

Associations Between Nitrate and Nitrite Intake and Cancer ............................................................ 81

3-3. Genotoxicity of Sodium Nitrite In Vivo ............................................................................................ 108

3-4. Genotoxicity of Sodium Nitrite In Vitro ........................................................................................... 110

3-5. Parameter Values for the Zeilmaker et al. (1996, 2010) PBPK Model of Nitrate and Nitrite in

Humans............................................................................................................................................. 120

4-1. Chemical Identity of Nitrate and Nitrite Ions ................................................................................... 153

4-2. Chemical Identity of Selected Inorganic Nitrate and Nitrite Compounds ........................................ 154

4-3. Chemical Identity of Ammonia and Urea ......................................................................................... 156

4-4. Physical and Chemical Properties of Selected Inorganic Nitrate and Nitrite Compounds ............... 157

4-5. Physical and Chemical Properties of Ammonia and Urea ................................................................ 158

5-1. Facilities that Produce, Process, or Use Nitrate Compounds ........................................................... 162

5-2. Facilities that Produce, Process, or Use Sodium Nitrite ................................................................... 164

5-3. Facilities that Produce, Process, or Use Ammonia ........................................................................... 166

5-4. Production of Ammonium Nitrate by the U.S. Chemical Industry................................................... 171

5-5. Production of Ammonia by the U.S. Chemical Industry .................................................................. 172

5-6. U.S. Imports and Exports (Metric Tons) of Selected Fertilizers 2000–2012 ................................... 174

5-7. U.S. Exports and Imports for Nitrate Fertilizers in 2012 (Short Tons) ............................................ 175

6-1. Releases to the Environment from Facilities that Produce, Process, or Use Nitrate Compounds .... 182

6-2. Releases to the Environment from Facilities that Produce, Process, or Use Sodium Nitrite............ 185

6-3. Releases to the Environment from Facilities that Produce, Process, or Use Ammonia ...................

187

6-4. Concentrations of Nitrate and Nitrite in Infant Food Products ......................................................... 202

6-5. Average Concentrations of Nitrate and Nitrite in Human Milk and Infant Formula ........................ 203

6-6. Geometric Mean and Selected Percentiles of Urine Concentrations of Urinary Nitrate (in mg/L) for

the U.S. P opulation from the National Health and Nutrition Examination Survey (NHANES) ...... 206

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6-7. Geometric Mean and Selected Percentiles of Urine Concentrations of Urinary Nitrate (Creatinine

Corrected) (in mg/g of creatinine) for the U.S. Population f rom the National Health and Nutrition

Examination Survey (NHANES) ..................................................................................................... 208

7-1. Analytical Methods for Determining Nitrate and Nitrite in Biological Materials ............................ 218

7-2. Analytical Methods for Determining Nitrate and Nitrite in Environmental Samples ...................... 221

8-1. Regulations, Advisories, and Guidelines Applicable to Nitrate and Nitrite ..................................... 230

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1 NITRATE AND NITRITE

1. PUBLIC HEALTH STATEMENT FOR NITRATE AND NITRITE

This Public Health Statement summarizes the Agency for Toxic Substances and Disease Registry’s

(ATSDR) findings on inorganic nitrate and nitrite, including chemical characteristics, exposure risks,

possible health effects from exposure, and ways to limit exposure. Nitrate and nitrite can be present in

organic or inorganic compounds, depending on their chemical structures. This profile pertains to

inorganic nitrate and nitrite, specifically the ionic forms of both nitrate and nitrite.

The U.S. Environmental Protection Agency (EPA) identifies the most serious hazardous waste sites in the

nation. These sites make up the National Priorities List (NPL) and are sites targeted for long-term federal

clean-up activities. Nitrate and nitrite are ubiquitous in the environment. Specific forms of nitrate and

nitrite have occasionally been identified in hazardous waste sites. Ammonium nitrate, sodium nitrate, and

sodium nitrite were identified in 7, 3, and 2 of the 1,832 hazardous waste sites, respectively, that have

been proposed for inclusion on the NPL. The total number of NPL sites evaluated for nitrate and nitrite is

not known. But the possibility remains that as more sites are evaluated, the number of sites at which

nitrate and/or nitrite are found may increase. This information is important because these future sites may

be sources of exposure, and overexposure to nitrate and/or nitrite may be harmful.

If you are exposed to nitrate and/or nitrite, many factors determine whether you’ll be harmed. These

include how much you are exposed to (dose), how long you are exposed (duration), how often you are

exposed (frequency), and how you are exposed (route of exposure). You must also consider the other

chemicals you are exposed to and your age, sex, diet, family traits, lifestyle, and state of health.

WHAT ARE NITRATE AND NITRITE?

Nitrate and nitrite are naturally occurring ionic species that are part of the earth’s nitrogen cycle. They

typically exist in the environment in highly water-soluble forms, in association with other ionic species

such as sodium and potassium. Nitrate and nitrite salts completely dissociate in aqueous environments.

Nitrite is readily oxidized (combines with oxygen) to form nitrate. Nitrate is generally stable in the

environment; however, it may be reduced to nitrite through biological processes involving plants,

microbes, etc.

In nature, plants utilize nitrate as an essential (key) nutrient. In commerce, the majority of nitrate is used

in inorganic fertilizers. Additional uses of commercial nitrate and nitrite include food preservation and

2 NITRATE AND NITRITE

1. PUBLIC HEALTH STATEMENT

the production of munitions and explosives. Sodium nitrite is also being used in medicines and

therapeutics; for example, as an antidote for cyanide poisoning and as a treatment for pulmonary arterial

hypertension.

WHAT HAPPENS TO NITRATE AND NITRITE WHEN THEY ENTER THE ENVIRONMENT?

Nitrate and nitrite ions naturally occur in the terrestrial (soil) and aquatic (water) environment as part of

the earth’s nitrogen cycle (see Figure 5-1) and can therefore be found in both soil and water. In nature,

nitrate and nitrite can also be found in igneous and volcanic rocks. Nitrate is formed naturally as an end

product of vegetable and animal decomposition, making this a principal source for nitrate ion in both

terrestrial (soil) and aquatic (water) environments. Nitrate and nitrite can also be released into the

atmospheric (air), terrestrial (soil), and aquatic (water) environments at places where human-made

materials such as fertilizers are produced or used. Human and animal wastes are important sources of

ammonia, a compound containing nitrogen, which undergoes chemical reaction to produce nitrite and

subsequently nitrate. In aerobic (containing oxygen) environments, ammonia is readily oxidized to nitrite

by ammonia-oxidizing bacteria; nitrite is oxidized to nitrate by nitrite-oxidizing bacteria. This two-stage

process is known as nitrification. Both human-made and natural sources of nitrogen may contribute to

nitrate aerosols in the atmosphere, as well as nitrate and nitrite ions in terrestrial (soil) and aquatic (water)

environments.

Nitrate and nitrite have been detected in surface waters, drinking water (including public and private

wells), and groundwater. Nitrate accounts for the majority of the total available nitrogen in surface

waters. Contamination of waters is a result of agricultural runoff (use of chemical fertilizer or animal

manure) and discharges from septic systems and municipal waste water treatment facilities. Nitrogen

exists naturally in soils, typically bound to organic matter or mineral soil material such as rocks.

Available forms of nitrogen, including nitrate and nitrite, are present in soils at a few kilograms

(kg)/hectare.

Nitrate and nitrite are a normal part of the human diet and can be found in vegetables, fruits, cured meats,

fish, dairy products, beers, cereals, and cereal products. Some salts, such as sodium nitrite, are

intentionally added to foods and beverages to preserve or cure them; inhibiting the formation of

microorganisms that may cause disease such as

botulism. Additionally, nitrites and nitrates may be

present in some medicines as they can be employed in medicinal and therapeutic uses.

3 NITRATE AND NITRITE

1. PUBLIC HEALTH STATEMENT

HOW MIGHT I BE EXPOSED TO NITRATE AND NITRITE?

The major source of overexposure of the general population to nitrate and nitrite is via ingestion of water,

foods, beverages, and/or medicines that contain nitrate and/or nitrite naturally or as an added preservative.

Nitrate and nitrite can be taken up by plants, especially leafy vegetables such as lettuce and spinach and

beet roots; vegetables account for about 80% of the nitrate in a typical human diet. Cured meats, meat

products, cheeses, and beverages may contain sodium nitrate and/or sodium nitrite as preservatives.

Relatively high nitrate concentrations are found in some privately owned wells with shallow depths and

permeable soils. Drinking of water from such sources, combined with nitrate intake from the diet, may

result in overexposure to nitrate in some individuals. Release of nitrate and/or nitrite to soil and water at

waste disposal sites could result in contamination of drinking water sources and increased uptake by

plants used for the human diet. Inhalation of nitrate or nitrite is not a likely exposure route of concern for

the general population, although inhalation of dust from fertilizer products containing nitrate salts is

possible. Dusts may also dissolve in sweat on skin, increasing the potential for dermal exposure.

HOW CAN NITRATE AND NITRITE ENTER AND LEAVE MY BODY?

Nitrate and nitrite could enter your body from the air you breathe; however, you are not likely to be

exposed to amounts of nitrate or nitrite in the air that might cause adverse health effects. Nitrate and

nitrite enter your body when you drink water or eat foods that contain these substances. Nitrate and

nitrite are also present in smokeless tobacco products. Certain bacteria and fungi in these products can

convert nitrate to nitrite, which can lead to the formation of carcinogenic nitrosamines. Neither nitrate

nor nitrite is likely to enter your body from soil. However, nitrate or nitrite in soil could enter the body of

young children if they put soil containing nitrate or nitrite in the mouth. Intake of some nitrate is a

normal part of the nitrogen cycle in humans. Both nitrate and nitrite can be produced inside the body as

well. Some of the nitrate in your body moves from blood to the salivary glands where some of it is

changed to nitrite. Nitrate and nitrite are widely distributed in the body. Nitrate and nitrite that enter your

body are no different chemically than nitrate and nitrite produced inside your body. Most nitrate in your

body leaves in the urine the same day it enters your body. Some nitrite in the stomach forms other

substances, some of which may be harmful. Nitrite in your blood can react with hemoglobin (which

carries oxygen to body tissues) and reduce the ability of hemoglobin to carry oxygen. Nitrite can also

form nitric oxide, which may be beneficial in some instances.

4 NITRATE AND NITRITE

1. PUBLIC HEALTH STATEMENT

HOW CAN NITRATE AND NITRITE AFFECT MY HEALTH?

Most people are not exposed to levels of nitrate and/or nitrite that would cause adverse health effects.

Young infants (<6 months of age) appeared to be particularly sensitive to the effects of nitrite on

hemoglobin after consuming formula prepared with drinking water that contained nitrate at levels higher

than recommended limits; some of these infants died. The cause of methemoglobinemia (a change to

hemoglobin that decreases the ability to transport oxygen to tissues) in many of these infants may have

been gastroenteritis from bacteria or viruses in the drinking water or from other sources not related to

nitrate. Some children and adults who ate food or drank fluids that contained unusually high levels of

nitrite experienced decreases in blood pressure, increased heart rate, reduced ability of the blood to carry

oxygen to tissues, headaches, abdominal cramps, vomiting, and even death.

There is limited evidence that nitrite may cause some cancers of the gastrointestinal tract in humans and

mice. Cancer could result from reactions between nitrite and certain other chemicals that may produce

cancer-causing substances. The International Agency for Research on Cancer (IARC) determined that

there is inadequate evidence for the carcinogenicity of nitrate in food or drinking water and limited

evidence for the carcinogenicity of nitrite in food (based on association with increased incidence of

stomach cancer). IARC determined that there is inadequate evidence for the carcinogenicity of nitrate,

limited evidence for the carcinogenicity of nitrite per se, and sufficient evidence for the carcinogenicity of

nitrite in combination with amines or amides. The overall conclusions of IARC were that “ingested

nitrate and nitrite under conditions that result in endogenous nitrosation is probably carcinogenic to

humans (Group 2A).” IARC noted that: (1) the endogenous nitrogen cycle in humans includes

interconversion of nitrate and nitrite; (2) nitrite-derived nitrosating agents produced in the acid stomach

environment can react with nitrosating compounds such as secondary amines and amides to generate

N-nitroso compounds; (3) nitrosating conditions are enhanced upon ingestion of additional nitrate, nitrite,

or nitrosatable compounds; and (4) some N-nitroso compounds are known carcinogens.

The U.S. EPA Integrated Risk Information System does not include a carcinogenicity evaluation for

nitrate or nitrite.

See Chapters 2 and 3 for more information on health effects of nitrate and nitrite.

5 NITRATE AND NITRITE

1. PUBLIC HEALTH STATEMENT

HOW CAN NITRATE AND NITRITE AFFECT CHILDREN?

This section discusses potential health effects of nitrate and nitrite exposure in humans from when they’re

first conceived to 18 years of age.

Children can experience the same effects as adults from overexposure to nitrate and/or nitrite. Young

infants (<6 months of age) who were fed formula prepared using nitrate-contaminated drinking water

sources appear to be particularly sensitive to the effects of nitrate on hemoglobin (i.e.,

methemoglobinemia), although bacterial infections may have been at least partially responsible for

increased sensitivity in these infants. It is not known whether nitrate or nitrite can cause birth defects.

Results of some studies suggest that ingestion of relatively high levels of nitrate or nitrite could cause

developmental effects, but other studies found no evidence for nitrate- or nitrite-related developmental

effects.

HOW CAN FAMILIES REDUCE THE RISK OF OVEREXPOSURE TO NITRATE AND

NITRITE?

If your doctor finds that you have been exposed to significant amounts of nitrate and/or nitrite, ask

whether your children might also be exposed. Your doctor might need to ask your state health department

to investigate. You may also contact the state or local health department with health concerns.

Much of the diet contains food with nitrate and possibly small amounts of nitrite. Some processed food

contains nitrate and/or nitrite as preservative. If you think that you are getting too much nitrate or nitrite

in your diet, consider eating less of those foods that contain high levels of nitrate or nitrite. This

consideration is particularly relevant to infants and small children. Don’t drink water containing levels of

nitrate or nitrite higher than guideline levels for drinking water.

ARE THERE MEDICAL TESTS TO DETERMINE WHETHER I HAVE BEEN OVEREXPOSED

TO NITRATE AND/OR NITRITE?

Methods are available to detect nitrate and nitrite in plasma and urine; however, these are usually not

available at a doctor’s office and are not clinically useful.

Routine blood tests are available to detect a condition known as methemoglobinemia, which is caused by

the presence of higher-than-normal levels of a form of hemoglobin. However, these tests cannot tell

6 NITRATE AND NITRITE

1. PUBLIC HEALTH STATEMENT

whether the high methemoglobin levels were caused by nitrate and nitrite or by some other substance or

disease.

For more information on the different substances formed by nitrate and nitrite breakdown and tests to

detect these substances in the body, see Chapters 3 and 7.

WHAT RECOMMENDATIONS HAS THE FEDERAL GOVERNMENT MADE TO PROTECT

HUMAN HEALTH?

The federal government develops regulations and recommendations to protect public health. Regulations

can be enforced by law. Federal agencies that develop regulations for toxic substances include the

Environmental Protection Agency (EPA), the Occupational Safety and Health Administration (OSHA),

and the Food and Drug Administration (FDA). Recommendations provide valuable guidelines to protect

public health but are not enforceable by law. Federal organizations that develop recommendations for

toxic substances include the Agency for Toxic Substances and Disease Registry (ATSDR) and the

National Institute for Occupational Safety and Health (NIOSH).

Regulations and recommendations can be expressed as “not-to-exceed” levels; that is, levels of a toxic

substance in air, water, soil, or food that do not exceed a critical value usually based on levels that affect

animals; levels are then adjusted to help protect humans. Sometimes these not-to-exceed levels differ

among federal organizations. Different organizations use different exposure times (e.g., an 8-hour

workday or a 24-hour day), different animal studies, or emphasize some factors over others, depending on

their mission.

Recommendations and regulations are also updated periodically as more information becomes available.

For the most current information, check with the federal agency or organization that issued the regulation

or recommendation.

The EPA lists maximum contaminant levels (MCL) and maximum contaminant level goals (MCLG) of

10 mg/L (or ppm) for nitrate (as nitrate-nitrogen; ~44 mg nitrate/L) and 1 mg/L (or ppm) for nitrite (as

nitrite-nitrogen; ~3.3 mg nitrite/L) in the 2012 Edition of the Drinking Water Standards and Health

Advisories. The FDA lists 10 mg/L nitrate (as nitrogen; ~44 mg nitrate/L), 1 mg/L nitrite (as nitrogen;

~3.3 mg nitrite/L), and 10 mg/L total nitrate and nitrite (as nitrogen) as allowable levels in bottled water.

OSHA has not set a legal limit for nitrate or nitrite in air. NIOSH has not set a recommended limit for

nitrate or nitrite in air.

7 NITRATE AND NITRITE

1. PUBLIC HEALTH STATEMENT

WHERE CAN I GET MORE INFORMATION?

If you have any questions or concerns, please contact your community or state health or environmental

quality department, or contact ATSDR at the address and phone number below. You may also contact

your doctor if experiencing adverse health effects or for medical concerns or questions. ATSDR can also

provide publicly available information regarding medical specialists with expertise and experience

recognizing, evaluating, treating, and managing patients exposed to hazardous substances.

• Call the toll-free information and technical assistance number at

1-800-CDCINFO (1-800-232-4636) or

• Write to:

Agency for Toxic Substances and Disease Registry

Division of Toxicology and Human Health Sciences

1600 Clifton Road NE

Mailstop F-57

Atlanta, GA 30329-4027

Toxicological profiles and other information are available on ATSDR’s web site:

http://www.atsdr.cdc.gov.

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2. RELEVANCE TO PUBLIC HEALTH

2.1 BACKGROUND AND ENVIRONMENTAL EXPOSURES TO NITRATE AND NITRITE IN

THE UNITED STATES

Nitrate and nitrite can be organic or inorganic chemicals depending on their chemical structures. This

profile pertains to inorganic nitrate and nitrite, specifically the nitrate anion and the nitrite anion. Nitrate

and nitrite occur naturally in the environment as part of the nitrogen cycle, and are produced both

endogenously and exogenously. Ammonia-oxidizing bacteria convert ammonia into nitrite; nitrite-

oxidizing bacteria convert nitrite into nitrate in aerobic environments. This two-stage process is known as

nitrification. Main sources of ammonia in the environment are decaying organic matter and human and

animal wastes. Nitrification, atmospheric fixation, and nitrogen fertilizers contribute to nitrite and nitrate

concentrations in the environment. In nature, salts of nitrate and nitrite completely dissociate and these

anions typically exist as ionic species. In the environment, nitrite is readily oxidized to nitrate. Nitrate is

generally stable in the environment; however, it may be reduced through biotic (living systems; plants,

microbes, etc.) processes to nitrite under anerobic conditions.

Nitrate and nitrite are ubiquitous in the environment and people are exposed to them primarily through the

ingestion of food and drinking water. Significant uptake of nitrate and nitrite occurs in all varieties of

plants; internal storage of nitrate (rather than metabolic conversion to ammonium and amino acids) can

occur in some plants, especially leafy vegetables such as lettuce and spinach. Vegetables account for

about 80% of the nitrate in a typical human diet. Nitrate and nitrite are also produced in the body as part

of the natural nitrate-nitrite-nitric oxide cycle.

2.2 SUMMARY OF HEALTH EFFECTS

Hematological Effects. In humans, ingested nitrate is nearly completely absorbed into the blood

from the small intestine and approximately 25% of the plasma nitrate enters the salivary glands where it is

secreted in saliva. As much as 20% of salivary nitrate (5% of ingested nitrate) is reduced to nitrite by

bacterial reductases in the mouth. This in vivo reduction of nitrate accounts for 80–85% of the body’s

nitrite and most of the rest comes from nitrite-containing food sources. Nitrite in the blood can react with

ferrous (Fe

2+

) hemoglobin (which transports oxygen) to form ferric (Fe

3+

) hemoglobin (methemoglobin, a

poor transporter of oxygen), and nitric oxide (which can also bind to deoxyhemoglobin) and nitrate.

10 NITRATE AND NITRITE

2. RELEVANCE TO PUBLIC HEALTH

Methemoglobinemia is a condition in which increased methemoglobin as a percentage of total

hemoglobin results in the expression of clinical signs that increase in severity with increasing percent

methemoglobin. In normal healthy individuals, methemoglobin levels are <1% of total hemoglobin.

Discoloration of the skin (cyanosis) is often observed at methemoglobin levels in the range of 3–15%;

most patients tolerate methemoglobin levels <10%. Tachycardia, weakness, and other signs of tissue

hypoxia may be observed at 10–20% methemoglobin levels. Symptoms involving the central nervous

system (e.g., headache, dizziness, fatigue) and dyspnea and nausea appear at >20% methemoglobin; the

severity of symptoms increases with increasing methemoglobin level. High risk of mortality occurs at

levels >70% methemoglobin. It should be noted that a patient with comorbidities that decrease oxygen

transport or delivery may develop moderate to severe symptoms at much lower methemoglobin levels

than a previously healthy patient. Furthermore, due to differences in the oxygen carrying capacity

between fetal hemoglobin and adult hemoglobin (which replaces fetal hemoglobin during the first year of

postnatal life), cyanosis in young infants with mostly fetal hemoglobin may not be detected at

methemoglobin levels eliciting clinical cyanosis in older infants with mostly adult hemoglobin.

As early as the mid-1900s, methemoglobinemia was reported in infants exposed to relatively large

amounts of nitrate from drinking water sources. Available data identify young bottle-fed infants (1–

3 months of age) as a subpopulation that is particularly susceptible to nitrate-induced

methemoglobinemia, especially those consuming formula prepared from drinking water sources

containing nitrate in excess of 10 mg nitrate-nitrogen/L (44 mg nitrate/L). Subsequent reports provide

additional evidence of associations between ingestion of nitrate from drinking water sources and elevated

methemoglobin levels in infants. Cyanosis and even death occurred in some of the reported cases.

Limited data are available regarding administration of controlled amounts of nitrate and methemoglobin

levels. A study reported methemoglobin levels as high as 5.3% of total hemoglobin in a group of four

infants (ages 11 days to 11 months) administered sodium nitrate in the formula for 2–18 days at a

concentration resulting in a dose of 50 mg nitrate/kg/day and as high as 7.5% in another group of four

infants (ages 2 days to 6 months) similarly treated at 100 mg nitrate/kg/day for 6–9 days. A study

reported methemoglobin levels as high as 6.9–15.9% among three infants (ages not specified) fed formula

prepared using water containing 108 mg nitrate/L.

Young children are somewhat less sensitive than infants to nitrate-induced methemoglobinemia. A study

evaluated methemoglobin levels in 102 children 1–8 years of age. Sixty-four of the children lived in

households where drinking water contained 22–111 mg nitrate-nitrogen/L (97–488 mg nitrate/L);

11 NITRATE AND NITRITE

2. RELEVANCE TO PUBLIC HEALTH

drinking water sources for the other 38 children (controls) contained <10 mg nitrate-nitrogen/L (<44 mg

nitrate/L). Methemoglobin measured 1.0–1.36% in those children 1–4 years of age and appeared to

increase with increasing nitrate intake, although there was no statistically significant change.

Methemoglobin levels in those children 5–8 years of age averaged 0.9–0.95%, independent of level of

exposure to nitrate.

Endocrine Effects. There is some evidence for nitrate-induced effects on thyroid function and

development. Nitrate is one of the substances that act as dose-dependent competitive inhibitors of the

sodium iodide symporter (NIS), which mediates the uptake of iodine by the thyroid. Sufficiently

decreased iodine uptake by the thyroid may result in decreased production of thyroid hormones

triiodothyronine (T3) and thyroxine (T4). Decreased thyroid hormone production causes increased

release of thyroid stimulating hormone (TSH) from the anterior pituitary gland leading to increased

uptake of iodine by the thyroid gland. Sufficiently inhibited uptake of iodine by the thyroid could result

in effects associated with thyroid dysfunction (e.g., hypothyroidism). Concern for nitrate-induced effects

on thyroid function has prompted scientists to perform studies designed to assess thyroid function relative

to drinking water and/or dietary nitrate levels. Some human studies provide suggestive evidence that

elevated levels of nitrate in drinking water and/or nitrate-rich diets may be associated with signs of

thyroid dysfunction. However, limitations of these studies include lack of individual dose-response data,

quantification of iodine intake, and control for other substances that may affect the thyroid; one study

relied on self-reported thyroid status and self-reported dietary nitrate intake. A study found no evidence

for nitrate-induced effects on thyroid function in adults ingesting sodium nitrate for 38 days at

15 mg/kg/day (which is 3 times the maximum acceptable daily intake of 5 mg sodium nitrate/kg/day set

by the Joint Expert Committee on Food Additives [JECFA] of the Food and Agriculture Organization of

the United Nations/World Health Organization and the European Commission's Scientific Committee on

Food).

Thyroid status has been assessed to some extent in animals consuming drinking water or food to which

nitrate salts had been added. There were no clinical signs of hypothyroidism or effects on serum T3 or T4

levels in adult Beagles or their puppies during exposure of the breeding dogs to sodium nitrate in the

drinking water for 1 year at concentrations in the range of 300–1,000 ppm (equivalent to 219–730 mg

nitrate/L). Decreased thyroidal

131

iodine uptake was noted in rats given food containing 0.5–2.5%

potassium nitrate (approximately 3,000–15,000 mg nitrate/kg food). Significantly increased uptake of

thyroidal

131

iodine; decreased serum T3, T4, and TSH levels; increased thyroid weight; and follicular

hyperplasia were noted in female Wistar rats administered sodium nitrate in the drinking water for

12 NITRATE AND NITRITE

2. RELEVANCE TO PUBLIC HEALTH

30 weeks at concentrations ≥250 mg/L (≥182 mg nitrate/L). In another study, significantly increased

serum T3 (34–44% lower than controls), increased thyroid weight (45–77% greater than controls), and

histopathologic thyroid lesions (glandular hypertrophy accompanied by vacuolization, increased colloidal

volume of the follicles, and flattened follicular epithelium) were observed in male Wistar rats receiving

drinking water for 5 months to which potassium nitrate had been added at concentrations ≥100 mg/L.

Significantly decreased serum T3 and T4 levels were observed in all groups of weanling male Wistar rats

with intakes in the range of 8.7–47.4 mg sodium nitrate/kg/day (equivalent to 6.4–34.6 mg

nitrate/kg/day). At doses ≥15.8 mg nitrate/kg/day, significantly increased serum TSH was also noted.

Groups of similarly-treated young adult male Wistar rats exhibited significantly decreased T3 and T4

levels and increased serum TSH at doses ≥15.8 mg nitrate/kg/day. Significantly increased thyroid gland

weight, increased TSH, decreased serum T3 and T4 levels, and decreased thyroid peroxidase activity

were reported in rats administered 3% potassium nitrate in the diet.

In a 13-week study of rats receiving drinking water to which potassium nitrite had been added, doses in

the range of 8.9–241.7 mg/kg/day (4.8–130.5 mg nitrite/kg/day), oral doses ≥13.3 mg nitrite/kg/day

(males) and ≥61.8 mg nitrite/kg/day (females) resulted in hypertrophy in the zona glomerulosa of the

adrenal gland. The effect on the adrenal gland was not observed in untreated controls or potassium

chloride controls. Similar results were obtained at estimated doses of 105.1 mg nitrite/kg/day (males) and

130.1 mg nitrite/kg/day (females) in a subsequent similarly-designed study. Results of a subsequent

study indicate that the effect on the adrenal gland of the rat is a physiological adaptation to repeated

episodes of hypotension caused by nitrite.

Metabolic Effects. Possible associations between nitrate and/or nitrite in drinking water and/or food

sources and risk of type 1 diabetes have been investigated in a number of case-control studies. Some

studies found no significant risk for childhood type 1 diabetes. In one case-control study that included

estimates of nitrate intake based on food frequency questionnaire results for children 0–14 years of age, a

significantly increased risk of type 1 diabetes was noted for children at the high end (≥75

th

percentile) of

estimated nitrate intake compared to those at the low end (<25

th

percentile). In an ecological study of

type 1 diabetes incidence rates by county in Colorado, children (<18 years of age) in counties with water

nitrate levels in the range of 0.77–8.2 mg/L had a significantly increased risk of type1 diabetes compared

to those in counties with water nitrate levels in the range of 0.0–0.084 mg/L. In another ecological study,

a significantly increased association between nitrate in drinking water (highest tertile versus lowest

tertile) and incidence of childhood type 1 diabetes was reported for children diagnosed between 1978 and

1994 in the Yorkshire Regional Health Authority in England. In a subsequent ecological study that

13 NITRATE AND NITRITE

2. RELEVANCE TO PUBLIC HEALTH

included portions of England and Scotland, the Drinking Water Inspectorate found no evidence for an

association between nitrate in the drinking water and incidence of childhood type 1 diabetes.

Cardiovascular Effects. Cardiovascular health is an end point of concern for nitrate and nitrite

because some nitrate is converted to nitrite in the body. Nitrite is a smooth muscle relaxant that can cause

hypotension and plasma nitrite is involved in the oxidation of hemoglobin to methemoglobin, which is

associated with hypotension, rapid pulse, and rapid breathing at high enough concentrations. Ingestion of

nitrite (from potassium nitrite or sodium nitrite sources) has been associated with severe

methemoglobinemia in adults and children; in some of these cases, symptoms included hypotension

and/or tachycardia. These cases were the result of consumption of food or drink that contained unusually

high levels of nitrite via contamination, inadvertent use of sodium nitrite instead of table salt, or ingestion

of a single sodium nitrite tablet (1 g; equivalent to 667 mg nitrite).

In a hospital-based study in Colorado that included 226 cases of hypertension among patients living in

areas where drinking water contained nitrate at concentrations ranging from 19 to 125 ppm (mean

52 ppm) and 261 cases from patients living in areas without nitrate in the drinking water, the mean annual

incidence rate for hypertension in the nitrate-exposed patients was only 5.9/1,000 compared to

7.9/1,000 for the control patients. However, the nitrate-exposed patients exhibited an earlier mean age at

hospitalization for hypertension (58.5 versus 65.2 years for controls); the toxicological significance of this

finding is uncertain because the incidence rate for hypertension was higher among control patients than

among patients exposed to nitrate in the drinking water.

In a study designed to evaluate the oral bioavailability of sodium nitrite in healthy volunteers (seven

females and two males; mean age 22.9 years), ingestion of 0.06 sodium nitrite per mmol hemoglobin

(~1.5–1.8 mg nitrite/kg) resulted in an average heart rate increase from 55 to 63 beats per minute (bpm)

and average mean arterial blood pressure decrease from 78 to 70 mmHg. At a higher intake (~2.9–3.6 mg

nitrite/kg), the average heart rate increased from 57 to 67 bpm and the average mean arterial blood

pressure decreased from 80 to 69 mmHg. The maximum effects on heart rate and blood pressure

occurred between 15 and 20 minutes following ingestion; heart rate and blood pressure returned to near-

baseline levels approximately 2 hours following ingestion at the low dose, but the effects had not returned

to baseline at 4 hours following ingestion at the high dose. The blood pressure-lowering effect of short-

term dietary supplementation of inorganic nitrate appears to be beneficial; however, there is some

uncertainty regarding potential health benefits of long-term nitrate supplementation to treat cardiovascular

diseases.

14 NITRATE AND NITRITE

2. RELEVANCE TO PUBLIC HEALTH

Gastrointestinal Effects. Ingestion of nitrite (from potassium nitrite or sodium nitrite sources) has

been associated with severe methemoglobinemia in adults and children; in many of these cases,

symptoms included abdominal cramps and vomiting. These cases were the result of consumption of food

or drink that contained unusually high levels of nitrite via contamination, inadvertent use of sodium nitrite

instead of table salt, or ingestion of a single sodium nitrite tablet (667 mg nitrite). In a study designed to

evaluate the oral bioavailability of sodium nitrite in healthy volunteers (seven females and two males;

mean age 22.9 years), one subject became nauseous and vomited within 20 minutes following ingestion

of 0.12 mmol sodium nitrite per mmol hemoglobin (~3.2 mg nitrite/kg); another subject reported nausea

within 30 minutes following ingestion of 0.12 mmol sodium nitrite per mmol hemoglobin (~2.9 mg

nitrite/kg).

Epithelial hyperplasia was noted in the forestomach of male and female B6C3F1 mice provided sodium

nitrite in the drinking water for 14 weeks at a concentrations resulting in estimated doses of 663.3 and

824.1 mg nitrite/kg/day, respectively); the no-observed-adverse-effect levels (NOAELs) for these lesions

in the males and females were 435.5 and 562.8 mg nitrite/kg/day, respectively. Similar results were noted

for male and female F344/N rats and male B6C3F1 mice treated for 104–105 weeks at estimated doses of

87.1, 100.5, and 147.4 mg nitrite/kg/day, respectively; the NOAELs for these lesions in the male and

female rats and male mice were 46.9, 53.6, and 80.4 mg nitrite/kg/day, respectively. Sodium nitrite

treatment did not result in increased incidences of forestomach lesions in other groups of male F344 rats

provided sodium nitrite in the drinking water at 2,000 mg/L (estimated dose of 208.4 mg nitrite/kg/day)

for 35 weeks or 51 weeks.

Neurological Effects. Neurological effects have been reported in humans and animals following

ingestion of nitrite; however, these effects may be secondary to nitrite-induced reductions in oxygen-

carrying capacity. Ingestion of nitrite (from potassium nitrite or sodium nitrite sources) has been

associated with severe methemoglobinemia in adults and children; in many of these cases, clinical signs

included dizziness, loss of consciousness, and/or convulsions. These cases were the result of

consumption of food or drink that contained unusually high levels of nitrite via contamination,

inadvertent use of sodium nitrite instead of table salt, or ingestion of a single sodium nitrite tablet

(667 mg nitrite).

Headache was induced in a male subject following consumption of a 10 mg sodium nitrite solution.

Headaches were induced in 8 out of 13 such tests. In a study designed to evaluate the oral bioavailability

15 NITRATE AND NITRITE

2. RELEVANCE TO PUBLIC HEALTH

of sodium nitrite in healthy volunteers (seven females and two males; mean age 22.9 years), headache

was reported by four out of the nine people after ingestion of 0.12 mmol sodium nitrite per mmol

hemoglobin (~2.9–3.6 mg nitrite/kg) and by four of nine subjects after ingestion of 0.06 mmol sodium

nitrite per mmol hemoglobin (~1.5–1.8 mg nitrite/kg).

Abnormalities in electroencephalograms (EEGs) were reported in male albino rats provided sodium nitrite

in the drinking water for 2 months at concentrations resulting in ≥9.38 mg nitrite/kg/day. The abnormal

readings persisted during up to 4.5 months following cessation of exposure to sodium nitrite. At the

highest dose (187.6 mg nitrite/kg/day), rats exhibited clinical signs of sedation and became motionless

during periods of electrical outbursts. Increased aggressive behavior was observed in male C57B1 mice

provided sodium nitrite in the drinking water at 1,000 mg/L for up to 13 weeks postweaning. The mice

had also been exposed via their parents during mating and their mothers during gestation and lactation.

Significantly reduced motor activity was reported in male mice provided sodium nitrite in the drinking

water. Sodium nitrite levels tested ranged from 100 to 2,000 mg/L; however, the study report did not

include specific information regarding the exposure levels that resulted in reduced motor activity.

Developmental Effects. A number of studies evaluated possible associations between

developmental end points and exposure to nitrate. The results provide some evidence of nitrate-related

developmental effects. The results are not adequate for quantitative risk assessment because estimations

of nitrate intakes were typically based on measurements of nitrate levels in drinking water sources at

selected time points and self-reported estimates of water consumption, possible confounding by other

potential toxicants was not evaluated, and most studies did not account for dietary nitrate or nitrite intake,

which is typically the major source of ingested nitrate and nitrite. Some studies reported significant

associations between selected developmental end points and nitrate in drinking water sources. One study

reported increased risk of intercalary limb defect associated with estimated total nitrite intake. Other

studies found no evidence of associations between nitrate and risk of developmental effects.

Cancer. Numerous case-control and cohort studies of carcinogenicity of ingested nitrate and nitrite in

humans have been reported. Many ecological studies have also been reported; however, interpretation of

outcomes of these studies is more uncertain because of various factors that contribute to ecologic bias

(group-based associations between exposure and cancer outcomes may not apply to individuals). In

general, outcomes of case-control and cohort studies have found no or weak associations between

exposure to nitrate and cancer in humans, with stronger associations with exposures to nitrite or intake of

high nitrite foods such as cured meat. Mechanistically, this outcome is consistent with nitrite being an

16 NITRATE AND NITRITE

2. RELEVANCE TO PUBLIC HEALTH

intermediate in the cancer mode of action of nitrate (see Section 3.5.2). This is further supported by

studies that have found interactions between cancer risk attributed to nitrite and exposure to antioxidants.

Uncertainties in estimates of cancer risk from exposure to nitrate or nitrite include those typical of

epidemiological studies in general: uncertainties in estimation of exposure (e.g., estimating long-term

dietary intakes from food frequency questionnaires or levels in public water supplies [PWS]), exposure

misclassification of individual outcomes (e.g., assigning group-level exposure estimates to individuals),

and adequacy of controlling for confounders (e.g., other factors contributing to the cancer). One

potentially important class of confounders is antioxidants that can influence the degree of nitrosation of

dietary amines and, thereby, the cancer risk from exposure to nitrate or nitrite.

The strongest and most consistent evidence of a carcinogenic role for nitrite is from studies of

gastrointestinal cancers and, in particular, gastric cancer. In general, these studies found significant

positive trends for cancer risk (risk increases with increasing intake), and three studies found elevated

cancer risk. Relative risks (RRs) were 1.71 (95% confidence interval [CI]: 1.24, 2.37) at a nitrite intake

of 1 mg/day and 2.5 (95% CI: 1.4, 4.3) at nitrite intakes ≥6 mg/day. Risk was modified by dietary

vitamin E and folate intake, with increased risk in association with higher nitrate/vitamin E or folate

ratios. Associations between exposure to nitrate or nitrite and colorectal cancer have been studied in

cohort and case-control studies and results are less consistent than for gastric cancer. Two studies found

elevated risk: 1.16 (95% CI: 1.04, 1.30) for colon cancer at nitrate-nitrogen levels >0.6 mg/L (>2.65 mg

nitrate/L drinking water; 1.5 (95% CI: 1.0, 2.1) for colon cancer at a dietary nitrite intake >1.26 mg/day,

and 1.7 (95% CI: 1.1, 2.5) at a dietary nitrite intake >1.26 mg/day. Risks were higher in populations

exposed to drinking water that had a calcium level >34.6 mg/L (odds ratio [OR] 1.37, 95% CI: 1.11; 1.69)

for nitrate <2.65 mg/L; or in populations exposed to nitrate in drinking water at levels >5 mg/L in

combination with a low vitamin C intake (OR 2.0, 95% CI: 1.2, 3.3).

Results have been mixed for other types of cancer. Some case-control or cohort studies found

associations between exposure to nitrite (or foods high in nitrite such as cured meat) and brain cancer in

children and adults, breast cancer, kidney cancer, testicular cancer, and non-Hodgkin’s lymphoma. Of

these studies, the highest risks were reported for brain cancers. Two case-control studies found elevated

relative risk of brain cancer in children in association with maternal exposure: 3.0 (95% CI: 1.2, 7.9) for

nitrite intakes >3.0 mg/day and 5.7 (95% CI: 1.2, 27.2) for astroglial tumors at drinking water exposures

≥5 mg/L. In general, case-control and cohort studies of cancers of larynx, liver, lung, mouth, pancreas,

and pharynx have found no consistent associations with exposures to nitrate or nitrite.

17 NITRATE AND NITRITE

2. RELEVANCE TO PUBLIC HEALTH

The potential carcinogenicity of nitrate has been investigated in several animal studies that employed the

oral exposure route. Studies in which negative results were reported include MCR-derived rats

(15/sex/group) provided 5,000 mg sodium nitrate/L (3,650 mg nitrate/L) in the drinking water for

84 weeks and sacrificed 20 weeks later, male white rats provided 4,000 mg sodium nitrate in the drinking

water for 273 days and sacrificed at 10 months, strain A male mice (n=40) provided 12,300 mg sodium

nitrate/L in the drinking water for 25 weeks and sacrificed 13 weeks later, female NMRI mice provided

1,000 mg calcium nitrate/L in the drinking water for 18 months, Fischer 344 rats (50/sex/group) fed diet

containing up to 5% sodium nitrate (1,517–1,730 mg nitrate/kg/day) for 2 years, and ICR mice

(10/sex/group) fed diets containing up to 5% sodium nitrate for 2 years. In one study, some groups of

male white rats were treated with drinking water containing 0.05% N-butyl-N-(4-hydroxybutyl)-

nitrosamine (BBNA, an inducer of urinary bladder cancer in laboratory animals) for 30 days, either alone

or followed by 4,000 mg sodium nitrate/L drinking water for 273 days. The group treated with BBNA

followed by sodium nitrate exhibited significantly increased incidence of urinary bladder carcinoma

(6/20 rats versus 1/18 rats treated with 0.05% BBNA only. These results indicate that sodium nitrate may

have promoted BBNA-induced bladder tumors.

The potential carcinogenicity of ingested nitrite has been investigated in numerous animal studies. Nitrite

treatment alone did not result in increased incidences of tumors in most studies. There was no evidence

of sodium nitrite-induced forestomach neoplasms among male and female F344/N rats provided sodium

nitrite in the drinking water for 2 years at concentrations of 750, 1,500, or 3,000 ppm (average doses in

the range of 35–150 mg sodium nitrite/kg/day or 23.3–100 mg nitrite/kg/day). Although the mid-dose

group of female rats exhibited a significantly increased incidence of mammary gland fibroadenoma, the

incidence in the high-dose group was not significantly different from that of controls; based on this

finding and the high historical background incidence of mammary gland fibroadenomas, the incidence in

the mid-dose group was not considered treatment related. Significantly decreased incidences of

mononuclear cell leukemia were observed in mid- and high-dose male and female rats. It was speculated

that increased methemoglobin concentrations may have played a role in the decreased incidences of

mononuclear cell leukemia. Significantly increased incidence of fibroma of the subcutis was noted in

mid-dose male rats; however, several factors (the incidence only slightly exceeded the historical range of

NTP controls, there was a lack of a dose-response characteristic, combined incidences of fibroma or

fibrosarcoma were within the historical range for NTP controls, and fibromas and fibrosarcomas are

common neoplasms in the skin of F344/N rats) suggested that the fibroma was not related to sodium

nitrite exposure. It was concluded that there was "no evidence of carcinogenic activity" of sodium nitrite

in the male or female F344/N rats under the conditions of the study.

18 NITRATE AND NITRITE

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In a similarly-designed study of B6C3F1 mice provided sodium nitrite in the drinking water (average

doses ranging from 45 to 220 mg sodium nitrite/kg/day or 30–146.7 mg nitrite/kg/day), female mice

exhibited a significantly positive trend for increased incidence of forestomach squamous cell papilloma or

carcinoma (combined) and the incidence in the high-dose female mice exceeded the historical range for

NTP controls; however, based on concurrent controls, incidences of squamous cell adenoma (1/50, 0/50,

1/50, and 3/50 for controls, 750, 1,500, and 3,000 ppm groups, respectively), squamous cell carcinoma

(0/50, 0/50, 0/50, and 2/50 for controls, 750, 1,500, and 3,000 ppm groups, respectively), and squamous

cell adenoma or carcinoma (1/50, 0/50, 1/50, and 5/50 for controls, 750, 1,500, and 3,000 ppm groups,

respectively) were not statistically significantly increased for any sodium nitrite exposure group. The

positive trend for incidences of forestomach squamous cell papilloma or carcinoma (combined) in the

female B6C3F1 mice was considered to provide "equivocal evidence of carcinogenic activity" of sodium

nitrite; there was "no evidence of carcinogenic activity" in the male B6C3F1 mice under the conditions of

the study. Incidences of alveolar/bronchiolar adenoma or carcinoma (combined) in sodium nitrite-

exposed groups of female mice were slightly greater than that of controls (incidences of 1/50, 6/50, 5/50,

and 6/50 for controls, 750, 1,500, and 3,000 ppm groups, respectively); however, incidences were within

that of historical NTP controls. Because the incidences did not exhibit exposure concentration-response

characteristics and were not accompanied by increased incidences of preneoplastic lesions, the study

authors did not consider them to be sodium nitrite exposure-related effects. Significantly increased

incidence of fibrosarcoma of the subcutis was noted in mid-dose female mice (incidences of 0/50, 5/50,

1/50, and 2/50 for 0, 750, 1,500, and 3,000 ppm groups, respectively) and exceeded the historical range

for controls; however, lack of exposure concentration-response characteristics and the fact that combined

incidence of fibroma or fibrosarcoma (0/50, 5/50, 1/50, and 3/50 for 0, 750, 1,500, and 3,000 ppm groups,

respectively) were within the historical range for controls suggest that these neoplasms were not related to

sodium nitrite exposure.

In two other studies of male and female F344 rats, addition of sodium nitrite to the drinking water at

concentrations as high as 2,000–3,000 ppm for up to 2 years did not result in significant increases in

tumor incidences at any site. Conversely, incidences of mononuclear cell leukemia were significantly

lower in the nitrite-treated groups relative to controls. In a 26-month study of male and female Sprague-

Dawley rats provided drinking water to which up to 2,000 ppm sodium nitrite was added, the study author

reported increased incidence of lymphomas, but not other types of tumors; however, two studies noted

that a working group sponsored by the U.S. FDA reevaluated the histology and did not confirm the results

of another study. A study reported that the working group considered the incidences of lymphomas to be

19 NITRATE AND NITRITE

2. RELEVANCE TO PUBLIC HEALTH

similar to those arising spontaneously in Sprague-Dawley rats. Increased incidences of total tumors and

lymphoreticular tumors were reported in rats fed diet to which sodium nitrite was added at 1,000 ppm

(total tumors: 58/96 versus 28/156 controls; lymphoreticular tumors 26/96 versus 9/156 controls); the

results were reported for F1 and F2 offspring that had been exposed via their mothers during gestation

and lactation and directly from the diet thereafter. In a 96-week study, a significantly increased incidence

of benign liver tumors among male CBA mice administered drinking water to which sodium nitrite was

added at a concentration resulting in author-estimated total dose of 1,600 mg sodium nitrite/mouse

compared to a group of untreated controls; however, there was no apparent sodium nitrite treatment-

related effect at a higher estimated dose (2,000 mg sodium nitrite/mouse).

Significantly increased incidences of forestomach squamous papillomas were reported for male and

female MRC Wistar rats provided drinking water to which sodium nitrite was added at 3,000 ppm on

5 days/week for life (5/22 males and 3/23 females versus 2/47 control males and 0/44 control females).

Dose-related decreases in time of onset and incidence of lymphomas, mononuclear cell leukemia, and

testicular interstitial-cell tumors were reported for male and female F344 rats administered reduced-

protein diet to which sodium nitrite was added for up to 115 weeks, compared to a group of controls

receiving reduced-protein only diet. There was no evidence of increased tumor incidences in male or

female ICR mice provided sodium nitrite in the drinking water for up to 109 weeks at concentrations as

high as 0.5% (5,000 ppm sodium nitrite), or in male or female Swiss mice or their offspring following a

single gavage administration of 10 mg/kg nitrite and subsequent exposure to 0.1% sodium nitrite

(1,000 ppm) in the drinking water during gestation days 15–21; terminal sacrifices occurred 10 months

following the initiation of treatment. There was no evidence of treatment-related effects on incidences of

nervous system tumors among male and female VM mice (susceptible to spontaneous development of

cerebral gliomas) provided drinking water to which sodium nitrite was added at 0.2% (2,000 ppm) from

weaning for a lifetime and others exposed via their mothers during gestation and lactation as well.

The potential carcinogenicity of combined exposure to sodium nitrite and selected nitrosatable substances

(oral exposures via combinations of drinking water, diet, and/or gavage dosing) has been well-studied in

laboratory animals. Many of the studies included sodium nitrite-only treatment groups for which there

was no evidence of sodium nitrite-induced carcinogenicity. However, one study reported significantly

increased incidence of hepatocellular neoplasms in female (but not male) F344 rats administered diet to

which sodium nitrite was added at 2,000 ppm for 2 years; significantly decreased incidence of

mononuclear-cell leukemia was observed as well.

20 NITRATE AND NITRITE

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Significantly increased incidences of selected tumor types were observed in some studies of laboratory

animals that employed coexposure to various amino compounds and sodium nitrite. These results were

typically attributed to in vivo nitrosation of amines by nitrite to produce carcinogenic N-nitrosoamines;

some of the studies did not include sodium nitrite-only treatment groups. Addition of sodium nitrite or

potassium nitrite to the food of rats in three other studies resulted in increased incidences of selected

tumors; analysis of the food revealed the presence of N-nitroso compounds (likely formed by nitrosation

in the presence of nitrite and selected amine compounds in the food), which were considered the probable

principal cause of the tumors. One study reported 30–70% incidences of malignant lymphomas, lung

adenomas, and hepatomas among maternal mice and their offspring following gavage treatment of the

dams with the fungicide, dodecylguanidine acetate, together with 0.05% sodium nitrite; the frequency of

spontaneous tumors in untreated controls was 6%. Dodecylguanidine acetate alone had no effect on

cancer incidence. One study found no significant increase in tumor incidences among male and female

MCR rats provided drinking water comprised of 0.5% nitrilotriacetic acid or iminodiacetic acid and 0.2 or

0.5% sodium nitrite on 5 days/week for a lifetime. There were no signs of treatment-related effects on

incidences of tumors at any site among groups of pregnant Syrian golden hamsters and their offspring fed

diets to which up to 1,000 ppm sodium nitrite and/or up to 1,000 ppm morpholine were added throughout

production of an F2 generation.

Based on available human data, one study determined that there is inadequate evidence for the

carcinogenicity of nitrate in food or drinking water and limited evidence for the carcinogenicity of nitrite

in food (based on association with increased incidence of stomach cancer). Evaluation of available

animal data resulted in the determination that there is inadequate evidence for the carcinogenicity of

nitrate, limited evidence for the carcinogenicity of nitrite per se, and sufficient evidence for the

carcinogenicity of nitrite in combination with amines or amides. The overall conclusions of a study were

that “ingested nitrate and nitrite under conditions that result in endogenous nitrosation is probably

carcinogenic to humans (Group 2A).” One study noted that: (1) the endogenous nitrogen cycle in

humans includes interconversion of nitrate and nitrite; (2) nitrite-derived nitrosating agents produced in

the acid stomach environment can react with nitrosating compounds such as secondary amines and

amides to generate N-nitroso compounds; (3) nitrosating conditions are enhanced upon ingestion of

additional nitrate, nitrite, or nitrosatable compounds; and (4) some N-nitroso compounds are known

carcinogens.

The U.S. EPA does not include a carcinogenicity evaluation for nitrate or nitrite.

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2.3 MINIMAL RISK LEVELS (MRLs)

Estimates of exposure levels posing minimal risk to humans (MRLs) have been established for nitrate and

nitrite. An MRL is defined as an estimate of daily human exposure to a substance that is likely to be

without an appreciable risk of adverse effects (noncarcinogenic) over a specified duration of exposure.

MRLs are derived when reliable and sufficient data exist to identify the target organ(s) of effect or the

most sensitive health effect(s) for a specific duration within a given route of exposure. MRLs are based

on noncancerous health effects only and do not consider carcinogenic effects. MRLs can be derived for

acute, intermediate, and chronic duration exposures for inhalation and oral routes. Appropriate

methodology does not exist to develop MRLs for dermal exposure.

Although methods have been established to derive these levels (Barnes and Dourson 1988; EPA 1990),

uncertainties are associated with these techniques. Furthermore, ATSDR acknowledges additional

uncertainties inherent in the application of the procedures to derive less than lifetime MRLs. As an

example, acute inhalation MRLs may not be protective for health effects that are delayed in development

or are acquired following repeated acute insults, such as hypersensitivity reactions, asthma, or chronic

bronchitis. As these kinds of health effects data become available and methods to assess levels of

significant human exposure improve, these MRLs will be revised.

Inhalation MRLs

Inhalation MRLs were not derived for nitrate or nitrite due to lack of adequate human or animal data.

Limited human data are available. Al-Dabbagh et al. (1986) evaluated mortality rates among a cohort of

1,327 male workers involved in the manufacture of nitrate fertilizer for at least 1 year between 1946 and

1981 for a chemical company in northeast England and found no evidence of an association between

exposure to nitrate dusts and death from all respiratory diseases, ischemic heart disease, or other

circulatory diseases compared to mortality rates for the northern region of England. There was no

evidence of an association between exposure to nitrate dust and death from ischemic heart disease,

cerebrovascular disease, or all circulatory diseases in a census-based (England and Wales) mortality study

of workers involved in the production of nitrate fertilizers (Fraser et al. 1982, 1989). The study included

a cohort of 866 men from the 1961 census and 651 men from the 1971 census. These cohorts were

followed through 1985. Studies of workers in which outcomes are compared to the general population

(e.g., observed versus expected deaths) may be biased by a healthy worker effect, which may lower

estimated risks.

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2. RELEVANCE TO PUBLIC HEALTH

Available animal data are limited to a study in which dogs and sheep were exposed to aerosols of sodium

nitrate for short periods (Sackner et al. 1979). No signs of exposure-related pulmonary effects (e.g.,

respiratory resistance, static lung performance, functional residual capacity) were seen in anesthetized

dogs exposed at 10 mg sodium nitrate/m

3

(2.88 ppm) for 7.5 minutes or anesthetized dogs and conscious

sheep exposed for 4 hours at 5 mg sodium nitrate/m

3

(1.44 ppm). There was no evidence of exposure-

related cardiac effects (pulmonary and systemic arterial pressure, cardiac output, heart rate, arterial blood

gases) in anesthetized dogs or conscious sheep exposed at 5 mg sodium nitrate/m

3

(1.44 ppm) for 4 hours.

Oral MRLs

Nitrate

• An MRL of 4 mg/kg/day has been derived for acute-duration oral exposure (14 days or less) to

nitrate.

• An MRL of 4 mg/kg/day has been derived for intermediate-duration oral exposure (15–364 days)

to nitrate.

• An MRL of 4 mg/kg/day has been derived for chronic-duration oral exposure (365 days or more)

to nitrate.

Results from studies in laboratory animals are not an appropriate basis for oral MRL derivation due to

significant interspecies differences in kinetics of the nitrate-nitrite-nitric oxide pathway.

Most human exposure to nitrate and nitrite is through the diet. Vegetables are the major source of

exposure to nitrate; both nitrate and nitrite may be found in some meat, fish, and dairy products as well.

Estimates of daily dietary intake in the United States range from 103 mg nitrate/day from the normal diet

to as high as 367 mg nitrate/day for a vegetarian diet and from 1.2 mg nitrite/day for the normal and

vegetarian diets to 2.6 mg nitrite/day for a diet high in cured meat (Gangolli et al. 1994). Nitrate-

contaminated drinking water is another source of exposure to nitrate and nitrite; estimated oral intake

from drinking water sources may be as high as 319 mg nitrate/day and 1.2 mg nitrite/day (Gangolli et al.

1994).

Methemoglobinemia is the hallmark effect of overexposure to nitrate or nitrite. Although available

human data are limited by lack of information regarding bacterial contamination in drinking water and its

possible influence on methemoglobin levels, the weight-of-evidence indicates that bottle-fed infants (0–

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2. RELEVANCE TO PUBLIC HEALTH

<3 months of age) ingesting formula prepared using drinking water sources containing >44 mg nitrate/L

are at risk of methemoglobinemia (e.g., Bosch et al. 1950; Walton 1951). Proposed explanations for

increased susceptibility of infants to methemoglobinemia following ingestion of nitrate include:

(1) increased reduction of nitrate to nitrite in the newborn, (2) increased tendency for nitrite-induced

methemoglobin formation by fetal hemoglobin compared to adult hemoglobin, (3) lower levels of

NADH-dependent methemoglobin reductase (the major enzyme responsible for reduction of

methemoglobin to normal hemoglobin; also termed NADH-diaphorase, a soluble form of cytochrome-b5

reductase) in the newborn compared to older infants and adults, and (4) incompletely developed hepatic

microsomal enzyme system in the infant and consequent lower rate of hepatic reduction of circulating

nitrite compared to that of older children and adults. A portion of ingested nitrate is reduced to nitrite by

commensal bacteria in the mouth; however, the acid environment of the normal stomach does not support

the growth of such bacteria and most of the nitrate that reaches the stomach passes to the small intestine

from which it is nearly completely absorbed into the blood. Although Kanady et al. (2012) reported little

or no bacterial conversion of nitrate to nitrite in the saliva of a group of 10 infants during the first

2 postnatal months (considered mainly due to lower numbers of major nitrate-reducing oral bacteria than

adults), a higher pH in the stomach of the newborn may favor growth of nitrate-reducing bacteria,

resulting in increased reduction of nitrate to nitrite and increased plasma methemoglobin. Most

hemoglobin in the newborn is in the form of fetal hemoglobin, which appears to be more readily oxidized

to methemoglobin than adult hemoglobin; fetal hemoglobin is replaced by adult hemoglobin during early

postnatal life. Levels of NADH-dependent methemoglobin reductase (the major enzyme responsible for

reduction of methemoglobin to normal hemoglobin) in the newborn increase approximately 2-fold during

the first 4 months of postnatal life to reach adult levels. During the period of relatively lower

methemoglobin reductase levels, methemoglobin would not be expected to be as readily reduced,

resulting in increased susceptibility to methemoglobinemia. In apparent contrast, Ibrahim et al. (2012)

reported that blood nitrite levels in newborns approximately 1–2 days of age were 35–55% lower than

that of adults. However, one study that evaluated reduction rates of methemoglobin in human adult blood

and cord blood from term newborns estimated methemoglobin half-lives of 162 and 210 minutes,

respectively, indicating that methemoglobin reduction occurs more slowly in newborns than adults

(Power et al. 2007). Although specific mechanisms have not been elucidated, the increased susceptibility

to nitrite-induced methemoglobinemia in infants is well-documented.

Available human data provide some evidence of nitrate-induced developmental effects, limited human

data provide only suggestive evidence that elevated levels of nitrate in drinking water and/or nitrate-rich

diets may be associated with signs of thyroid dysfunction (Aschebrook-Kilfoy et al. 2012; Gatseva and

24 NITRATE AND NITRITE

2. RELEVANCE TO PUBLIC HEALTH

Argirova 2008; Rádiková et al. 2008; Tajtáková et al. 2006; Ward et al. 2010). Significant associations

between nitrate levels in drinking water and risk of childhood type 1 diabetes were reported by some

investigators (Kostraba et al. 1992; Parslow et al. 1997; Virtanen et al. 1994); others found no evidence

for such an association (Casu et al. 2000; Dahlquist et al. 1990; Moltchanova et al. 2004; van Maanen et

al. 2000; Zhao et al. 2001).

Although available data suggest that reports of methemoglobinemia among infants exposed to nitrate

from the drinking water may involve factors other than (or in addition to) nitrate exposure, the study of

Walton (1951) is selected as the principal study and methemoglobinemia is selected as the critical effect

for deriving acute-, intermediate-, and chronic-duration oral MRLs for nitrate to be protective of

particularly sensitive subpopulations (e.g., infants from birth to <3 months of age), including those with

gastrointestinal infections. Following ingestion of relatively large amounts of nitrate by healthy normal

individuals, blood methemoglobin levels increase rapidly, followed by a return to normal within several

hours following intake. Repeated ingestion for intermediate- or chronic-duration time periods would be

expected to result in changes in methemoglobin levels similar to those elicited from a single exposure.

Therefore, the acute-, intermediate- and chronic-duration oral MRL values are equivalent.

There is some evidence that methemoglobinemia in infants drinking formula prepared using drinking

water with relatively high levels of nitrate may be related to bacterial contamination of such water sources

and consequent gastrointestinal disorders, as well as overproduction of nitric oxide due to gastrointestinal

infection and inflammation (Avery 1999; Gupta et al. 1998; L’hirondel and L’hirondel 2002; Yano et al.

1982). On behalf of the World Health Organization (WHO), Fewtrell (2004) performed a literature-based

investigation of methemoglobinemia and drinking water concentrations >50 mg nitrate/L and concluded

that nitrate may be one of a number of cofactors in causing methemoglobinemia. Fewtrell (2004) noted a

paucity of information since the early 1990s linking methemoglobinemia to nitrate in drinking water,

although numerous reports describe water supplies worldwide that contain nitrate at levels >50 mg/L.

The acute-, intermediate-, and chronic-duration oral MRLs were calculated using estimated mean values

for drinking water ingestion rates (Kahn and Stralka 2009) and body weight (EPA 2008) and the

assumption that a drinking water level of 44 mg nitrate/L is a concentration not expected to cause

methemoglobinemia. A NOAEL of 4.33 mg nitrate/kg/day for infants <3 months of age was calculated

based on a drinking water NOAEL of 44 mg nitrate/L and estimations of water intake (0.525 L/day) and

body weight (5.33 kg) (i.e., [44 mg nitrate/L x 0.525 L/day] / 5.33 kg = 4.33 mg nitrate/kg/day). The

dose of 4.33 mg nitrate/kg/day for infants from birth to <3 months of age is selected as the point of

25 NITRATE AND NITRITE

2. RELEVANCE TO PUBLIC HEALTH

departure for deriving acute-, intermediate-, and chronic-duration oral MRLs for nitrate because infants

<3 months of age are particularly sensitive to nitrate-induced adverse effects. Application of a total

uncertainty factor of 1 is justified because the point of departure is a NOAEL for nitrate-induced effects

on methemoglobin in a sensitive human subpopulation (i.e., <3 month-old infants, which in many cases

may have been at increased risk of methemoglobinemia due to microbial contamination and associated

gastrointestinal infection, or which may have had gastroenteritis-associated methemoglobinemia unrelated

to nitrate intake). The resulting acute-, intermediate-, and chronic-duration oral MRLs for nitrate are

4 mg/kg/day and are considered to be highly conservative because they were derived using results from a

particularly sensitive population exhibiting nitrate-induced methemoglobinemia (infants <3 months of

age), and because increased risk of methemoglobinemia in the most sensitive population may have been

due in part to exposure to contaminants other than nitrate in the drinking water (refer to Appendix A for

additional details regarding derivation of oral MRLs for nitrate).

A physiologically based pharmacokinetic (PBPK) model approach to derivation of oral MRLs for nitrate

was initially considered, in which case a methemoglobin level of 10% of total hemoglobin would have

been considered a threshold for nitrate-induced methemoglobinemia in infants. However, although the

model of Zeilmaker et al. (1996, 2010b) simulates the kinetics of methemoglobin formation resulting

from gastrointestinal absorption of nitrate in adult humans, the model is not considered adequate for the

purpose of simulating the kinetics in infants.

Nitrite

• An MRL of 0.1 mg/kg/day has been derived for acute-duration oral exposure (14 days or less) to

nitrite.

• An MRL of 0.1 mg/kg/day has been derived for intermediate-duration oral exposure (15–

364 days) to nitrite.

• An MRL of 0.1 mg/kg/day has been derived for chronic-duration oral exposure (365 days or

more) to nitrite.

Ingestion of nitrite (from potassium nitrite or sodium nitrite sources) has been associated with severe

methemoglobinemia in adults and children (Aquanno et al. 1981; CDC 1997, 2002; Gautami et al. 1995;

Gowans 1990; Greenberg et al. 1945; Kaplan et al. 1990; Ringling et al. 2003; Sevier and Berbatis 1976;

Ten Brink et al. 1982; Walley and Flanagan 1987). In many of these cases, clinical signs included

dizziness, loss of consciousness, and/or convulsions (CDC 1997, 2002; Gautami et al. 1995; Greenberg et

26 NITRATE AND NITRITE

2. RELEVANCE TO PUBLIC HEALTH

al. 1945; Sevier and Berbatis 1976; Ten Brink et al. 1982). All cases were the result of consumption of

food or drink that contained unusually high levels of nitrite via contamination, inadvertent use of sodium

nitrite instead of table salt, or ingestion of a single sodium nitrite tablet (667 mg nitrite). Headache was

induced in a male subject following consumption of a 10 mg sodium nitrite solution (Henderson and

Raskin 1972). Headaches were induced in 8 out of 13 such tests. No information was located regarding

methemoglobin concentrations in infants following oral exposure to nitrite. The ingestion of nitrate

results in the formation of nitrite, which is the moiety responsible for methemoglobinemia. The study of

Walton (1951) is selected as the principal study and methemoglobinemia as the critical effect for deriving

acute-, intermediate-, and chronic-duration oral MRLs for nitrite to be protective of particularly sensitive

subpopulations (e.g., infants from birth to <3 months of age), including those with gastrointestinal

infections. On average, approximately 25% of an ingested dose of nitrate enters the saliva of an adult

where a portion (ca. 20% g/g) is reduced by commensal bacteria to nitrite (i.e., approximately 5% g/g of

ingested nitrate is reduced to nitrite in the saliva of an adult) (Spiegelhalder et al. 1976); most salivary

nitrite is absorbed into the blood in the small intestine. Therefore, the ingestion of nitrate at the oral MRL

of 4 mg/kg/day would be expected to result in the production of 0.2 mg nitrite/kg/day by an adult (i.e.,

0.2 mg nitrite/kg/day is 5% (g/g) of an oral dose of nitrate at the oral MRL of 4 mg/kg/day). Although

quantitative data are lacking regarding the effective blood nitrite level in a young infant from an ingested

dose of nitrate, young infants exhibit increased susceptibility to methemoglobinemia following nitrate

ingestion. Mechanisms responsible for increased susceptibility in infants may include greater reduction

of nitrate to nitrite (which may be higher in the stomach of an infant due to a higher pH), lower levels of

NADH-dependent methemoglobin reductase, slower rate of hepatic reduction of circulating nitrite, and/or

increased tendency for nitrite-induced methemoglobin formation by fetal hemoglobin compared to adult

hemoglobin. To account for increased susceptibility to methemoglobinemia following ingestion of nitrate

by infants, a modifying factor of 2 is applied to the point of departure (0.2 mg nitrite/kg/day ÷ 2 =

0.1 mg/kg/day). The modifying factor assumes that the effective methemoglobin level from a given

intake of nitrate by an infant is twice that of an adult (e.g., approximately 5% of an oral dose of nitrate is

converted to nitrite in the adult; the modifying factor of 2 accounts for up to 10% conversion in the

infant). The resulting acute-, intermediate-, and chronic-duration oral MRLs of 0.1 mg nitrite/kg/day are

considered protective of nitrite-induced methemoglobinemia for particularly sensitive subpopulations

(e.g., infants <3 months of age). The oral MRLs for nitrite are considered to be highly conservative

because they were derived using results from a particularly sensitive population exhibiting nitrate-induced

methemoglobinemia (infants <3 months of age), and because increased risk of methemoglobinemia in the

infants may have been due in part to exposure to contaminants other than nitrate in the drinking water

(refer to Appendix A for additional details regarding derivation of oral MRLs for nitrite).

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Drinking water and dietary sources may contain both nitrate and nitrite; furthermore, as discussed in

Section 3.4, some nitrate is converted to nitrite in the body and nitrite can be converted to nitrate as well.

Overexposure to either nitrate or nitrite can result in elevated methemoglobin levels. At a worldwide

level, WHO (2011a, 2011b) provides guidance for combined exposure to nitrate and nitrite in drinking

water, which states that the sum of the ratios of the concentration of each to its guideline value should not

exceed 1.

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29 NITRATE AND NITRITE

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3.1 INTRODUCTION

The primary purpose of this chapter is to provide public health officials, physicians, toxicologists, and

other interested individuals and groups with an overall perspective on the toxicology of nitrate and nitrite.

It contains descriptions and evaluations of toxicological studies and epidemiological investigations and

provides conclusions, where possible, on the relevance of toxicity and toxicokinetic data to public health.

A glossary and list of acronyms, abbreviations, and symbols can be found at the end of this profile.

3.2 DISCUSSION OF HEALTH EFFECTS BY ROUTE OF EXPOSURE

To help public health professionals and others address the needs of persons living or working near

hazardous waste sites, the information in this section is organized first by route of exposure (inhalation,

oral, and dermal) and then by health effect (e.g., death, systemic, immunological, neurological,

reproductive, developmental, and carcinogenic effects). These data are discussed in terms of three

exposure periods: acute (14 days or less), intermediate (15–364 days), and chronic (365 days or more).

Levels of significant exposure for each route and duration are presented in tables and illustrated in

figures. The points in the figures showing no-observed-adverse-effect levels (NOAELs) or lowest-

observed-adverse-effect levels (LOAELs) reflect the actual doses (levels of exposure) used in the studies.

LOAELs have been classified into "less serious" or "serious" effects. "Serious" effects are those that

evoke failure in a biological system and can lead to morbidity or mortality (e.g., acute respiratory distress

or death). "Less serious" effects are those that are not expected to cause significant dysfunction or death,

or those whose significance to the organism is not entirely clear. ATSDR acknowledges that a

considerable amount of judgment may be required in establishing whether an end point should be

classified as a NOAEL, "less serious" LOAEL, or "serious" LOAEL, and that in some cases, there will be

insufficient data to decide whether the effect is indicative of significant dysfunction. However, the

Agency has established guidelines and policies that are used to classify these end points. ATSDR

believes that there is sufficient merit in this approach to warrant an attempt at distinguishing between

"less serious" and "serious" effects. The distinction between "less serious" effects and "serious" effects is

considered to be important because it helps the users of the profiles to identify levels of exposure at which

major health effects start to appear. LOAELs or NOAELs should also help in determining whether or not

30 NITRATE AND NITRITE

3. HEALTH EFFECTS

the effects vary with dose and/or duration, and place into perspective the possible significance of these

effects to human health.

The significance of the exposure levels shown in the Levels of Significant Exposure (LSE) tables and

figures may differ depending on the user's perspective. Public health officials and others concerned with

appropriate actions to take at hazardous waste sites may want information on levels of exposure

associated with more subtle effects in humans or animals (LOAELs) or exposure levels below which no

adverse effects (NOAELs) have been observed. Estimates of levels posing minimal risk to humans

(Minimal Risk Levels or MRLs) may be of interest to health professionals and citizens alike.

A User's Guide has been provided at the end of this profile (see Appendix B). This guide should aid in

the interpretation of the tables and figures for Levels of Significant Exposure and the MRLs.

Nitrate (NO

3

-

) and nitrite (NO

2

-

) are naturally-occurring oxidation products of nitrogen. Nitrate may be

expressed in terms of ionic concentration (i.e., mg nitrate/L), or elemental concentration (i.e., mg nitrate-

nitrogen/L or mg nitrogen as nitrate/L). A concentration of nitrate expressed in elemental concentration

can be converted to its ionic concentration according to the following relationship: 1 mg nitrate-nitrogen

is equivalent to 4.4 mg nitrate. In aqueous environments, nitrate and nitrite salts such as sodium nitrate,

potassium nitrate, sodium nitrite, and potassium nitrite rapidly ionize. Sodium nitrate is approximately

27% sodium and 73% nitrate. To determine a nitrate dose from a sodium nitrate source, the quantity of

sodium nitrate is multiplied by the nitrate proportion (0.73). Thus a nitrate dose from a 5 mg sodium

nitrate source is 5x0.73=3.65 mg nitrate. The conversion factor for nitrate from a potassium nitrate

source is 0.61. Conversion factors for nitrite from sodium nitrite and potassium nitrite are 0.67 and 0.54,

respectively.

3.2.1 Inhalation Exposure

3.2.1.1 Death

No information was located regarding death in humans following inhalation exposure to nitrate or nitrite.

An inhalation LC

50

is an exposure level expected to result in 50% mortality. RTECS (2014) lists a rat

4-hour LC

50

of 5.5 mg/m

3

(1.95 ppm) for sodium nitrite and a rat 2-hour LC

50

of 85 mg/m

3

(24.42 ppm)

for potassium nitrite. No additional information was located regarding death in animals exposed to nitrate

or nitrite.

31 NITRATE AND NITRITE

3. HEALTH EFFECTS

3.2.1.2 Systemic Effects

No studies were located regarding gastrointestinal, hematological, musculoskeletal, hepatic, renal,

endocrine, dermal, ocular, or body weight effects in humans or animals after inhalation exposure to nitrate

or nitrite.

Respiratory Effects. Limited human data are available. Al-Dabbagh et al. (1986) evaluated the

mortality of a cohort of 1,327 male workers involved in the manufacture of nitrate fertilizer for at least

1 year between 1946 and 1981 for a chemical company in northeast England. There was no evidence of

an association between exposure to nitrate dusts and death from all respiratory diseases compared to

mortality rates for the northern region of England.

Available information in animals is limited to a study in which dogs and sheep were exposed to aerosols

of sodium nitrate for short periods (Sackner et al. 1979). There was no evidence of exposure-related

pulmonary effects (e.g., respiratory resistance, static lung performance, functional residual capacity) in

anesthetized dogs exposed at up to 10 mg sodium nitrate/m

3

(2.88 ppm) for 7.5 minutes or anesthetized

dogs or conscious sheep exposed at 5 mg sodium nitrate/m

3

(1.44 ppm) for 4 hours.

Cardiovascular Effects. Available information in humans is limited to results of mortality studies of

workers involved in the production of nitrate fertilizers. In general, studies of workers in which outcomes

are compared to the general population (e.g., observed versus expected deaths) may be biased by a

healthy worker effect, which may lower estimated risks. There was no evidence of an association

between exposure to nitrate dust and death from ischemic heart disease, cerebrovascular disease, or all

circulatory diseases in a census-based (England and Wales) mortality study of workers involved in the

production of nitrate fertilizers (Fraser et al. 1982, 1989). The study included a cohort of 866 men from

the 1961 census and 651 men from the 1971 census. These cohorts were followed through 1985. Al-

Dabbagh et al. (1986) evaluated the mortality of a cohort of 1,327 male workers involved in the

manufacture of nitrate fertilizer for at least 1 year between 1946 and 1981 for a chemical company in

northeast England. There was no evidence of an association between exposure to nitrate dusts and death

from ischemic heart disease or other circulatory diseases compared to mortality rates for the northern

region of England.

32 NITRATE AND NITRITE

3. HEALTH EFFECTS

Available information in animals is limited to a study in which dogs and sheep were exposed to aerosols

of sodium nitrate for short periods (Sackner et al. 1979). There was no evidence of exposure-related

cardiac effects (pulmonary and systemic arterial pressure, cardiac output, heart rate, arterial blood gases)

in anesthetized dogs or conscious sheep exposed at 5 mg sodium nitrate/m

3

(1.44 ppm) for 4 hours.

No information was located regarding the following effects in humans or animals exposed to nitrate or

nitrite via the inhalation route:

3.2.1.3 Immunological and Lymphoreticular Effects

3.2.1.4 Neurological Effects

3.2.1.5 Reproductive Effects

3.2.1.6 Developmental Effects

3.2.1.7 Cancer

Available information in humans is limited to results of mortality studies of workers involved in the

production of nitrate fertilizers. In general, studies of workers in which outcomes are compared to the

general population (e.g., observed versus expected deaths) may be biased by a healthy worker effect,

which may lower estimated risks. A census-based (England and Wales) mortality study of workers

involved in the production of nitrate fertilizers included 866 men from the 1961 census and 651 men from

the 1971 census; mortality rates among these workers were compared to mortality rates of men from

England and Wales (Fraser et al. 1982). At follow-up until 1978, slight excess of death from intestinal

cancer was noted among men from the 1961 census (6 observed versus 4.5 expected); excess of death

from all cancers, (19 versus 14.4 expected), esophageal cancer (1 versus 0.4 expected), gastric cancer

(2 versus 1.5 expected), intestinal cancer (1 versus 0.9 expected), rectal cancer (2 versus 0.6 expected),

and lung cancer (9 versus 6.4 expected) were observed in the 1971 census cohort. However, follow-up

through 1985 revealed no significant increased risk for cancer at any site (Fraser et al. 1989).

Al-Dabbagh et al. (1986) evaluated mortality rates within a cohort of 1,327 male workers involved in the

manufacture of nitrate fertilizer for at least 1 year between 1946 and 1981 for a chemical company in

northeast England; mortality rates were compared with those of the male population of the region.

Among 537 workers described as having been heavily exposed to nitrate dust (i.e., working in an

environment likely to have contained >10 mg nitrate/m

3

[>2.88 ppm]), slight excesses were noted for

deaths from lung cancer (25 observed versus 21.04 expected) and death from all malignant neoplasms

(59 observed versus 51.36 expected), but not for cancers of the esophagus, stomach, or bladder. After

categorizing the heavily-exposed workers by duration of exposure and time since first exposure, excess

33 NITRATE AND NITRITE

3. HEALTH EFFECTS

death from lung cancer was noted for those exposed for ≥10 years, with a lag time of ≥20 years since first

exposure (13 observed versus 8.11 expected). The study authors indicated that they were unable to adjust

for smoking.

Hagmar et al. (1991) evaluated mortality rates within a cohort of 2,131 male workers at a nitrate fertilizer

production facility in Sweden and compared them to mortality rates for men in the same county. Death

from prostate cancer (26 observed versus 16.1 expected) was in excess (standardized mortality ratio

[SMR] 161, 95% CI: 107, 239); however, risk of prostate cancer within this cohort was not enhanced

following application of a ≥10-year latency period. There was no significant increase in death from

tumors of the lips, oral cavity, pharynx, salivary glands, gastrointestinal tract, stomach, respiratory tract,

lung, urinary bladder, blood, or all sites combined.

Fandrem et al. (1993) evaluated incidences of selected cancers among 2,023 male workers who had been

employed for >1 year at a Norwegian nitrate fertilizer plant between 1945 and 1979. The average

historical concentration of nitrate in the workplace air was estimated to have been 10 mg/m

3

. The cohort

was followed from 1953 through 1988 and incidences of cancer among the workers were compared to

national rates. The study authors reported 30 incidences of lung cancer (27.5 expected: standardized

incidence ratio [SIR] 1.09; 95% CI 0.73, 1.53), 9 incidences of kidney cancer (7.6 expected: SIR 1.18;

95% CI 0.54, 2.25), and 9 incidences of pancreatic cancer (7.3 expected: SIR 1.23; 95% CI 0.56, 2.34).

There were fewer than expected cancers of the oesophagus, stomach, colon/rectum, pleura, bladder,

malignant melanoma, and all cancers combined. No association was found between gastric cancer and

cumulative exposure to nitrate, duration of employment, or time since first exposure.

Rafnsson and Gunnarsdóttir (1990) evaluated mortality rates among 603 male workers at a nitrate

fertilizer plant in Iceland who had been employed for >1 year between 1954 and 1985. Mortality data

were compared to national rates for men. The study authors reported nonstatistically significant excesses

of cancers of the large intestine (2 observed versus 1.25 expected: SMR 160; 95% CI 19, 578), rectum

(1 observed versus 0.61 expected: SMR 164; 95% CI 4, 913), pancreas (3 observed versus 1.31 expected:

SMR 229; 95% CI 47, 669), and respiratory tract (4 observed versus 2.88 expected: SMR 139; 95 CI 38,

356). There was no excess of death from stomach cancer (4 observed versus 4.32 expected: SMR 93;

95% CI 25, 237). This study is limited by low incidences of selected cancers and possible confounding

by the healthy worker effect.

34 NITRATE AND NITRITE

3. HEALTH EFFECTS

3.2.2 Oral Exposure

3.2.2.1 Death

As early as the mid-1900s, methemoglobinemia was reported in infants exposed to relatively large

amounts of nitrate from drinking water sources (e.g., Bosch et al. 1950; Bucklin and Myint 1960; Chapin

1947; Comly 1987; Donahoe 1949; Faucett and Miller 1946; Ferrant 1946; McLetchie and Robertson

1949; Medovy 1948; Robertson and Riddell 1949; Stafford 1947). Deaths occurred in some of these

cases. Ingestion of nitrite (from potassium nitrite or sodium nitrite sources) has been associated with

severe methemoglobinemia in adults and children (Aquanno et al. 1981; CDC 1997, 2002; Gautami et al.

1995; Gowans 1990; Greenberg et al. 1945; Kaplan et al. 1990; Ringling et al. 2003; Sevier and Berbatis

1976; Ten Brink et al. 1982; Walley and Flanagan 1987). Deaths occurred in some of these cases

following consumption of food or drink that contained unusually high levels of nitrite via contamination,

inadvertent use of sodium nitrite instead of table salt, or ingestion of a single sodium nitrite tablet

(667 mg nitrite).

An oral LD

50

is the dose expected to result in 50% mortality. Single oral doses of sodium nitrite at

multiple dose levels resulted in LD

50

values of 150 mg/kg (100 mg nitrite/kg) in rats (Imaizumi et al.

1980) and 265 mg/kg (178.2 mg nitrite/kg) in mice (Sheehy and Way 1974). RTECS (2014) lists oral

LD

50

values for sodium nitrate of 1,267, 3,500, and 2,680 mg/kg for the rat, mouse, and rabbit,

respectively; LD

50

values for sodium nitrite of 157.9, 175, and 186 mg/kg for the rat, mouse, and rabbit,

respectively; LD

50

values for potassium nitrate of 3,540 and 3,750 for the rat and 1,901 mg/kg for the

rabbit; and an LD

50

for potassium nitrite of 200 mg/kg. Among rats provided sodium nitrate in the

drinking water for 6 weeks, concentrations of sodium nitrate resulting in an estimated dose of 14,600 mg

nitrate/kg/day was lethal to 7/10 male rats; an estimated dose of 16,483.9 mg nitrate/kg/day was lethal to

10/10 female rats. Among male rats similarly treated with sodium nitrite, an estimated dose of

1,080.6 mg nitrite/kg/day was lethal to 4/10 rats. Inai et al. (1979) reported 100% mortality in male and

female mice (10/sex) provided sodium nitrite in the drinking water at concentrations resulting in

estimated doses of 330.8 and 354.1 mg nitrite/kg/day, respectively; the deaths occurred within the first

3 weeks of a 6-week study.

3.2.2.2 Systemic Effects

No studies were located regarding musculoskeletal or ocular effects in humans or animals after oral

exposure to nitrate or nitrite.

35 NITRATE AND NITRITE

3. HEALTH EFFECTS

The highest NOAEL values and all LOAEL values from each reliable study for systemic effects in each

species and duration category are recorded in Table 3-1 and plotted in Figure 3-1.

Respiratory Effects. No studies were located regarding respiratory effects in humans or animals

following oral exposure to nitrate or nitrite.

Cardiovascular Effects. Malberg et al. (1978) investigated possible associations between

hypertension and levels of nitrate in the drinking water in a hospital-based study in Colorado that included

226 cases of hypertension among patients living in areas where drinking water contained nitrate at

concentrations ranging from 19 to 125 ppm (mean 52 ppm) and 261 cases from patients living in areas

without nitrate in the drinking water. The mean annual incidence rate for the nitrate-exposed patients was

5.9/1,000 population versus 7.9/1,000 for the control patients. However, the nitrate-exposed patients

exhibited an earlier mean age at hospitalization for hypertension (58.5 years versus 65.2 years for

controls); the toxicological significance of this finding is uncertain because the incidence rate for

hypertension was higher among control patients than among patients exposed to nitrate in the drinking

water.

Cardiovascular health is an end point of concern for nitrate because some nitrate is converted to nitrite in

the body. Nitrite is a smooth muscle relaxant that can cause hypotension and plasma nitrite is involved in

the oxidation of hemoglobin to methemoglobin, which is associated with hypotension, rapid pulse, and

rapid breathing at high enough concentrations. Ingestion of nitrite (from potassium nitrite or sodium

nitrite sources) has been associated with severe methemoglobinemia in adults and children; in some of

these cases, symptoms included hypotension and/or tachycardia (Gowans 1990; Sevier and Berbatis 1976;

Ten Brink et al. 1982). These cases were the result of consumption of food or drink that contained

unusually high levels of nitrite via contamination, inadvertent use of sodium nitrite instead of table salt, or

ingestion of a single sodium nitrite tablet (667 mg nitrite).

In a study designed to evaluate the oral bioavailability of sodium nitrite in healthy volunteers (seven

females and two males; mean age 22.9 years), ingestion of 0.06 sodium nitrite per mmol hemoglobin

(~2.2–2.7 mg sodium nitrite/kg, or 1.5–1.8 mg nitrite/kg) resulted in an average heart rate increase from

55 to 63 bpm and average mean arterial blood pressure decrease from 78 to 70 mmHg (Kortboyer et al.

1997b). At a higher intake (0.12 mmol sodium nitrite per mmol hemoglobin; ~4.4–5.4 mg sodium

nitrite/kg, or 2.9–3.6 mg nitrite/kg), the average heart rate increased from 57 to 67 bpm and the average

83

100.5

92

178.2

104

4.33

106

0.2

84

6.7 16.75

88

104.2

NITRATE AND NITRITE

3. HEALTH EFFECTS

36

Table 3-1 Levels of Significant Exposure to Nitrate And Nitrite - Oral

a

Key to

Figure

Species

(Strain)

Exposure/

Duration/

Frequency

(Route)

ACUTE EXPOSURE

Death

1

Rat

(Sprague-

Dawley)

Once

(GW)

System

NOAEL

(mg/kg/day)

Less Serious

(mg/kg/day)

LOAEL

Serious

(mg/kg/day)

100.5 (LD50)

Reference

Chemical Form

Imaizumi et al. 1980

Sodium Nitrite

Comments

2

Mouse

(Swiss-

Webster)

Once

(GW)

178.2 M (LD50)

Sheehy and Way 1974

Sodium Nitrite

Systemic

3

Human NS

(F)

Hemato

b

4.33

Walton 1951

Nitrate

Dose based on a

drinking water level (44

mg nitrate/L) above

which nitrate could

cause

methemoglobinemia in

infants <3 months old.

4

Human NS

(F)

Hemato

c

0.2

Walton 1951

Nitrite

The NOAEL represents

the estimated nitrite

dose to an infant <3

months of age

consuming nitrate from

drinking water at up to

44 mg/L.

5

Rat

(Sprague-

Dawley)

Once

(GW)

Hemato 6.7

16.75 (8.6% methemoglobin)

Imaizumi et al. 1980

Sodium Nitrite

6

Rat

(Wistar)

1 or 3 d

1 x/d

(GW)

Hepatic 104.2 M

Lijinsky and Greenblatt 1972

Sodium Nitrite

78

150

86

53.6

80

13

93

113.2

12

1080.6

13

14600

16483.9

50

330.8

354.1

NITRATE AND NITRITE

3. HEALTH EFFECTS

37

Table 3-1 Levels of Significant Exposure to Nitrate And Nitrite - Oral (continued)

a

Key to

Figure

Species

(Strain)

System

Exposure/

Duration/

Frequency

(Route)

NOAEL

(mg/kg/day)

Less Serious

(mg/kg/day)

LOAEL

Serious

(mg/kg/day)

Reference

Chemical Form

Comments

7

Hepatic

Mouse

HaM/ICR

Once

(GW)

Bd Wt

Developmental

8

Rat

(NS)

Gd 15

Once

(GW)

150 M

53.6

A

sahina et al. 1971

Sodium Nitrite

Khera 1982

Sodium Nitrite

9

Mouse

(CD-1)

Gd 1-14, 16, or

18

1 x/d

(GW)

13

Globus and Samuel 1978

Sodium Nitrite

10

Mouse

(ICR)

Gd 7-18

(W)

INTERMEDIATE EXPOSURE

Death

11

Rat

(Fischer- 344)

6 wk

(W)

113.2

1080.6 F (4/10 died)

Shimada 1989

Sodium Nitrite

Maekawa et al. 1982

Sodium Nitrite

12

Rat

(Fischer- 344)

6 wk

(F)

14600 M (7/10 died)

16483.9 F (10/10 died)

Maekawa et al. 1982

Sodium Nitrate

13

Mouse

(ICR)

6 wk

(W)

330.8 M (death during first 3

treatment weeks)

Inai et al. 1979

Sodium Nitrite

354.1 F (death during first 3

treatment weeks)

101

4972

107

4.33

109

0.2

111

80

32

28.14 187.6

56

40.5

NITRATE AND NITRITE

3. HEALTH EFFECTS

38

Table 3-1 Levels of Significant Exposure to Nitrate And Nitrite - Oral (continued)

a

Key to

Figure

Species

(Strain)

Exposure/

Duration/

Frequency

(Route)

System

LOAEL

NOAEL

(mg/kg/day)

Less Serious

(mg/kg/day)

Serious

(mg/kg/day)

Reference

Chemical Form

Comments

14

Gn Pig

(NS)

143-204 d

(W)

Systemic

15

Human NS

(F)

Hemato

Sleight and Atallah 1968

potassium nitrate

4972 F (1/3 died)

Walton 1951

Nitrate

b

4.33

Dose based on a

drinking water level (44

mg nitrate/L) above

which nitrate could

cause

methemoglobinemia in

infants <3 months old.

16

Human NS

(F)

Hemato

Walton 1951

Nitrite

c

0.2

The NOAEL represents

the estimated nitrite

dose to an infant <3

months of age

consuming nitrate from

drinking water at up to

44 mg/L.

17

Rat

(Sprague-

Dawley)

12wk

1x/d

(G)

Metab

A

l-Gayyar et al. 2015

Sodium Nitrite

80 M (hyperglycemia, insulin

resistance)

18

Rat

(albino)

2 mo

(W)

Hemato

Behroozi et al. 1972

Sodium Nitrite

28.14 M

187.6 M (12.16% methemoglobin)

19

Rat

(Sprague-

Dawley)

16 wk

(W)

Hemato

Chow et al. 1980

Sodium Nitrate

40.5 M

58

18.6

98

6.4

15.8

6.4

113

34.8 34.8

34.8

20

60.16

158.77

85

167.5

NITRATE AND NITRITE

3. HEALTH EFFECTS

39

Table 3-1 Levels of Significant Exposure to Nitrate And Nitrite - Oral (continued)

a

Key to Species

Figure (Strain)

20

Rat

(Sprague-

Dawley)

21

Rat

(Wistar)

22

Rat

(Wistar)

23

Rat

(Wistar)

Exposure/

Duration/

Frequency

(Route)

System

NOAEL

(mg/kg/day)

Less Serious

(mg/kg/day)

LOAEL

Serious

(mg/kg/day)

Reference

Chemical Form

Comments

16 wk

(W)

Hemato 18.6 M

Chow et al. 1980

Sodium Nitrite

4 mo

(W)

Renal 6.4 M

15.8 M (increased urinary urea

and creatinine levels)

El-Wakf et al. 2008

Sodium Nitrate

Endocr 6.4 M (decreased serum T3

and T4; increased serum

TSH)

Bd Wt 6.4 M (11-12% depressed

mean body weight and

body weight gain)

4mo

Continuous

(W)

Bd Wt 34.8 M (9 and 30% depressed

mean body weight and

body weight gain,

respectively, among adult

rats)

34.8 M (24 and 39% depressed

mean body weight and

body weight gain,

respectively, among

young rats)

El-Wakf et al. 2015

Sodium Nitrate

Metab 34.8 M (hyperglycemia)

30 wk

(W)

Endocr 60.16 F

158.77 F (decreased serum T3,

T4, and TSH levels;

increased thyroid weight;

follicular hyperplasia)

Eskiocak et al. 2005

Sodium Nitrate

Rat 6 mo

Hemato 167.5 (peak methemoglobin of

Imaizumi et al. 1980

(Sprague-

(W)

33-88%)

Sodium Nitrite

Dawley)

24

74

208.4

71

183.1

10

3650

4121

7300

8241.9

7300

4121

14600

8241.9

NITRATE AND NITRITE

3. HEALTH EFFECTS

40

Table 3-1 Levels of Significant Exposure to Nitrate And Nitrite - Oral (continued)

a

Key to

Figure

Species

(Strain)

Exposure/

Duration/

Frequency

(Route)

System

NOAEL

(mg/kg/day)

Less Serious

(mg/kg/day)

LOAEL

Serious

(mg/kg/day)

Reference

Chemical Form

Comments

25

Rat

(Fischer- 344)

51 wk

(W)

Gastro 208.4 M

Kawabe et al. 1994

Sodium Nitrite

26

Rat

(Sprague-

Dawley)

10 mo

(F)

Hepatic 183.1

Lin and Ho 1992

Sodium Nitrite

Bd Wt 183.1

27

Rat

(Fischer- 344)

6 wk

(F)

Hemato 3650 M

4121 F

7300 M (discolored blood and

spleen indicative of

methemoglobinemia)

Maekawa et al. 1982

Sodium Nitrate

8241.9 F (discolored blood and

spleen indicative of

methemoglobinemia)

Bd Wt 7300 M

4121 F

14600 M (at least 10% depressed

body weight gain)

8241.9 F (at least 10% depressed

body weight gain)

9

186.1

270.2

372.2

540.3

372.2

540.3

744.4

1080.6

76

208.4

22

2416.6

NITRATE AND NITRITE

3. HEALTH EFFECTS

41

Table 3-1 Levels of Significant Exposure to Nitrate And Nitrite - Oral (continued)

a

Key to

Figure

Species

(Strain)

Exposure/

Duration/

Frequency

(Route)

System

NOAEL

(mg/kg/day)

Less Serious

(mg/kg/day)

LOAEL

Serious

(mg/kg/day)

Reference

Chemical Form

Comments

28

Rat

(Fischer- 344)

6 wk

(W)

Hemato 186.1 M

270.2 F

372.2 M (discolored blood and

spleen indicative of

methemoglobinemia)

Maekawa et al. 1982

Sodium Nitrite

540.3 F (discoloration of blood

and spleen indicative of

methemoglobinemia)

Bd Wt 372.2 M

540.3 F

744.4 M (at least 10% depressed

body weight gain)

1080.6 F (at least 10% depressed

body weight gain)

29

Rat

(Fischer- 344)

35 wk

(W)

Gastro 208.4 M

Miyauchi et al. 2002

Sodium Nitrite

30

Rat

(Wistar)

4 wk

(F)

Endocr 2416.6 (increased thyroid

weight, decreased

thyroid peroxidase

activity, decreased serum

T3 and T4, increased

serum TSH)

Mukhopadhyay et al. 2005

potassium nitrate

Bd Wt 2416.6

1

77.1

53.6

134

87.1

17

41.9

61.8

107.6

130.5

4.8

16.8

13.3

61.8

NITRATE AND NITRITE

3. HEALTH EFFECTS

42

Table 3-1 Levels of Significant Exposure to Nitrate And Nitrite - Oral (continued)

a

Key to

Figure

Species

(Strain)

Exposure/

Duration/

Frequency

(Route)

System

NOAEL

(mg/kg/day)

Less Serious

(mg/kg/day)

LOAEL

Serious

(mg/kg/day)

Reference

Chemical Form

Comments

31

Rat

(Fischer- 344)

14 wk

(W)

Hemato 77.1 M

53.6 F

134 M (up to 10%

methemoglobin)

NTP 2001

Sodium Nitrite

87.1 F (up to 13%

methemoglobin)

32

Rat

(Wistar)

13 wk

(W)

Hemato 41.9 M

61.8 F

107.6 M (5.7% methemoglobin)

130.5 F (7.6% methemoglobin)

Til et al. 1988

potassium nitrite

Endocr 4.8 M

16.8 F

13.3 M (hypertrophy in zona

glomerulosa of adrenal

gland)

61.8 F (hypertrophy in zona

glomerulosa of adrenal

gland)

18

4.59

5.94

105.1

130.1

19

5.2

7.1

106.3

124.8

5.2

7.1

106.3

124.8

95

28.1

NITRATE AND NITRITE

3. HEALTH EFFECTS

43

Table 3-1 Levels of Significant Exposure to Nitrate And Nitrite - Oral (continued)

a

Key to Species

Figure (Strain)

33

Rat

(Wistar)

34

Rat

(Wistar)

35

Rat

(Sprague-

Dawley)

Exposure/

Duration/

Frequency

(Route)

System

NOAEL

(mg/kg/day)

Less Serious

(mg/kg/day)

LOAEL

Serious

(mg/kg/day)

Reference

Chemical Form

Comments

13 wk

(W)

Endocr 4.59 M

5.94 F

105.1 M (hypertrophy in zona

glomerulosa of adrenal

gland)

Til et al. 1997

potassium nitrite

130.1 F (hypertrophy in zona

glomerulosa of adrenal

gland)

13 wk

(W)

Hemato 5.2 M

7.1 F

106.3 M (increased

methemoglobin,

magnitude not specified)

Til et al. 1997

Sodium Nitrite

124.8 F (increased

methemoglobin,

magnitude not specified)

Endocr 5.2 M

7.1 F

106.3 M (hypertrophy in zona

glomerulosa of adrenal

gland)

124.8 F (hypertrophy in zona

glomerulosa of adrenal

gland)

F0 males:

15-28 d

F0 females:

58-71 d

F1 pups: 69 d

(F)

Bd Wt 28.1

Vorhees et al. 1984

Sodium Nitrite

21

9

13.5

9

13.5

66

82.5

35

118.1

36

1583

39

236.3

NITRATE AND NITRITE

3. HEALTH EFFECTS

44

Table 3-1 Levels of Significant Exposure to Nitrate And Nitrite - Oral (continued)

a

Key to Species

Figure (Strain)

36

Rat

(Wistar)

37

Mouse

Swiss

38

Mouse

Strain A

39

Mouse

Strain A

40

Mouse

Strain A

Exposure/

Duration/

Frequency

(Route)

System

NOAEL

(mg/kg/day)

Less Serious

(mg/kg/day)

LOAEL

Serious

(mg/kg/day)

Reference

Chemical Form

Comments

5 mo

(W)

Endocr 9 M

13.5 M (increases in serum T3

and thyroid weight;

nonneoplastic lesions in

thyroid gland)

Zaki et al. 2004

potassium nitrate

Bd Wt 9 M

13.5 M (16% lower mean body

weight than controls)

26 wk

(W)

Bd Wt 82.5

Greenblatt and Lijinsky 1974

Sodium Nitrite

25 wk

5 d/wk

(W)

Bd Wt 118.1 M

Greenblatt and Mirvish 1973

Sodium Nitrite

25 wk

5 d/wk

(W)

Bd Wt 1583 M

Greenblatt and Mirvish 1973

Sodium Nitrate

20 wk

5 d/wk

(W)

Bd Wt 236.3 M

Greenblatt and Mirvish 1973

Sodium Nitrite

2

435.5

562.8

663.3

824.1

231.2

160.8

435.5

298.1

435.5

824.1

663.3

31

9.38

82

165.4

NITRATE AND NITRITE

3. HEALTH EFFECTS

45

Table 3-1 Levels of Significant Exposure to Nitrate And Nitrite - Oral (continued)

a

Key to Species

Figure (Strain)

41

Mouse

(B6C3F1)

Neurological

42

Rat

(albino)

43

Rat

C57B1

Exposure/

Duration/

Frequency

(Route)

System

NOAEL

(mg/kg/day)

Less Serious

(mg/kg/day)

LOAEL

Serious

(mg/kg/day)

Reference

Chemical Form

Comments

14 wk

(W)

Gastro 435.5 M

562.8 F

663.3 M (focal hyperplasia in

forestomach)

NTP 2001

Sodium Nitrite

824.1 F (focal hyperplasia in

forestomach)

Hemato 231.2 M

160.8 F

435.5 M (extramedullary

hematopoiesis in spleen)

298.1 F (extramedullary

hematopoiesis in spleen)

Bd Wt 435.5 M

824.1 F

663.3 M (10% depressed final

mean body weight and

body weight gain)

2 mo

(W)

9.38 M (altered EEG)

Behroozi et al. 1972

Sodium Nitrite

F0: Mating,

gestation,

lactation

F1: 14 wk

postweaning

(W)

165.4 M (increased aggressive

behavior)

Gruener 1974

Sodium Nitrite

112

80

114

80

99

160

5

77.1

134

3

231.2

435.5

NITRATE AND NITRITE

3. HEALTH EFFECTS

46

Table 3-1 Levels of Significant Exposure to Nitrate And Nitrite - Oral (continued)

Exposure/

LOAEL

Duration/

a

Frequency

Key to Species

NOAEL Less Serious Serious

(Route)

Figure (Strain)

System

(mg/kg/day) (mg/kg/day) (mg/kg/day)

Reproductive

44

Rat

(Sprague-

Dawley)

12wk

1x/d

(G)

80 M

45

Rat

(Sprague-

Dawley)

12wk

1x/d

(G)

80 M

46

47

Rat

(Wistar)

2 generations

(F)

Rat

(Fischer- 344)

14 wk

(W)

160 F

77.1 M

134 M (7% decreased sperm

motility)

48

Mouse

(B6C3F1)

14 wk

(W)

231.2 M

435.5 M

(Increases in testicular

weight and serum FSH,

LH, and prolactin;

decreases in sperm

count and serum

testosterone)

(decreased serum

testosterone; increases

in testicular weight;

increased testicular

levels of

pro-inflammatory

cytokines, oxidative

stress markers, and

enzymes involved in

programmed cell death)

(degeneration in testis,

characterized by

increased size of residual

bodies within the lumen

of the seminiferous

tubules)

Reference

Chemical Form

Comments

A

lyoussef and Al-Gayya

r

2016a

Sodium Nitrite

A

lyoussef and Al-Gayya

r

2016b

Sodium Nitrite

Hugot et al. 1980

Sodium Nitrite

NTP 2001

Sodium Nitrite

NTP 2001

Sodium Nitrite

102

2230.8 4972

103

59.4

148.5

100

160

94

7.2

14.4

108

4.33

NITRATE AND NITRITE

3. HEALTH EFFECTS

47

Table 3-1 Levels of Significant Exposure to Nitrate And Nitrite - Oral (continued)

a

Key to

Figure

Species

(Strain)

Exposure/

Duration/

Frequency

(Route)

System

NOAEL

(mg/kg/day)

Less Serious

(mg/kg/day)

LOAEL

Serious

(mg/kg/day)

Reference

Chemical Form

Comments

49

Gn Pig

(NS)

143-204 d

(W)

2230.8 F

4972 F

Sleight and Atallah 1968

potassium nitrate

50

Gn Pig

(NS)

100-240 d

(W)

59.4 F

148.5 F (decreased number of

litters and live fetuses)

Sleight and Atallah 1968

potassium nitrite

Developmental

51

Rat

(Wistar)

2 generations

(F)

160

Hugot et al. 1980

Sodium Nitrite

52

Rat

(Sprague-

Dawley)

F0 males:

15-28 d

F0 females:

58-71 d

F1 pups: 69 d

(F)

CHRONIC EXPOSURE

Systemic

53

Human NS

(F)

Hemato

7.2

b

4.33

14.4 (increased pup mortality,

depressed preweaning

pup body weight, delayed

swimming development)

Vorhees et al. 1984

Sodium Nitrite

Walton 1951

Nitrate

Dose based on a

drinking water level (44

mg nitrate/L) above

which nitrate could

cause

methemoglobinemia in

infants <3 months old.

110

0.2

45

60.5

178.2

97

14.5

22.6

14

82.4

60.3

101

15

1517

832

1730

NITRATE AND NITRITE

3. HEALTH EFFECTS

48

a

Key to

Figure

54

55

56

57

58

Exposure/

Duration/

Frequency

Species

(Route)

(Strain)

Human NS

(F)

Table 3-1 Levels of Significant Exposure to Nitrate And Nitrite - Oral (continued)

LOAEL

System

Hemato

Bd Wt

NOAEL

(mg/kg/day)

c

0.2

60.5 M

14.5 M

22.6 F

82.4 M

60.3 F

1517 M

832 F

Less Serious

(mg/kg/day)

178.2 M (approximately 15%

depressed mean body

weight)

101 F (more than 10% lower

mean body weight than

controls)

1730 F (up to 13% lower mean

body weight than

controls)

Serious

(mg/kg/day)

Reference

Chemical Form

Walton 1951

Nitrite

Grant and Butler 1989

Sodium Nitrite

Greenblatt et al. 1973

Sodium Nitrite

Maekawa et al. 1982

Sodium Nitrite

Maekawa et al. 1982

Sodium Nitrate

Comments

The NOAEL represents

the estimated nitrite

dose to an infant <3

months of age

consuming nitrate from

drinking water at up to

44 mg/L.

Study authors did not

specify whether

reported nitrite

consumption was nitrite

or sodium nitrite

Rat

(Fischer- 344)

Rat

(Wistar)

Rat

(Fischer- 344)

Rat

(Fischer- 344)

115 wk

(F)

67 wk

5 d/wk

(W)

104 wk

(W)

104 wk

(F)

4

46.9

53.6

87.1

100.5

25

172.8

86.4

69

176.8

204.5

176.8

204.5

176.8

204.5

176.8

NITRATE AND NITRITE

3. HEALTH EFFECTS

49

Table 3-1 Levels of Significant Exposure to Nitrate And Nitrite - Oral (continued)

Exposure/

Duration/

a

Key to Species

Frequency

Figure (Strain)

(Route)

59

Rat

(Fischer- 344)

105 wk

(W)

60

Rat

NS

24 mo

(W)

61

Rat

(Wistar)

29 mo

(F)

System

Gastro

Hemato

Hepatic

Gastro

Hemato

Hepatic

Bd Wt

LOAEL

NOAEL

(mg/kg/day)

Less Serious

(mg/kg/day)

Serious

(mg/kg/day)

Reference

Chemical Form

Comments

46.9 M

53.6 F

87.1 M (epithelial hyperplasia in

the forestomach)

NTP 2001

Sodium Nitrite

100.5 F (epithelial hyperplasia in

the forestomach)

86.4 M

172.8 M (12% methemoglobin)

Shuval and Gruener 1972

Sodium Nitrite

176.8 M

204.5 F

van Logten et al. 1972

Sodium Nitrite

176.8 M

204.5 F

176.8 M

204.5 F

204.5 F 176.8 M (10% lower mean body

weight)

6

80.4

147.4

110.6

23

38.5

39

24

38.5

39

43

108.4

87

110.4

53

298

NITRATE AND NITRITE

3. HEALTH EFFECTS

50

Table 3-1 Levels of Significant Exposure to Nitrate And Nitrite - Oral (continued)

a

Key to

Figure

Species

(Strain)

Exposure/

Duration/

Frequency

(Route)

System

NOAEL

(mg/kg/day)

Less Serious

(mg/kg/day)

LOAEL

Serious

(mg/kg/day)

Reference

Chemical Form

Comments

62

Mouse

(B6C3F1)

104-105 wk

(W)

Gastro 80.4 M

147.4 M (epithelial hyperplasia in

glandular stomach)

NTP 2001

Sodium Nitrite

Bd Wt 147.4 M

110.6 F

63

Dog

(Beagle)

1 yr

(W)

Endocr 38.5 M

39 F

Kelley et al. 1974

Sodium Nitrate

Reproductive

64

Dog

(Beagle)

1 yr

(W)

38.5 M

39 F

Kelley et al. 1974

Sodium Nitrate

Cancer

65

Rat

(Fischer- 344)

106 wk

(F)

108.4 F (CEL; hepatocellular

neoplasms)

Lijinsky 1984a; Lijinsky et al.

1983

Sodium Nitrite

66

Rat

(Fischer- 344)

104 wk

(F)

110.4 F (CEL: hepatocellular

neoplasms)

Lijinsky 1984b; Lijinsky et al.

1983

Sodium Nitrite

67

Rat

(Wistar)

Lifetime

(W)

298 (CEL; forestomach

tumors)

Mirvish et al. 1980

Sodium Nitrite

48

207.7

NITRATE AND NITRITE

3. HEALTH EFFECTS

51

Table 3-1 Levels of Significant Exposure to Nitrate And Nitrite - Oral (continued)

Exposure/

LOAEL

Duration/

a

Key to Species

Frequency

NOAEL Less Serious Serious

Reference

(Route)

Figure (Strain)

System

(mg/kg/day) (mg/kg/day) (mg/kg/day)

Chemical Form

Comments

68

Mouse Lifetime

207.7 M (CEL: lung carcinoma)

A

nderson et al. 1985

(Hybrid)

(W)

Sodium Nitrite

a The number corresponds to entries in Figure 3-1.

b NOAEL of of 4 mg/kg/day for nitrate was used to derive acute-, intermediate, and chronic-duration oral minimal risk levels (MRLs) of 4 mg/kg/day for nitrate, as described in detail in

Chapter 2 and Appendix A. The NOAEL was divided by an uncertainty factor of 1 for human variability because the NOAEL accounted for exposure of a particularly sensitive

subpopulation (infants <3 months of age).

c NOAEL of 0.2 mg/kg/day for nitrite was used to derive acute-, intermediate, and chronic-duration oral minimal risk levels (MRLs) of 0.1 mg/kg/day for nitrite, as described in detail in

Chapter 2 and Appendix A. The NOAEL represents the dose of nitrite that would be expected to enter the blood following ingestion of nitrate by an adult at the oral MRL value of 4

mg nitrate/kg/day assuming 5% reduction of an oral dose of nitrate to nitrite in the adult saliva complete absorption of nitrite from the digestive tract. The NOAEL of 0.2 mg/kg/day for

nitrite was divided by an uncertainty factor of 1 for human variability because the NOAEL was for exposure of a particularly sensitive subpopulation (infants <3 months of age). A

modifying factor of 2 was applied based on the assumption that the effective methemoglobin level from a given intake by an infant may be up to twice that of an adult.

Bd Wt = body weight; CEL = cancer effect level; d = day(s); EEG = electroencephalogram; Endocr = endocrine; (F) = feed; F = Female; Gastro = gastrointestinal; Gd = gestational

day; Gn pig = guinea pig; (GW) = gavage in water; Hemato = hematological; LD50 = lethal dose, 50% kill; LOAEL = lowest-observed-adverse-effect level; M = male; mo = month(s);

NOAEL = no-observed-adverse-effect level; NS = not specified; T3 = triiodothyronine ; T4 = thyroxine; TSH = thyroid stimulating hormone ; (W) = drinking water; wk = week(s); x =

time(s); yr = year(s)

Death

1000

2m

7m 7m

10m

6r

1r

100

8r

5r

9m

10

3

5r

←MRL for Nitrate

1

4

←MRL for Nitrite

0.1

c-Cat

d-Dog

r-Rat

p-Pig

q-Cow

k-Monkey

m-Mouse

h-Rabbit

a-Sheep

f-Ferret

j-Pigeon

e-Gerbil

s-Hamster

g-Guinea Pig

n-Mink

o-Other

Cancer Effect Level-Animals

LOAEL, More Serious-Animals

LOAEL, Less Serious-Animals

NOAEL - Animals

Cancer Effect Level-Humans

LOAEL, More Serious-Humans

LOAEL, Less Serious-Humans

NOAEL - Humans

LD50/LC50

Minimal Risk Level

for effects

other than

Cancer

Hematological

Hepatic

Body Weight

Developmental

NITRATE AND NITRITE

3. HEALTH EFFECTS

52

Figure 3-1 Levels of Significant Exposure to Nitrate And Nitrite - Oral

Acute (≤14 days)

Systemic

mg/kg/day

Death

Gastroint

Hematol

Hepatic

Renal

Endocrin

mg/kg/day

100000

12r

10000

27r

14g

27r

30r

11r

1000

41m

28r

41m 41m

15

←MRL for Nitrate

28r

13m

41m

28r

41m

25r 29r

18r

28r

26r

24r

41m

23r

31r

32r

33r

34r 34r

32r

34r 34r

33r

100

31r

32r 32r

23r

31r

32r

18r

20r

19r

32r

21r

36r

32r

10

36r

34r 34r

21r

33r

34r 34r

32r

33r

1

16

←MRL for Nitrite

0.1

c-Cat

d-Dog

r-Rat

p-Pig

q-Cow

k-Monkey

m-Mouse

h-Rabbit

a-Sheep

f-Ferret

j-Pigeon

e-Gerbil

s-Hamster

g-Guinea Pig

n-Mink

o-Other

Cancer Effect Level-Animals

LOAEL, More Serious-Animals

LOAEL, Less Serious-Animals

NOAEL - Animals

Cancer Effect Level-Humans

LOAEL, More Serious-Humans

LOAEL, Less Serious-Humans

NOAEL - Humans

LD50/LC50

Minimal Risk Level

for effects

other than

Cancer

estinal

ogical

e

NITRATE AND NITRITE

3. HEALTH EFFECTS

53

Figure 3-1 Levels of Significant Exposure to Nitrate And Nitrite - Oral (Continued)

Intermediate (15-364 days)

Systemic

Body Weight

Metabolic

Neurological

Reproductive

Developmental

NITRATE AND NITRITE

3. HEALTH EFFECTS

54

mg/kg/day

100000

10000

1000

100

10

1

0.1

Figure 3-1 Levels of Significant Exposure to Nitrate And Nitrite - Oral (Continued)

Intermediate (15-364 days)

Systemic

27r

49g

39m

30r

49g

28r

41m

28r

48m

38m

40m

26r

43r

50g

48m

46r

47r

51r

37m

17r

50g

44r 45r

47r

22r 22r

35r

22r

36r

52r

21r

36r

42r

52r

c-Cat

d-Dog

r-Rat

p-Pig

q-Cow

k-Monkey

m-Mouse

h-Rabbit

a-Sheep

f-Ferret

j-Pigeon

e-Gerbil

s-Hamster

g-Guinea Pig

n-Mink

o-Other

Cancer Effect Level-Animals

LOAEL, More Serious-Animals

LOAEL, Less Serious-Animals

NOAEL - Animals

Cancer Effect Level-Humans

LOAEL, More Serious-Humans

LOAEL, Less Serious-Humans

NOAEL - Humans

LD50/LC50

Minimal Risk Level

for effects

other than

Cancer

G

H

E

B

R

C

mg/kg/day

10000

58r

1000

58r

67r

61r

68m

61r

61r 61r

55r

53

←MRL for Nitrate

61r

60r

61r

61r 61r

62m 62m

62m

66r

65r

57r

59r

100

59r

60r

57r

55r

62m

57r

59r

63d 64d

63d

64d

56r

10

1

54

*Doses represent the lowest dose tested per study that produced a tumorigenic

←MRL for Nitrite

response and do not imply the existence of a threshold for the cancer endpoint.

0.1

c-Cat

d-Dog

r-Rat

p-Pig

q-Cow

k-Monkey

m-Mouse

h-Rabbit

a-Sheep

f-Ferret

j-Pigeon

e-Gerbil

s-Hamster

g-Guinea Pig

n-Mink

o-Other

Cancer Effect Level-Animals

LOAEL, More Serious-Animals

LOAEL, Less Serious-Animals

NOAEL - Animals

Cancer Effect Level-Humans

LOAEL, More Serious-Humans

LOAEL, Less Serious-Humans

NOAEL - Humans

LD50/LC50

Minimal Risk Level

for effects

other than

Cancer

astrointestinal

ematological

epatic

ndocrine

ody Weight

eproductive

ancer*

NITRATE AND NITRITE

3. HEALTH EFFECTS

55

Figure 3-1 Levels of Significant Exposure to Nitrate And Nitrite - Oral (Continued)

Chronic (≥365 days)

Systemic

56 NITRATE AND NITRITE

3. HEALTH EFFECTS

mean arterial blood pressure decreased from 80 to 69 mmHg. The maximum effects on heart rate and

blood pressure occurred between 15 and 20 minutes following ingestion; heart rate and blood pressure

returned to near-baseline levels approximately 2 hours following ingestion at the low dose, but the effects

had not returned to baseline at 4 hours following ingestion at the high dose. The blood pressure-lowering

effect of short-term dietary supplementation of inorganic nitrate appears to be beneficial; however, there

is some uncertainty regarding potential health benefits of long-term nitrate supplementation to treat

cardiovascular diseases (Maccha and Schecter 2012; Siervo et al. 2013).

Gastrointestinal Effects. Ingestion of nitrite (from potassium nitrite or sodium nitrite sources) has

been associated with severe methemoglobinemia in adults and children; in many of these cases,

symptoms included abdominal cramps and vomiting (CDC 1997, 2002; Gautami et al. 1995; Gowans

1990; Greenberg et al. 1945; Sevier and Berbatis 1976; Ten Brink et al. 1982). These cases were the

result of consumption of food or drink that contained unusually high levels of nitrite via contamination,

inadvertent use of sodium nitrite instead of table salt, or ingestion of a single sodium nitrite tablet

(667 mg nitrite). In a study designed to evaluate the oral bioavailability of sodium nitrite in healthy

volunteers (seven females and two males; mean age 22.9 years), one subject became nauseous and

vomited within 20 minutes following ingestion of 0.12 mmol sodium nitrite per mmol hemoglobin

(~4.8 mg sodium nitrite/kg, or 3.2 mg nitrite/kg); another subject reported nausea within 30 minutes

following ingestion of 0.12 mmol sodium nitrite per mmol hemoglobin (~4.4 mg sodium nitrite/kg, or

2.9 mg nitrite/kg) (Kortboyer et al. 1997b).

In a population-based study, Nasseri-Moghaddam et al. (2011) evaluated the prevalence of acid

regurgitation and/or heartburn in regions of Tehran categorized by nitrate levels in drinking water

sources. The study authors reported a significantly increased prevalence of frequent (at least weekly) acid

regurgitation among residents living in areas with drinking water nitrate concentrations >100 mg/L

compared to those living in areas with drinking water nitrate concentrations <100 mg/L (OR 3.65; 95%

CI 1.32, 10.09).

NTP (2001) observed epithelial hyperplasia in the forestomach of male and female B6C3F1 mice

provided sodium nitrite in the drinking water for 14 weeks at a concentration (5,000 ppm) that resulted in

estimated sodium nitrite doses of 990 and 1,230 mg/kg/day, respectively (663.3 and 824.1 mg

nitrite/kg/day, respectively); NOAELs for these lesions in the males and females were 435.5 and

562.8 mg nitrite/kg/day, respectively. Similar results were noted for male and female F344/N rats and

male B6C3F1 mice treated for 104–105 weeks at estimated doses of 87.1, 100.5, and 147.4 mg

57 NITRATE AND NITRITE

3. HEALTH EFFECTS

nitrite/kg/day, respectively; NOAELs for these lesions in the male and female rats and male mice were

46.9, 53.6, and 80.4 mg nitrite/kg/day, respectively. Sodium nitrite treatment did not result in increased

incidences of forestomach lesions in other groups of male F344 rats provided sodium nitrite in the

drinking water at 2,000 mg/L (estimated dose of 208.4 mg nitrite/kg/day) for 35 weeks (Miyauchi et al.

2002) or 51 weeks (Kawabe et al. 1994).

Hematological Effects. As discussed in detail in Section 3.4 (Toxicokinetics) and Section 3.5

(Mechanisms of Action), some plasma nitrite, arising from reduction of ingested nitrate and via

endogenous production, is involved in the oxidation of hemoglobin-Fe

2+

(which transports oxygen) to

hemoglobin-Fe

3+

(methemoglobin, incapable of binding oxygen).

Methemoglobinemia is a condition in which increased methemoglobin as a percentage of total

hemoglobin results in the expression of clinical signs that increase in severity with increasing percent

methemoglobin (ATSDR 2013a; Bloom et al. 2013; Denshaw-Burke et al. 2013; Haymond et al. 2005).

In normal healthy individuals, methemoglobin levels are <1% of total hemoglobin. Discoloration (e.g.,

pale, gray blue) of the skin is often observed at methemoglobin levels in the range of 3–15%; most

patients tolerate methemoglobin levels <10%. Tachycardia, weakness, and other signs of tissue hypoxia

may be observed at 10–20% methemoglobin levels. Effects on the central nervous system (e.g.,

headache, dizziness, fatigue) and dyspnea and nausea appear at >20% methemoglobin; the severity of

symptoms increases with increasing methemoglobin level. High risk of mortality occurs at levels >70%

methemoglobin).

As early as the mid-1900s, methemoglobinemia was reported in infants exposed to relatively large

amounts of nitrate from drinking water sources (e.g., Bailey 1966; Bosch et al. 1950; Bucklin and Myint

1960; Chapin 1947; Comly 1987; Donahoe 1949; Faucett and Miller 1946; Ferrant 1946; McLetchie and

Robertson 1949; Medovy 1948; Robertson and Riddell 1949; Stafford 1947; Walton 1951). Available

data identify young bottle-fed infants (1–3 months of age) as a subpopulation that is particularly

susceptible to nitrate-induced methemoglobinemia, especially those consuming formula prepared from

drinking water sources containing nitrate in excess of 10 mg nitrate-nitrogen/L (44 mg nitrate/L)

(e.g., Bosch et al. 1950; Walton 1951); EPA established a maximum contaminant level (MCL) of

10 mg/L for nitrate-nitrogen in drinking water (EPA 2009c). Subsequent reports provide additional

evidence of associations between ingestion of nitrate from drinking water sources and elevated

methemoglobin levels in infants (e.g., Craun et al. 1981; Fan and Steinberg 1996; Fan et al. 1987;

Gruener and Toeplitz 1975; Gupta et al. 1999; Johnson et al. 1987; Jones et al. 1973; Miller 1971; Shuval

58 NITRATE AND NITRITE

3. HEALTH EFFECTS

and Gruener 1972; Simon et al. 1964; Super et al. 1981; Winton et al. 1971; Zeman et al. 2002).

Cyanosis and even death occurred in some of the reported cases. However, there is some evidence that

methemoglobinemia in infants who drank formula prepared using drinking water with relatively high

levels of nitrate may be related to bacterial contamination of such water sources and consequent

gastrointestinal disorders, as well as endogenous overproduction of nitric oxide due to gastrointestinal

infection and inflammation (Avery 1999; Gupta et al. 1998; Hegesh and Shiloah 1982; L’hirondel and

L’hirondel 2002; Yano et al. 1982).

Walton (1951) reviewed available literature and found 278 reported cases of infant methemoglobinemia.

Among those infants for whom data on nitrate levels in water sources used to prepare infant formula were

available (n=214), levels >50 mg nitrate-nitrogen/L (220 mg nitrate/L) were associated with 173 cases

(81%), levels of 21–50 mg/L (92–220 mg nitrate/L) were associated with 36 cases (17%), and levels of

11–20 mg nitrate-nitrogen (48–88 mg nitrate/L) were associated with 5 cases (2%). There were no cases

among those infants consuming water containing <10 mg nitrate-nitrogen/L (<44 mg nitrate/L).

Limitations include lack of information regarding the actual ages of the infants, total nitrate doses, and

other water source contaminants (e.g., bacterial levels).

Bosch et al. (1950) evaluated 139 reported cases of cyanosis among infants in Minnesota (90% of which

were <2 months of age; range 8 days to 5 months). Samples from 129 wells that served as water sources

to the cases revealed nitrate-nitrogen concentrations >100 mg/L (>440 mg nitrate/L) in 49 wells, 50–

100 mg/L (220–440 mg nitrate/L) in 53 wells, 21–50 mg/L (92–220 mg nitrate/L) in 25 wells, and 10–

20 mg/L (44–88 mg nitrate/L) in the other 2 wells. A major limitation of this study was the detection of

coliform organisms in 45 of 51 well water samples tested for bacterial contamination; bacteria in the

water source might have been a causal factor for gastrointestinal tract disturbances in some of the infants

and may have been at least partially responsible for increased susceptibility to nitrate-induced cyanosis

(e.g., gastrointestinal tract disturbances could have influenced conversion of ingested nitrate to nitrite or

absorption of nitrite).

Subsequent reports provide additional evidence of associations between ingestion of nitrate from drinking

water sources and elevated methemoglobin levels in infants (e.g., Craun et al. 1981; Fan and Steinberg

1996; Fan et al. 1987; Gruener and Toeplitz 1975; Gupta et al. 1999; Johnson et al. 1987; Jones et al.

1973; Miller 1971; Shuval and Gruener 1972; Simon et al. 1964; Super et al. 1981; Winton et al. 1971;

Zeman et al. 2002). Cyanosis and even death occurred in some of the reported cases.

59 NITRATE AND NITRITE

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Simon et al. (1964) evaluated methemoglobin levels from 89 healthy infants with a nitrate-free water

source (group 1), 38 infants whose water source contained 50–100 mg nitrate/L (group 2), and 25 infants

whose water source contained >100 mg nitrate/L (group 3). Nitrite levels in the water sources measured

less than 0.3 mg/L (with the exception of a single measurement of 1 mg nitrite/L). For groups 1, 2, and 3,

methemoglobin levels averaged 1.0, 1.3, and 2.9%, respectively, in the first postnatal trimester (0–

3 months of age) and 0.8, 0.8, and 0.7 %, respectively, in the second trimester. Significantly increased

methemoglobin was observed only in the highest exposure group (>100 mg nitrate/L) and only during the

first trimester.

Super et al. (1981) evaluated associations between methemoglobin levels among infants 1–12 months of

age (relatively evenly distributed by month) and estimates of nitrate intake (based on measured drinking

water nitrate levels and considerations of liquid intake from other sources). When divided into two

groups according to estimated nitrate intake (310 infants ingesting ≤2.93 mg nitrate/kg/day and

102 infants ingesting >2.93 mg nitrate/kg/day), mean methemoglobin levels were 1.54 and 3.03%,

respectively. There were no striking age-related differences in frequency of infants with methemoglobin

levels >3%.

A nested case-control study included 26 cases of infants diagnosed with methemoglobinemia at

≤2 months of age and 45 age-matched controls (Zeman et al. 2002). Nitrate exposure levels were

categorized as low (<0.5 ppm), medium (1–10 ppm), or high (>10 ppm) according to estimated nitrate

levels reconstructed from parental responses to dietary questionnaires and environmental sampling

(1 ppm in the diet is equivalent to 1 mg/kg diet; 1 ppm in drinking water is equivalent to 1 mg/L).

Numbers of methemoglobinemia cases in the low-, medium-, and high-exposure categories were 0/26,

4/26, and 22/26, respectively, and estimated dietary nitrate intake ranged from 2.83 to 451.20 mg/kg/day

(mean 103.6 mg nitrate/kg/day); diarrheal disease was reported for 14/26 methemoglobinemia cases.

Numbers of controls in the low-, medium-, and high-exposure categories were 21/45, 11/45, and 13/45,

respectively, and estimated dietary nitrate intake ranged from 0 to 182 mg/kg/day (mean 11.2 mg

nitrate/kg/day) for the controls; diarrheal disease was reported for 13/45 controls. Univariate and

multifactorial analysis of risk factors for methemoglobinemia indicated that methemoglobinemia was

most strongly associated with dietary exposure to nitrate/nitrite (p=0.0318), but also significantly

associated with diarrheal disease (p=0.0376). Controls in the high-exposure category were less likely

than high-exposure methemoglobinemia cases to have experienced severe diarrhea and were more likely

to have been breastfed for >2 weeks. Major limitations to the study include the collection of information

60 NITRATE AND NITRITE

3. HEALTH EFFECTS

contributing to the exposure estimates several years following the occurrences of methemoglobinemia and

reliance on parental recollection of infant nutritional intake.

Sadeq et al. (2008) measured methemoglobin levels in children ranging in age from birth to 8 years of age

who either lived in a region where nitrate levels in 78 tested wells ranged from 15.39 to 246.9 mg/L or a

region supplied by municipal water with a mean nitrate level of 2.99 mg/L. The mean methemoglobin

level (0.205 g/dL) among 100 children in the region supplied by well water was slightly higher than that

of 37 children in the region supplied by municipal water (0.166 g/dL). The study authors stated that

0.24 g methemoglobin/dL is the equivalent of 2% methemoglobin, in which case mean methemoglobin

among the children in the region supplied by well water was approximately 1.7% of total hemoglobin

compared to a mean of 1.4% for the children in the region supplied by municipal water. The slight

increases in mean methemoglobin among the children in the region supplied by well water were

consistent within various age ranges (0–6, 7–11, 13–35, 36–71, and 72–95 months). The study authors

stated that methemoglobin ≤0.24 g/dL (2%) was considered to be within normal limits.

Craun et al. (1981) evaluated methemoglobin levels in 102 children 1–8 years of age. Sixty-four of the

children lived in households where drinking water contained 22–111 mg nitrate-nitrogen/L (97–488 mg

nitrate/L); drinking water sources for the other 38 children (controls) contained <10 mg nitrate-nitrogen/L

(<44 mg nitrate/L). Methemoglobin measured 1.0–1.36% in those children 1–4 years of age and

appeared to increase with increasing nitrate intake, although there was no statistically significant change.

Methemoglobin levels in those children 5–8 years of age averaged 0.9–0.95% independent of level of

exposure to nitrate.

In one longitudinal study of 357 pregnant women in south-central Minnesota, there was no apparent

association between estimated intake of nitrate from tap water and methemoglobin levels (Manassaram et

al. 2010). However, only four of the women used tap water with nitrate-nitrogen content above the EPA

(2009c) MCL of 10 mg/L.

Elevated methemoglobin levels and methemoglobinemia have been associated with consumption of foods

high in nitrate (e.g., borage, carrots, kohlrabi, spinach) by infants and small children (Greer and Shannon

2005; Keating et al. 1973; Martinez et al. 2013; Sanchez-Echaniz et al. 2001). In the study of Sanchez-

Echaniz et al. (2001), a homemade purée of mixed vegetables with high nitrate content was considered

the source of elevated methemoglobin levels (10–58% of total hemoglobin) among seven infants 7–

13 months of age.

61 NITRATE AND NITRITE

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Limited data are available regarding administration of controlled amounts of nitrate and methemoglobin

levels. Cornblath and Hartmann (1948) administered sodium nitrate in the formula fed to four infants

(ages 11 days to 11 months) for 2–18 days at a concentration resulting in a dose of 50 mg nitrate/kg/day.

The highest observed level of methemoglobin was 5.3% of total hemoglobin; there was no evidence of

cyanosis. Among four other infants (ages 2 days to 6 months) similarly treated at 100 mg nitrate/kg/day

for 6–9 days, the only reported effect was that of 7.5% methemoglobin in a 10-day-old infant following

8 days of treatment in the absence of clinical cyanosis. Gruener and Toeplitz (1975) fed 104 infants

(1 week to 10 months of age) for 1 day with formula prepared using water containing 15 mg nitrate/L

(~0.8–1.5 mg nitrate/kg, based on age-specific values for water consumption [Kahn and Stralka 2009] and

body weight [EPA 2008]), increased to 108 mg nitrate/L for the next 3 days (~5.5–10.6 mg nitrate/kg/day,

based on age-specific values for water consumption [Kahn and Stralka 2009] and body weight [EPA

2008], and returned to 15 mg nitrate/L for one additional day. Mean methemoglobin levels were 0.89%

after the first day of feeding, 1.3, 0.91, and 0.93% after days 2, 3, and 4, and dropped to 0.8% on the fifth

day. Among three of these infants (ages not specified), methemoglobin levels reached 6.9, 13.9, and

15.9% during the high-dose days. Limitations of this study include the use of a wide range of ages and

the fact that only 57 of the 104 infants supplied blood samples on all 5 treatment days.

Ingestion of nitrite (from potassium nitrite or sodium nitrite sources) has been associated with severe

methemoglobinemia in adults and children (Aquanno et al. 1981; CDC 1997, 2002; Finan et al. 1998;

Gautami et al. 1995; Gowans 1990; Greenberg et al. 1945; Kaplan et al. 1990; Ringling et al. 2003;

Sevier and Berbatis 1976; Ten Brink et al. 1982; Walley and Flanagan 1987). These cases were the result

of consumption of food or drink that contained unusually high levels of nitrite via contamination,

inadvertent use of sodium nitrite instead of table salt, inadvertent use of sodium nitrite-contaminated

sugar, or ingestion of a single sodium nitrite tablet (667 mg nitrite).

In a study designed to evaluate the oral bioavailability of sodium nitrite in healthy volunteers (seven

females and two males; mean age 22.9 years), ingestion of 0.06 sodium nitrite per mmol hemoglobin

(~2.2–2.7 mg sodium nitrite/kg, or 1.5–1.8 mg nitrite/kg) resulted in a mean maximum methemoglobin

concentration of 0.309 mmol/L (range of 3.4–4.5% of total hemoglobin) at approximately 0.70 hours

following ingestion, and a mean half-life of approximately 1.07 hours for methemoglobin reduction

(Kortboyer et al. 1997b). At a higher intake (0.12 mmol sodium nitrite per mmol hemoglobin; ~4.4–

5.4 mg sodium nitrite/kg, or 2.9–3.6 mg nitrite/kg), the mean maximum methemoglobin concentration

62 NITRATE AND NITRITE

3. HEALTH EFFECTS

was 0.727 mmol/L (range of 7.7–10.9% of total hemoglobin) at approximately 1.14 hours following

ingestion, and a mean half-life of approximately 1.13 hours for methemoglobin reduction.

Increased methemoglobin levels have been reported in rats administered sodium nitrite orally. Imaizumi

et al. (1980) administered aqueous sodium nitrite to fasted Sprague-Dawley rats by gavage at 20, 25, 50,

100, or 150 mg/kg (6.7, 16.75, 33.5, 67, and 100.5 mg nitrite/kg, respectively) and observed

methemoglobin levels of 4.3, 8.6, 40.3, 64.7, and 45–80%, respectively, at 1 hour posttreatment. The

highest dose resulted in 50% mortality. Among surviving rats, methemoglobin levels returned to normal

after 24 hours. Imaizumi et al. (1980) administered sodium nitrite in the drinking water of other rats for

6 months at 0.5% (5,000 mg sodium nitrite/L or 3,333 mg nitrite/L). Methemoglobin levels as high as

88% were observed during evening hours of treatment day 18 when the rats were likely drinking water

and as low as 4% during morning and afternoon hours of the following day. The study authors did not

provide information regarding clinical signs or mortality, but stated that there was no effect on growth.

In a 14-week study of male and female Fischer-344 rats administered sodium nitrite in the drinking water,

clinical signs of cyanosis and brownish discoloration of mucous membranes and skin were noted at

concentrations ≥1,500 ppm (≥130 mg/kg or 87.1 mg nitrite/kg) in the females and ≥3,000 ppm

(≥225 mg/kg or 134 mg nitrite/kg) in the males (NTP 2001). The clinical signs were consistent with

increased methemoglobin, which measured as high as 13, 24, and 50% in the 1,500, 3,000, and 5,000 ppm

groups, respectively. Til et al. (1988) reported methemoglobin levels of 5.7 and 7.6% in male and female

Wistar rats, respectively, administered potassium nitrite in the drinking water for 13 weeks at

concentrations resulting in approximate doses of 107.6 and 130.5 mg nitrite/kg/day, respectively. Til et

al. (1997) reported similar effects on methemoglobin in rats similarly exposed to either potassium nitrite

or sodium nitrite; however, quantitative data were not included in the study report.

Behroozi et al. (1972) provided sodium nitrite in the drinking water of male albino rats for 2 months at

concentrations resulting in sodium nitrite doses of 0, 14, 42, and 280 mg/kg/day (0, 9.38, 28.14, and

187.6 mg nitrite/kg/day, respectively). Methemoglobin in all groups was approximately 0.5% prior to the

initiation of sodium nitrite treatment and remained at that level in the control group throughout the study.

Methemoglobin in the low-, mid- and high-dose groups averaged 1.1, 3.0, and 12.16%, respectively,

during the treatment period; following cessation of sodium nitrite exposure, methemoglobin levels in all

sodium nitrite-treated groups decreased to 0.3–0.7%.

63 NITRATE AND NITRITE

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Chow et al. (1980) provided drinking water to male Sprague-Dawley rats for 16 weeks that contained 0 or

200 mg sodium nitrite/L (calculated dose of 18.6 mg nitrite/kg/day, based on EPA [1988] subchronic

reference values for body weight and food consumption). Methemoglobin averaged 0.5–3.0% in the

sodium nitrite-treated group and 0–1.2% in the controls.

Shuval and Gruener (1972) provided sodium nitrite in the drinking water to male rats for 24 months at 0,

100, 1,000, 2,000, or 3,000 mg/L (calculated doses of 0, 8.64, 86.4, 172.8, and 259.2 mg nitrite/kg/day,

based on EPA [1988] chronic default reference values for body weight and food consumption).

Methemoglobin levels in the three highest exposure groups averaged 5, 12, and 22% of total hemoglobin;

there were no treatment-related effects on hemoglobin levels.

Maekawa et al. (1982) added sodium nitrite to the food of male and female F-344 rats for 6 weeks at

concentrations ranging from 0.06 to 1% and sodium nitrate to the food of other rats at concentrations

ranging from 1.25 to 20%. Discoloration in blood and spleen were noted in rats from the two highest

exposure levels for sodium nitrite and sodium nitrate. These exposure levels were equivalent to doses

≥370 mg nitrite/kg/day and ≥7,300 mg nitrate/kg/day (based on EPA [1988] subchronic reference values

for body weight and food consumption in male and female F-344 rats). The study report did not include

information regarding methemoglobin levels.

Chow et al. (1980) provided drinking water to male Sprague-Dawley rats for 16 weeks that contained 0 or

400 mg sodium nitrate/L (calculated dose of 40.5 mg nitrate/kg/day, based on EPA [1988] subchronic

reference values for body weight and food consumption). There were no treatment-related effects on

mean methemoglobin levels.

Other hematological effects were noted in some animal studies that employed exposure to sodium nitrite

or potassium nitrite in the drinking water for periods of 13–115 weeks. Imaizumi et al. (1980) reported

decreased hemoglobin and irregularities in erythrocytes (irregular sizes and marked Heinz body

formation) in rats receiving 167.5 mg nitrite/kg/day. Til et al. (1988, 1997) noted slightly decreased

hemoglobin in male rats at ≥42 mg nitrite/kg/day, decreased packed cell volume and erythrocyte count at

approximately 108 mg nitrite/kg/day, and decreases in erythrocyte count, mean corpuscular volume and

mean corpuscular hemoglobin in female rats at 130 mg nitrite/kg/day. Initially decreased erythrocyte

counts were noted in male rats at ≥60 mg nitrite/kg/day (as much as 44% lower than controls at 8 weeks

of treatment, but returning to control levels by 52 weeks); significant decreases in mean corpuscular

volume, and hemoglobin in these rats were noted throughout the 115-week treatment period (Grant and

64 NITRATE AND NITRITE

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Butler 1989). Significantly increased spleen weights were noted in male mice receiving sodium nitrite for

14 weeks at ≥ 435 mg nitrite/kg/day (39% greater than that of controls) and in male and female mice at

663 or 824 mg nitrite/kg/day (approximately 66% greater than their controls). The study authors

suggested that the increased spleen weights may have represented increased erythropoietic activity in

response to increased methemoglobin; however, methemoglobin data were not included in the study

report.

Hepatic Effects. No information was located regarding hepatic effects in humans following oral

exposure to nitrate or nitrite.

No indications of sodium nitrite-induced liver effects were observed in animal studies that included

assessment of liver function and/or histopathology (Asahina et al. 1971; Lijinsky and Greenblatt 1972;

Lin and Ho 1992; Shuval and Gruener 1972; van Logten et al. 1972).

Renal Effects. No information was located regarding renal effects in humans following oral exposure

to nitrate or nitrite.

El-Wakf et al. (2008) reported significantly increased urinary levels of urea and creatinine in male rats

provided sodium nitrate in the drinking water for 4 months at author-estimated doses of 21.7 and 47.4 mg

sodium nitrate/kg/day (15.8 and 34.6 mg nitrate/kg/day, respectively).

Endocrine Effects. Nitrate acts as a dose-dependent competitive inhibitor of the sodium iodide

symporter (NIS) that mediates the uptake of iodine by the thyroid. Sufficiently decreased iodine uptake

by the thyroid may result in decreased production of thyroid hormones T3 and T4. Decreased thyroid

hormone production causes increased release of TSH from the anterior pituitary gland and consequent

increased uptake of iodine by the thyroid gland. Sufficiently inhibited uptake of iodine by the thyroid

could result in effects associated with thyroid dysfunction (e.g., hypothyroidism). Concern for nitrate-

induced effects on thyroid function has prompted scientists to perform studies designed to assess thyroid

function relative to drinking water and/or dietary nitrate levels. Available human data provide suggestive

evidence that elevated levels of nitrate in drinking water and/or nitrate-rich diets may be associated with

signs of thyroid dysfunction. However, limitations of these studies include lack of individual dose-

response data, quantification of iodine intake, and control for other potential substances that may affect

the thyroid; one study relied on self-reported thyroid status and self-reported dietary nitrate intake.

65 NITRATE AND NITRITE

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Tajtáková and coworkers evaluated thyroid function among schoolchildren (boys and girls 10 or 13 years

of age) from three areas in Slovakia; an agricultural area with drinking water sources containing nitrate at

51–274 mg/L (n=324), an area from a neighboring area where drinking water sources contained <2 mg

nitrate/L (n=168), and the city of Košice supplied with drinking water reported to be low in nitrate

(n=596) (Rádiková et al. 2008; Tajtáková et al. 2006). At the time of the study, measurements of urinary

iodine indicated that the children in the high- and low-nitrate areas were ingesting sufficient iodine.

Thyroid volume and density were estimated with the assistance of ultrasound equipment. Mean thyroid

volume was significantly higher in the 10- and 13-year-old children from the high-nitrate area

(5.10±0.14 mL for the 10-year-olds and 5.97±0.11 mL for the 13-year-olds) compared to that of the

children from the low-nitrate area (4.58±0.17 and 5.23±0.15 mL, respectively) and from the city of

Košice (4.77±0.10 and 4.87±0.10 mL, respectively). The frequency of hypoechogenicity (ultrasound

indicator of decreased thyroid density typically indicating destruction of normal thyroid tissue) was

significantly greater in children from the high-nitrate area compared to those from the low-nitrate areas

(13.7 versus 4.7% for the 10-year-olds and 10.6 versus 5.7% for the 13-year-olds). Blood samples

revealed TSH in the range of subclinical hypothyroidism in 13/324 children and positive anti-

thyroperoxidase antibodies (an indicator of subclinical thyroid disorder) in 8/324 of the children from the

high-nitrate area versus no cases in 109 children from the low-nitrate area. There were no significant

differences between children from the low- and high-nitrate areas regarding serum T3 or T4 levels.

Iodine status and goiter prevalence were evaluated in 156 schoolchildren (7–14 years of age) in an area of

rural Bulgaria where nitrate in the drinking water averaged 75 mg/L and 163 schoolchildren in a nearby

area drinking water nitrate averaged 8 mg/L at the time of the study (Gatseva and Argirova 2008). At the

time of the study, perchlorate was below the detection limit (1 µg/mL). Urinary iodine measurements

indicated that iodine intake was satisfactory for most children from each group. The goiter rate within the

high-nitrate areas was significantly higher than the goiter rate within the low-nitrate area (13.5 versus

5.9%). Familial thyroid disorders and chronic diseases were reported by families of 7.7% of the children

from the high-nitrate area and only 3.06% of the children from the low-nitrate area. In a similar study

that included two areas of Bulgaria, one with high nitrate in the drinking water (average of 93 mg/L) and

one with low nitrate in the drinking water (average 8 mg/L), pregnant women from the high- (n=26) and

low- (n=22) nitrate areas and children (3–6 years of age) from the high- (n=50) and low- (n=49) areas

were evaluated for iodine status and goiter frequency. Mean urinary iodine in the women from the high-

nitrate area was significantly lower than that of the women from the low-nitrate area (147.85±56.38

versus 230.55±61.56 µg/L). Iodine deficiency was indicated for 5/26 women and 11/50 of the children

from the high-nitrate area and 1/22 women and 5/49 children from the low-nitrate area. Goiter was

66 NITRATE AND NITRITE

3. HEALTH EFFECTS

reported for 9/26 women and 14/50 children from the high-nitrate area and 2/22 women and 7/49 children

from the low-nitrate area. Familial thyroid disorders and chronic diseases were reported for 9/26 women

and 3/50 children from the high-nitrate area and 2/22 women and 1/49 children from the low-nitrate area.

The differences in goiter rates may be the result of differences in iodine intake and reported familial

thyroid disorder and chronic disease prevalence.

Aschebrook-Kilfoy et al. (2012) reported an association between nitrate in private wells at estimated

levels >6.5 mg nitrate-nitrogen /L (>28.6 mg nitrate/L) and elevated serum TSH in women (but not men)

as an indicator of subclinical hypothyroidism (OR 1.60, 95% CI: 1.11, 2.32). The study included

2,543 Old Order Amish residing in several counties in Pennsylvania for whom TSH levels were available.

Nitrate levels in the wells were estimated by modeling data provided by the U.S. Geological Survey

(USGS) that monitored nitrate levels in 3,613 wells in the study area.

In one cohort of 21,977 older women in Iowa who had used the same water supply for >10 years, there

were no significant differences in prevalence of self-reported hypothyroidism or hyperthyroidism between

those using private wells as drinking water source (n=5,436) and those using public water sources

(n=16,541) (Ward et al. 2010). Sufficient data for public water sources were available from which to

evaluate prevalence of thyroid disorders by quartile of nitrate concentration in public water sources

defined as mean concentrations <0.36, 0.36–1.00, 1.01–2.46, and >2.46 mg nitrate-nitrogen/L (<1.58,

1.58–4.4, 4.41–10.82, and >10.82 mg nitrate/L, respectively). There was no apparent association between

nitrate in the drinking water and prevalence of self-reported hypothyroidism or hyperthyroidism when

comparing results by quartile. No nitrate measurement data were available for women using private

wells. Data for these women were compared to data for women in the lowest quartile of public water

sources, although it was estimated at the time of the study that 18% of the rural private wells in Iowa had

nitrate levels >10 mg nitrate-nitrogen/L (>44 mg nitrate/L). In the same study (Ward et al. 2010), dietary

nitrate intake was estimated using a food frequency questionnaire and published nitrate levels for various

food sources and the study subjects (3,018 cases of hypothyroidism and 937 cases of hyperthyroidism)

were divided into quartiles according to dietary nitrate intake (≤17.4, 17.5–27.7, 27.8–41.1, and >41.1 mg

nitrate-nitrogen/day; approximately equivalent to <77, 77–121.9, 122–181, and >181 mg nitrate mg/day,

respectively). Using the lowest quartile as a referent, associations were found for prevalence of

hypothyroidism (but not hyperthyroidism) for the second quartile (OR 1.13, 95% CI: 1.01, 1.27), third

quartile (OR 1.19, 95% CI: 1.06, 1.33), and fourth quartile (OR 1.24, 95% CI: 1.10, 1.40). A significant

trend was noted as well for increasing prevalence of hypothyroidism with increasing quartile of dietary

nitrate (p=0.001).

67 NITRATE AND NITRITE

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In a randomized controlled study, 10 volunteers consumed sodium nitrate in aqueous solution at a dose of

15 mg/kg/day for 28 days; 10 other volunteers receiving distilled water served as controls. There were no

sodium nitrate treatment-related effects on thyroidal

131

iodine uptake or plasma thyroid hormone

concentrations (Hunault et al. 2007).

Thyroid status has been assessed to some extent in animals consuming drinking water or food to which

nitrate salts had been added. There were no clinical signs of hypothyroidism or effects on serum T3 or T4

levels in adult Beagles or their puppies during exposure of the breeding dogs to sodium nitrate in the

drinking water for 1 year at concentrations in the range of 300–1,000 ppm (equivalent to 219–730 mg

nitrate/L) (Kelley et al. 1974).

Decreased thyroidal

131

iodine uptake was noted in rats given food

containing 0.5–2.5% potassium nitrate (equivalent to 3,000–15,000 mg nitrate/kg food) (Bloomfield et al.

1961). Significantly increased uptake of thyroidal

131

iodine; decreased serum T3, T4, and TSH levels;

increased thyroid weight; and follicular hyperplasia were noted in female Wistar rats administered sodium

nitrate in the drinking water for 30 weeks at concentrations ≥250 mg/L (≥159 mg nitrate/kg/day, based on

reported average water intake and EPA [1988] subchronic reference body weight of 0.156 kg for the

female Wistar rat) (Eskiocak et al. 2005). In another study (Zaki et al. 2004), significantly decreased

serum T3 (34–44% lower than controls), increased thyroid weight (45–77% greater than controls), and

histopathologic thyroid lesions (glandular hypertrophy accompanied by vacuolization, increased colloidal

volume of the follicles, and flattened follicular epithelium) were observed in male Wistar rats receiving

drinking water for 5 months to which potassium nitrate had been added at concentrations resulting in

estimated doses ≥13.5 mg nitrate/kg/day (based on EPA [1988] subchronic reference values for body

weight and water consumption for the male Wistar rat).

El-Wakf et al. (2008) reported significantly decreased serum T3 and T4 levels (17–41% lower than

controls) in all groups of weanling male Wistar rats provided sodium nitrate in the drinking water for

4 months at concentrations resulting in author-estimated intakes in the range of 8.7–47.4 mg sodium

nitrate/kg/day (equivalent to 6.4–34.6 mg nitrate/kg/day). At estimated doses ≥15.8 mg nitrate/kg/day,

significantly increased serum TSH was also noted (26–30% higher than that of controls). Groups of

similarly-treated young adult male Wistar rats exhibited significantly decreased T3 and T4 levels (24–

47% lower than controls) and increased serum TSH (30–35% higher than controls) at estimated doses

≥15.8 mg nitrate/kg/day.

68 NITRATE AND NITRITE

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In a 28-day study of rats receiving food to which potassium nitrate had been added to constitute 3% of the

diet, thyroid effects included significantly increased thyroid gland weight (45% greater than controls),

increased TSH (nearly 7-fold higher than that of controls), decreased serum T3 and T4 levels (61–63%

lower than controls), and decreased thyroid peroxidase activity (84% lower than controls)

(Mukhopadhyay et al. 2005). Based on reported body weight data and the EPA (1988) allometric

equation for calculating a food consumption rate for laboratory mammals (0.056 x body weight

0.6611

), an

estimated dose was 2,416 mg nitrate/kg/day.

Til et al. (1988) added potassium nitrite to the drinking water of male and female rats for 13 weeks at

concentrations resulting in estimated doses in the range of 8.9–199.2 mg/kg/day (4.8–108 mg

nitrite/kg/day) to the males and 10.9–241.7 mg/kg/day (5.9–130.5 mg nitrite/kg/day) to the females.

Doses ≥13.3 mg nitrite/kg/day (males) and ≥61.8 mg nitrite/kg/day (females) resulted in hypertrophy in

the zona glomerulosa of the adrenal gland. In this study, potassium was added to the drinking water of

each treatment group up to the level of potassium in the drinking water of the highest dose group.

Controls included groups with untreated drinking water and groups with potassium chloride-treated water.

The effect on the adrenal gland was not observed in the untreated controls or the potassium chloride

controls, indicating that the effect was the result of nitrite ion. Similar results were obtained at estimated

doses of 105.1 mg nitrite/kg/day (males) and 130.1 mg nitrite/kg/day (females) in a subsequent similarly-

designed study (Til et al. 1997) to evaluate effects at lower doses than those employed in the earlier study

(Til et al. 1988). Results of a subsequent study indicate that the effect on the adrenal gland of the rat is a

physiological adaptation to repeated episodes of hypotension caused by nitrite (RIVM 1996).

Dermal Effects. Available information regarding dermal effects following oral exposure to nitrate or

nitrite is limited to a case report in which ingestion of ammonium nitrate was considered a possible cause

of erythema dyschromicum perstans (ashy dermatosis) (Jablonska 1975).

Body Weight Effects. No information was located regarding body weight effects in humans

following ingestion of nitrate or nitrite.

No body weight effects were observed in some studies of laboratory animals provided sodium nitrate,

sodium nitrite, or potassium nitrite in the drinking water for intermediate exposure durations (4 weeks to

10 months) at concentrations resulting in estimated doses in the range of 1,583–7,300 mg nitrate/kg/day

(Maekawa et al. 1982; Mukhopadhyay et al. 2005) or 28–435.5 mg nitrite/kg/day (Greenblatt and

Lijinsky 1974; Greenblatt and Mirvish 1973; Greenblatt et al. 1971; Lin and Ho 1992; Maekawa et al.

69 NITRATE AND NITRITE

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1982; NTP 2001; Vorhees et al. 1984). Depressed body weight and/or body weight gain (approximately

10% less that of controls) were observed in other studies at estimated doses of 8,241.9–14,600 mg

nitrate/kg/day (Maekawa et al. 1982) and 663.3–1,080.6 mg nitrite/kg/day (Maekawa et al. 1982; NTP

2001). In chronic-duration studies (≥365 days), doses in the range of 101–178.2 mg nitrite/kg/day and

1,730 mg nitrate/kg/day resulted in 10–15% depressed body weight in rats and mice (Grant and Butler

1989; Maekawa et al. 1982; van Logten et al. 1972).

Body weight data in the study report of Zaki et al. (2004) indicate as much as 16–25% depressed mean

body weight among male Wistar rats provided drinking water for 5 months that contained 150 or 500 mg

potassium nitrate/L (estimated doses of 13.5 and 45 mg nitrate/kg/day); however, data regarding food and

water consumption were not included in the study report. El-Wakf et al. (2008) provided drinking water

to weanling male Wistar rats for 4 months that contained 100, 250, or 500 mg sodium nitrate/L (estimated

doses of 6.4, 15.8, and 34.6 mg nitrate/kg/day) and reported mean final body weights that were 11, 29,

and 46%, respectively, less than that of control; however, data regarding food and water consumption

were not included in the study report. El-Wakf et al. (2015) provided young (3-week-old) and adult

(12-week-old) male Wistar rats with drinking water to which sodium nitrate was added at 550 mg/L

(estimated daily intake of 47.7 mg sodium nitrate/kg/day or 34.8 mg nitrate/kg/day) for 4 months;

controls received drinking water without added sodium nitrate. The sodium nitrate treatment resulted in

depressed body weight (24 and 9% less among the young and adult rats, respectively, compared to

controls) and depressed body weight gain (39 and 30% less among the young and adult rats, respectively,

compared to controls).

Metabolic Effects. Possible associations between nitrate and/or nitrite in drinking water and/or food

sources and risk of type 1 diabetes have been investigated in a number of epidemiological studies (Casu et

al. 2000; Dahlquist et al. 1990; Kostraba et al. 1992; Moltchanova et al. 2004; Parslow et al. 1997; van

Maanen et al. 2000; Zhao et al. 2001). Statistically significant associations between estimated nitrate

and/or nitrite intake were reported by some investigators, but were not observed by others. Limitations of

studies include the lack of quantitative dose-response data and the likelihood of confounding by other

potential toxicants. Therefore, there is considerable uncertainty regarding nitrate or nitrite intake and risk

of type 1 childhood diabetes.

A study in the Netherlands involved 1,064 cases of type 1 diabetes in a total of 2,829,020 children (0–

14 years of age) included in the analysis (van Maanen et al. 2000). Nitrate levels in drinking water were

determined by postal code. Two exposure categories were used. One category was based on equal

70 NITRATE AND NITRITE

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numbers of children exposed to various levels of nitrate in the drinking water (0.25–2.08, 2.10–6.42, and

6.44–41.19 mg nitrate/L); the other category was based on cutoff values of 10 and 25 mg nitrate/L. The

study authors concluded that there was little evidence that nitrate in the drinking water was a risk factor

for childhood type 1 diabetes under the conditions of the study.

Zhao et al. (2001) found no significant association between nitrate in the drinking water and risk for

childhood type 1 diabetes in a study of 517 cases (0–15 years of age). The mean concentration of nitrate

in the drinking water was 6.62 mg/L (range 0.49–31.9 mg/L). Casu et al. (2000) found no significant

association between nitrate in tap water or bottled water and risk of type 1 diabetes among 1,975 cases

(0–29 years of age), 1,142 of which were <15 years of age. In this study, nitrate concentrations in tap and

bottled water were below the acceptable maximal concentration of 50 mg/L established by the European

Community and the recommended level of 25 mg/L. Moltchanova et al. (2004) found no significant

association between childhood type 1 diabetes and nitrate in the groundwater in Finland. The study

included 3,598 cases of childhood type 1 diabetes (ages 0–14 years) and 9,601,164 children at risk;

drinking water nitrate levels averaged 6.228 mg/L.

Dahlquist et al. (1990) evaluated a variety of nutrients and food additives (including nitrate) as possible

risk factors for type 1 diabetes among 339 children under 15 years of age and matched with 528 control

children in Sweden. Estimates of intake of the various nutrients and food additives were made based on

parental responses to food frequency questionnaires. Upon dividing the subjects into three groups

according to estimated nitrate intake (low=<25

th

percentile; medium=25–75

th

percentile; high=>75

th

percentile), a significant nonlinear trend for increased risk of type 1 diabetes with increasing nitrate intake

was noted. The high-nitrate intake group exhibited a significantly increased risk (crude OR 2.14, 95% CI:

1.64, 3.54) compared to the low-nitrate intake group; adjustment for age, sex, maternal age, maternal

education, and family history of type 1 diabetes did not significantly alter the results.

Kostraba et al. (1992) calculated incidence rates by county in Colorado (63 counties) for type 1 diabetes

in children (<18 years of age at diagnosis during the years 1978 and 1988; n=1,280) and compared the

rates to nitrate levels in potable water supplies. Children in counties with water nitrate levels in the range

of 0.77–8.2 mg/L had a significantly increased risk of type1 diabetes compared to those in counties with

water nitrate levels in the range of 0.0–0.084 mg/L.

Parslow et al. (1997) reported a significant increase association (SIR 115, 95% CI: 107,124) between

nitrate in drinking water (highest tertile versus lowest tertile) and incidence of childhood type 1 diabetes

71 NITRATE AND NITRITE

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diagnosed between 1978 and 1994 in the Yorkshire Regional Health Authority in England. The study

subjects were 0–16 years of age, and nitrate levels in the drinking water were divided into tertiles (1.48–

<3.22, 3.22–<14.85, 14.85–40.01 mg/L). The study included 498 cases in a population of

225,708 children in the lowest tertile, 591 cases in a population of 232,373 children in the middle tertile,

and 708 cases in a population of 237,951 children in the highest tertile.

Virtanen et al. (1994) reported a significant association between estimated dietary nitrite intake by

children and mothers and risk for type 1 diabetes in all age groups of boys and girls (ages 0–4, 5–9, and

10–14 years). The study included 684 children with Type 1 diabetes, 595 control children, 548 case-

control pairs of fathers, and 620 case-control pairs of mothers in a nationwide Finnish study. Nitrate and

nitrite levels were estimated based on results from food frequency questionnaires and household water

data provided by the Finnish waterworks. Nitrate intake of the mother was associated with decreased risk

for childhood type 1 diabetes.

El-Wakf et al. (2015) provided young (3-week-old) and adult (12-week-old) male Wistar rats with

drinking water to which sodium nitrate was added at 550 mg/L (estimated daily intake of 47.7 mg sodium

nitrate/kg/day or 34.8 mg nitrate/kg/day) for 4 months; controls received drinking water without added

sodium nitrate. The sodium nitrate treatment induced hyperglycemia in both age groups. In a study of

Sprague-Dawley rats administered sodium nitrite by gavage at 80 mg/kg/day for 12 weeks, nitrite-induced

effects included inhibition of liver glycogenesis (generation of glycogen from glucose molecules) and

enhanced liver glycogenolysis (breakdown of glycogen) and gluconeogenesis (generation of glucose from

non-carbohydrate carbon substrates), accompanied by hyperglycemia and insulin resistance (Al-Gayyar et

al. 2015).

3.2.2.3 Immunological and Lymphoreticular Effects

No information was located regarding immunological or lymphoreticular effects in humans or animals

following oral exposure to nitrate or nitrite.

3.2.2.4 Neurological Effects

Ingestion of nitrite (from potassium nitrite or sodium nitrite sources) has been associated with severe

methemoglobinemia in adults and children; in many of these cases, clinical signs included dizziness, loss

of consciousness, and/or convulsions (CDC 1997, 2002; Gautami et al. 1995; Greenberg et al. 1945;

Sevier and Berbatis 1976; Ten Brink et al. 1982). These cases were the result of consumption of food or

72 NITRATE AND NITRITE

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drink that contained unusually high levels of nitrite via contamination, inadvertent use of sodium nitrite

instead of table salt, or ingestion of a single sodium nitrite tablet (667 mg nitrite).

Headache was induced in a male subject following consumption of a 10 mg sodium nitrite solution

(Henderson and Raskin 1972). Headaches were induced in 8 out of 13 such tests. The tests were

performed to evaluate whether nitrite in frankfurters that the subject had previously ingested might be

cause for the headache he had developed shortly thereafter. In a study designed to evaluate the oral

bioavailability of sodium nitrite in healthy volunteers (seven females and two males; mean age

22.9 years), headache was reported by four of the nine people ingesting 0.12 mmol sodium nitrite per

mmol hemoglobin (~4.4–5.4 mg sodium nitrite/kg, or 2.9–3.6 mg nitrite/kg) and by four of the nine

subjects ingesting 0.06 mmol sodium nitrite per mmol hemoglobin (~2.2–2.7 mg sodium nitrite/kg, or

1.5–1.8 mg nitrite/kg) (Kortboyer et al. 1997b).

Abnormalities in electroencephalograms (EEGs) were reported in male albino rats provided sodium nitrite

in the drinking water for 2 months at concentrations resulting in author-reported doses ≥14 mg sodium

nitrite (≥9.38 mg nitrite/kg/day) (Behroozi et al. 1972). The abnormal readings persisted during up to

4.5 months following cessation of exposure to sodium nitrite. At the highest dose (187.6 mg

nitrite/kg/day), rats exhibited clinical signs of sedation and became motionless during periods of electrical

outbursts.

Gruener (1974) reported increased aggressive behavior in male C57B1 mice provided sodium nitrite in

the drinking water at 1,000 mg/L (estimated dose of 165.4 mg nitrite/kg/day) for up to 13 weeks

postweaning. The mice had also been exposed via their parents during mating and their mothers during

gestation and lactation. Shuval and Gruener (1972) reported significantly reduced motor activity in male

mice provided sodium nitrite in the drinking water. Sodium nitrite levels tested ranged from 100 to

2,000 mg/L; however, the study report did not include specific information regarding the exposure levels

that resulted in reduced motor activity.

3.2.2.5 Reproductive Effects

See Section 3.2.2.6 for information regarding results of case-control studies that evaluated reproductive/

developmental end points.

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Several animal studies included evaluation of selected reproductive end points. Among three female

guinea pigs provided potassium nitrate in the drinking water for up to 204 days of cohabitation at a

concentration resulting in estimated intake of 4,972 mg nitrate/kg/day, one female died and the other two

females produced a total of two litters (one live birth per litter) (Sleight and Atallah 1968). During

191 days of cohabitation, four control females produced eight litters and a total of 31 live births. There

was no gross or histopathologic evidence of treatment-related effects on reproductive organs. Sleight and

Atallah (1968) provided other guinea pigs with drinking water that contained potassium nitrite at

concentrations ranging from 300 to 10,000 ppm. Exposure levels ≥1,000 ppm potassium nitrite

(estimated doses ≥148.5 mg nitrite/kg/day) resulted in decreases in number of litters and live births;

histopathologic evaluations of reproductive organs revealed placental, uterine, and cervical lesions.

No treatment-related effects on implantations or resorptions were seen in female Wistar rats provided

sodium nitrite in the food throughout the production of two litters at concentrations resulting in estimated

doses as high as 160 mg nitrite/kg/day (Hugot et al. 1980). No treatment-related effects on fertility were

seen in breeding dogs provided sodium nitrate in the drinking water for 1 year at concentrations resulting

in doses as high as 39 mg nitrate/kg/day (Kelley et al. 1974).

Alavantić et al. (1988a) treated male mice with sodium nitrate or sodium nitrite by gavage for 3 days at

doses of 0, 600, or 1,200 mg/kg/day (sodium nitrate) or 0, 60, or 120 mg/kg/day (sodium nitrite); sperm-

head abnormalities were evaluated at 11 and 17 days following treatment. Frequencies of sperm-head

abnormalities in the low- and high-dose sodium nitrate-treated and the low-dose sodium nitrite-treated

groups were not significantly different from controls. However, the high-dose group of sodium nitrite-

treated mice exhibited significantly increased frequency of sperm-head abnormalities at 11 and 17 days

following treatment (approximately 1.5-fold greater than controls). Alavantić et al. (1988b) treated male

mice with sodium nitrate or sodium nitrite by gavage for 2 weeks at doses of 0, 600, or 1,200 mg/kg/day

(sodium nitrate) or 0, 60, or 120 mg/kg/day (sodium nitrite) and subsequently mated them to virgin

females. Evaluation of primary spermatocytes from parental males revealed significantly increased

frequency of sperm-head abnormalities in the high-dose sodium nitrate-treated group (1.4-fold greater

than controls) and the low- and high-dose sodium nitrite-treated groups (1.2- and 1.4-fold greater,

respectively, than controls). There was no treatment-related effect on frequency of sperm-head

abnormalities in F1 males. Fertility in the high-dose sodium nitrite-treated group was significantly

affected; only 31 of 49 females mated to the high-dose males became pregnant compared to 45 of

50 females mated to control males.

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Alyoussef and Al-Gayyar (2016a, 2016b) administered sodium nitrite to male Sprague-Dawley rats by

gavage at 0 or 80 mg/kg/day for 12 weeks. Sodium nitrite treatment resulted in increased testicular

weight (1.6–1.7-fold greater than controls); decreased serum testosterone levels (36–44% less than

controls); decreased epididymal sperm count (48% less than controls); decreased testicular anti-

inflammatory cytokine levels; increased serum luteinizing hormone (LH), follicle stimulating hormone

(FSH), and prolactin levels; and increased testicular levels of pro-inflammatory cytokines, oxidative stress

markers, and enzymes involved in programmed cell death.

NTP (2001) reported degeneration of the testis (characterized by increased size of residual bodies within

the lumen of the seminiferous tubules) in male mice provided sodium nitrite in the drinking water for

14 weeks at concentrations resulting in estimated doses ≥435.5 mg nitrite/kg/day; the biological

significance of this lesion was uncertain. In similarly-treated female mice, estrous cycles were

significantly increased (11 and 15%, respectively, longer than controls) at estimated doses of 298.1 and

824.1 mg nitrite/kg/day, but not at 562.8 mg nitrite/kg/day. Among similarly-treated male and female

rats, the males exhibited 7–18% decreased sperm motility at doses ≥134 mg nitrite/kg/day; there were no

treatment-related effects on vaginal cytology end points in the females at doses as high as 231 mg

nitrite/kg/day.

3.2.2.6 Developmental Effects

Several population-based, case-control studies evaluated possible associations between developmental

end points and exposure to nitrate from drinking water sources. The results are not adequate for

quantitative risk assessment because estimations of nitrate intakes were typically based on measurements

of nitrate levels in drinking water sources at selected time points and self-reported estimates of water

consumption, possible confounding by other potential toxicants was not evaluated, and most studies did

not account for dietary nitrate or nitrite intake which is typically the major source of ingested nitrate and

nitrite. Statistically significant associations between nitrate in the drinking water and selected

developmental end points (e.g., birth defects, spontaneous abortions) were reported by some investigators,

but were not observed by others.

Brender et al. (2013) evaluated possible relationships between prenatal exposure to nitrate in drinking

water and selected birth defects in a large population-based, case-control study that included 3,300 case

mothers and 1,121 control mothers who were participants in the National Birth Defects Prevention Study.

Nitrate levels were measured in public water supplies and in representative samples of bottled water sold

75 NITRATE AND NITRITE

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in local stores; daily nitrate consumption was estimated from self-reported water consumption at home

and work. The lowest tertile of nitrate intake from water (<0.91 mg/day at conception or <1.0 mg/day

during preconception and the first trimester of pregnancy) represented the referent group. Within the

highest tertile (≥5.0 mg/day at conception; ≥5.42 mg/day during preconception and the first trimester of

pregnancy), significant associations were noted for risk of spina bifida (OR 2.02, 95% CI: 1.01, 2.04), any

limb deficiency (OR 1.79, 95% CI: 1.05, 3.08), any oral cleft defect (OR 1.45, 95% CI: 1.10, 1.92), cleft

lip without cleft palate (OR 1.82, 95% CI: 1.08, 3.07), cleft palate (OR 1.90, 95% CI: 1.17, 3.09), and any

neural tube defect (OR 1.43, 95% CI: 1.01, 2.04). Cases in the various tertiles ranged in number from

23 to 173. The study authors noted that higher estimated nitrate intakes from drinking water did not

increase associations between reported maternal intake of nitrosatable drugs and birth defects.

Dorsch et al. (1984) evaluated 218 cases of congenital malformations and matched controls between 1951

and 1979 in an area of South Australia. In an analysis of data by estimated level of nitrate in the drinking

water, the risk of malformations was significantly greater at nitrate levels of 5–15 mg/L (OR 2.6, 95% CI:

1.6, 4.1; 138 cases, 106 controls) and >15 mg/L (OR 4.1, 95% CI: 1.3, 13.1; 10 cases, 5 controls)

compared to those with nitrate levels <5 mg/L (70 cases, 107 controls).

Scragg et al. (1982) evaluated possible associations between maternal water source and frequency of

congenital malformations (mainly neural tube defects) in a locality in South Australia (258 cases and

matched controls). A referent group consisted of those women who used rainwater as the drinking water

source. Significantly increased risk of occurrence of a malformation was noted for those women who

drank water from a lake source (RR 2.8, 95% CL: 1.6, 5.1) and for women who used water from private

wells with nitrate levels typically >15 ppm (RR 4.1, 95% CL: 1.7, 10.0).

Cedergren et al. (2002) reported nonstatistically significant increased risk of cardiac defects among

infants of mothers exposed to nitrate in the drinking water at levels ≥2 mg/L (OR 1.18, 95% CI: 0.97,

1.44; 392 cases, 27,962 controls) compared to those with nitrate levels <2 mg/L; all measured nitrate

concentrations were below the Swedish maximum contaminant level. The study population included

75,832 infants born in a Swedish county between January 1982 and December 1996.

Croen et al. (2001) evaluated 538 cases of neural tube defects and 539 normal controls in an area of

California between June 1989 and May 1991. Exposure to nitrate in drinking water at concentrations

>45 mg/L was associated with statistically significantly increased risk of anencephaly (OR 4.0, 95% CI:

76 NITRATE AND NITRITE

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1.0, 15.4), but no increased risk for spina bifida. Increased risk was also noted at nitrate levels <45 mg/L

among groundwater drinkers.

Arbuckle et al. (1988) evaluated mothers in the area of New Brunswick where private wells averaged

26 mg/L nitrate and public municipal sources averaged 0.1 mg/L nitrate. There was no statistically

significant increased risk for delivering a central nervous system-malformed infant by mothers using

private wells (OR 2.30, 95% CI: 0.73, 7.29). The study included 130 cases of central nervous system

birth defects for the years 1973–1983, each matched to 2 controls.

Aschengrau et al. (1993) found no statistically significant association between drinking water nitrate

levels (up to 4.5 mg/L) or nitrite levels (up to 0.15 mg/L) and frequency of congenital anomalies,

stillbirth, or neonatal death among 1,171 cases and 1,177 controls who delivered at a Massachusetts

hospital between August 1977 and March 1980.

Holtby et al. (2014) evaluated possible associations between nitrate in drinking water sources and

incidence of congenital anomalies in the agricultural region of Kings County, Nova Scotia, Canada

between 1988 and 2006. A mean level of 6.44 mg nitrate-nitrogen/L was calculated for rural wells

(equivalent to 28.34 mg nitrate/L), based on 1,113 water samples from 140 wells. A mean level of

2.03 mg nitrate-nitrogen/L was calculated for municipal water supplies (equivalent to 8.93 mg nitrate/L),

based on 53 water samples from 20 water sources (19 groundwater sources and 1 surface water source).

Nitrate-nitrogen concentration estimates were divided into tertiles (<1, 1–5.56, and >5.56 mg nitrate-

nitrogen/L; equivalent to <4.4, 4.4–24.46, and >24.46 mg nitrate/L). Overall, no significant association

was found between nitrate levels in drinking water sources and incidences of congenital malformations.

However, stratification of the data by conception before or after the onset of food fortification with folate

in Canada (instituted in 1998) resulted in an OR of 2.44 (95% CI 1.05, 5.66) for risk of congenital

anomalies with exposure of 1–5.56 mg nitrate-nitrogen/L (4.4–24.46 mg nitrate/L) for the time period

(1998–2006).

Ericson et al. (1988) found no association between frequency of neural tube defects and levels of nitrate

in the drinking water in a case-control study that included 1,458 cases of neural tube defects and

280 matched controls. The reported average nitrate levels in the water were 4.9 mg/L among the cases

and 5.1 mg/L among the controls.

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Super et al. (1981) evaluated the status of 486 infants in a geographical area of southwest Africa served

by 153 wells divided into regions of high nitrate (>20 mg/L) and low nitrate (≤20 mg/L). There was no

significant association between nitrate levels in drinking water sources and incidence of stillbirths,

prematurity, or birth size; however, an increased incidence of deaths during the first year of life was noted

for the high-nitrate region.

Winchester et al. (2009) investigated whether U.S. live births are at increased risk for birth defects when

conception occurs during months when surface water agrichemicals (including nitrate, atrazine, and other

pesticides) are at greatest concentrations (April–July). For the years 1996–2002, monthly agrichemical

concentrations were calculated using USGS’s National Water Quality Assessment data and live birth data

collected from the Centers for Disease Control and Prevention (CDC) natality data sets. Birth defects

were more likely to occur in live births conceived between April and July. However, this finding does not

necessarily implicate nitrate in the drinking water.

Brender et al. (2004) found no significant association between dietary nitrate or nitrite intake and risk of

offspring with neural tube defects at estimated total nitrite doses (dietary nitrite plus 5% dietary nitrate)

ranging from <7.5 to >10.53 mg/day. However, the risk of neural tube defect was significant among

those women with total nitrite doses >10.53 mg/day who also reported taking nitrosatable drugs (OR 7.5,

95% CI: 1.8, 45).

Huber et al. (2013) estimated daily nitrate and nitrite intakes among 6,544 mothers of infants with neural

tube defects, oral clefts, or limb deficiencies and 6,807 mothers of unaffected control infants, based on

results of food frequency questionnaires. The study included areas of 10 U.S. states, and the population

was divided into quartiles of estimated nitrate intake and nitrite intake. There was no statistically

significant increased risk of neural tube defect with any estimate of nitrate or nitrite intake. Similar

results were obtained for oral cleft and limb deficiency, with the exceptions of increased risk at the

highest quartile of cleft lip only (OR 1.32, 95% CI: 1.01, 1.72) and cleft lip with or without cleft palate

(OR 1.24, 95% CI: 1.05, 1.48) at the highest quartile of animal-based nitrite intake, and increased risk of

intercalary limb defect (OR 4.70, 95% CI: 1.23, 17.93) at the highest quartile of total nitrite intake.

Aschengrau et al. (1989) found no nitrate-related increased risk of spontaneous abortion in a study of

286 women who presented at a Massachusetts hospital between July 1976 and February 1978 with a

spontaneous loss through gestation week 27 and 1,391 controls.

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The CDC (1996) investigated a small cluster of spontaneous abortions (three women, six spontaneous

abortions) in close proximity to one another and to a hog farm in LaGrange County, Indiana during 1991–

1993. Well water on the hog farm contained >50 mg nitrate/L. Water samples from wells supplying the

women who aborted contained 19–26 mg nitrate/L. A mean concentration of 3.1 mg nitrate/L (1.6–

8.4 mg/L) was determined for well water supplies to residences of a comparison group of five women,

each having full-term birth within the same time period. During the investigation, another case was

identified in which a 35-year-old woman, living approximately 10 miles from the other three women, had

two spontaneous abortions after having five previous live births. Well water during the first four

pregnancies was found to contain 1.2 mg nitrate-nitrogen/L (5.3 mg nitrate/L); the spontaneous abortions

occurred after installation of a new well that was found to contain 28.7 mg nitrate-nitrogen/L (126 mg

nitrate/L). Although all four women delivered full-term, live-born infants after changing to nitrate-free

drinking water sources, the occurrences of spontaneous abortion may have been unrelated to nitrate-

containing drinking water.

George et al. (2001) evaluated the geographical and seasonal distribution of sudden infant death

syndrome (SIDS) in Sweden during the period 1990–1996 in relation to nitrate levels in drinking water

and changes in groundwater nitrate content. The local incidence of SIDS was correlated to maximally

recorded concentrations of nitrate in the drinking water. However, in addition to lack of dose-response

data for individuals, the SIDS incidence was declining during the study period, numbers of SIDS cases

were small in scarcely populated areas, and nitrate concentrations in groundwater sources may have

changed rapidly with weather changes and other factors.

Tabacova et al. (1997) evaluated maternal health among 61 pregnant women who lived near an

ammonium nitrate fertilizer plant and presented at a local prenatal care clinic. Tabacova et al. (1998)

evaluated the status of 51 mother-infant pairs in the same region. Nitrogen oxides in the air averaged

23.1 µg/m

3

with short-term peak levels as high as 238.5; nitrate concentrations in the public drinking

water supply measured 8–54 mg/L; nitrate levels in private wells measured as much as 13–400 mg/L. Of

the 61 pregnant mothers in the sample of Tabacova et al. (1997), only 10 had “normal” pregnancies.

Mothers diagnosed with anemia (41 cases), toxemia (20 cases), and/or threatened abortion/premature

labor (20 cases) exhibited ≥2-fold higher serum methemoglobin than those with “normal” pregnancies.

Of the 51 mothers in the sample of Tabacova et al. (1998), there were 38 full-term and normal-weight

infants, 7 full-term and low-weight infants, 6 premature deliveries, 1 Caesarean delivery, and 1 breech

delivery. Elevated methemoglobin was observed in serum from 28/51 of the mothers, and 24/51 cord

blood samples. Both maternal and cord blood methemoglobin levels were higher in cases of abnormal

79 NITRATE AND NITRITE

3. HEALTH EFFECTS

birth outcome. These results could not be directly linked to elevated nitrate intake from drinking water or

food sources.

Developmental end points have been assessed in some animal studies. No indications of treatment-

related developmental toxicity were seen in fetuses from pregnant mice administered sodium nitrite in the

drinking water during gestation days 7–18 at concentrations as high as 1,000 mg/L (approximate doses as

high as 113.2 mg nitrite/kg/day) (Shimada 1989). There were no signs of toxicity in offspring of pregnant

rats administered 80 mg sodium nitrite/kg (53.6 mg nitrite/kg) on gestation day 15; offspring were

observed for up to 140 days postpartum (Khera 1982). There were no signs of treatment-related

developmental effects during the production of two litters by female Wistar rats provided sodium nitrite

in the food at concentrations resulting in estimated doses as high as 160 mg nitrite/kg/day (Hugot et al.

1980). Among three female guinea pigs provided potassium nitrate in the drinking water for up to

204 days of cohabitation at a concentration resulting in estimated intake of 4,972 mg nitrate/kg/day, one

female died and the other two females produced a total of two litters (one live birth per litter) (Sleight and

Atallah 1968). During 191 days of cohabitation, four control females produced eight litters and a total of

31 live births. The only indication of a treatment-related effect on the offspring of pregnant mice

administered sodium nitrite by gavage at 0.5 mg/mouse/day (approximate dose of 13 mg nitrite/kg/day)

on gestation days 1–14, 16, or 18 was increased fetal hepatic erythropoiesis at gestation days 14 and 16,

which was thought to have been a response to nitrite-induced fetal methemoglobinemia (Globus and

Samuel 1978).

Significantly impaired auditory and visual discrimination learning behavior and retention of passive

avoidance responses (Nyakas et al. 1990), and delay in cholinergic and serotonergic fiber outgrowth in

cortical target areas of the brain (Nyakas et al. 1994), presumably due to nitrite-induced hypoxia, were

reported in offspring of Wistar rats provided sodium nitrite in the drinking water at 2,000 mg/L (1,334 mg

nitrite/L) during gestation day 13 until parturition. However, lack of information regarding body weight

and water consumption of the pregnant rats precludes estimation of nitrite doses to the pregnant dams.

Shuval and Gruener (1972) provided sodium nitrite in the drinking water of pregnant rats for 6 weeks

(that presumably included gestation and lactation) at concentrations of 2,000 or 3,000 mg/L (1,334 or

2,001 mg nitrite/L, respectively). There were no treatment-related effects on group litter sizes or pup

birth weights. However, during 3 weeks postpartum, 30 and 53% of the low- and high-dose pups died

(compared to 6% of control pups); surviving pups from the low- and high-dose groups exhibited 43 and

66% lower mean body weight than controls at 3 weeks postpartum. Lack of information regarding body

80 NITRATE AND NITRITE

3. HEALTH EFFECTS

weight and water consumption of the pregnant rats precludes estimation of nitrite doses to the pregnant

dams.

Increased pup mortality, depressed preweaning pup body weight, and delayed swimming development

were observed in offspring of male and female rats provided sodium nitrite in the diet at 0.025 or 0.05%

(estimated dose levels of 14.4 and 28.1 mg nitrite/kg/day, based on author-reported dose of 43 mg sodium

nitrite/kg/day for the high-dose group) (Vorhees et al. 1984). There were no treatment-related effects on

preweaning behavior that included surface righting, pivoting, negative geotaxis, or auditory startle and no

effects on postweaning survival, body weight, or most behavioral indices, with the exception of decreased

open-field behavior on days 40–45 in groups from dams exposed to 0.0125 or 0.05% (but not 0.025%)

sodium nitrite in the diet.

3.2.2.7 Cancer

Human Data. Numerous studies are available in which the carcinogenicity of ingested nitrate and nitrite

in humans was assessed. A comprehensive review of the cancer epidemiology studies of nitrate and

nitrite, published up to approximately 2007, is provided in IARC (2010). Up to that point, most studies

employed ecological designs and fewer case-control or cohort studies were available on cancers other

than gastrointestinal cancers. Since then, several cohort and case-control studies have been reported that

examine a variety of different cancer types (Aschebrook-Kilfoy et al. 2011, 2013a, 2013b; DellaValle et

al. 2013; Espejo-Herrera et al. 2015, 2016a, 2016b; Inoue-Choi et al. 2012, 2015; Kilfoy et al. 2010,

2011; Kim et al. 2007; Michaud et al. 2009; Ward et al. 2007, 2008; Wu et al. 2013; Yang et al. 2010;

Zeegers et al. 2006; Table 3-2). Ecological studies measure exposure and outcomes at the group level

rather than the individual level. Interpretation of outcomes of these studies is more uncertain because of

various factors that contribute to ecologic bias (group-based associations between exposure and cancer

outcomes may not apply to individuals). Ecological studies can be valuable for exploring causal

relationships when the exposures within exposure groups have low variability (homogenous), differences

in exposure are large between exposure groups, and when groups are assigned based on geography and

migration in and out of exposure areas is minimal (IARC 2010). A typical example of an ecological

design assigns group exposures based on residence within a public water supply (PWS) district, where the

average (or median) concentration of nitrate or nitrite in the PWS is the exposure metric and outcomes are

measured at the level of the PWS area (e.g., cancer incidences in two areas served by public water

supplies that have different nitrate or nitrite levels). The major limitation of this approach is that the

group-based exposure estimate may (and probably does not) apply to individuals and their cancer

81 NITRATE AND NITRITE

3. HEALTH EFFECTS

Table 3-2. Selected Cohort and Case-Control Studies Published Since 2006

Examining Possible Associations Between Nitrate and Nitrite Intake and

Cancer

Nitrate and nitrite

Reference

type

Study design

intakes

Outcomes

a

Aschebrook-

Pancreatic

Cohort from NIH-

Quintile median

Kilfoy et al. AARP Diet and intake:

2011 Health Study, Nitrate from food:

1995–2006 34.8, 56.9, 75.0,

95.3, 150.3 mg/day;

303,156 cohort,

19.3, 29.9, 40.9,

1,728 cases

57.4,

94.8 mg/1,000 kcal

Nitrite from food: 0.8

,

1.0, 1.2, 1.2,

1.6 mg/day; 0.45,

0.57, 0.65, 0.74,

0.9 mg/1,000 kcal

Based on food

frequency

questionnaire,

24-hour recall, and

published food

nitrate and nitrite

levels

Aschebrook-

Thyroid

Cohort from

Quartile median

Kilfoy et al. Shanghai intake:

2013a Women’s Health Nitrate from food:

Study, 1996–2000 165.8, 257.8, 350.6,

506.8 mg/day;

73,317 cohort,

108.6, 164.2, 217.6,

164 cases

250.9 mg/1,000 kcal

Nitrite from food:

0.89, 1.27, 1.61,

2.14 mg/day; 0.62,

0.81, 0.95,

1.12 mg/1,000 kcal

Based on food

frequency

questionnaire,

24-hour dietary

recall, and published

food nitrate and

nitrite levels

Highest quintile vs lowest quintile:

Nitrate:

OR 1.01 (95% CI 0.85, 1.20)

Nitrite:

OR 0.92 (95% CI 0.78, 1.08)

No increased risk when accounting

for nitrite intake from plant sources,

animal sources, or processed meats

Adjustments: age, race, caloric

intake, smoking, family history of

cancer and diabet

es, BMI; intakes of

saturated fat, folate, vitamin C

Highest quartile vs lowest quartile:

Nitrate:

RR 0.93 (95% CI 0.42, 2.07)

Nitrite: RR 2.05 (95% CI 1.20, 3.51)

with total nitrite intake

RR 1.96 (95% CI 1.28, 2.99) for

nitrite intake from processed meat

Adjustments: age, caloric intake,

education, history of thyroid disease;

intakes of vitamin C, carotene, folate

82 NITRATE AND NITRITE

3. HEALTH EFFECTS

Table 3-2. Selected Cohort and Case-Control Studies Published Since 2006

Examining Possible Associations Between Nitrate and Nitrite Intake and

Cancer

Nitrate and nitrite

Reference

type

Study design

intakes

Outcomes

a

Aschebrook-

Non-

Case-control with

Quartile median

Kilfoy et al. Hodgkin’s subjects from intake:

2013b lymphoma Nebraska

between 1999 and

Nitrate from food:

2002

22.0, 39.1, 57.5,

106.1 mg/day; 22.2,

348 cases,

38.2, 55.5, 88.3

470 controls

mg/1,000 kcal

Nitrite from food: 0.5,

0.6, 0.7, 0.9 mg/day;

0.49, 0.61, 0.71,

0.86 mg/1,000 kcal

Based on food

frequency

questionnaire and

published food

nitrate and nitrite

levels

Chiu et al.

Colon

Case-control from

Tertile median

2011 Taiwan Provincial Nitrate-nitrogen

Department of ranges in drinking

Health, 2003– water: <0.38, 0.39–

2007 0.57, ≥0.60 mg/L

(<1.67, 1.72–2.51,

3,707 cases,

≥2.64 mg nitrate/L)

3,707 controls

Based on PWS data

Highest quartile vs lowest quartile:

Nitrate:

OR 0.8 (95% CI 0.5, 1.3; p-trend

0.6)

Nitrite:

OR 1.3 (95% CI 0.8, 1.9; p-trend

0.4)

No significant associations for nitrate

or nitrite by lymphoma subtype

t(14;18)-positive or -negative)

Adjustments: sex, age, BMI, caloric

intake, education, family history of

cancer, vitamin C intake

Highest tertile vs lowest tertile:

OR 1.16 (95% CI 1.04, 1.30; p-trend

0.001)

OR 1.37 (95% CI 1.11, 1.69) with

drinking water calcium levels <34.6

mg/L

Adjustments: age, gender, marital

status, urbanization level of

residence

83 NITRATE AND NITRITE

3. HEALTH EFFECTS

Table 3-2. Selected Cohort and Case-Control Studies Published Since 2006

Examining Possible Associations Between Nitrate and Nitrite Intake and

Cancer

Nitrate and nitrite

Reference

type

Study design

intakes

Outcomes

a

DellaValle et

al. 2013

Kidney

(RCC)

Cohort from NIH-

AARP Diet and

Health Study,

1995–2006

491,841cohort,

488 cases

Quintile median

intake:

Nitrate intake from

food: 19.3, 40.9, and

94.8 mg/1,000 kcal,

for quintiles 1, 3, and

5, respectively

Highest quintile vs lowest quintile:

Nitrate:

No increased risk (HR 0.98; 95% CI

0.84, 1.14 for total RCC)

Nitrite:

No increased risk for total nitrite (HR

1.02; 95% CI 0.87, 1.19 for total

Nitrite intake from

food: 0.5, 0.7, and

0.9 mg/1,000 kcal,

for quintiles 1, 3, and

5, respectively

Based on food

frequency

questionnaire and

published food

nitrate and nitrite

levels

RCC) or nitrite from plant sources

(HR 0.89; 95% CI 0.76, 1.04 for total

RCC)

Increased risk for nitrite from animal

sources (HR 1.28; 95% CI 1.10,

1.49; p-trend <0.01 for total RCC),

nitrite from processed meat sources

(HR 1.16; 95% CI 1.00, 1.35; p-

trend

0.04 for total RCC), nitrite from

animal sources other than

processed meat (HR 1.23 (95% CI

1.06, 1.43; p-trend 0.02 for total

RCC), nitrate and nitrite from

processed meat sources (HR 1.17;

95% CI 1.00, 1.37; p-trend 0.03 for

total RCC)

Risk of RCC mainly associated with

clear cell histological subtype (e.g.,

HR 1.68; 95% CI 1.25, 2.27; p-trend

<0.01 for nitrite from animal sources

and clear cell subtype)

Adjustments: age, sex, caloric

intake, race, smoking, family history

of cancer, BMI, alcohol intake,

education; history of hypertension,

diabetes

84

2015

NITRATE AND NITRITE

3. HEALTH EFFECTS

Table 3-2. Selected Cohort and Case-Control Studies Published Since 2006

Examining Possible Associations Between Nitrate and Nitrite Intake and

Cancer

Nitrate and nitrite

Reference

type

Study design

intakes

Outcomes

a

Espejo-

Bladder

Case-control.

Herrera et al.

Spain, 1998–2001

556 controls,

531 cases

Espejo-

Breast

Case-control.

Herrera et al.

Spain, 2008–2013

2016b

1,520 controls,

1,245 cases

Average residential

No associations between risk of

ranges in drinking bladder cancer and average nitrate

water by tertiles: ≤5 level (OR 1.09; 95% CI 0.63, 1.87)

mg/L, >5–10 mg/L, for highest versus lowest level

>10 mg/L

For subjects with longest exposure

Based on historical duration (>20 years) to highest

records of nitrate levels (>9.5 mg/L), OR=1.42; 95%

levels in municipal CI 0.989 2.26

water sources

Stratification by intake of vitamin C,

vitamin E, meat, and gastric ulcer

did not modify the results

Adjustments: age, sex and area of

residence smoking status, NSAIDs

use, night-time urinary frequency,

time working in farm/agriculture

activities, tap water and vitamin C

daily intake, urinary infections (ever)

Average waterborne

No associations between dietary

ingested nitrate

nitrate intake or waterborne ingested

ranged from 2.9 ±1.9

nitrate and risk of breast cancer

mg/day (mean ± SD) overall, but increased risk (OR 1.64;

to 13.5 ±7.5 mg/day 95% CI 1.08, 2.49) among

postmenopausal women with both

Based on historical

high waterborne nitrate intake (>6

records of nitrate

mg/day) and high red meat intake

levels in municipal

(≥20 g/day)

water sources and

monitoring of other

Adjustments: study area, age,

sources

education, BMI, family history of

breast cancer, age at first birth, age

Average dietary

at menopause, oral contraceptives

nitrate intake ranged

use, energy intake

from 88.5±48.7

mg/day to 154±87.8

mg/day

Based on food

frequency

questionnaire and

published food

nitrate and nitrite

levels

85 NITRATE AND NITRITE

3. HEALTH EFFECTS

Table 3-2. Selected Cohort and Case-Control Studies Published Since 2006

Examining Possible Associations Between Nitrate and Nitrite Intake and

Cancer

Nitrate and nitrite

Reference

type

Study design

intakes

Outcomes

a

Espejo-

Colorectal

Case-control.

Herrera et al.

Spain and Italy,

2016a 2008–2013

3,530 controls,

1,869 cases

(1,285 colon; 557

rectal)

Average waterborne

ingested nitrate

ranged from 3.4±3.3

mg/day to 19.7±22.6

mg/day

Tertiles: ≤5, <5–10,

>10 mg/day

Based on historical

records of nitrate

levels in municipal

water sources and

monitoring of other

sources

Mean dietary nitrate

intake was 118±72

mg/day overall

(102±70.5 mg/day

from vegetables and

6.2±3.3 mg/day from

animal sources)

Tertiles: <4.5, 4.5–

6.8, >6.8 mg/day

Based on food

frequency

questionnaire and

published food

nitrate and nitrite

levels

For highest versus lowest

waterborne nitrate intake:

OR 1.49 (95% CI 1.24, 1.78) for

colorectal cancer

OR 1.52 (95% CI 1.24, 1.86) for

colon cancer

OR 1.62 (95% CI 1.23, 2.14) for

rectal cancer

For risk of rectal cancer among

subjects with dietary intake of nitrate

from animal sources:

OR 1.59 (95% CI 1.22, 2.06) for mid

tertile

OR 1.55 (95% CI 1.17, 2.05) for

highest tertile

Greater risk among men than

women

Adjustments: sex, age, education,

physical activity, BMI, family history

of colorectal cancer, NSAIDs use,

energy intake, oral contraceptives

use

86 NITRATE AND NITRITE

3. HEALTH EFFECTS

Table 3-2. Selected Cohort and Case-Control Studies Published Since 2006

Examining Possible Associations Between Nitrate and Nitrite Intake and

Cancer

Nitrate and nitrite

Reference

type

Study design

intakes

Outcomes

a

Inoue-Choi et

Breast

Cohort of post-

al. 2012 menopausal

women from Iowa

Women’s Health

Study, 1989–2008

34,388 cohort,

2,875 cases

Quintile median

nitrate intake from

drinking water: 1.6,

4.1, 9.4, 21.2, and

57.8 mg/2 L

Based on historical

database of Iowa

municipal water

supplies

Quintile median

nitrate intake from

food: 49.3, 78.7,

106.1, 140.2, 209.9

mg/day

Quintile median

nitrite intake from

food: 0.6, 0.9, 1.1,

1.4, 1.8 mg/day

Based on food

frequency

questionnaire and

published food

nitrate and nitrite

levels

Highest quintile vs lowest quintile:

Overall, no increased risk of breast

cancer with intake of nitrate and/or

nitrite from diet and/or drinking water

Significant trend for increasing HR

with increasing nitrite intake

HR 1.40 (95% CI 1.05, 1.87) for

nitrate and folate ≥400 µg/day

HR 1.0 95% CI

(0.79, 125) for nitrate

and folate <400 µg/day

Adjustments: age; caloric intake;

BMI; waist-hip ratio; education;

smoking; physical activity level;

alcohol intake; family history of

breast cancer; age at menopause;

age at first live birth; estrogen use;

intakes of alcohol, vitamin C, vitamin

E, flavonoids, cruciferae, red meat

87 NITRATE AND NITRITE

3. HEALTH EFFECTS

Table 3-2. Selected Cohort and Case-Control Studies Published Since 2006

Examining Possible Associations Between Nitrate and Nitrite Intake and

Cancer

Nitrate and nitrite

Reference

type

Study design

intakes

Outcomes

a

Inoue-Choi et

Ovarian

Cohort of post-

Quartile median

al. 2015 menopausal nitrate levels in

women from Iowa drinking water: 0.31,

Women’s Health 0.75, 1.68, 3.81 mg

Study, 1986–2010 nitrate-nitrogen/L

(1.36, 3.3, 7.39,

28,555 cohort,

16.76 mg nitrate/L)

315 cases

Historical database

of Iowa municipal

water supplies

Quintile median

nitrate intake from

food: 49.5, 78.9,

106.2, 140.2, 209.2

mg/day

Quintile medium

nitrite dietary intake:

0.7, 0.9, 1.1, 1.4, 1.8

mg/day

Based on food

frequency

questionnaire and

published food

nitrate and nitrite

levels

Kilfoy et al.

Non-

Case-control of

Quartile ranges:

2010 Hodgkin’s women in

lymphoma Connecticut Nitrate from food:

between 1995 and

<63.9, 63.9 to <93.0,

2001 93.0 to <140.5,

≥140.5 mg/day

594 cases,

710 controls Nitrite from food:

<0.77, 0.77 to <0.99,

0.99 to <1.32, ≥1.32

mg/day

Based on food

frequency

questionnaire and

published food

nitrate and nitrite

levels

Highest quartile/quintile vs lowest

quartile/quintile:

HR 2.03 (95% CI 1.22, 3.38) for

highest quartile of nitrate in public

drinking water; association was

stronger when vitamin C intake was

≤190 mg/day and when red meat

servings exceeded 5 per week

Overall, no increased risk of ovarian

cancer with total intake of nitrate or

nitrite.

Adjustments: age, BMI, family

history of ovarian cancer, number of

live births, age at menarche, age at

menopause, age at first live birth,

oral contraceptive use, estrogen

use, history of unilateral

oophorectomy, and/or total energy

intake

Highest quartile vs lowest quartile:

Overall non-Hodgkin’s lymphoma:

OR 0.9 (95% CI 0.6, 1.2) for nitrate

OR 1.4 (95% CI 0.9, 2.2) for nitrite

Significant trend (p=0.04) for

follicular lymphoma with increasing

nitrate intake

OR 2.3 (95% CI 1.1, 4.9; p-trend

0.008) for follicular lymphoma with

nitrite intake

Adjustments: age; family history of

cancer; calories; intakes of vitamins

C, vitamin E, protein

88 NITRATE AND NITRITE

3. HEALTH EFFECTS

Table 3-2. Selected Cohort and Case-Control Studies Published Since 2006

Examining Possible Associations Between Nitrate and Nitrite Intake and

Cancer

Nitrate and nitrite

Reference

type

Study design

intakes

Outcomes

a

Kilfoy et al.

2011

Thyroid

Cohort from NIH-

AARP Diet and

Health Study,

1995–1996 with

Quintile median

ranges:

Nitrate from food:

Highest quintile versus lowest

quintile:

Nitrate:

average of 7 years

of follow-up

490,194 cohort

(292,125 men;

198,069 women),

370 cases (170

men; 200 women)

29.6, 49.8, 70.2,

100.9, 166.8 mg/day

(19.4, 29.9, 40.9,

57.4,

94.8 mg/1,000 kcal)

Nitrite from food: 0.

6,

0.9, 1.1, 1.4, 1.9

mg/day (0.5, 0.6,

0.7, 0.7,

Men: RR 2.28 (95% CI 1.29, 4.04; p-

trend <0.01) for thyroid cancer

RR 2.10 (95% CI 1.09, 4.05; p-trend

0.05) for papillary thyroid cancer

RR 3.42 (95% CI 1.03, 11.4; p-

trend

<0.01) for follicular thyroid cancer

Women: RR 0.76 (95% CI 0.48,

1.10) for thyroid cancer

Nitrite:

0.9 mg/1,000 kcal)

Based on food

frequency

questionnaire and

published food

nitrate and nitrite

Men: RR 1.36 (95% CI 0.78, 2.37)

for thyroid cancer

RR 2.74 (95% CI 0.86, 8.77; p-trend

0.04) for follicular thyroid cancer

Women: RR 1.19 (95% CI 0.71,

1.98) for thyroid cancer

levels

Adjustments: sex; age; smoking

status; race; physical activity;

alcohol use; BMI; caloric intake;

education; family history of cancer;

intakes of vitamin C, beta-carotene,

folate

Kim et al.

2007

Stomach

Case-control with

subjects from two

Korean hospitals,

1997–1998

136 controls,

136 cases

Tertile median

values:

Nitrate from food:

240, 458, and 811

mg/day

Based on food

Highest tertile vs lowest tertile:

OR 1.13 (95% CI 0.42, 3.06)

OR 2.78 (95% CI 1.01, 7.67) for

nitrate/86.7 mg/mg vitamin E

OR 3.37 (95% CI 1.28, 8.87) for

nitrate/2.47 mg/µg folate

frequency

questionnaire and

published food

nitrate and nitrite

levels

Adjustments: age; sex; SES; family

history of gastric cancer; refrigerator

use; Helicobacter pylori infection;

intakes of charcoal grilled beef,

Korean cabbage kimichi, Dongchimi

,

spinach, garlic, mushroom, salty

foods

89 NITRATE AND NITRITE

3. HEALTH EFFECTS

Table 3-2. Selected Cohort and Case-Control Studies Published Since 2006

Examining Possible Associations Between Nitrate and Nitrite Intake and

Cancer

Nitrate and nitrite

Reference

type

Study design

intakes

Outcomes

a

McElroy et

al. 2008

Colorectal

Wisconsin, United

States, 1990–

2001

Quintile cutoff range:

Nitrate in water:

Highest quintile versus lowest

quintile:

4,297 controls,

<0.5, 0.5–1.9, 2.0–

5.9, 6.0–9.9,

OR 1.52 (95% CI 0.95, 2.44) for all

colon cancer sites

1,476 cases,

all females

≥10 mg/L

Based on

OR 2.91 (95% CI 1.52, 5.56) for all

proximal colon sites

groundwater nitrate

data and spatial

interpolation to

individual residences

Adjustments: age

Michaud et

al. 2009

Brain

(glioma)

Combined

analysis of cohorts

from NHS I

(1980–2004),

NHS II (1991–

2005), and HPFS

(1986–2004)

230,655 cohort,

335 cases

Quintile cutoff

ranges, based on

baseline values:

Nitrate from food:

43–205 mg/day

Nitrite from food:

1.1–2.4 mg/day

NDMA from food:

0.02–0.09 mg/day

Highest tertile vs lowest tertile:

Nitrate: RR 1.02 (95% CI 0.66, 1.58)

Nitrite: RR 1.26 (95% CI 0.89, 1.79)

NDMA: RR 0.88 (95% CI: 0.57,

1.36)

Processed meat: RR 0.92 (95% CI

0.48, 1.77)

No effect of vitamin C, vitamin E, or

Based on food

frequency

questionnaire and

published food

nitrate and nitrite

ferric-reducing ability of plasma

Adjustments: age, caloric intake

levels

90 NITRATE AND NITRITE

3. HEALTH EFFECTS

Table 3-2. Selected Cohort and Case-Control Studies Published Since 2006

Examining Possible Associations Between Nitrate and Nitrite Intake and

Cancer

Nitrate and nitrite

Reference

type

Study design

intakes

Outcomes

a

Ward et al.

Kidney

Case-control,

2007 (RCC) Iowa, United

States, 1986–

1989

2,434 controls,

406 cases

Quartile cutoff

ranges:

Nitrate from food:

<59.32, 59.32–

86.62, 86.63–

122.00,

≥122.01 mg/day

Nitrite from food:

<0.70, 0.70–0.93,

0.94–1.25,

≥1.26 mg/day

Nitrate in water:

<0.62, 0.62–<1.27,

1.27–≤2.78,

≥2.78 mg/L

Based on food

frequency

questionnaire and

published food

nitrate and nitrite

levels, and PWS

data

Highest quartile versus lowest

quartile:

Nitrate: OR 0.41 (

95% CI 0.28, 0.60)

for dietary nitrate

OR 0.89 (95% CI 0.57, 1.39) for

nitrate in water

Nitrite: OR 0.82 (95% CI 0.50, 1.33)

OR 1.00 (95% CI 0.63, 1.59) for

nitrite from animal sources

OR 1.91 (95% CI 1.04, 3.51) for red

meat intake ≥1.2 servings/day and

PWS nitrate >5 mg/L for >10 years

Adjustments: age, sex, BMI, caloric

intake, intakes of sodium and fat

91 NITRATE AND NITRITE

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Table 3-2. Selected Cohort and Case-Control Studies Published Since 2006

Examining Possible Associations Between Nitrate and Nitrite Intake and

Cancer

Nitrate and nitrite

Reference

type

Study design

intakes

Outcomes

a

Ward et al.

2008

Esophagus,

Stomach

Case-control,

Nebraska, United

States, 1988–

Quartile cutoff

ranges:

Highest quartile versus lowest

quartile:

1993

321 controls,

79 stomach

cases,

Nitrate-nitrogen in

water: <2.45, 2.45–

<2.58, 2.58–4.32,

>4.32 mg/L (<10.78,

10.78–<11.35,

Nitrate in water: OR 1.2 (95% CI 0.5,

2.7) for stomach cancer

OR 1.2 (95% CI 0.6, 2.7) for

esophageal cancer

84 esophagus

cases

11.35–19.01,

>19.01 mg nitrate/L)

Dietary nitrate from

plant sources: <16.9,

16.9–<26.2, 26.2–

Nitrate from plant sources: OR 1.6

(95% CI 0.7, 3.6) for stomach cancer

OR 0.8 (95% CI 0.3, 1.8) for

esophageal cancer

Nitrate and nitrite from animal

<38.8, >38.8 mg/day

nitrate-nitrogen

(<74.4, 74.4–

<115.3,

115.3–<170.7,

>170.7 mg

nitrate/day)

Nitrate and nitrite

sources: OR 1.6 (95% CI 0.7, 3.7)

for stomach cancer

OR 2.2 (95% CI 0.9, 5.7; p-trend

0.015) for esophageal cancer

Adjustments: year of birth; sex; BMI;

smoking; alcohol; caloric intake;

intakes of vitamin A, folate,

from animal sources:

<3.8, 3.8–<5.7, 5.7–

riboflavin, zinc, protein, carbohydrate

<8.3, ≥8.3 mg/day

Based on food

frequency

questionnaire and

published food

nitrate and nitrite

levels, and PWS

data

Wu et al.

2013

Prostate

Case-control with

subjects from

HPFS (1997–

2005)

630 controls,

630 cases

Quartile median

range:

Plasma nitrate

(cases): 29.39, and

51.47 µmol/L (1.82

and 3.19 mg/L)

Adjusted RR not significant; no

significant trend

RR 0.99 (95% CI 0.68, 1.48) for

highest plasma nitrate quintile

Adjustments: age, BMI, caloric

intake, time of blood draw, hours

since last meal before blood draw

year of blood draw, family history of

prostate cancer, smoking, history of

hypertension, history of diabetes,

physical activity

92 NITRATE AND NITRITE

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Table 3-2. Selected Cohort and Case-Control Studies Published Since 2006

Examining Possible Associations Between Nitrate and Nitrite Intake and

Cancer

Nitrate and nitrite

Reference

type

Study design

intakes

Outcomes

a

Yang et al.

2010

Breast

Case-control.

Seoul, South

Korea, 2004–2006

362 controls,

362 cases

Quintile median

nitrate intake from

food: 179.4, 299.7,

372.1, 492.5,

716.1 mg/day

Based on food

Adjusted OR not significant for

dietary nitrate, no significant trend.

Significant trend for increasing OR

with increasing nitrate/folate ratio;

OR significantly elevated in highest

nitrate/folate quintile.

frequency

questionnaire and

published food

nitrate levels

OR 1.54 (95% CI 0.88, 2.70) for

nitrate

OR 2.03 (95% CI 1.16, 3.54) for

nitrate/folate intake ratio (2.10)

Adjustments: age, education,

physical activity, family history of

breast cancer, parity, breast feeding,

menopause, oral contraceptive use;

intakes of soy protein, mushroom,

fat

Zeegers et

al. 2006

Bladder

Cohort from

Netherlands

Cohort Study,

1986–1996

Quintile median

nitrate intakes:

Nitrate from food:

Adjusted RR not significant for

nitrate in food or water; no significant

trend

cohort 120,852,

889 cases,

4,441 subcohort

57.4, 78.6, 97.8,

119.5, 158.9 mg/day

Nitrate from water:

Highest quintile vs lowest quintile:

RR 1.06 (95% CI 0.81, 1.37) for

nitrate from food

0.5, 1.4, 3.4, 5.6,

10.6 mg/day RR 1.06 (95% CI 0.82, 1.37) for

nitrate from water

Based on food

frequency

questionnaire and

published food

nitrate and nitrite

levels, and

RR 1.09 (95% CI

0.84, 1.42) for total

nitrate intake

Adjustments: age, sex, smoking

PWS data

a

Risk estimates (95% confidence limits)

AARP = American Association of Retired Persons; BMI = body mass index; HPFS = Health Professionals Follow-Up

Study; HR = multivariate hazard ratio; NDMA = nitrosodimethylamine; NIH = National Institutes of Health;

NHS = Nurses’ Health Study; NSAIDs = non-steroidal anti-inflammatories; OR = odds ratio; PWS = public water

supply; RCC = renal cell carcimoma; RR = relative risk; SD = standard deviation; SES = socioeconomic status

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outcomes. Exposure misclassification can occur for various reasons including dietary factors that

contribute to variability in dose of nitrosation precursors (e.g., nitrate or nitrite in fish, meat, and

vegetables) and nitrosatable compounds; consumption of antioxidants that can inhibit nitrosation (e.g.,

vitamin C, flavenoids, polyphenols); migration in and out of the PWS district; and ingestion of other

water sources (e.g., bottled water). Estimates of exposure from drinking water can be made at the

individual subject level. This can be accomplished with surveys of the individual’s residence history and

consumption patterns (e.g., percentage of drinking water consumed from the PWS and other sources, such

as bottled water), along with data on nitrate concentrations in the water supply (Inoue-Choi et al. 2012).

Dietary surveys (e.g., food frequency questionnaires, 24-hour recalls), coupled with data from residue

monitoring studies of market basket foods, can be used to estimate individual exposures to nitrosation

precursors in foods. However, in this approach, exposure misclassification can occur as a result of

ingestion of nitrosation precursors from non-market basket foods. Also, the diet survey is typically cross-

sectional, even in longitudinal studies, and results may not accurately reflect the average diets during the

entire follow-up period. Exposure misclassification can also occur in studies that examine associations at

the individual level. However, in these studies, exposure misclassification is likely to be non-differential

or independent of cancer status. As a result, exposure comparisons (exposed versus unexposed) would

tend to be biased towards the null if there truly is an effect of the exposure on cancer outcome, and if

more than two levels of exposure are being evaluated (e.g., high, low, versus no exposure), then the bias

can be in either direction for the middle levels of exposure and tend to be biased towards the null at the

highest level so that exposure-response relationships are distorted (e.g., the risk would be attenuated or

fall at the highest levels of exposure because of this bias). Most of the nitrate and nitrite ingested comes

from the diet (Zeegers et al. 2006); therefore, studies that quantify exposure only from drinking water are

weak designs for assessing cancer risk unless the water supply is extraordinarily contaminated (>20 mg

nitrate/L). Some studies have employed biomarkers (blood, plasma, saliva, or urine) as exposure metrics

(Armijo et al. 1981; Cuello et al. 1976; Forman et al. 1985; Joossens et al. 1996; Kamiyama et al. 1987;

Knight et al. 1990; Lin et al. 2003; Lu et al. 1986; Sierra et al. 1993; Tsugane et al. 1992; Wu et al. 1993,

2013). Biomarkers can provide more accurate estimates of the steady-state levels of nitrate (or nitrite) in

an individual; however, they may not reflect the cumulative absorbed dose or the dose of nitrosation

products that may contribute to cancers (e.g., N-nitrosodimethylamine) (Zeilmaker et al. 2010a). An

additional uncertainty that applies to all studies described in this summary is that cancer risk may be mis-

attributed to nitrite (or nitrosation precursors) as a result of other factors that contribute cancer risk that

co-vary with exposure to nitrite or nitrite precursors. These may include other carcinogens in drinking

water or diet. However, unless these risk factors have extremely strong associations with exposures to

nitrate or nitrite (or nitrosation precursors), confounding from these factors is unlikely to be a major

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source of uncertainty in interpretation of cancer risk estimates. One potentially important class of

confounders is anti-oxidants, which can interfere with nitrosation of dietary amines and, thereby, the

mode of carcinogenicity of nitrite, and may also interfere with other carcinogenic process that involve

reactive intermediates. In the discussions of individual studies, the terms “statistically significant” refer

to relative risks that are estimated to be ≥1 or trends that were reported by the investigators to be

statistically significant, typically p<0.05).

In general, outcomes of cohort and case-control studies have found no or weak associations between

nitrate intakes and cancer in humans, with stronger associations for exposures to nitrite or intake of high-

nitrite foods such as cured meat (Aschebrook et al. 2013; DellaValle et al. 2013; IARC 2010; Inoue-Choi

et al. 2012). Mechanistically, this outcome is consistent with nitrite being a reactive intermediate in the

cancer mode of action of nitrate (see Section 3.5.2).

Studies that form the basis for evidence of carcinogenicity of nitrate or nitrite are briefly described below.

Most of these studies are described in greater detail in IARC (2010). Studies published since IARC

(2010) are summarized in Table 3-2. Studies included in Table 3-2 estimated nitrate or nitrite intakes

from dietary survey instruments of individuals, in some cases, supplemented with estimates from drinking

water based on well water or PWS data and geographic location of the residence, or with biomarkers of

exposure. The table summarizes major features of the design of each study and the major outcomes.

Complete details of the outcomes for various design strata can be obtained from the cited references.

This summary of carcinogenicity of nitrate and nitrite in humans is intentionally biased for the sake of

brevity, in that it is restricted to case-control and cohort studies and emphasizes studies that have found

associations between nitrate or nitrite and cancer, while most studies that found no associations are not

described. Descriptions of important ecological studies and negative outcome studies can be found in

IARC (2010). In the summary below, reported risks are adjusted for co-variables, which differed across

studies. Most studies adjusted for age, sex, body mass index (BMI), caloric intake, family history of

cancer, smoking, and alcohol consumption. Some studies also adjusted for socioeconomic status,

education, and various dietary intakes (e.g., vitamin C, vitamin E, flavenoids, folate), as well as cancer

specific-adjustments (e.g., reproductive history in breast cancer studies). Estimates of risk for studies not

included in Table 3-2 were those reported in IARC (2010) where they were expressed as relative risk

(RR) without specification of the actual risk metric estimated in the study. Risk metrics reported in

Table 3-2 are ORs for case-control studies and RR or hazard ratio (HR) for cohort studies.

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Gastrointestinal Cancer. Associations between intake of nitrite and a variety of cancer types has been

studied; however, the strongest and most consistent evidence for carcinogenicity of nitrite derives from

studies of gastrointestinal cancers and, in particular, gastric cancer (Buiatti et al. 1990; Engel et al. 2003;

La Vecchia et al. 1994, 1997; Mayne et al. 2001; Palli et al. 2001; Risch et al. 1985; Rogers et al. 1995;

Ward et al. 2007, 2008). In general, these studies have found significant positive trends for cancer risk

(risk increases with increasing intake), and three studies found elevated cancer risk (Engel et al. 2003;

Kim et al. 2007; Risch et al. 1985). In the Risch et al. (1985) case-control study (246 cases,

246 controls), relative risk was 1.71 (95% CI: 1.24, 2.37) for a nitrite intake of 1 mg/day. In another case-

control study (369 cases, 695 controls) (Engel et al. 2003; Mayne et al. 2001), risk for stomach cancer

(non-cardia) was elevated at nitrite intakes ≥6 mg/day (OR 2.5, 95% CI: 1.4, 4.3). Risk increased with

decreasing vitamin C intake (RR 2.95, 95% CI: 1.90, 4.59). Additional support for antioxidants as effect

modifiers comes from a case-control study (136 cases, 136 controls) in which stomach cancer risk

increased in association with increasing ratio of nitrate to antioxidants in the diet (e.g., vitamin C, vitamin

E, folate) (Kim et al. 2007). Risk (OR) at the highest nitrate/vitamin E ratio (86.7 mg nitrate/mg vitamin

E) was 2.78 (95% CI: 1.01, 7.67). At the highest nitrate/folate ratio (2.47 mg nitrate/µg folate), an OR of

3.37 (95% CI: 1.28, 8.87) was determined.

Associations between exposure to nitrate or nitrite and colorectal cancer have been studied in cohort and

case-control studies (Chiu et al. 2011; De Roos et al. 2003; Knekt et al. 1999; Weyer et al. 2001). The

largest of the case-control studies (3,707 cases, 3,707 controls) (Chiu et al. 2011) found a significant

positive trend (chi-square for trend =13.26, p=0.001) for mortality from colon cancer with increasing

nitrate levels in drinking water (OR 1.16, 95% CI: 1.04, 1.30 at nitrate-nitrogen levels >0.6 mg/L;

>2.65 mg nitrate/L). Risks were higher in a stratum exposed to drinking water that had a calcium level

>34.6 mg/L (OR 1.37, 95% CI: 1.11, 1.69 for nitrate <2.64 mg/L). The De Roos et al. (2003) case-

control study (685 cases of colon cancer, 655 cases of rectal cancer, 2,434 controls) found elevated risk of

colon (RR 1.5, 95% CI: 1.0, 2.1) and rectal cancer (RR 1.7, 95% CI: 1.1, 2.5) at a dietary nitrite intake

>1.26 mg/day. Risk of colon cancer was higher in a stratum exposed to nitrate in drinking water at levels

>5 mg/L in combination with a low vitamin C intake (RR 2.0, 95% CI: 1.2, 3.3). Two meta-analyses

reported in IARC (2010) concluded that ingestion of cured meats was associated with increased risk of

colorectal cancer (Norat et al. 2002; Sandhu et al. 2001).

Central Nervous System Cancer. Cancer of the central nervous system has been studied extensively in

case-control studies (IARC 2010). Some studies found significant positive trends with nitrite and/or

cured meat intake; elevated risk was reported in a few studies (Blowers et al. 1997; Giles et al. 1994, Lee

96 NITRATE AND NITRITE

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et al. 1997; Mueller et al. 2004; Pogoda and Preston-Martin 2001a, 2001b; Preston-Martin et al. 1996).

Risks increased with higher nitrite intake or cured meat/antioxidant ratios (Blowers et al. 1997; Preston-

Martin et al. 1996). The study of Preston-Martin and coworkers (Pogoda and Preston-Martin 2001a,

2001b; Preston-Martin et al. 1996) included 540 cases and 801 controls. Significantly increased risk (OR

3.0, 95% CI: 1.2, 7.9) was observed for central nervous system cancers (brain, cranial nerves, or cranial

meninges) in children of mothers reporting a nitrite intake >3.0 mg/day from cured meat during

pregnancy. The Mueller et al. (2004) case-control study (1,218 cases, 2,223 controls) found elevated risk

(RR 5.7, 95% CI: 1.2, 27.2) for astroglial tumors in children in association with maternal exposure to

drinking water to nitrite concentrations ≥5 mg/L during pregnancy. Risks for other types of brain tumors

were not elevated. A smaller case-control study (94 cases, 94 controls) found elevated risk of glioma in

women (with trend p=0.07) in association with intake of nitrite from cured meat (RR 2.1, 95% CI: 1.0,

4.6). Results of meta-analyses of brain cancer studies also support associations between intake of cured

meat during pregnancy and brain tumors in children and cured meat ingestion and brain tumors in adults

(Huncharek and Kupelnick 2004; Huncharek et al. 2003). A large cohort study (230,655 subjects,

335 cases) of associations between intakes of nitrate, nitrite, and nitrosodimethylamine (NDMA) and

glioma in adults did not find significant trends or elevated risk for glioma (Michaud et al. 2009,

Table 3-2).

Urinary Tract Cancer. Cancer of the urinary tract has been studied in several case-control and large

cohort studies (DellaValle et al. 2013; Espejo-Herrera et al. 2015; IARC 2010; Ward et al. 2007, Zeegers

et al. 2006). Positive trends for risk or elevated risk were found in some studies (DellaValle et al. 2013;

Ward et al. 2007; Wilkens et al. 1996). In the Wilkens et al. (1996) case-control study (272 cases,

522 controls), risk was elevated (trend p=0.05) in association with dietary nitrite intake (RR 2.0, 95% CI:

1.0, 4.0). In the Ward et al. (2007) case-control study (406 cases, 2,434 controls), risk of kidney cancer

was elevated in the strata who consumed >1.2 servings of red meat/day and who resided for >10 years in

a PWS district that had nitrate concentrations >5 mg/L (OR 1.91, 95% CI: 1.04, 3.51; see Table 3-2). A

large cohort study (491,841 subjects, 488 cases) found a significant positive trend and elevated risk for

renal cell carcinoma in association with nitrite intake from animal sources (HR 1.28, 95% CI: 1.10, 1.49

for renal cell carcinoma; HR 1.68, 95% CI: 1.25, 2.27 for clear cell carcinoma, both at 0.9 mg

nitrite/1,000 kcal) (DellaValle et al. 2013). The Zeegers et al. (2006) cohort study (120,852 subjects,

889 cases) found no association between bladder cancer and intake of nitrate from food or drinking water.

Wang et al. (2012) evaluated possible association between nitrate in drinking water and risk of bladder

cancer in a meta-analysis that included results from one ecological study (Morales et al. 1993), two cohort

studies (Weyer et al. 2001; Zeegers et al. 2006), and two case-control studies (Chiu et al.2007; Ward et al.

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2003) and found no evidence that nitrate in the drinking water was associated with risk of bladder cancer

(combined RR 1.27; 95% CI 0.75, 2.15) based on data for highest nitrate levels reported relative to

reference values from each study.

Reproductive Organ Cancer. A small number of case-control and cohort studies have examined

associations between exposure to nitrate or nitrite and cancers of breast, ovary, uterus, prostate, and testis

(Barbone et al. 1993; IARC 2010; Espejo-Herrera et al. 2016b; Inoue-Choi et al. 2012, 2015; Moller

1997;Wu et al. 2013; Yang et al. 2010). A cohort study of post-menopausal women (34,388 subjects,

2,875 cases) found a significant positive trend (p=0.04) and elevated risk (HR 1.40, 95% CI: 1.05, 1.87)

for breast cancer in association with consumption of public drinking water at ≥33.5 mg nitrate/2 L

(median 57.8 mg nitrate/2 L) among women who consumed folate at rates ≥400 µg/day; risk was not

elevated among those women who ingested folate at <400 µg/day (Inoue-Choi et al. 2012). Similarly

increased risk (HR 1.38, 95% CI: 1.05, 1.82) was noted for private well users who ingested folate at

>400 µg/day when compared to the lowest quintile of users of the public drinking water sources who

ingested folate at >400 µg/day. In contrast, Yang et al. (2010) reported elevated risk for breast cancer in

association with increasing dietary nitrate/folate ratio, with significantly elevated risk (OR 2.03, 95% CI:

1.16, 3.54) at nitrate/folate ratios in the range of 1.79–8.19. The contrasting effects of folate in these two

studies may reflect dose-dependent effect modification: an antioxidant effect at lower folate intakes and a

tumor promoting effect of folate at higher folate intakes (Inoue-Choi et al. 2012). Inoue-Choi et al.

(2015) reported increased risk of ovarian cancer (HR 2.03; 95% CI 1.22, 3.38) among subjects with

public water containing ≥2.98 mg nitrate-nitrogen/L (≥13.1 mg nitrate/L) in a cohort study of

28,555 post- menopausal women (315 ovarian cancer cases) in the Iowa Women’s Health Study.

Associations were stronger when vitamin C intake was ≤190 mg/day and when red meat servings

exceeded five per week. Espejo-Herrera et al. (2016b) reported increased risk (OR 1.64; 95% CI 1.08,

2.49) of breast cancer among postmenopausal women with both high waterborne nitrate intake

(>6 mg/day) and high red meat intake (≥20 g/day) in a case control study in Spain (1,245 cases,

1,520 controls). A case-control study of prostate cancer (630 cases, 630 controls) did not find significant

associations between prostate cancer risk and plasma nitrate concentrations (1.8–3.8 mg/L) (Wu et al.

2013). In the Moller (1997) case-control study (514 cases, 720 controls), elevated risk of testicular cancer

(OR 1.51, 95% CI: 1.03, 2.20) was found among men who had lived in areas during childhood with

drinking water containing >25 mg nitrate/L. Barbone et al. (1993) conducted a case-control study of

endometrial cancer (168 cases, 334 controls) and found a negative trend for risk (risk decreased with

increasing dietary nitrate intake).

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Reticuloendothelial Cancer. Associations between exposure to nitrate or nitrite and leukemia or non-

Hodgkin’s lymphoma have been studied in population-based case-control studies (Aschebrook-Kilfoy et

al. 2013; Chiu et al. 2008; Freedman et al. 2000; Kilfoy et al. 2010; Ward et al. 1996, 2006) and a

prospective cohort study (Weyer et al. 2001). One case-control study (181 cases, 142 controls) reported

elevated risk (OR 3.1, 95% CI: 1.7, 5.5) of non-Hodgkin’s lymphoma in association with dietary nitrite

(but not nitrate) at dietary nitrite intake >1.21 mg/day (Ward et al. 2006). Another case-control study

(156 cases, 527 controls) reported elevated risk (OR 2.0; 95% CI 1.1, 3.6) of non-Hodgkin’s lymphoma in

association with average nitrate levels ≥4 mg/L nitrate-nitrogen (17.6 mg nitrate/L) in the community

drinking water supply (Ward et al. 1996). Chiu et al. (2008) evaluated possible associations between diet

and non-Hodgkin’s lymphoma according to t(14;18) status (one of the most common chromosomal

abnormalities in non-Hodgkin’s lymphoma. Dietary factors in 60 t(14;18)-positive and 87 t(14; 18)-

negative cases were compared with 1,075 controls. The study authors reported increased risk (OR 2.8;

95% CI 1.3, 6.1) of t(14;18)-positive non-Hodgkin’s lymphoma for the highest tertile of dietary nitrite

(>1 mg/day) versus the lowest tertile (<1 mg/day). The Freedman et al. (2000) case-control study

(73 cases, 147 controls) found no association between non-Hodgkin’s lymphoma and nitrate levels in

public drinking water. Kilfoy et al. (2010) evaluated risk of non-Hodgkin’s lymphoma overall and by

histological type in relation to self-reported dietary nitrate and nitrite intake in a case-control study of

1,304 women. No significant association was found between risk of non-Hodgkin’s lymphoma overall

and dietary nitrate or nitrite. Significant positive trends were reported for follicular lymphoma and

increasing intakes of nitrate (p-trend =0.04) and nitrite (p-trend <0.01); a significant association (OR 2.3;

95% CI 1.1, 4.9) was noted for the highest nitrite intake quartile (≥1.32 mg/day). Aschebrook-Kilfoy et

al. (2013) estimated dietary intake of nitrate and nitrite intake via food frequency questionnaire among

348 non-Hodgkin’s lymphoma cases and 470 controls in Nebraska in 1999–2002 and reported

nonsignificant excess risk of non-Hodgkin’s lymphoma (OR 1.6; 95% CI 0.8, 2.9) among women in the

highest quartile of nitrite intake (median nitrite intake 0.86 mg/1,000 kcal) compared to the lowest

quartile (median nitrite intake 0.49 mg/kcal). An OR of 1.9 (95% CI 1.0, 3.4) was estimated for the

highest quartile based on nitrite intake from animal sources (median nitrite intake 0.41 mg/kcal versus

0.16 mg/kcal for the lowest quartile). There were no significant associations between estimated nitrate or

nitrite intake and risk of non-Hodgkin’s lymphoma subtypes. The Weyer et al. (2001) cohort study

(21,977 subjects, 105 cases of non-Hodgkin’s lymphoma, 94 cases of leukemia) did not find positive

associations or elevated risk of non-Hodgkin’s lymphoma or leukemia in association with dietary or

drinking water nitrate.

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Thyroid Cancer. Kilfoy et al. (2011) evaluated possible associations between dietary intake of nitrate and

nitrite and risk of thyroid cancer in a cohort of 292,125 men (170 thyroid cancer cases) and

198,069 women (200 thyroid cancer cases) from the NIH-AARP Diet and Health Study 1995–1996. The

study authors reported increased risk of thyroid cancer overall with nitrate intake among men (RR 2.28;

95% CI 1.29, 4.04; p-trend <0.01), but not women (RR 0.69; 95% CI 0.42, 1.15; p-trend 0.61). For

nitrate intake among the men, thyroid cancer risk was increased by subtype as well; RR 2.10; 95% CI

1.09, 4.05; p-trend 0.05 for papillary cancer and RR 2.74; 95% CI 0.86, 8.77; p-trend 0.04 for follicular

cancer. There were no significant associations between nitrite intake and risk of thyroid cancer among

men or women. Aschebrook-Kilfoy et al. (2013a) evaluated possible associations between dietary intake

of nitrate and nitrite and risk of thyroid cancer in a cohort of 73,317 women enrolled in the Shanghai

Women’s Health Study in 1996–2000 and followed-up for 11 years (164 thyroid cancer cases). The study

authors reported increased risk of thyroid cancer among the group with highest nitrite intake (RR 2.05;

95% CI 1.20, 3.51). The risk was strongest for nitrite intake from processed meats (RR 1.96; 95% CI

1.28, 2.99). Nitrate intake was not associated with increased risk (RR 0.93; 95% CI 0.42, 2.07). Meta-

analysis of the results from selected studies that evaluated risk of thyroid cancer with nitrate intake

(Aschebrook-Kilfoy et al. 2013a; Kilfoy et al. 2011; Ward et al. 2010) or nitrite intake (Aschebrook-

Kilfoy et al. 2013a; Kilfoy et al. 2011) indicated increased risk of thyroid cancer with nitrite intake (RR

1.48; 95% CI 1.09, 2.02), but not with nitrate intake (RR 1.36; 95% CI 0.67, 2.75) (Bahadoran et al.

2015).

Other Cancers. In general, case-control and cohort studies of cancers of larynx, liver, lung, mouth,

pancreas, or pharynx have found no consistent associations with exposure to nitrate or nitrite

(Aschebrook-Kilfoy et al. 2011; IARC 2010).

Studies of Laboratory Animals. The potential carcinogenicity of nitrate has been investigated in several

animal studies that employed the oral exposure route. Studies in which negative results were reported

include MCR-derived rats (15/sex/group) provided 5,000 mg sodium nitrate/L (3,650 mg nitrate/L) in the

drinking water for 84 weeks and sacrificed 20 weeks later (Lijinsky et al. 1973a), male white rats

provided 4,000 mg sodium nitrate in the drinking water for 273 days and sacrificed at 10 months (Pliss

and Frolov 1991), strain A male mice (n=40) provided 12,300 mg sodium nitrate/L in the drinking water

for 25 weeks and sacrificed 13 weeks later (Greenblatt and Mirvish 1973), female NMRI mice provided

1,000 mg calcium nitrate/L in the drinking water for 18 months (Mascher and Marth 1993), Fischer 344

rats (50/sex/group) fed diets containing up to 5% sodium nitrate (1,517–1,730 mg nitrate/kg/day) for

2 years (Maekawa et al. 1982), and ICR mice (10/sex/group) fed diets containing up to 5% sodium nitrate

100 NITRATE AND NITRITE

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for 2 years (IARC 2010). In the study of Pliss and Frolov (1991) some groups of male white rats were

treated with drinking water containing 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBNA, an

inducer of urinary bladder cancer in laboratory animals) for 30 days, either alone or followed by 4,000 mg

sodium nitrate/L drinking water for 273 days. The group treated with BBNA followed by sodium nitrate

exhibited a significantly increased incidence of urinary bladder carcinoma (6/20 rats versus 1/18 rats

treated with 0.05% BBNA only). These results indicate that sodium nitrate promoted BBNA-induced

bladder tumors.

The potential carcinogenicity of ingested nitrite has been investigated in numerous animal studies. Nitrite

treatment alone did not result in increased incidences of tumors in most studies. Nitrite doses (expressed

as nitrite/kg/day) reported in this Toxicological Profile for Nitrate and Nitrite were either provided by the

study authors or estimated using available body weight and oral intake data; otherwise, EPA (1988)

default reference values for body weight, food consumption, and water intake were used to calculate

doses.

NTP (2001) performed a cancer bioassay of male and female F344/N rats (50/sex/group) provided sodium

nitrite in the drinking water for 2 years at concentrations of 0, 750, 1,500, or 3,000 ppm. Author-reported

average doses were 35–130 mg sodium nitrite/kg/day (23.5–87.1 mg nitrite/kg/day) to the males and 40–

150 mg sodium nitrite/kg/day (26.8–100.5 mg nitrite/kg/day) to the females. There was no evidence of

sodium nitrite-induced forestomach neoplasms. Although the mid-dose group of female rats exhibited a

significantly increased incidence of mammary gland fibroadenoma, the incidence in the high-dose group

was not significantly different from that of controls; based on this finding and the high historical

background incidence of mammary gland fibroadenomas, the incidence in the mid-dose group was not

considered treatment related. Significantly decreased incidences of mononuclear cell leukemia were

observed in mid- and high-dose male and female rats. It was speculated that increased methemoglobin

concentrations may have played a role in the decreased incidences of mononuclear cell leukemia.

Significantly increased incidence of fibroma of the subcutis was noted in mid-dose male rats; however,

several factors (the incidence only slightly exceeded the historical range of NTP controls, lacked a dose-

response characteristic, combined incidences of fibroma or fibrosarcoma were within the historical range

for NTP controls, and fibromas and fibrosarcomas are common neoplasms in the skin of F344/N rats)

suggested that the fibroma was not related to sodium nitrite exposure. NTP (2001) concluded that there

was "no evidence of carcinogenic activity" of sodium nitrite in the male or female F344/N rats under the

conditions of the study.

101 NITRATE AND NITRITE

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NTP (2001) also provided sodium nitrite in the drinking water of B6C3F1 mice (50/sex/group) for 2 years

at concentrations of 0, 750, 1,500, or 3,000 ppm. Author-reported average doses were 60–220 mg sodium

nitrite/kg/day (40.2–107.2 mg nitrite/kg/day) to the males and 45–160 mg sodium nitrite/kg/day (30.2–

107.2 mg nitrite/kg/day) to the females. Female mice exhibited a significant positive trend for increased

incidence of forestomach squamous cell papilloma or carcinoma (combined) and the incidence in the

high-dose female mice exceeded the historical range for NTP controls; however, based on concurrent

controls, incidences of squamous cell adenoma (1/50, 0/50, 1/50, and 3/50 for controls, 750, 1,500, and

3,000 ppm groups, respectively), squamous cell carcinoma (0/50, 0/50, 0/50, and 2/50 for controls, 750,

1,500, and 3,000 ppm groups, respectively), and squamous cell adenoma or carcinoma (1/50, 0/50, 1/50,

and 5/50 for controls, 750, 1,500, and 3,000 ppm groups, respectively) were not statistically significantly

increased for any sodium nitrite exposure group. NTP (2001) considered the positive trend for incidences

of forestomach squamous cell papilloma or carcinoma (combined) in the female B6C3F1 mice to provide

"equivocal evidence of carcinogenic activity" of sodium nitrite and noted that there was "no evidence of

carcinogenic activity" in the male B6C3F1 mice under the conditions of the study. Incidences of

alveolar/bronchiolar adenoma or carcinoma (combined) in sodium nitrite-exposed groups of female mice

were slightly greater than that of controls (incidences of 1/50, 6/50, 5/50, and 6/50 for controls, 750,

1,500, and 3,000 ppm groups, respectively); however, incidences were within that of historical NTP

controls. Because the incidences did not exhibit exposure concentration-response characteristics and

were not accompanied by increased incidences of preneoplastic lesions, the study authors did not consider

them to be sodium nitrite exposure-related effects. Significantly increased incidence of fibrosarcoma of

the subcutis was noted in mid-dose female mice (incidences of 0/50, 5/50, 1/50, and 2/50 for 0, 750,

1,500, and 3,000 ppm groups, respectively) and exceeded the historical range for NTP controls; however,

lack of exposure concentration-response characteristics and the fact that combined incidence of fibroma

or fibrosarcoma (0/50, 5/50, 1/50, and 3/50 for 0, 750, 1,500, and 3,000 ppm groups, respectively) were

within the historical range for NTP controls suggest that these neoplasms were not related to sodium

nitrite exposure.

In two other studies of male and female F344 rats, addition of sodium nitrite to the drinking water at

concentrations as high as 2,000–3,000 ppm for up to 2 years did not result in significant increases in

tumor incidences at any site (Lijinsky 1984a, 1984b; Lijinsky et al. 1983; Maekawa et al. 1982).

Conversely, incidences of mononuclear cell leukemia were significantly lower in the nitrite-treated

groups relative to controls. In a 26-month study of male and female Sprague-Dawley rats provided

drinking water to which up to 2,000 ppm sodium nitrite was added, the study author reported increased

incidence of lymphomas, but not other types of tumors (Newberne 1979); however, IARC (2010) and

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NTP (2001) noted that a working group sponsored by the U.S. FDA reevaluated the histology and did not

confirm the results of Newberne (1979). IARC (2010) reported that the working group considered the

incidences of lymphomas to be similar to those arising spontaneously in Sprague-Dawley rats. Shank and

Newberne (1976) reported increased incidences of total tumors and lymphoreticular tumors in rats fed

diet to which sodium nitrite was added at 1,000 ppm (total tumors: 58/96 versus 28/156 controls;

lymphoreticular tumors: 26/96 versus 9/156 controls); the results were reported for F1 and F2 offspring

that had been exposed via their mothers during gestation and lactation and directly from the diet

thereafter. In a 96-week study, Iurchenko et al. (1986) reported significantly increased incidences of

benign liver tumors among male CBA mice administered drinking water to which sodium nitrite was

added at a concentration resulting in author-estimated total dose of 1,600 mg sodium nitrite/mouse

compared to a group of untreated controls; however, there was no apparent sodium nitrite treatment-

related effect at a higher estimated dose (2,000 mg sodium nitrite/mouse).

Significantly increased incidences of forestomach squamous papillomas (by the life-table method) were

reported for male and female MRC Wistar rats provided drinking water to which sodium nitrite was

added at 3,000 ppm on 5 days/week for life (5/22 males and 3/23 females versus 2/47 control males and

0/44 control females) (Mirvish et al. 1980). The study authors stated that the sodium nitrite-treated rats

received a total dose of 63 g sodium nitrite/kg. Total numbers of rats and incidences of rats with

papillomas were small.

Grant and Butler (1989) added sodium nitrite to a reduced-protein diet and administered the diet to male

and female F344 rats for up to 115 weeks; a control group received reduced-protein diet alone. The study

authors reported dose-related decreases in time of onset and incidence of lymphomas, mononuclear cell

leukemia, and testicular interstitial-cell tumors in the nitrite-treated groups.

There was no evidence of increased tumor incidences in male or female ICR mice provided sodium nitrite

in the drinking water for up to 109 weeks at concentrations as high as 0.5% (5,000 ppm sodium nitrite)

(Inai et al. 1979), or in male or female Swiss mice or their offspring following a single gavage

administration of 10 mg/kg nitrite and subsequent exposure to 0.1% sodium nitrite (1,000 ppm) in the

drinking water during gestation days 15–21; terminal sacrifices occurred 10 months following the

initiation of treatment (Börzsönyi et al. 1978). Hawkes et al. (1992) found no evidence of treatment-

related effects on incidences of nervous system tumors among male and female VM mice (susceptible to

spontaneous development of cerebral gliomas) provided drinking water to which sodium nitrite was

103 NITRATE AND NITRITE

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added at 0.2% (2,000 ppm) from weaning for a lifetime and others exposed via their mothers during

gestation and lactation as well.

The potential carcinogenicity of combined exposure to sodium nitrite and selected nitrosatable substances

(oral exposures via combinations of drinking water, diet, and/or gavage dosing) has been well-studied in

laboratory animals. Many of the studies included sodium nitrite-only treatment groups for which there

was no evidence of sodium-nitrite induced carcinogenicity (Anderson et al. 1985; Börzsönyi and Pintér

1977; Börzsönyi et al. 1976; Greenblatt and Lijinsky 1972, 1974; Greenblatt and Mirvish 1973;

Greenblatt et al. 1971, 1973; Hirose et al. 2002; Ivankovic 1979; Ivankovic and Preussman 1970; Kitano

et al. 1997; Murthy et al. 1979; Lijinsky 1984a, 1984b; Lijinsky and Reuber 1980; Mirvish et al. 1972;

Miyauchi et al. 2002; Rijhsinghani et al. 1982; Scheunig et al. 1979; Taylor and Lijinsky 1975a, 1975b;

van Logten et al. 1972; Yada et al. 2002; Yoshida et al. 1993, 1994). However, Lijinsky et al. (1983)

reported significantly increased incidences of hepatocellular neoplasms in female (but not male) F344 rats

administered diet to which sodium nitrite was added at 2,000 ppm for 2 years; significantly decreased

incidences of mononuclear-cell leukemia was observed as well.

Significantly increased incidences of selected tumor types were observed in some studies of laboratory

animals that employed coexposure to various amino compounds and sodium nitrite (Anderson et al. 1985;

Börzsönyi and Pintér 1977; Börzsönyi et al. 1976, 1978; Chan and Fong 1977; Greenblatt and Mirvish

1973; Greenblatt et al. 1971; Hirose et al. 1990; Iurchenko et al. 1986; Ivankovic 1979; Ivankovic and

Preussmann 1970; Kawabe et al. 1994; Murthy 1979; Lijinsky 1984a, 1984b; Lijinsky and Reuber 1980;

Lijinsky and Taylor 1977; Lijinsky et al. 1973b; Lin and Ho 1992; Maekawa et al. 1977; Mirvish et al.

1972, 1976, 1980; Miyauchi et al. 2002; Mokhtar et al. 1988; Newberne and Shank 1973; Nishiyama et

al. 1998; Nixon et al. 1979; Oka et al. 1974; Rijhsinghani et al. 1982; Rustia and Shubik 1974; Scheunig

et al. 1979; Shank and Newberne 1976; Tahira et al. 1988; Taylor and Lijinsky 1975a, 1975b; Weisburger

et al. 1980; Xiang et al. 1995; Yada et al. 2002; Yamamoto et al. 1989; Yoshida et al. 1993, 1994). These

results were typically attributed to in vivo nitrosation of amines by nitrite to produce carcinogenic

N-nitrosoamines; some of the studies did not include sodium nitrite-only treatment groups. Addition of

sodium nitrite or potassium nitrite to the food of rats in three other studies resulted in increased incidences

of selected tumors; analysis of the food revealed the presence of N-nitroso compounds (likely formed by

nitrosation in the presence of nitrite and selected amine compounds in the food), which were considered

the probable principal cause of the tumors (Aoyagi et al. 1980; Matsukura et al. 1977; Olsen et al. 1984).

Börzsönyi et al. (1978) reported 30–70% incidences of malignant lymphomas, lung adenomas, and

hepatomas among maternal mice and their offspring following gavage treatment of the dams with the

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fungicide, dodecylguanidine acetate, together with 0.05% sodium nitrite; the frequency of spontaneous

tumors in untreated controls was 6%. Dodecylguanidine acetate alone had no effect on cancer incidence.

Lijinsky et al. (1973a) found no significant increase in tumor incidences among male and female MCR

rats provided drinking water comprised of 0.5% nitrilotriacetic acid or iminodiacetic acid and 0.2 or 0.5%

sodium nitrite on 5 days/week for a lifetime.

There were no signs of treatment-related effects on incidences of tumors at any site among groups of

pregnant Syrian golden hamsters and their offspring fed diets to which sodium nitrite and/or morpholine

were added throughout production of an F2 generation (Shank and Newberne 1976). Fresh diet was

prepared every 2–7 days and 25% of the initial concentration of sodium nitrite was lost during 7 days

after preparation of the diet.

Based on available human data, IARC (2010) determined that there is inadequate evidence for the

carcinogenicity of nitrate in food or drinking water and limited evidence for the carcinogenicity of nitrite

in food (based on association with increased incidence of stomach cancer). Evaluation of available

animal data by IARC (2010) resulted in the determination that there is inadequate evidence for the

carcinogenicity of nitrate, limited evidence for the carcinogenicity of nitrite per se, and sufficient evidence

for the carcinogenicity of nitrite in combination with amines or amides. The overall conclusions of IARC

(2010) were that “ingested nitrate and nitrite under conditions that result in endogenous nitrosation is

probably carcinogenic to humans (Group 2A).” IARC (2010) noted that: (1) the endogenous nitrogen

cycle in humans includes interconversion of nitrate and nitrite; (2) nitrite-derived nitrosating agents

produced in the acid stomach environment can react with nitrosating compounds such as secondary

amines and amides to generate N-nitroso compounds; (3) nitrosating conditions are enhanced upon

ingestion of additional nitrate, nitrite, or nitrosatable compounds; and (4) some N-nitroso compounds are

known carcinogens.

The U.S. EPA IRIS (2002) does not include a carcinogenicity evaluation for nitrate or nitrite.

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3.2.3 Dermal Exposure

No relevant information was located regarding the following effects in humans or animals exposed to

nitrate or nitrite via the dermal route:

3.2.3.1 Death

3.2.3.2 Systemic Effects

3.2.3.3 Immunological and Lymphoreticular Effects

3.2.3.4 Neurological Effects

3.2.3.5 Reproductive Effects

3.2.3.6 Developmental Effects

3.2.3.7 Cancer

3.3 GENOTOXICITY

No studies were located regarding genotoxicity in human populations exposed to exogenous nitrite.

Limited information is available for nitrate. Kleinjans et al. (1991) examined the association between

nitrate levels in drinking water and frequency of sister chromatid exchanges (SCEs) in peripheral

lymphocytes from women from the Netherlands. Three groups were formed, low- (n=30), medium-

(n=30), and high- (n=18) exposure groups, based on the levels of nitrate in their drinking water. The

corresponding nitrate levels were 0.13, 32.0, and 133.5 mg/L. Regression analysis showed a good

correlation between levels on nitrate in water and nitrate body burden monitored by 24-hour urine levels

of nitrate. Examination of peripheral lymphocytes showed no significant association between 24-hour

urine excretion of nitrate and frequency of SCEs. Another study examined the frequency of

hypoxanthine-guanine phosphoribosyltransferase (HPRT) variants (an index of genetic risk) in peripheral

lymphocytes in groups of women from the Netherlands in relation to levels of nitrate in drinking water

(van Maanen et al. 1996a). A total of 50 subjects were exposed to concentrations of nitrate of 0.02 mg/L

(n=14), 17.5 mg/L (n=21), 25 mg/L (n=6), or 135 mg/L (n=9). The two lower concentrations were from

PWS, whereas the two highest originated from private wells. Analysis of 24-hour urine samples showed

a positive correlation between nitrate in drinking water and urinary nitrate. Also, salivary nitrate and

nitrite were similarly increased. Results of multiple regression analysis showed that the mean log

frequency of HPRT variants was significantly higher in the group exposed to 25 mg/L nitrate than in the

groups exposed to 0.02 and 17.5 mg/L nitrate. The analyses also showed a significant correlation

between frequency of HPRT variants and 24-hour urinary nitrate and salivary nitrite levels and between

24-hour urinary excretion of N-nitrosopyrrolidine and 24-hour urinary excretion of nitrate. The results

suggested that drinking water with nitrate poses a genetic risk due to the potential formation of

nitrosamines after endogenous reduction of nitrate to nitrite and reaction with amino compounds. A third

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study examined the frequency of SCEs and chromosomal aberrations in peripheral blood lymphocytes

from70 male and female Greek children (12–15 years of age) who were exposed to high nitrate in

drinking water (55.7–88.0 mg/L) (Tsezou et al. 1996). Controls consisted of 20 children from areas with

low nitrate content in the drinking water (0.70 mg/L). No measurements of nitrate or nitrite in biological

fluids were conducted in this study. Analyses of the results showed a significant increase in chromatid

and chromosome breaks in children exposed to nitrate levels ≥70.5 mg/L of drinking water. However,

levels of SCEs showed no significant increase with increasing nitrate levels. IARC (2010) noted that the

possibility that chemicals other than nitrate could have been responsible for the elevated chromosomal

aberrations could not be ruled out.

A limited number of studies have examined the in vivo genotoxicity of nitrate in laboratory animals.

Gavage administration of up to 500 mg/kg/day sodium nitrate to pregnant Syrian Golden hamsters on

gestation days 11 and 12 did not significantly affect the frequency of micronuclei, chromosomal

aberrations, morphological or malignant cell transformation, or drug-resistant mutations in embryonic

cells (Inui et al. 1979). In another in vivo study, oral administration of 150 mg/kg sodium nitrate (only

dose tested) to male Swiss mice did not inhibit testicular DNA synthesis measured 3.5 hours after dosing

(Friedman and Staub 1976). Gavage administration of up to 2,120 mg/kg/day sodium nitrate for 2 days to

male Wistar rats did not induce chromosomal aberrations in bone marrow cells examined 24 hours after

the last dose (Luca et al. 1985). A similar experiment with male Swiss mice showed induction of

chromosomal aberrations at 706.6 mg/kg/day sodium nitrate but not at 2,120 mg/kg/day (Luca et al.

1985). Daily administration of ≥78.5 mg/kg sodium nitrate for 2 weeks to rats resulted in a significant

dose-dependent increase in chromosomal aberrations in bone marrow cells 24 hours after the last dose

(Luca et al. 1985). Evaluation of micronuclei in mice treated daily for 2 weeks showed significant

increases (approximately 2-fold greater than controls) at the low concentrations tested, 78.5 and

235.5 mg/kg/day sodium nitrate, but not at 706.6 or 2,120 mg/kg/day, which the investigators attributed

to possible induction of cytotoxic effects (Luca et al. 1985). Alavantić et al. (1988a) treated male mice

with sodium nitrate by gavage for 3 days at doses of 0, 600, or 1,200 mg/kg/day; there was no sign of

treatment-related unscheduled DNA synthesis in spermatids analyzed 17 days following treatment.

Alavantić et al. (1988b) treated male mice with sodium nitrate by gavage for 2 weeks doses of 0, 600, or

1,200 mg/kg/day and subsequently mated them to virgin females; evaluation of primary spermatocytes

from F1 males revealed no sign of treatment-related heritable translocations.

In studies in vitro, neither potassium nitrate nor sodium nitrate in concentrations of up to 20 and

5 mg/plate, respectively, was mutagenic in various strains of Salmonella typhimurium (TA92, TA94,

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TA98, TA100, TA1535) (Ishidate et al. 1984), tested with and without metabolic activation. Lanthanum

nitrate hexahydrate also yielded negative results in S. typhimurium strains TA100 and TA1535 (Zeiger et

al. 1992). Tests for chromosomal aberrations in Chinese hamster fibroblast cells were positive for sodium

nitrate, but negative for potassium nitrate (Ishidate et al. 1984). IARC (2010) noted that since sodium

chlorite also yielded positive results in the same assay, the chromosomal aberrations induced by sodium

nitrate could have been due to the high osmotic pressure and sodium ion concentration. In another study,

incubation of Chinese hamster ovary cells with up to 10 mM ammonium nitrate for up to 24 hours in the

presence of metabolic activation or up to 48 hours without metabolic activation did not induce

chromosomal aberrations (Kim et al. 2011).

Several studies have examined the in vivo genotoxicity of nitrite using a variety of tests, a summary is

shown in Table 3-3. The results have been mixed, and at times inconsistent, between laboratories that

used the same tests. Administration of up to 7.3 mg sodium nitrite to pregnant mice (~290 mg/kg

assuming 0.025 kg body weight) on gestation days 7–18 via the drinking water did not induce

chromosomal aberrations in maternal bone marrow cells or in fetal liver cells (Shimada 1989). Negative

results for chromosomal aberrations were also reported in embryonic hamster cells after administration of

a single dose of up to 500 mg/kg sodium nitrite on gestation day 11 or 12 (Inui et al. 1979). However,

significantly increased incidences of chromosomal aberrations were reported in bone marrow cells from

male rats (ca. 2.1–2.4 times greater than controls), mice (ca. 4–5 times greater than controls), and rabbits

(ca. 2–3.6 times greater than controls) dosed with ≥1.7 mg/kg sodium nitrite (Luca et al. 1987). Rats and

mice were dosed twice by gavage, whereas rabbits received sodium nitrite via the drinking water for

3 months. No dose-response was apparent in the studies by Luca et al. (1987) over an approximately

27-fold dose range, suggesting that maximum response was already achieved with the lowest dose,

1.7 mg/kg. Sodium nitrite also induced micronuclei in polychromatic erythrocytes of mice dosed twice at

≥1.7 mg/kg (Luca et al. 1987) and in embryonic hamster cells after a single administration of 250 mg/kg

sodium nitrite to the pregnant dams (Inui et al. 1979). However, in another study (NTP 2001), sodium

nitrite did not induce micronuclei in male rat or mouse bone marrow cells after three intraperitoneal

injections at nonlethal doses up to 50 mg/kg/day (rats) and 125 mg/kg/day (mice). Evaluation of SCEs

also provided seemingly conflicting results. In a study by Giri et al. (1986), single doses of ≥5 mg/kg

sodium nitrite by gavage induced dose-related significant increases in SCEs in mouse bone marrow cells,

but Bambrilla et al. (1983) reported that a single gavage dose of 80 mg/kg sodium nitrite did not induce

SCEs in mouse bone marrow cells. Results from assays for DNA repair, DNA damage, or DNA

synthesis in mammalian cells from rats or mice generally yielded negative results (Bambrilla et al. 1983;

Friedman and Staub 1976; Hellmér and Bolcsfoldi 1992; Robbiano et al. 1990). Sodium nitrite induced

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Table 3-3. Genotoxicity of Sodium Nitrite In Vivo

Species (test system)

End point

Results

Reference

Mammalian cells:

Pregnant mouse bone marrow Chromosomal aberrations − Shimada 1989

cells

Mouse fetal liver cells

Chromosomal aberrations

–

Shimada 1989

Embryonic hamster cells

Chromosomal aberrations

−

Inui et al. 1979

Rat bone marrow cells

Chromosomal aberrations

+

Luca et al. 1987

Mouse bone marrow cells

Chromosomal aberrations

+

Luca et al. 1987

Rabbit bone marrow cells

Chromosomal aberrations

+

Luca et al. 1987

Mouse polychromatic

Micronuclei

+

Luca et al. 1987

erythrocytes

Rat bone marrow cells

Micronuclei

−

NTP 2001

Mouse bone marrow cells

Micronuclei

−

NTP 2001

Embryonic hamster cells

Micronuclei

+

Inui et al. 1979

Embryonic hamster cells

Malignant cell transformation

+

Inui et al. 1979

Embryonic hamster cells

Drug-resistant mutations

+

Inui et al. 1979

Mouse bone marrow cells

Sister chromatid exchange

+

Giri et al. 1986

Mouse bone marrow cells

Sister chromatid exchange

−

Brambrilla et al. 1983

Mouse host-mediated assay

Mutations in Salmonella

−

Couch and Friedman 1975

Mouse host-mediated assay

DNA repair in E. coli K-12

−

Hellmér and Bolcsfoldi 1992

uvrb/recA

Rat liver cells

DNA damage

−

Robbiano et al. 1990

Rat liver and gastric mucosa

DNA damage

−

Brambrilla et al. 1983

cells

Mouse testicular cells

DNA synthesis

−

Friedman and Staub 1976

Male mouse germ cells

Unscheduled DNA synthesis

−

Alavantić et al. 1988a

Male mouse germ cells

Heritable translocations

−

Alavantić et al. 1988b

Insect systems:

Drosophila melanogaster (wing

Somatic mutation + Graf et al. 1989

spot test)

+ = positive results; – = negative results

109 NITRATE AND NITRITE

3. HEALTH EFFECTS

malignant cell transformation and produced drug-resistant mutations in embryonic hamster cells

following treatment of the pregnant dams on gestation day 11 or 12 with a single dose of ≥125 mg/kg

(Inui et al. 1979). Alavantić et al. (1988a) treated male mice with sodium nitrite by gavage for 3 days at

doses of 0, 60, or 120 mg/kg/day; there was no sign of treatment-related unscheduled DNA synthesis in

spermatids analyzed 17 days following treatment. Alavantić et al. (1988b) treated male mice with sodium

nitrite by gavage for 2 weeks doses of 0, 60, or 120 mg/kg/day and subsequently mated them to virgin

females; evaluation of primary spermatocytes from F1 males revealed no sign of treatment-related

heritable translocations. In a host-mediated assay, mice were intraperitoneally inoculated with

S. typhimurium strain G46 and gavaged with sodium nitrite (Couch and Friedman 1975); the sodium

nitrite treatment did not induced increased frequency in S. typhimurium mutation rate in this host-

mediated assay. Finally, feeding sodium nitrite to larvae of Drosophila melanogaster induced somatic

mutations as assessed by the wing spot test (Graf et al. 1989).

Numerous studies have examined the genotoxicity of nitrite in in vitro assays. As shown in Table 3-4,

there seem to be more positive results than negative results in tests of gene mutations in prokaryotic

organisms, but it is difficult to draw a firm conclusion (Andrews et al. 1980, 1984; Balimandawa et al.

1994; Brams et al. 1987; De Flora 1981, De Flora et al. 1984; Ehrenberg et al. 1980; Ishidate et al. 1981,

1984; McCann et al. 1975; Törnqvist et al. 1983; Zeiger et al. 1992). However, it appears that the

addition of metabolic activation systems to the incubation mixtures did not make a difference in the

results. That is, tests that were positive without activation were also positive with activation; tests that

were negative without activation were also negative with activation. This would indicate that nitrite can

be a direct mutagenic chemical. In vitro tests that assessed chromosomal aberrations, SCEs, DNA repair,

and cell transformations in sodium nitrite-treated mammalian cells yielded positive results (Inoue et al.

1985; Ishidate et al. 1984; Luca et al. 1987; Lynch et al. 1983; Tsuda and Kato 1977; Tsuda et al. 1973,

1981). Nitrite enhanced neutrophil-induced DNA strand breakage in rat lung type II epithelial cells; the

enhancement was associated with an inhibition of neutrophil-derived myeloperoxidase (Knaapen et al.

2005).

3.4 TOXICOKINETICS

No information was located regarding the pharmacokinetics of nitrate or nitrite following inhalation or

dermal exposure. However, numerous reports are available regarding the pharmacokinetics of ingested

nitrate and nitrite. Comprehensive reviews of the available data (Bailey et al. 2012; Bryan and van

Grinsven 2013; IARC 2010; JECFA 2003a, 2003b; Lundberg and Weitzberg 2013; Lundberg and Govoni

110 NITRATE AND NITRITE

3. HEALTH EFFECTS

Table 3-4. Genotoxicity of Sodium Nitrite In Vitro

Results

With

Without

Species (test system)

End point

activation

Reference

Prokaryotic organisms:

Salmonella typhimurium

Gene mutation

–

TA98

S. typhimurium TA100

Gene mutation

+

S. typhimurium TA98, TA100,

Gene mutation

+

TA1537

S. typhimurium TA100,

Gene mutation

+

TA1535

S. typhimurium TA98, TA100,

Gene mutation

−

TA1535, TA1537, TA1538

S. typhimurium TA100,

Gene mutation

+

TA1530, TA1535

S. typhimurium TA102,

Gene mutation

−

YG1024, DJ400, DJ460

S. typhimurium TA100

Gene mutation

+

S typhimurium TA97, TA98

Gene mutation

−

S. typhimurium TA1530

Gene mutation

NT

S typhimurium TA100,

Gene mutation

−

a

TA1535

S typhimurium TA98,

Gene mutation

−

TA1537, TA1538

S. typhimurium TA1535

Gene mutation

NT

Escherichia coli WP2, WP67,

DNA repair

+

CM871

Eukaryotic organisms:

Cultured human lymphocytes

Sister chromatid

NT

exchange

Chinese hamster ovary cells

Sister chromatid

NT

exchange

Chinese hamster ovary cells

Chromosomal

NT

aberrations

Monkey BS-C-1 fetal liver

Chromosomal

NT

cells

aberrations

HeLa cells

Chromosomal

NT

aberrations

Chinese hamster fibroblasts

Chromosomal

NT

aberrations

Syrian hamster embryo cells

Chromosomal

NT

aberrations

–

NTP 2001; Zeiger et al.

1992

+

NTP 2001; Zeiger et al.

1992

+

Ishidate et al. 1981

+

Ishidate et al. 1984

−

Andrews et al. 1980,

1984

+

Balimandawa et al. 1994

−

Balimandawa et al. 1994

NT

Brams et al. 1987

NT

Brams et al. 1987

+

Ehrenberg et al. 1980

+

De Flora 1981, 1984

−

De Flora 1981, 1984

(+)

McCann et al. 1975

+

De Flora et al. 1984

+

Inoue et al. 1985

+

Tsuda et al. 1981

+

Tsuda et al. 1981

+

Luca et al. 1987

+

Luca et al. 1987

+

Ishidate et al.1984

+

Tsuda and Kato 1977

111 NITRATE AND NITRITE

3. HEALTH EFFECTS

Table 3-4. Genotoxicity of Sodium Nitrite In Vitro

Results

With

Without

Species (test system)

End point

activation

Reference

HeLa S3 carcinoma cells

DNA repair

NT

+

Lynch et al. 1983

Syrian hamster embryo cells

Cell transformation

NT

+

Tsuda et al. 1973

a

Reported as a decrease in mutagenicity in the presence of S9 mix; however, it was not specified whether the

decrease was relative to controls or sodium nitrite treatment in the absence of S9 mix.

+ = positive results; (+) = weakly positive; – = negative results; DNA = deoxyribonucleic acid; NT = not tested

112 NITRATE AND NITRITE

3. HEALTH EFFECTS

2004; Lundberg et al. 2008, 2009; Weitzberg and Lundberg 2013; Weitzberg et al. 2010; WHO 2011b)

serve as references for the major portion of toxicokinetic data presented in this section of the ATSDR

Toxicological Profile for Nitrate and Nitrite.

Ingestion is the major source of exposure to nitrate and nitrite. Vegetables are the main source of nitrate

in the diet (approximately 60–80% of total nitrate intake); nitrate in some drinking water sources may

contribute 15–20% of total nitrate intake. Small amounts of nitrate and nitrite are added to some animal-

based products to serve as preservatives and to enhance taste. Approximately 80–85% of nitrite in

humans is produced from in vivo reduction of nitrate.

The nitrate-nitrite-nitric oxide pathway in mammals includes a dietary component and an endogenous

component. Figure 3-2 depicts the metabolic pathways for ingested nitrate and nitrite, as well as the

endogenous production of nitric oxide via nitric oxide synthase (NOS). Numbers in brackets in the figure

coincide with those in the following description of the pathways. Ingested nitrate passes through the

stomach [1] to the small intestine [2] where it is nearly completely absorbed into the blood [3]. Following

a nitrate-containing meal, circulating nitrate concentrations are normally in the range of 20–40 µM,

depending on the type of diet and activity of nitric oxide synthases. Peak plasma nitrate levels are

reached 15–30 minutes following ingestion; the half-time of plasma nitrate is on the order of 5–6 hours.

Most nitrate that passes through the kidney [4] is reabsorbed into the blood [5]. However, some is

excreted in the urine [6]. In humans, approximately 25% of plasma nitrate is taken up by the salivary

glands and secreted in the saliva [7]; concentrations salivary nitrate can be as much as 10–20 times that of

plasma nitrate. Approximately 20% of the nitrate in saliva undergoes anaerobic, nitrate reductase-

catalyzed reduction to nitrite by commensal bacteria; thus, salivary secretion and reduction in saliva

results in conversion of approximately 5% of ingested nitrate to nitrite (Gangolli et al. 1994; Walker

1996). In vitro results using selected rat and mouse tissues and human liver tissues suggest a possible

metabolic pathway whereby some plasma nitrate could be reduced to nitrite by enzymes such as xanthine

oxidase (Jansson et al. 2008). Most salivary nitrate, however, passes to the small intestine and is

absorbed into the blood. A portion of nitrite (either produced from reduction of nitrate or ingested from

food sources) that enters the stomach is rapidly protonated to nitrous acid (HNO

2

), which decomposes

spontaneously to nitric oxide and other biologically active nitrogen oxides (e.g., nitrogen dioxide [NO

2

];

dinitrogen trioxide [N

2

O

3

]) in the acid environment of the stomach [8]; this process is enhanced in the

presence of reducing compounds such as ascorbic acid and polyphenols. Nitrite can also react with

proteins, amines, and amides in the stomach. Reaction of nitrite with some low-molecular-weight amines

NitrateNitrate

Nitrate

Nitrite

N

113 NITRATE AND NITRITE

3. HEALTH EFFECTS

Figure 3-2. The Nitrate-Nitrite-Nitric Oxide Cycle in Humans*

Kidney

Mouth

Nitrate

Nitrite

Small intestine

Nitrate Nitrite

Blood, tissues

Urine

[4]

[5]

[6]

Nitrate in

saliva

Nitric oxide

Physiological

functions

25% of blood nitrate taken up by salivary gland

HNO

2

NO

2

N

2

O

3

N-nitroso

compounds

Nitrate

[1]

[2]

Nitrate Nitrite

[3]

L-arginine

NOS

[7]

[8]

[13]

[14]

[15]

Nitrate

deoxyHb

metHb

[10]

[11]

Nitrite

Stomach

[16]

oxyHb

metHb

[12]

oxyHb

metHb

Oral intake

Nitrate

Nitrite

*Numbers in brackets coincide with those in the descriptive text.

deoxyHb = deoxyhemoglobin; HNO

2

= nitrous acid; metHb = methemoglobin; NO = nitric oxide; NO

2

= nitrogen

dioxide; N

2

O

3

= dinitrogen trioxide; NOS = nitric oxide synthase; oxyHb = oxyhemoglobin

114 NITRATE AND NITRITE

3. HEALTH EFFECTS

(nitrosation) produces N-nitroso derivatives [9], including carcinogenic compounds, portions of which

can be absorbed and distributed via systemic circulation. However, most nitrite passes to the small

intestine where it is absorbed into the blood. Plasma levels of nitrite increase within 30 minutes

following ingestion of nitrate. Although the biological half-time of plasma nitrite is only 20–30 minutes,

plasma levels remain elevated for several hours due to the enterosalivary circulation of nitrate. Plasma

nitrite concentrations, which are normally 50–100 nM, may increase as much as 5 times after a nitrate-

rich meal. The production of N-nitroso derivatives from plasma nitrite occurs to some extent in selected

tissues.

Nitrite in the blood and tissues can be reduced to nitric oxide, which is involved in a variety of

physiological processes. In the presence of deoxyhemoglobin, reduction of nitrite to nitric oxide occurs

via oxidation of ferrous (Fe

2+

) hemoglobin (which transports oxygen) to ferric (Fe

3+

) hemoglobin

(methemoglobin, a poor transporter of oxygen) [10]. Methemoglobin is converted to deoxyhemoglobin

[11] in a reaction catalyzed by methemoglobin reductase. Nitrite can also react with oxyhemoglobin to

form nitrate and methemoglobin [12].

In addition to exogenous sources of nitrate and nitrite (e.g., diet), nitrate, nitrite, and nitric oxide are

produced endogenously. A major endogenous production mechanism is oxygen-dependent reduction of

L-arginine (a biologically-relevant amino acid) to nitric oxide [13], which occurs in most cells of the body

in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and cofactors flavin adenine

dinucleotide (FAD), tetrahydrobiopterin (BH

4

), heme, and calmodulin. Nitric oxide is involved in a

variety of physiological functions that include regulation of blood flow, platelet function, pulmonary

function, nerve function, host defense, and metabolic control. Nitric oxide may also be formed via an

oxygen-independent one-electron reduction of nitrite in acidic and hypoxic tissues [14]. Nitrite may serve

as an important source of nitric oxide under such acidic and hypoxic conditions because the half-time for

plasma nitrite (15–20 minutes) is much longer than that of nitric oxide (<6 seconds). Nitric oxide is

rapidly oxidized to nitrite in the presence of oxygen and ceruloplasmin [15]. Nitric oxide can also react

with oxyhemoglobin to form nitrate and methemoglobin [16]. Various physiological processes are

involved in maintaining a balance between systemic levels of nitrate, nitrite, and nitric oxide. The

endogenous nitrate-nitrite-nitric oxide pathway provides baseline levels of nitrate and nitrite in the body

which are supplemented by dietary intake. The total plasma nitrate and nitrite content consists of portions

entering the blood from oral intake and portions generated endogenously from nitric oxide in the body.

115 NITRATE AND NITRITE

3. HEALTH EFFECTS

As much as 60–75% of plasma nitrate is excreted unchanged in the urine within 24 hours following

ingestion. Under normal physiological conditions, nitrite is not detected in the urine and its presence in

urine is an indication of infection by nitrate-reducing organisms. Zhou et al. (2014) reported increased

urinary excretion of N-nitroso compounds following ingestion of nitrite from the drinking water of rats.

Minor urinary products of nitrate and nitrite metabolism include ammonia and urea. Nitrate and nitrite

are secreted to some extent in breast milk and perspiration. Fecal excretion of nitrate and nitrite is

negligible.

3.4.1 Physiologically Based Pharmacokinetic (PBPK)/Pharmacodynamic (PD) Models

Physiologically based pharmacokinetic (PBPK) models use mathematical descriptions of the uptake and

disposition of chemical substances to quantitatively describe the relationships among critical biological

processes (Krishnan et al. 1994). PBPK models are also called biologically based tissue dosimetry

models. PBPK models are increasingly used in risk assessments, primarily to predict the concentration of

potentially toxic moieties of a chemical that will be delivered to any given target tissue following various

combinations of route, dose level, and test species (Clewell and Andersen 1985). Physiologically based

pharmacodynamic (PBPD) models use mathematical descriptions of the dose-response function to

quantitatively describe the relationship between target tissue dose and toxic end points.

PBPK/PD models refine our understanding of complex quantitative dose behaviors by helping to

delineate and characterize the relationships between: (1) the external/exposure concentration and target

tissue dose of the toxic moiety, and (2) the target tissue dose and observed responses (Andersen and

Krishnan 1994; Andersen et al. 1987). These models are biologically and mechanistically based and can

be used to extrapolate the pharmacokinetic behavior of chemical substances from high to low dose, from

route to route, between species, and between subpopulations within a species. The biological basis of

PBPK models results in more meaningful extrapolations than those generated with the more conventional

use of uncertainty factors.

The PBPK model for a chemical substance is developed in four interconnected steps: (1) model

representation, (2) model parameterization, (3) model simulation, and (4) model validation (Krishnan and

Andersen 1994). In the early 1990s, validated PBPK models were developed for a number of

toxicologically important chemical substances, both volatile and nonvolatile (Krishnan and Andersen

1994; Leung 1993). PBPK models for a particular substance require estimates of the chemical substance-

specific physicochemical parameters, and species-specific physiological and biological parameters. The

116 NITRATE AND NITRITE

3. HEALTH EFFECTS

numerical estimates of these model parameters are incorporated within a set of differential and algebraic

equations that describe the pharmacokinetic processes. Solving these differential and algebraic equations

provides the predictions of tissue dose. Computers then provide process simulations based on these

solutions.

The structure and mathematical expressions used in PBPK models significantly simplify the true

complexities of biological systems. However, if the uptake and disposition of the chemical substance(s)

are adequately described, this simplification is desirable because data are often unavailable for many

biological processes. A simplified scheme reduces the magnitude of cumulative uncertainty. The

adequacy of the model is, therefore, of great importance, and model validation is essential to the use of

PBPK models in risk assessment.

PBPK models improve the pharmacokinetic extrapolations used in risk assessments that identify the

maximal (i.e., the safe) levels for human exposure to chemical substances (Andersen and Krishnan 1994).

PBPK models provide a scientifically sound means to predict the target tissue dose of chemicals in

humans who are exposed to environmental levels (for example, levels that might occur at hazardous waste

sites) based on the results of studies where doses were higher or were administered in different species.

Figure 3-3 shows a conceptualized representation of a PBPK model.

If PBPK models for nitrate and nitrite exist, the overall results and individual models are discussed in this

section in terms of their use in risk assessment, tissue dosimetry, and dose, route, and species

extrapolations.

Kinetics of absorption of nitrate from the gastrointestinal tract and elimination in urine can be described

mathematically with simple one-compartment first-order models (Schultz et al. 1985; Wagner et al.

1983). The complex kinetics of salivary secretion of nitrate, reduction and absorption in the

gastrointestinal tract, and binding to hemoglobin and formation of methemoglobin have been described

with a multicompartment model (Zeilmaker et al. 1996, 2010b).

117 NITRATE AND NITRITE

3. HEALTH EFFECTS

Figure 3-3. Conceptual Representation of a Physiologically Based

Pharmacokinetic (PBPK) Model for a

Hypothetical Chemical Substance

Inhaled chemical Exhaled chemical

Lungs

Liver

Fat

Slowly

perfused

tissues

Richly

perfused

tissues

Kidney

Skin

V

E

N

O

U

S

B

L

O

D

A

R

T

E

R

I

A

L

B

L

O

D

V

max

K

m

Ingestion

GI

Tract

Feces

Urine

Chemicals

contacting skin

Note: This is a conceptual representation of a physiologically based pharmacokinetic (PBPK) model for a

hypothetical chemical substance. The chemical substance is shown to be absorbed via the skin, by inhalation, or by

ingestion, metabolized in the liver, and excreted in the urine or by exhalation.

Source: adapted from Krishnan and Andersen 1994

118 NITRATE AND NITRITE

3. HEALTH EFFECTS

The Zeilmaker et al. (1996, 2010b) Model

Description of the Model. Zeilmaker et al. (1996, 2010b) developed a PBPK model for simulating

kinetics of methemoglobin formation resulting from absorption of nitrate in adult humans. The structure

of the model is depicted in Figure 3-4. Parameters and parameter values for the model are presented in

Table 3-5.

The model simulates absorption of nitrate from the gastrointestinal tract as a first-order transfer to a

central nitrate distribution compartment, which is assumed to be in equilibrium with blood plasma (k

a,NO

3

,

hour

-1

). The fraction of ingested nitrate that is absorbed is assumed to be 100% (F

a

=1). The model also

simulates delivery of endogenously produced nitrate to blood (zero-order K

end

= 162 mg NO

3

/24 hours).

Absorbed nitrate is eliminated from the central compartment by excretion into urine, metabolism (tissues

and gastrointestinal bacteria), and secretion into saliva. The metabolism and urinary pathways are

combined in the model into a single first-order pathway (k

el

, hour

-1

), a fraction of which goes to urine

(f

u

=0.56). Secretion of nitrate into saliva is simulated as a separate pathway. Secretion occurs by

capacity-limited transport mediated by a sodium/iodide (Na

+

/I

-

symporter, NIS) in the salivary gland

epithelium. Although the NIS has limited capacity for nitrate, relatively large nitrate doses and blood

nitrate concentrations are required to exceed linear blood-to-saliva kinetics in vivo, indicative of

saturation of the carrier. The model can simulate salivary secretion of nitrate as either a first-order

process (k

sec,NO

3

, hour

-1

), or a capacity-limited process (K

m

, mM; C

s,max,NO

3

, mg/L), depending on the dose

(<1,000 mg/70 kg; 14 mg/kg) or plasma nitrate concentration (<34 mg NO

3

/L). Nitrate is eliminated

from saliva by transfer to the gastrointestinal tract (flow-limited B, L/hour) or reduction to nitrite (first-

order k

conv

, hour

-1

). Nitrite in saliva undergoes transfer to the gastrointestinal tract (flow-limited B,

L/hour), from where it can be absorbed into blood (first-order k

a,NO

2

, hour

-1

) or be converted to other

metabolites and reaction products (first-order k

dec

, hour

-1

). Nitrite in blood is secreted into saliva (first-

order k

sec,NO

2

, hour

-1

) or reacts with hemoglobin to produce methemoglobin (first-order k

NO

2

, hour

-1

) and

nitrate. Methemoglobin is regenerated as a product of methemoglobin reductase (capacity-limited K

m,r

,

mM). Nitrate formed in the reaction of nitrite with hemoglobin is returned to blood (first-order z

⋅

k

NO

2

,

hour

-1

). Background production of methemoglobin from reactants other than nitrite is accounted for as a

background concentration of reactants (C

bg

, mM), which combines additively with the concentration of

nitrite (C

NO

2

, mM) to react with hemoglobin.

119 NITRATE AND NITRITE

3. HEALTH EFFECTS

Figure 3-4. Structure of the Zeilmaker et al. (1996, 2010b) Model*

*See Table 3-5 for explanation of symbols; solid lines = mass flows; dotted lines = functional relationships

Hb = hemoglobin; MetHb = methemoglobin

Source: Adapted from Zeilmaker et al. (2010b)

120 NITRATE AND NITRITE

3. HEALTH EFFECTS

Table 3-5. Parameter Values for the Zeilmaker et al. (1996, 2010) PBPK Model of

Nitrate and Nitrite in Humans

Parameter

Value (standard deviation or range)

Physiological parameters

Volume of saliva compartment (V

s

)

0.001 L

Salivary flow (B)

0.069 L/hour (0.042–0.120)

Nitrate parameters

Volume fraction (of body weight) of central nitrate distribution

0.30 (0.29–0.33)

compartment (V

NO3

)

Nitrate dose averaging time (

∆

t)

0.1 hours (drinking water); 0.8 hours

(vegetables)

Nitrate gastrointestinal absorption rate (k

a,NO3

)

>5 hour

-1

Nitrate gastrointestinal absorption fraction (F

a,NO3

)

1

Nitrate endogenous production (K

end

)

162 mg/24 hours

Nitrate elimination rate (k

el

)

0.14±0.01 hour

-1

Nitrate urinary elimination fraction (f

u

)

0.56±0.029

Nitrate blood-to-saliva secretion rate (k

sec,NO3

)

0.045±0.003 hour

-1

Nitrate blood-to-saliva half-maximum (K

M,s

)

104 mg/L

Nitrate blood-to-saliva maximum (C

max,s

)

2,258 mg/L

Nitrate-to-nitrite conversion rate in saliva (k

conv

)

19.95±1.75 hour

-1

Nitrite parameters

Volume fraction (of body weight) of central nitrite distribution

0.65±0.03

compartment (V

NO2

)

Nitrite gastrointestinal absorption rate (k

a,NO2

)

>5 hour

-1

Nitrite gastrointestinal absorption fraction (F

a,NO2

)

1

Nitrite blood-to-saliva secretion rate (k

sec,NO2

)

0.045±0.003 hour

-1

Nitrite gastrointestinal conversion rate to other products (k

dec

)

0.67 hour

-1

(at pH 1.5)

Hemoglobin/methemoglobin parameters

Nitrite reaction rate with hemoglobin (k

NO2

)

4.23±0.15 mM

-1

hour

-1

Methemoglobin reductase half maximum (K

M,r

)

0.012±0.0018 mM

Methemoglobin reductase maximum (V

max,r

)

4.23±0.15 mM/hour

Stoichiometric constant for regeneration of nitrate from

0.5±0.01

methemoglobin (z)

Hemoglobin concentration in blood (C

Hg

)

8 mM

Background methemoglobin concentration in blood (C

MetHb,bg

)

0.03 mM

Background concentration of hemoglobin oxidizing reactants in

0.0058 mM

blood(C

bg

)

a

Based on Zeilmaker et al. (2010b)

121 NITRATE AND NITRITE

3. HEALTH EFFECTS

Following ingestion of nitrate in a given medium (e.g., drinking water or vegetables), the ingested nitrate

dose is assumed to enter the absorption compartment at a rate (mg/hour) given by the oral dose (mg)

divided by a dose averaging time,

∆

t (hour), where the parameter, ∆t, is assigned a value specific for the

ingested medium.

Sources for model parameter estimates are presented in Table 3-5. Nine parameters were derived by

statistical optimization to experimental in vivo data (Kortboyer et al. 1997b, 1998b; Wagner et al. 1983).

Data from Wagner et al. (1983) were used to optimize parameters

∆

t

water

, k

a,NO

3

, k

sec,NO

3

, and k

conv

. Wagner

et al. (1983) measured plasma, saliva, and urine, and nitrite in plasma and saliva, in 12 healthy adults

following a single oral dose of

15

N-nitrate in drinking water. The parameter,

∆

t

veg

(for vegetables), was

optimized with data from a study in which plasma nitrate was measured in six adults before and following

a vegetable meal (Kortboyer et al. 1998b). The parameter, V

NO

2

, was optimized with data from a study in

which plasma nitrite concentrations were measured in nine adults before and following an intravenous

dose of sodium nitrite (Kortboyer et al. 1997b). Parameters describing reactions with hemoglobin and

methemoglobin (k

NO

2

, [K

m,r

, V

max,r

, z]) were derived by statistical optimization to experimental in vitro

studies in which reaction kinetics of nitrite with hemoglobin were measured in whole human blood

(Kosaka et al. 1979; Rodkey 1976). The remaining parameters were estimated from reported literature or

calculated from other parameters (Cortas and Wakid 1991; Kortboyer et al. 1995, 1997a, 1997b, 1998a,

1998b; Lambers et al. 2000

; McKnight et al. 1997; Mirvish et al. 1975; Schultz et al. 1985; Wagner et al.

1983).

Validation of the Model. The optimized model was evaluated by comparing predictions of plasma

nitrate and nitrite concentrations and blood methemoglobin concentrations in nine adults who consumed a

single oral dose of sodium nitrite (2.42 or 4.84 mg sodium nitrite/kg) (Kortboyer et al. 1997b). The

results of the evaluation are reported in Zeilmaker et al. (2010b) as overlay plots of observations and

predictions. Statistical evaluations of the agreement between predictions and observations were not

reported.

Risk Assessment. The model has been used to predict concentrations of methemoglobin that would

result from a vegetable meal and to evaluate whether the average daily intake of nitrate would result in

clinically significant methemoglobinemia (JECFA 2003a). JECFA (2003a) applied the model to make

predictions in adults and infants. In order to apply the model to infants, blood volume and volumes of the

central nitrate and nitrite compartments were scaled to infants (the exact scaling procedure or scaled

parameter values were not reported). JECFA (2003a) also applied the model to predict methemoglobin

122 NITRATE AND NITRITE

3. HEALTH EFFECTS

concentrations that might occur in patients who have inflammatory reactions to absorbed nitrite.

Absorbed doses of nitrate in patients were simulated in the model as an intravenous infusion of nitrite.

Target Tissues. The model was calibrated to predict concentrations of nitrate and nitrite in plasma

and blood methemoglobin concentrations in humans.

Species Extrapolation. The model simulates nitrate and nitrite kinetics in humans. Applications to

other species would require development of appropriate scaling methods, optimization, and validation.

Interroute Extrapolation. The model is currently configured to simulate kinetics associated with

intravenous and oral dosing. Simulation of other potential routes of exposure (e.g., inhalation, dermal)

would require development of models for the absorption of inhaled nitrate or nitrate deposited on the skin.

3.5 MECHANISMS OF ACTION

3.5.1 Pharmacokinetic Mechanisms

Ingestion is the major route of exposure to exogenous nitrate and nitrite. Nitrate is assumed to enter the

blood from the upper small intestinal tract via active transport (EPA 1990b), which may involve active

transport systems such as the sodium iodide symporter (NIS) because nitrate has been shown to be a

relatively weak competitive inhibitor of NIS (e.g., Eskandari et al. 1997) and the NIS-mediated uptake of

iodine from the intestine has been demonstrated (Nicola et al. 2009). Nitrite is readily absorbed via

diffusion across the gastric mucosa and wall of the small intestine (EPA 1990b). As described in detail in

Section 3.4, nitrate and nitrite are readily distributed throughout the body and a portion of plasma nitrate

is concentrated in the salivary gland at concentrations as much as 10 times that of plasma nitrate. Qin et

al. (2012) demonstrated that the scialic acid (SA)/H

+

cotransporter, sialin, is endogenously localized in the

plasma membrane of salivary gland cells and functions as an electrogenic 2NO

3

-

/H

+

cotransporter; this

active transport mechanism may be responsible for high concentrations of nitrate in the salivary gland.

Refer to Section 3.4 for information regarding metabolic pathways involved in the nitrate-nitrite-nitric

oxide cycle. No information was located regarding specific mechanisms involved in transfer of nitrate to

the urine.

123 NITRATE AND NITRITE

3. HEALTH EFFECTS

3.5.2 Mechanisms of Toxicity

The most sensitive and widely-recognized toxic effect of nitrate and nitrite is that of nitrite-induced

methemoglobinemia in which nitrite (ingested as nitrite, formed via bacterial reduction of ingested nitrate,

and/or produced as an endogenous product of the nitric oxide oxidation) reacts with ferrous (Fe

2+

)

hemoglobin (which transports oxygen) to form ferric (Fe

3+

) hemoglobin (methemoglobin, a poor

transporter of oxygen) (refer to Section 3.4 for additional information regarding the nitrate-nitrite-nitric

oxide pathway).

As stated in Section 3.2.2.2 (Endocrine Effects), nitrate is a dose-dependent competitive inhibitor of the

NIS, which mediates the uptake of iodine by the thyroid. Sufficiently decreased iodine uptake by the

thyroid might result in decreased production of thyroid hormones T3 and T4 and consequent adverse

effects associated with thyroid dysfunction (e.g., hypothyroidism), including effects on developing

fetuses.

Proposed mechanisms of carcinogenicity involve the production of N-nitrosamines via nitrosating

reactions that involve nitrite and amines or amides. Such reactions may occur within some food items

during storage or preparation or in the body (usually in the stomach) (Mirvish 1975). The National

Toxicology Program’s 12

th

Report on Carcinogens (NTP 2011) lists 17 N-nitroso compounds (mostly

nitrosamines) as reasonably anticipated to be a human carcinogen based on sufficient evidence of

carcinogenicity from studies in experimental animals and one nitrosourea compound as known to be a

human carcinogen and one nitrosourea compound (1-(2-chloroethyl)-3-(4-methylcyclohexyl)-

1-nitrosourea) as known to be a human carcinogen based on sufficient evidence of carcinogenicity from

studies in humans. The International Agency for Research on Cancer (IARC 2014) lists eight of these

compounds in Group 2A (probably carcinogenic to humans), another eight in Group 2B (possibly

carcinogenic to humans), and two compounds (N-nitrosopiperadine and 4-(N-nitrosomethylamino)-

1-(3-pyridyl)-1-butanone) in Group 1 (carcinogenic to humans). Interactions between nitrite and a variety

of drugs have been shown to result in the formation of carcinogenic N-nitroso compounds (Brambilla and

Martelli (2007).

3.5.3 Animal-to-Human Extrapolations

Interspecies differences in nitrate-nitrite-nitric acid pathways indicate that laboratory animals do not

represent reliable models of nitrate-nitrite-nitric oxide pathways for humans (EPA 1990b; Health Canada

2012; Kortboyer et al. 1997a, 1997b; Walker 1995; WHO 2011b). For example, Til et al. (1988) reported

124 NITRATE AND NITRITE

3. HEALTH EFFECTS

that the rate of conversion of nitrate to nitrite is much lower in rats than humans. Cohen and Myant

(1959) reported that the rat lacks the active transport mechanism (sodium iodide symporter) responsible

for secretion of plasma nitrate to the salivary gland in humans. Therefore, the rate of reduction of salivary

nitrate to nitrite in the rat is likely much less than the estimate of 25% reduction in human saliva.

3.6 TOXICITIES MEDIATED THROUGH THE NEUROENDOCRINE AXIS

Recently, attention has focused on the potential hazardous effects of certain chemicals on the endocrine

system because of the ability of these chemicals to mimic or block endogenous hormones. Chemicals

with this type of activity are most commonly referred to as endocrine disruptors. However, appropriate

terminology to describe such effects remains controversial. The terminology endocrine disruptors,

initially used by Thomas and Colborn (1992), was also used in 1996 when Congress mandated the EPA to

develop a screening program for “...certain substances [which] may have an effect produced by a

naturally occurring estrogen, or other such endocrine effect[s]...”. To meet this mandate, EPA convened a

panel called the Endocrine Disruptors Screening and Testing Advisory Committee (EDSTAC), and in

1998, the EDSTAC completed its deliberations and made recommendations to EPA concerning endocrine

disruptors. In 1999, the National Academy of Sciences released a report that referred to these same types

of chemicals as hormonally active agents. The terminology endocrine modulators has also been used to

convey the fact that effects caused by such chemicals may not necessarily be adverse. Many scientists

agree that chemicals with the ability to disrupt or modulate the endocrine system are a potential threat to

the health of humans, aquatic animals, and wildlife. However, others think that endocrine-active

chemicals do not pose a significant health risk, particularly in view of the fact that hormone mimics exist

in the natural environment. Examples of natural hormone mimics are the isoflavinoid phytoestrogens

(Adlercreutz 1995; Livingston 1978; Mayr et al. 1992). These chemicals are derived from plants and are

similar in structure and action to endogenous estrogen. Although the public health significance and

descriptive terminology of substances capable of affecting the endocrine system remains controversial,

scientists agree that these chemicals may affect the synthesis, secretion, transport, binding, action, or

elimination of natural hormones in the body responsible for maintaining homeostasis, reproduction,

development, and/or behavior (EPA 1997). Stated differently, such compounds may cause toxicities that

are mediated through the neuroendocrine axis. As a result, these chemicals may play a role in altering,

for example, metabolic, sexual, immune, and neurobehavioral function. Such chemicals are also thought

to be involved in inducing breast, testicular, and prostate cancers, as well as endometriosis (Berger 1994;

Giwercman et al. 1993; Hoel et al. 1992).

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As discussed in detail in Section 3.2.2.2 (Endocrine Effects), available human data provide some evidence

that elevated levels of nitrate in drinking water and/or nitrate-rich diets may be associated with signs of

thyroid dysfunction (Aschebrook-Kilfoy et al. 2012; Gatseva and Argirova 2008; Rádiková et al. 2008;

Tajtáková et al. 2006; Ward et al. 2010). In animals, orally-administered nitrate has been demonstrated to

cause decreased iodine uptake by the thyroid and changes in serum thyroid hormone levels (e.g.,

Bloomfield et al. 1961; El-Wakf et al. 2008; Eskiocak et al. 2005; Mukhopadhyay et al. 2005; Zaki et al.

2004).

No in vitro studies were located regarding endocrine disruption of nitrate or nitrite.

3.7 CHILDREN’S SUSCEPTIBILITY

This section discusses potential health effects from exposures during the period from conception to

maturity at 18 years of age in humans, when most biological systems will have fully developed. Potential

effects on offspring resulting from exposures of parental germ cells are considered, as well as any indirect

effects on the fetus and neonate resulting from maternal exposure during gestation and lactation.

Relevant animal and in vitro models are also discussed.

Children are not small adults. They differ from adults in their exposures and may differ in their

susceptibility to hazardous chemicals. Children’s unique physiology and behavior can influence the

extent of their exposure. Exposures of children are discussed in Section 6.6, Exposures of Children.

Children sometimes differ from adults in their susceptibility to adverse health effects from exposure to

hazardous chemicals, but whether there is a difference depends on the chemical(s) (Guzelian et al. 1992;

NRC 1993). Children may be more or less susceptible than adults to exposure-related health effects, and

the relationship may change with developmental age (Guzelian et al. 1992; NRC 1993). Vulnerability

often depends on developmental stage. There are critical periods of structural and functional

development during both prenatal and postnatal life that are most sensitive to disruption from exposure to

hazardous substances. Damage from exposure in one stage may not be evident until a later stage of

development. There are often differences in pharmacokinetics and metabolism between children and

adults. For example, absorption may be different in neonates because of the immaturity of their

gastrointestinal tract and their larger skin surface area in proportion to body weight (Morselli et al. 1980;

NRC 1993); the gastrointestinal absorption of lead is greatest in infants and young children (Ziegler et al.

1978). Distribution of xenobiotics may be different; for example, infants have a larger proportion of their

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bodies as extracellular water, and their brains and livers are proportionately larger (Altman and Dittmer

1974; Fomon 1966; Fomon et al. 1982; Owen and Brozek 1966; Widdowson and Dickerson 1964). Past

literature has often described the fetus/infant as having an immature (developing) blood-brain barrier that

is leaky and poorly intact (Costa et al. 2004). However, current evidence suggests that the blood-brain

barrier is anatomically and physically intact at this stage of development, and the restrictive intracellular

junctions that exist at the blood-CNS interface are fully formed, intact, and functionally effective

(Saunders et al. 2008, 2012).

However, during development of the brain, there are differences between fetuses/infants and adults that

are toxicologically important. These differences mainly involve variations in physiological transport

systems that form during development (Ek et al. 2012). These transport mechanisms (influx and efflux)

play an important role in the movement of amino acids and other vital substances across the blood-brain

barrier in the developing brain; these transport mechanisms are far more active in the developing brain

than in the adult. Because many drugs or potential toxins may be transported into the brain using these

same transport mechanisms—the developing brain may be rendered more vulnerable than the adult.

Thus, concern regarding possible involvement of the blood-brain barrier with enhanced susceptibility of

the developing brain to toxins is valid. It is important to note however, that this potential selective

vulnerability of the developing brain is associated with essential normal physiological mechanisms; and

not because of an absence or deficiency of anatomical/physical barrier mechanisms.

The presence of these unique transport systems in the developing brain of the fetus/infant is intriguing;

whether these mechanisms provide protection for the developing brain or render it more vulnerable to

toxic injury is an important toxicological question. Chemical exposure should be assessed on a case-by-

case basis. Research continues into the function and structure of the blood-brain barrier in early life

(Kearns et al. 2003; Saunders et al. 2012; Scheuplein et al. 2002).

Many xenobiotic metabolizing enzymes have distinctive developmental patterns. At various stages of

growth and development, levels of particular enzymes may be higher or lower than those of adults, and

sometimes unique enzymes may exist at particular developmental stages (Komori et al. 1990; Leeder and

Kearns 1997; NRC 1993; Vieira et al. 1996). Whether differences in xenobiotic metabolism make the

child more or less susceptible also depends on whether the relevant enzymes are involved in activation of

the parent compound to its toxic form or in detoxification. There may also be differences in excretion,

particularly in newborns given their low glomerular filtration rate and not having developed efficient

tubular secretion and resorption capacities (Altman and Dittmer 1974; NRC 1993; West et al. 1948).

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Children and adults may differ in their capacity to repair damage from chemical insults. Children also

have a longer remaining lifetime in which to express damage from chemicals; this potential is particularly

relevant to cancer.

Certain characteristics of the developing human may increase exposure or susceptibility, whereas others

may decrease susceptibility to the same chemical. For example, although infants breathe more air per

kilogram of body weight than adults breathe, this difference might be somewhat counterbalanced by their

alveoli being less developed, which results in a disproportionately smaller surface area for alveolar

absorption (NRC 1993).

As discussed in detail in Section 3.4 (Toxicokinetics), a portion of ingested nitrate is reduced to nitrite by

commensal bacteria in the mouth; however, the acid environment of the normal stomach does not support

the growth of such bacteria. Most nitrite (ingested or reduced from nitrate) is absorbed from the upper

gastrointestinal tract and enters the blood; plasma nitrite readily reacts with hemoglobin to form

methemoglobin. Sufficiently high levels of methemoglobin levels result in poor oxygen supply to tissues.

Clinical methemoglobinemia is generally indicated at methemoglobin levels >10% of total hemoglobin

and cyanosis is an early clinical sign. The first 6 months of postnatal life is a period of increased

susceptibility to methemoglobinemia (termed infantile methemoglobinemia or blue baby syndrome);

possible contributing factors to this increased susceptibility (pH of the infant stomach, proportion of fetal

hemoglobin to adult hemoglobin, and concentration of NADH-dependent methemoglobin reductase)

(Greer and Shannon 2005) are discussed below.

A portion of ingested nitrate is reduced to nitrite by commensal bacteria in the mouth; however, the acid

environment of the normal stomach does not support the growth of such bacteria and most of the nitrate

that reaches the stomach passes to the small intestine from which it is nearly completely absorbed into the

blood. However, a higher pH in the stomach of the newborn may favor growth of nitrate-reducing

bacteria and increased reduction of nitrate to nitrite and consequent increased plasma methemoglobin.

Most hemoglobin in the newborn is in a form termed fetal hemoglobin, which appears to be more readily

oxidized to methemoglobin than adult hemoglobin; fetal hemoglobin is replaced by adult hemoglobin

during early postnatal life. Levels of NADH-dependent methemoglobin reductase (the major enzyme

responsible for reduction of methemoglobin to normal hemoglobin) in the newborn increase

approximately 2-fold during the first 4 month of postnatal life to reach adult levels.

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There is some evidence that methemoglobinemia in infants drinking formula prepared using drinking

water with relatively high levels of nitrate may be related to bacterial contamination of such water sources

and consequent gastrointestinal disorders, as well as gastrointestinal infection and inflammation and the

ensuing overproduction of nitric oxide (Avery 1999). Kanady et al. (2012) reported little or no bacterial

conversion of nitrate to nitrite in the saliva of a group of 10 infants during the first 2 postnatal months that

was considered mainly due to lower numbers of major nitrate-reducing oral bacteria than adults. Ibrahim

et al. (2012) found that blood nitrite levels of newborns approximately 1–2 days of age were 35–55%

lower than that of adults.

Some investigators have reported significant associations between nitrate levels in drinking water (or

living in areas presumed to have elevated nitrate levels in drinking water sources) and risk of childhood

type 1 diabetes (Dahlquist et al. 1990; Kostraba et al. 1992; Parslow et al. 1997; Virtanen et al. 1994).

However, no such relationship was observed in two other studies (van Maanen et al. 2000; Zhao et al.

2001). Refer to Section 3.2.2.2 (Metabolic Effects) for summaries of these study reports.

Results of studies designed to assess possible associations between nitrate levels in drinking water

sources and developmental end points in humans provide equivocal evidence of nitrate-related effects on

the developing fetus and infant (see Section 3.2.2.6, Developmental Effects). There is limited evidence of

nitrate-induced thyroid dysfunction (see Section 3.2.2.2, Endocrine Effects), which could result in adverse

effects on the developing fetus of a pregnant mother.

3.8 BIOMARKERS OF EXPOSURE AND EFFECT

Biomarkers are broadly defined as indicators signaling events in biologic systems or samples. They have

been classified as markers of exposure, markers of effect, and markers of susceptibility (NAS/NRC

1989).

The National Report on Human Exposure to Environmental Chemicals provides an ongoing assessment

of a generalizable sample of the exposure of the U.S. population to environmental chemicals using

biomonitoring. This report is available at http://www.cdc.gov/exposurereport/. The biomonitoring data

for nitrate from this report is discussed in Section 6.5. A biomarker of exposure is a xenobiotic substance

or its metabolite(s) or the product of an interaction between a xenobiotic agent and some target

molecule(s) or cell(s) that is measured within a compartment of an organism (NAS/NRC 1989). The

preferred biomarkers of exposure are generally the substance itself, substance-specific metabolites in

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readily obtainable body fluid(s), or excreta. However, several factors can confound the use and

interpretation of biomarkers of exposure. The body burden of a substance may be the result of exposures

from more than one source. The substance being measured may be a metabolite of another xenobiotic

substance (e.g., high urinary levels of phenol can result from exposure to several different aromatic

compounds). Depending on the properties of the substance (e.g., biologic half-life) and environmental

conditions (e.g., duration and route of exposure), the substance and all of its metabolites may have left the

body by the time samples can be taken. It may be difficult to identify individuals exposed to hazardous

substances that are commonly found in body tissues and fluids (e.g., essential mineral nutrients such as

copper, zinc, and selenium). Biomarkers of exposure to nitrate and nitrite are discussed in Section 3.8.1.

Biomarkers of effect are defined as any measurable biochemical, physiologic, or other alteration within an

organism that, depending on magnitude, can be recognized as an established or potential health

impairment or disease (NAS/NRC 1989). This definition encompasses biochemical or cellular signals of

tissue dysfunction (e.g., increased liver enzyme activity or pathologic changes in female genital epithelial

cells), as well as physiologic signs of dysfunction such as increased blood pressure or decreased lung

capacity. Note that these markers are not often substance specific. They also may not be directly

adverse, but can indicate potential health impairment (e.g., DNA adducts). Biomarkers of effects caused

by nitrate and nitrite are discussed in Section 3.8.2.

A biomarker of susceptibility is an indicator of an inherent or acquired limitation of an organism's ability

to respond to the challenge of exposure to a specific xenobiotic substance. It can be an intrinsic genetic or

other characteristic or a preexisting disease that results in an increase in absorbed dose, a decrease in the

biologically effective dose, or a target tissue response. If biomarkers of susceptibility exist, they are

discussed in Section 3.10, Populations That Are Unusually Susceptible.

3.8.1 Biomarkers Used to Identify or Quantify Exposure to Nitrate and Nitrite

There are no biomarkers of exposure that are specific to nitrate or nitrite. Although nitrate and nitrite can

be detected in blood, saliva, and urine (mostly nitrate), nitrate and nitrite are also produced endogenously

via the nitrate-nitrite-nitric oxide pathway. Sources for nitrate and nitrite levels in the body may therefore

include not only ingested food and drinking water, but also oxidation of nitric oxide produced

endogenously. Similarly, N-nitroso compounds that may be detected in the blood or urine may indicate

exposure to nitrate or nitrite; however, these compounds may also be products of the endogenous nitrate-

nitrite-nitric oxide pathway.

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3.8.2 Biomarkers Used to Characterize Effects Caused by Nitrate and Nitrite

Biomarkers of effects from exposure to nitrate or nitrite are not specific to nitrate or nitrite. Blood

methemoglobin level has been used as a biomarker of nitrate and nitrite toxicity; however,

methemoglobinemia may be elicited by other substances such as selected drugs, pesticides, industrial and

commercial products, and medical conditions such as pediatric gastrointestinal infection, sepsis, and sickle

cell crisis (ATSDR 2013a). Methemoglobinemia may also be inherited (genetic conditions that result in

decreased activity of enzymes that reduce methemoglobin or the presence of hemoglobin M). Jansen et al.

(1995) reported a rapid 6-fold increase in urinary N-methylnicotinamide (a metabolite of tryptophan) in

four of eight volunteers following the ingestion of sodium nitrate at 10 mg/kg; however, little to no

increase in urinary N-methylnicotinamide was observed in the other four volunteers. Urinary levels of

various other N-nitroso compounds (e.g., nitrosoproline) have been measured as an index of nitrosation

(Ohshima and Bartsch 1988); however, N-nitroso compounds can form via endogenous nitrosation and do

not require the intake of nitrate or nitrite.

3.9 INTERACTIONS WITH OTHER CHEMICALS

Information regarding interactions between nitrate or nitrite and other substances is comprised mainly of

studies that assessed the tumorigenicity of oral exposure to sodium nitrite in the presence of selected

amino compounds or other substances suspected or known to cause cancer and studies that assessed

modulation of tumorigenicity by selected antioxidants. As discussed in Section 3.5 (Mechanisms of

Action), nitrosating reactions that involve nitrite and amines or amides may result in the production of

N-nitrosamines, some of which may be carcinogenic. Interactions between nitrite and a variety of drugs

may also result in the formation of carcinogenic N-nitroso compounds (Brambilla and Martelli (2007).

Adverse effects elicited in laboratory animals exposed to selected substances were enhanced or

diminished upon co-exposure to nitrite, although mechanisms for such nitrite-induced enhanced or

diminished responses have not been identified. For example, Kawabe et al. (1994) observed increased

severity of forestomach hyperplasia in groups of catechol- or 3-methoxycatechol-treated rats

coadministered sodium nitrite and increased thickness of forestomach mucosa (indication of cellular

proliferation) in rats treated with sodium nitrite in combination with phenolic compounds such as

t-butylhydroquinone, catechol, gallic acid, 1,2,4-benzenetriol, dl-3-(3,4-dihydroxyphenyl)-alanine, and

hydroquinone. Coadministration of sodium nitrite with catechol resulted in enhanced cellular

proliferation. Pregnant Syrian golden hamsters fed a diet containing nitrite and morpholine exhibited a

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higher incidence of liver-cell carcinoma (5/16 hamsters) compared to those fed diets containing

morpholine in the absence of nitrite (0/22) (Shank and Newberne 1976). Sodium nitrite treatment

resulted in increased incidences of forestomach papillomas and decreased incidences of glandular

stomach epithelial adenomas in rats provided drinking water to which sodium nitrite and either catechol

or 3-methoxycatechol were added either with or without coexposure to known carcinogens (Hirose et al.

1990, 1993). IARC (2010) summarized results from a Russian study (Ilnitsky and Kolpakova 1997) in

which sodium nitrite appeared to enhance the carcinogenic effect of leukemia viruses in mice. Hirose et

al. (2002) observed a reduction of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced mammary

gland tumors in rats coexposed to sodium nitrite in the drinking water. Commoner et al. (1970) reported

an inhibition of the tumorigenic action of 2-acetylaminofluorene in rats co-treated with nitrite.

Nitrate, thiocyanate, and perchlorate are dose-dependent competitive inhibitors of the sodium-iodide

symporter (NIS), which mediates the uptake of iodine by the thyroid (De Groef et al. 2006).

Overexposure to any one of these competitive inhibitors could decrease iodine uptake and result in

thyroid dysfunction; this effect could be more severe during exposures to combinations of these

substances (and possibly other NIS competitive inhibitors).

3.10 POPULATIONS THAT ARE UNUSUALLY SUSCEPTIBLE

A susceptible population will exhibit a different or enhanced response to nitrate and nitrite than will most

persons exposed to the same level of nitrate and nitrite in the environment. Factors involved with

increased susceptibility may include genetic makeup, age, health and nutritional status, and exposure to

other toxic substances (e.g., cigarette smoke). These parameters result in reduced detoxification or

excretion of nitrate and nitrite, or compromised function of organs affected by nitrate and nitrite.

Populations who are at greater risk due to their unusually high exposure to nitrate and nitrite are discussed

in Section 6.7, Populations with Potentially High Exposures.

Infants 1–6 months of age appear to be particularly sensitive to nitrite-induced methemoglobinemia

following ingestion of formula prepared from drinking water containing elevated levels of nitrate (see

Section 3.7 for detailed discussion of biological factors that may be responsible for increased sensitivity

of infants). Infants with gastroenteritis may be at increased risk for nitrite-induced methemoglobinemia,

although nitrite and nitrate generation from oxidation of endogenous nitric oxide produced under

inflammatory conditions may be a major contributory factor (Avery 1999). Individuals with higher-than-

normal gastric pH (e.g., achlorhydria, a condition whereby gastric acid production is low or absent;

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individuals taking antacids) may be at increased risk of methemoglobinemia if the gastric environment

supports survival of nitrate-reducing bacteria.

Some epidemiological studies provide suggestive evidence of associations between exposure to nitrates in

drinking water and spontaneous abortions, intrauterine growth restriction, and selected birth defects (e.g.,

Brender et al. 2013; Bukowski et al. 2001; CDC 1996; Dorsch et al. 1984; Schmitz 1961; Tabacova et al.

1997, 1998). Results from these studies suggest that the pregnant mother and her developing fetus might

be particularly susceptible to nitrate/nitrite toxicity. However, estimates of nitrate intakes were typically

based on measurements of nitrate levels in drinking water sources at selected time points and self-

reported estimates of water consumption. Furthermore, possible confounding by other potential toxicants

was not evaluated and studies did not typically account for dietary nitrate or nitrite.

Other factors that may contribute to increased risk of methemoglobinemia include glucose-6-phosphate

dehydrogenase deficiency (which can result in decreased numbers of red blood cells); deficiency in

NADH-dependent methemoglobin reductase (the major enzyme responsible for the reduction of

methemoglobin to normal hemoglobin); diseases such as anemia, cardiovascular disease, lung disease,

and sepsis; and abnormal hemoglobin species including carboxyhemoglobin, sulfhemoglobin, and sickle

hemoglobin. Individuals consuming diets deficient in selected antioxidants (e.g., vitamin C, vitamin E)

might be at increased risk of cancer associated with the production of potentially carcinogenic N-nitroso

compounds (WHO 2011b).

3.11 METHODS FOR REDUCING TOXIC EFFECTS

This section will describe clinical practice and research concerning methods for reducing toxic effects of

exposure to nitrate and nitrite. Because some of the treatments discussed may be experimental and

unproven, this section should not be used as a guide for treatment of exposures to nitrate and nitrite.

When specific exposures have occurred, poison control centers, board certified medical toxicologists,

board-certified occupational medicine physicians and/or other medical specialists with expertise and

experience treating patients overexposed to nitrate and nitrite can be consulted for medical advice. The

following texts provide specific information about treatment following exposures to nitrate and nitrite:

Barclay PJ. 1998. Nitrates and nitrites. In: Viccellio P, ed. Emergency toxicology. 2

nd

ed.

Philadelphia, PA: Lippincott-Raven Publishers, 315-323.

Leikin JB, Paloucek FP, eds. 2008. Poisoning and toxicology handbook. 4th ed. Boca Raton, FL: CRC

Press, 830.

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Seifert SA. 2004. Nitrates and nitrites. In: Dart RC, ed. Medical toxicology. 3

rd

ed. Philadelphia, PA:

Lippincott Williams & Williams, 1174-1180.

Additional relevant information can be found in the front section of this profile under QUICK

REFERENCE FOR HEALTH CARE PROVIDERS.

3.11.1 Reducing Peak Absorption Following Exposure

Ingestion is the most likely route of overexposure to nitrate or nitrite. Nitrate and nitrite bind to activated

charcoal, which may be administered (1 g/kg without cathartic) within 1–2 hours following significant

ingestion (Seifert 2004). Use of mouthwash containing chlorhexidine (an active antibacterial) resulted in

a large decrease in the mean percent reduction of salivary nitrate to nitrite (van Maanen et al. 1996b).

3.11.2 Reducing Body Burden

No information was located regarding methods to reduce the body burden of nitrate or nitrite.

3.11.3 Interfering with the Mechanism of Action for Toxic Effects

Severe methemoglobinemia (methemoglobin levels generally >30% of total hemoglobin) can be reduced

by intravenous administration of methylene blue (1–2 mg/kg) (Barclay 1998; Leikin and Paloucek 2008;

Seifert 2004). Exchange transfusions may be considered for patients who do not respond to methylene

blue (particularly patients with glucose-6-phosphate dehydrogenase deficiency or hemoglobin M), and

patients where methylene blue is contraindicated (e.g., patients on serotonin uptake inhibitors) (ATSDR

2013a; Barclay 1998). In symptomatic patients, 100% oxygen and assisted ventilation should be

considered; seizures can be treated with oxygen and benzodiazepines, followed by phenobarbital (Seifert

2004). Hyperbaric oxygen therapy may be of some benefit, but has not been demonstrated in controlled

studies (Leikin and Paloucek 2008; Seifert 2004). Management of nitrite-induced hypotension involves

placement of the patient in Trendelenburg position, administration of intravenous isotonic fluids at 10–

20 mL/kg bolus and as required thereafter, and pressors such as dopamine or norepinephrine, as needed

(Seifert 2004).

In several rat studies, tumorigenicity associated with concurrent exposure to nitrite and various amino

compounds was modulated by coexposure to selected antioxidants such as ascorbic acid, catechol,

3-methoxycatechol, tert-butylhydroquinone, α-tocopherol, and propyl gallate (Chan and Fong 1977;

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Mirvish et al. 1976, 1983; Miyauchi et al. 2002; Mohktar et al. 1988; Yada et al. 2002; Yoshida et al.

1994); thioproline (which may serve as a nitrite scavenger when nitrosated to nitrosothioproline) (Tahira

et al. 1988); or soy bean (Mokhtar et al. 1988).

3.12 ADEQUACY OF THE DATABASE

Section 104(I)(5) of CERCLA, as amended, directs the Administrator of ATSDR (in consultation with the

Administrator of EPA and agencies and programs of the Public Health Service) to assess whether

adequate information on the health effects of nitrate and nitrite is available. Where adequate information

is not available, ATSDR, in conjunction with the National Toxicology Program (NTP), is required to

assure the initiation of a program of research designed to determine the adverse health effects (and

techniques for developing methods to determine such health effects) of nitrate and nitrite.

The following categories of possible data needs have been identified by a joint team of scientists from

ATSDR, NTP, and EPA. They are defined as substance-specific informational needs that if met would

reduce the uncertainties of human health risk assessment. This definition should not be interpreted to

mean that all data needs discussed in this section must be filled. In the future, the identified data needs

will be evaluated and prioritized, and a substance-specific research agenda will be proposed.

3.12.1 Existing Information on Health Effects of Nitrate and Nitrite

The existing data on health effects of inhalation, oral, and dermal exposure of humans and animals to

nitrate and nitrite are summarized in Figures 3-5 and 3-6. The purpose of this figure is to illustrate the

existing information concerning the health effects of nitrate and nitrite. Each dot in the figure indicates

that one or more studies provide information associated with that particular effect. The dot does not

necessarily imply anything about the quality of the study or studies, nor should missing information in

this figure be interpreted as a “data need”. A data need, as defined in ATSDR’s Decision Guide for

Identifying Substance-Specific Data Needs Related to Toxicological Profiles (Agency for Toxic

Substances and Disease Registry 1989), is substance-specific information necessary to conduct

comprehensive public health assessments. Generally, ATSDR defines a data gap more broadly as any

substance-specific information missing from the scientific literature.

135 NITRATE AND NITRITE

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Figure 3-5. Existing Information on Health Effects of Nitrate

Death

Acute

Intermediate

Chronic

Immunologic/Lymphoretic

Neurologic

Reproductive

Developmental

Genotoxic

Cancer

Syste mic

Inhalation

Oral

Dermal

Human

Death

Acute

Intermediate

Chronic

Immunologic/Lymphoretic

Neurologic

Reproductive

Developmental

Genotoxic

Cancer

Syste mic

Inhalation

Oral

Dermal

Animal

Existing Studies

136 NITRATE AND NITRITE

3. HEALTH EFFECTS

Figure 3-6. Existing Information on Health Effects of Nitrite

Death

Acute

Intermediate

Chronic

Immunologic/Lymphoretic

Neurologic

Reproductive

Developmental

Genotoxic

Cancer

Syste mic

Inhalation

Oral

Dermal

Human

Death

Acute

Intermediate

Chronic

Immunologic/Lymphoretic

Neurologic

Reproductive

Developmental

Genotoxic

Cancer

Syste mic

Inhalation

Oral

Dermal

Animal

Existing Studies

137 NITRATE AND NITRITE

3. HEALTH EFFECTS

3.12.2 Identification of Data Needs

Acute-Duration Exposure. No information was located regarding the effects of acute-duration

inhalation exposure to nitrate or nitrite in humans. Available information in laboratory animals is limited.

RTECS (2014) lists a rat 4-hour LC

50

of 5.5 mg/m

3

(1.95 ppm) for sodium nitrite and a rat 2-hour LC

50

of

85 mg/m

3

(24.42 ppm) for potassium nitrite. There was no evidence of exposure-related pulmonary or

cardiac effects in anesthetized dogs exposed at up to 10 mg sodium nitrate/m

3

(2.88 ppm) for 7.5 minutes

or anesthetized dogs or conscious sheep exposed at 5 mg sodium nitrate/m

3

(1.44 ppm) for 4 hours.

Additional information regarding the effects of acute-duration inhalation exposure to nitrate or nitrite is

not considered necessary because the general population is not likely to be exposed to airborne nitrate or

nitrite concentrations at levels that might cause adverse health effects.

Refer to the section titled “Epidemiological and Human Dosimetry Studies” for a summary of available

information regarding noncancer effects in humans following oral exposure to nitrate or nitrite.

Among laboratory animals, acute oral LD

50

values range from 1,267 to 3,750 mg/kg for selected nitrate

salts (RTECS 2014) and from 150 to 200 mg/kg for selected nitrite salts (Imaizumi et al. 1980; RTECS

2014; Sheehy and Way 1974). Imaizumi et al. (1980) administered aqueous sodium nitrite to fasted

Sprague-Dawley rats by gavage and observed dose-related increased methemoglobin levels. Additional

studies regarding the effects of acute-duration oral exposure of laboratory animals to nitrate or nitrite are

not considered necessary, in part due to interspecies differences in kinetics of the nitrate-nitrite-nitric

oxide pathway.

No information was located regarding health effects in humans or animals following acute-duration

dermal exposure to nitrate or nitrite. Information regarding the effects of acute-duration dermal exposure

to nitrate or nitrite is not considered necessary because the general population is not likely to be dermally-

exposed to nitrate or nitrite concentrations at levels that might cause adverse health effects.

Intermediate-Duration Exposure. No information was located regarding the effects of

intermediate-duration inhalation exposure to nitrate or nitrite in humans or animals. The general

population is not likely to be exposed to airborne nitrate or nitrite concentrations at levels that might

cause adverse health effects.

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Refer to the section titled “Epidemiological and Human Dosimetry Studies” for a summary of available

information regarding noncancer effects in humans following oral exposure to nitrate or nitrite.

Epithelial hyperplasia was noted in the forestomach of mice provided sodium nitrite in the drinking water

for 14 weeks (NTP 2001). Another study found no evidence of treatment-related forestomach lesions in

male rats provided sodium nitrite in the drinking water for 35 weeks (Kawabe et al. 1994). Increased

methemoglobin levels and other evidence of hematological effects have been reported in laboratory

animals administered sodium nitrite or potassium nitrite orally for intermediate-duration time periods

(Behroozi et al. 1972; Chow et al. 1980; Grant and Butler 1989; Imaizumi et al. 1980; NTP 2001; Shuval

and Gruener 1972; Til et al. 1988, 1997). Several animal studies found no indications of sodium nitrite-

induced effects on liver function or histopathology (Asahina et al. 1971; Lijinsky and Greenblatt 1972;

Lin and Ho 1992; Shuval and Gruener 1972; van Logten et al. 1972). El-Wakf et al. (2008) reported

significantly increased urinary levels of urea and creatinine in male rats provided sodium nitrate in the

drinking water for 4 months. Sodium or potassium nitrate-induced effects on the endocrine system of

laboratory animals have been reported by several groups of investigators; effects include decreased serum

thyroidal iodine uptake, decreased serum thyroid hormone levels, increased thyroid weight, and follicular

hyperplasia (El-Wakf et al. 2008; Eskiocak et al. 2005; Mukhopadhyay et al. 2005; Zaki et al. 2004). Til

et al. (1988, 1997) observed adrenal gland hypertrophy in rats administered potassium nitrite in the

drinking water for 13 weeks; results of a subsequent study indicated that this effect was a physiological

adaptation to repeated episodes of hypotension caused by nitrite (RIVM 1996). Depressed body weight

and/or body weight gain were observed in some laboratory animals receiving nitrate or nitrite from the

drinking water for intermediate exposure durations (El-Wakf et al. 2008; Maekawa et al. 1982; Zaki et al.

2004). Intermediate-duration oral exposure to sodium nitrite in the drinking water of laboratory animals

has been associated with neurological effects such as abnormalities in EEGs (Behroozi et al. 1972),

increased aggressive behavior (Gruener 1974), and reduced motor activity (Shuval and Gruener 1972).

Available intermediate-duration oral studies in laboratory animals adequately characterize nitrate- and

nitrite-induced effects; additional animal studies do not appear necessary.

No information was located regarding health effects in humans or animals following intermediate-

duration dermal exposure to nitrate or nitrite. Information regarding the effects of intermediate-duration

dermal exposure to nitrate or nitrite is not considered necessary because the general population is not

likely to be dermally-exposed to nitrate or nitrite concentrations at levels that might cause adverse health

effects.

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Chronic-Duration Exposure and Cancer. Information regarding the effects of chronic-duration

inhalation exposure is limited. A cohort mortality study of male workers involved in the manufacture of

nitrate fertilizer for at least 1 year between 1946 and 1981 found no evidence of associations between

exposure to nitrate dusts and death from respiratory or circulatory diseases (Al-Dabbagh et al. 1986).

Among workers described as having been heavily exposed to nitrate dust, slight excesses were noted for

death from lung cancer and death from all malignant neoplasms, but not for cancers of the esophagus,

stomach, or bladder. After categorizing the heavily-exposed workers by duration of exposure and time

since first exposure, excess death from lung cancer was noted for those exposed for ≥10 years, with a lag

time of ≥20 years since first exposure. The study authors indicated that they were unable to adjust for

smoking. In a census-based mortality study of workers involved in production of nitrate fertilizer, there

was no evidence of associations between exposure to nitrate dust and death from circulatory diseases;

slight excesses were noted for deaths from lung cancer and death from all malignant neoplasms, but not

for cancers of the esophagus, stomach, or bladder (Fraser et al. 1982, 1989). No significant increased risk

for cancer at any site was observed at 7-year follow-up evaluation. In yet another cohort of workers at a

nitrate fertilizer production facility (Hagmar et al. 1991), death from prostate cancer was in excess;

however, risk of prostate cancer within this cohort was not enhanced following application of a ≥10-year

latency period, and there was no significant increase in death from tumors of the lips, oral cavity,

pharynx, salivary glands, gastrointestinal tract, stomach, respiratory tract, lung, urinary bladder, blood, or

all sites combined. The general population is not likely to be exposed to airborne nitrate or nitrite

concentrations at levels that might cause adverse health effects.

Refer to the section titled “Epidemiological and Human Dosimetry Studies” for a summary of available

information regarding noncancer effects in humans following oral exposure to nitrate or nitrite.

Numerous case-control and cohort studies regarding the carcinogenicity of ingested nitrate and nitrite in

humans have been reported (IARC 2010). Many ecological studies have also been reported; however,

interpretation of outcomes of these studies is more uncertain because of various factors that contribute to

ecologic bias (group-based associations between exposure and cancer outcomes may not apply to

individuals). In general, outcomes of case-control and cohort studies have found no or weak associations

between exposure to nitrate and cancer in humans, with stronger associations with exposures to nitrite or

intake of high nitrite foods such as cured meat (Aschebrook et al. 2013; DellaValle et al. 2013; IARC

2010; Inoue-Choi et al. 2012). Mechanistically, this outcome is consistent with nitrite and being a

reactive intermediate in the cancer mode of action of nitrate. This is further supported by studies that

found interactions between cancer risk attributed to nitrite and exposure to antioxidants (IARC 2010;

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Inoue-Choi et al. 2012; Kim et al. 2007; Yang et al. 2010). Uncertainties in estimates of cancer risk from

exposure to nitrate or nitrite include those typical of epidemiological studies in general: uncertainties in

estimation of exposure (e.g., estimating long-term dietary intakes from food frequency questionnaires or

levels in PWS), exposure misclassification of individual outcomes (e.g., assigning group-level exposure

estimates to individuals), and adequacy of controlling for confounders (e.g., other factors contributing to

the cancer). One potentially important class of confounders is antioxidants that can interfere with

nitrosation of dietary amines, and thereby the mode of carcinogenicity of nitrite, and may also interfere

with other carcinogenic processes that involve reactive intermediates.

The strongest and most consistent evidence for carcinogenicity of nitrite is from studies of gastrointestinal

cancers and, in particular, gastric cancer (Buiatti et al. 1990; Engel et al. 2003; La Vecchia et al. 1994,

1997; Mayne et al. 2001; Palli et al. 2001; Risch et al. 1985; Rogers et al. 1995; Ward et al. 2007, 2008).

Results have been mixed for other types of cancer. Some case-control or cohort studies found

associations between exposure to nitrite (or foods high in nitrite such as cured meat) and brain cancer in

children and adults (Blowers et al. 1997; Giles et al. 1994, Huncharek and Kupelnick 2004; Huncharek et

al. 2003; Lee et al. 1997; Pogoda and Preston-Martin 2001a, 2001b; Preston-Martin et al. 1996; Mueller

et al. 2004), breast cancer (Inoue-Choi et al. 2012; Yang et al. 2010), kidney cancer (DellaValle et al.

2013; Ward et al. 2007; Wilkens et al. 1996), testicular cancer (Moller 1997), and non-Hodgkin’s

lymphoma (Ward et al. 2006). Of these studies, the highest risks were reported for brain cancers. Two

case-control studies found elevated relative risk of brain cancer in children in association with maternal

nitrite intake (Mueller et al. 2004; Pogoda and Preston-Martin 2001a, 2001b; Preston-Martin et al. 1996).

In general, case-control and cohort studies of cancers of larynx, liver, lung, mouth, pancreas, and pharynx

have found no consistent associations with exposures to nitrate or nitrite (IARC 2010).

The potential carcinogenicity of nitrate has been investigated in several animal studies that employed the

oral exposure route. Studies in which negative results were reported include MCR-derived rats provided

sodium nitrate in the drinking water for 84 weeks (Lijinsky et al. 1973a), male white rats provided sodium

nitrate in the drinking water for 273 days (Pliss and Frolov 1991), strain A male mice provided sodium

nitrate in the drinking water for 25 weeks (Greenblatt and Mirvish 1973), female NMRI mice provided

calcium nitrate in the drinking water for 18 months (Mascher and Marth 1993), Fischer 344 rats fed diet

containing sodium nitrate for 2 years (Maekawa et al. 1982), and ICR mice fed diets containing sodium

nitrate for 2 years (IARC 2010). In the study of Pliss and Frolov (1991), some groups of male rats were

treated with drinking water containing BBNA (an inducer of urinary bladder cancer in laboratory animals)

for 30 days, either alone or followed by sodium nitrate in the drinking water for 273 days. The group

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treated with BBNA followed by sodium nitrate exhibited significantly increased incidence of urinary

bladder carcinoma. These results indicate that sodium nitrate promoted BBNA-induced bladder tumors.

The potential carcinogenicity of ingested nitrite has been investigated in numerous animal studies. Nitrite

treatment alone was not associated with tumor incidences in most studies (Börzsönyi et al. 1978; Hawkes

et al. 1992; Inai et al. 1979; Lijinsky 1984a, 1984b; Lijinsky et al. 1983; Maekawa et al. 1982; NTP

2001). Significantly increased incidences of forestomach squamous papillomas were reported for male

and female MRC Wistar rats provided drinking water to which sodium nitrite was added for life (Mirvish

et al. 1980). Dose-related decreases in time of onset and incidence of lymphomas, mononuclear cell

leukemia, and testicular interstitial-cell tumors were reported for male and female F344 rats administered

reduced-protein diet to which sodium nitrite was added for up to 115 weeks (Grant and Butler 1989). In a

96-week study, Iurchenko et al. (1986) reported a significantly increased incidence of benign liver tumors

among male CBA mice receiving sodium nitrite from the drinking water at an author-estimated total dose

of 1,600 mg sodium nitrite/mouse; however, there was no apparent sodium nitrite treatment-related effect

at a higher estimated dose (2,000 mg sodium nitrite/mouse). Increased incidences of total tumors and

lymphoreticular tumors were reported in rats fed diet to which sodium nitrite was added; the results were

reported for F1 and F2 offspring that had been exposed via their mothers during gestation and lactation

and directly from the diet thereafter (Shank and Newberne 1976). A positive trend for incidences of

forestomach squamous cell papilloma or carcinoma (combined) in female B6C3F1 mice administered

sodium nitrite in the drinking water for 2 years was considered to provide "equivocal evidence of

carcinogenic activity" of sodium nitrite (NTP 2001). In a 26-month study of male and female Sprague-

Dawley rats provided drinking water to which sodium nitrite was added, the study author reported

increased incidence of lymphomas, but not other types of tumors (Newberne 1979); however, IARC

(2010) and NTP (2001) noted that a working group sponsored by the U.S. FDA reevaluated the histology

and did not confirm the results of Newberne (1979). IARC (2010) reported that the working group

considered the incidences of lymphomas to be similar to those arising spontaneously in Sprague-Dawley

rats.

The potential carcinogenicity of combined exposure to sodium nitrite and selected nitrosatable substances

(oral exposures via combinations of drinking water, diet, and/or gavage dosing) has been well-studied in

laboratory animals. Many of the studies included sodium nitrite-only treatment groups for which there

was no evidence of sodium-nitrite induced carcinogenicity (Anderson et al. 1985; Börzsönyi and Pintér

1977; Börzsönyi et al. 1976; Greenblatt and Lijinsky 1972, 1974; Greenblatt and Mirvish 1973;

Greenblatt et al. 1971, 1973; Hirose et al. 2002; Ivankovic 1979; Ivankovic and Preussman 1970; Kitano

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et al. 1997; Murthy et al. 1979; Lijinsky 1984a, 1984b; Lijinsky and Reuber 1980; Mirvish et al. 1972;

Miyauchi et al. 2002; Rijhsinghani et al. 1982; Scheunig et al. 1979; Taylor and Lijinsky 1975a, 1975b;

van Logten et al. 1972; Yada et al. 2002; Yoshida et al. 1993, 1994). However, Lijinsky et al. (1983)

reported significantly increased incidences of hepatocellular neoplasms in female (but not male) F344 rats

administered diet to which sodium nitrite was added for 2 years.

Significantly increased incidences of selected tumor types were observed in some studies of laboratory

animals that employed coexposure to various amino compounds and sodium nitrite (Anderson et al. 1985;

Aoyagi et al. 1980; Börzsönyi and Pintér 1977; Börzsönyi et al. 1976, 1978; Chan and Fong 1977;

Greenblatt and Mirvish 1973; Greenblatt et al. 1971; Hirose et al. 1990; Iurchenko et al. 1986; Ivankovic

1979; Ivankovic and Preussmann 1970; Kawabe et al. 1994; Matsukura et al. 1977; Murthy 1979;

Lijinsky 1984a, 1984b; Lijinsky and Reuber 1980; Lijinsky and Taylor 1977; Lijinsky et al. 1973b; Lin

and Ho 1992; Maekawa et al. 1977; Mirvish et al. 1972, 1976, 1980; Miyauchi et al. 2002; Mokhtar et al.

1988; Newberne and Shank 1973; Nishiyama et al. 1998; Nixon et al. 1979; Oka et al. 1974; Olsen et al.

1984; Rijhsinghani et al. 1982; Rustia and Shubik 1974; Scheunig et al. 1979; Shank and Newberne 1976;

Tahira et al. 1988; Taylor and Lijinsky 1975a, 1975b; Weisburger et al. 1980; Xiang et al. 1995; Yada et

al. 2002; Yamamoto et al. 1989; Yoshida et al. 1993, 1994). These results were typically attributed to in

vivo nitrosation of amines by nitrite to produce carcinogenic N-nitrosoamines; some of the studies did not

include sodium nitrite-only treatment groups.

Based on available human data, IARC (2010) determined that there is inadequate evidence for the

carcinogenicity of nitrate in food or drinking water and limited evidence for the carcinogenicity of nitrite

in food (based on association with increased incidence of stomach cancer). Evaluation of available

animal data by IARC (2010) resulted in the determination that there is inadequate evidence for the

carcinogenicity of nitrate, limited evidence for the carcinogenicity of nitrite per se, and sufficient

evidence for the carcinogenicity of nitrite in combination with amines or amides. The overall conclusions

of IARC (2010) were that “ingested nitrate and nitrite under conditions that result in endogenous

nitrosation is probably carcinogenic to humans (Group 2A).” IARC (2010) noted that: (1) the

endogenous nitrogen cycle in humans includes interconversion of nitrate and nitrite; (2) nitrite-derived

nitrosating agents produced in the acid stomach environment can react with nitrosating compounds such

as secondary amines and amides to generate N-nitroso compounds; (3) nitrosating conditions are

enhanced upon ingestion of additional nitrate, nitrite, or nitrosatable compounds; and (4) some N-nitroso

compounds are known carcinogens. The U.S. EPA IRIS (2002) does not include a carcinogenicity

evaluation for nitrate or nitrite.

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No information was located regarding health effects in humans or animals following chronic-duration

dermal exposure to nitrate or nitrite. Information regarding the effects of chronic-duration dermal

exposure to nitrate or nitrite is not considered necessary because the general population is not likely to be

dermally-exposed to nitrate or nitrite concentrations at levels that might cause adverse health effects.

Genotoxicity. Limited information is available regarding the potential genotoxicity of nitrate in

human studies. One study found no significant association between urinary excretion of nitrate and

frequency of SCEs in peripheral lymphocytes (Kleinjans et al. 1991). In another study, frequency of

HPRT variants in peripheral lymphocytes was associated with nitrate levels in drinking water, urinary and

salivary nitrite levels, and urinary excretion of nitrate and N-nitrosopyrrolidine (van Maanen et al. 1996a).

The results suggest that drinking water with nitrate poses a genetic risk due to the potential formation of

nitrosamines after endogenous reduction of nitrate to nitrite and reaction with amino compounds. Tsezou

et al. (1996) reported a significant increase in chromatid and chromosome breaks in children exposed to

nitrate in drinking water.

A limited number of studies have examined the in vivo genotoxicity of nitrate in laboratory animals;

results were negative for frequency of micronuclei, chromosomal aberrations, morphological or malignant

cell transformation, or drug-resistant mutations in embryonic cells in one study (Inui et al. 1979),

inhibition of testicular DNA synthesis in another study (Friedman and Staub 1976), and chromosomal

aberrations in bone marrow cells in a 2-day study (Luca et al. 1985). However, daily administration of

sodium nitrate for 2 weeks resulted in significant dose-dependent increase in chromosomal aberrations in

bone marrow cells (Luca et al. 1985). Gavage administration of 706.6 mg/kg/day sodium nitrate for

2 days to male Swiss mice showed induction of chromosomal aberrations; however, this effect was not

observed at a much higher dose (Luca et al. 1985). Evaluation of micronuclei in mice treated daily for

2 weeks showed significant increases at the low concentrations tested (78.5 and 235.5 mg/kg/day sodium

nitrate), but not at 706.6 or 2,120 mg/kg/day; the investigators attributed the result to possible induction

of cytotoxic effects (Luca et al. 1985).

Neither potassium nitrate, sodium nitrate, nor lanthanum nitrate hexahydrate were mutagenic to multiple

strains of S. typhimurium either with or without metabolic activation (Ishidate et al. 1984; Zeiger et al.

1992). Tests for chromosomal aberrations in Chinese hamster fibroblast cells were positive for sodium

nitrate, but negative for potassium nitrate (Ishidate et al. 1984). IARC (2010) noted that since sodium

chlorite also yielded positive results in the same assay, the chromosomal aberrations induced by sodium

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nitrate could have been due to the high osmotic pressure and sodium ion concentration. Ammonium

nitrate did not induce chromosomal aberrations in Chinese hamster ovary cells with or without metabolic

activation (Kim et al. 2011).

In vivo tests for nitrite conducted in mammalian cells yielded negative results for chromosomal

aberrations, SCEs, DNA repair, and cell transformations (Inoue et al. 1985; Ishidate et al. 1984; Lynch et

al. 1983; Tsuda and Kato 1977; Tsuda et al. 1973, 1981). Numerous studies have examined the in vitro

genotoxicity of nitrite; more positive results than negative results were found in tests of gene mutations in

prokaryotic organisms, but it is difficult to draw a firm conclusion (Andrews et al. 1980, 1984;

Balimandawa et al. 1994; Brams et al. 1987; De Flora 1981, De Flora et al. 1984; Ehrenberg et al. 1980;

Ishidate et al. 1984; Törnqvist et al. 1983; Zeiger et al. 1992). However, it appears that the addition of

metabolic activation systems to the incubation mixtures did not make a difference in the results. This

would indicate that nitrite could be a direct mutagenic chemical.

Additional in vivo and in vitro studies could be designed to further assess the genotoxicity of nitrate and

nitrite.

Reproductive Toxicity. Refer to the section titled “Developmental Toxicity” for information

regarding results of case-control studies that evaluated reproductive/developmental end points.

Several animal studies included evaluation of selected reproductive end points. Sleight and Atallah

(1968) reported death and reduced litter production among female guinea pigs provided potassium nitrate

in the drinking water for up to 204 days of cohabitation at a concentration resulting in estimated intake of

4,972 mg nitrate/kg/day. Reduced litter production was the likely result of maternal toxicity rather than

reproductive toxicity per se. Sleight and Atallah (1968) also reported decreases in number of litters and

live births and histopathologic lesions in reproductive organs (placenta, uterus, and cervix) of guinea pigs

administered sodium nitrite in the drinking water. No treatment-related reproductive effects were seen in

female Wistar rats provided sodium nitrite in the food throughout the production of two litters (Hugot et

al. 1980) or in breeding dogs provided sodium nitrate in the drinking water for 1 year (Kelley et al. 1974).

NTP (2001) reported degeneration of the testis in male mice provided sodium nitrite in the drinking water

for 14 weeks, and significantly increased estrous cycles in similarly-treated female mice. Among

similarly-treated male and female rats, the males exhibited decreased sperm motility.

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A multi-generation reproductive toxicity study in laboratory animals could be designed to more

comprehensively assess the reproductive toxicity potential of ingested nitrate and nitrite.

Developmental Toxicity. A number of studies evaluated possible associations between

developmental end points and exposure to nitrate in humans. The results provide some evidence of

nitrate-related developmental effects. The results are not adequate for quantitative risk assessment

because (1) estimations of nitrate intakes were typically based on measurements of nitrate levels in

drinking water sources at selected time points and self-reported estimates of water consumption;

(2) possible confounding by other potential toxicants was not evaluated; and (3) most studies did not

account for dietary nitrate or nitrite intake, which is typically the major source of ingested nitrate and

nitrite. Some studies reported significant associations between selected developmental end points and

nitrate in drinking water sources (Brender et al. 2013; Croen et al. 2001; Dorsch et al. 1984; Scragg et al.

1982). One study reported increased risk of intercalary limb defect associated with estimated total nitrite

intake (Huber et al. 2013). Other studies found no evidence of associations between nitrate and risk of

developmental effects (Arbuckle et al. 1988; Aschengrau et al. 1989, 1993; Brender et al. 2004;

Cedergren et al. 2002; Ericson et al. 1988; Huber et al. 2013; Super et al. 1981). Tabacova et al. (1997,

1998) evaluated maternal health among pregnant women and their infants who lived near an ammonium

nitrate fertilizer plant. Nitrogen oxides in the air averaged 23.1 µg/m

3

with short-term peak levels as high

as 238.5; nitrate concentrations in the public drinking water supply measured 8–54 mg/L and nitrate

levels in private wells measured as much as 13–400 mg/L. Results indicated that both maternal and cord

blood methemoglobin levels were higher in cases of abnormal birth outcome.

Developmental end points have been assessed in some animal studies. Some studies found no indication

of nitrite treatment-related developmental toxicity (Hugot et al. 1980; Khera 1982; Shimada 1989). One

study reported increased fetal hepatic erythropoiesis, which was thought to have been a response to

nitrite-induced fetal methemoglobinemia (Globus and Samuel 1978). Significantly impaired auditory and

visual discrimination learning behavior and retention of passive avoidance responses (Nyakas et al. 1990),

and delay in cholinergic and serotonergic fiber outgrowth in cortical target areas of the brain (Nyakas et

al. 1994), presumably due to nitrite-induced hypoxia, were reported in offspring of Wistar rats provided

sodium nitrite in the drinking water. Shuval and Gruener (1972) reported decreases in postpartum

survival and pup body weight during 3 weeks postpartum following addition of sodium nitrite to the

drinking water of pregnant rats for 6 weeks; no treatment-related effects were observed regarding group

litter sizes or pup birth weights. Increased pup mortality, depressed preweaning pup body weight, and

delayed swimming development were observed in offspring of male and female rats provided sodium

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nitrite in the diet (Vorhees et al. 1984). There were no treatment-related effects on preweaning behavior

(surface righting, pivoting, negative geotaxis, or auditory startle) and no effects on postweaning survival,

body weight, or most behavioral indices among pups from dams exposed to sodium nitrite in the diet.

Additional human data are needed to comprehensively assess the developmental toxicity potential of

ingested nitrate and nitrite.

Immunotoxicity. No information was located regarding immunological or lymphoreticular effects in

humans or animals following exposure to nitrate or nitrite by any route. An animal study could be

designed to assess the potential immunotoxicity of ingested nitrate and nitrite.

Neurotoxicity. No information was located regarding the neurotoxicity of nitrate in humans or

animals. Ingestion of nitrite has been associated with severe methemoglobinemia in adults and children;

in many of these cases, clinical signs included dizziness, loss of consciousness, and/or convulsions (CDC

1997, 2002; Gautami et al. 1995; Greenberg et al. 1945; Sevier and Berbatis 1976; Ten Brink et al. 1982).

These cases were the result of consumption of food or drink that contained unusually high levels of nitrite

via contamination, inadvertent use of sodium nitrite instead of table salt, or ingestion of a single sodium

nitrite tablet (667 mg nitrite). Headache was induced in a male subject following consumption of a 10 mg

sodium nitrite solution (Henderson and Raskin 1972). In a study designed to evaluate the oral

bioavailability of sodium nitrite in healthy volunteers, headache was reported after ingestion of nitrite at

doses as low as approximately 1.5–1.8 mg nitrite/kg (Kortboyer et al. 1997b).

Abnormalities in EEGs were reported in male albino rats provided sodium nitrite in the drinking water for

2 months at concentrations resulting in ingestion of ≥9.38 mg nitrite/kg/day (Behroozi et al. 1972). At the

highest dose (187.6 mg nitrite/kg/day), rats exhibited clinical signs of sedation and became motionless

during periods of electrical outbursts. Increased aggressive behavior was observed in male C57B1 mice

provided sodium nitrite in the drinking water at 1,000 mg/L for up to 13 weeks postweaning (Gruener

1974). The mice had also been exposed via their parents during mating and via their mothers during

gestation and lactation. Significantly reduced motor activity was reported in male mice provided sodium

nitrite in the drinking water (Shuval and Gruener 1972).

The nervous system is not expected to be a particularly sensitive target of nitrate toxicity; available data

for nitrite appear adequate for the purpose of hazard identification. Additional neurotoxicity studies do

not appear necessary.

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Epidemiological and Human Dosimetry Studies. Oral exposure to nitrate and nitrite is

ubiquitous because nitrate and nitrite are part of the normal diet. Elevated methemoglobin levels are

commonly associated with levels of nitrate in drinking water sources or ingestion of nitrate; clinical signs

of methemoglobinemia may be observed at sufficiently high nitrate levels, particularly among newborn

infants (e.g., Bosch et al. 1950; Chapin 1947; Comly 1987; Craun et al. 1981; Donahoe 1949; Fan and

Steinberg 1996; Fan et al. 1987; Faucett and Miller 1946; Ferrant 1946; Gruener and Toeplitz 1975;

Gupta et al. 1999; Johnson et al. 1987; Jones et al. 1973; Medovy 1948; Miller 1971; Robertson and

Riddell 1949; Sadeq et al. 2008; Shuval and Gruener 1972; Simon et al. 1964; Stafford 1947; Super et al.

1981; Walton 1951; Winton et al. 1971; Zeman et al. 2002). Although oral exposure to nitrate has been

associated with methemoglobinemia in bottle-fed infants receiving drinking water containing measurable

levels of nitrate, available studies are limited by lack of accounting for substances in the drinking water

(e.g., bacteria) that may have contributed to the methemoglobinemia and the fact that many of the infants

exhibited gastroenteritis, which in itself can trigger increased methemoglobin levels. Therefore,

additional information regarding the effects of oral exposure of infants to nitrate would serve to reduce

uncertainty as to the role of nitrate in the observed methemoglobinemia cases reported in the literature.

Available human data provide suggestive evidence that elevated levels of nitrate in drinking water and/or

nitrate-rich diets may be associated with signs of thyroid dysfunction (Aschebrook-Kilfoy et al. 2012;

Gatseva and Argirova 2008; Rádiková et al. 2008; Tajtáková et al. 2006; Ward et al. 2010). However,

limitations of these studies include lack of individual dose-response data, quantification of iodine intake,

and control for other potential substances that may affect the thyroid; one study relied on self-reported

thyroid status and self-reported dietary nitrate intake. Additional studies should focus on possible

associations between nitrate and/or nitrite and thyroid status.

Possible associations between nitrate and/or nitrite in drinking water and/or food sources and risk of type

1 diabetes have been investigated in a number of epidemiological studies. Significant associations were

reported in some studies (Dahlquist et al. 1990; Kostraba et al. 1992; Parslow et al. 1997; Virtanen et al.

1994), but not in other studies (Casu et al. 2000; Moltchanova et al. 2004; van Maanen et al. 2000; Zhao

et al. 2001). Limitations of studies include the lack of quantitative dose-response data and the likelihood

of confounding by other potential toxicants. Additional studies should focus on possible associations

between nitrate and/or nitrite and risk of type 1 diabetes.

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Ingestion of nitrite has been associated with severe methemoglobinemia in adults and children (Aquanno

et al. 1981; CDC 1997, 2002; Gautami et al. 1995; Gowans 1990; Greenberg et al. 1945; Kaplan et al.

1990; Ringling et al. 2003; Sevier and Berbatis 1976; Ten Brink et al. 1982; Walley and Flanagan 1987),

typically following consumption of food or drink that contained unusually high levels of nitrite via

contamination, inadvertent use of sodium nitrite instead of table salt, or ingestion of a single sodium

nitrite tablet (667 mg nitrite). Other effects noted in some of these cases include hypotension and/or

tachycardia, abdominal cramps, vomiting, dizziness, loss of consciousness, convulsions, and even death.

In a study designed to evaluate the oral bioavailability of sodium nitrite in healthy volunteers, ingestion of

approximately 1.5–1.8 mg nitrite/kg resulted in increased percent methemoglobin and average heart rate,

and decreased mean arterial blood pressure (Kortboyer et al. 1997b). Higher ingested doses resulted in

more pronounced effects and included nausea and vomiting. Additional information regarding effects of

oral exposure to nitrite at lower dose levels would be useful in determining minimal risk levels for nitrite

toxicity if populations with such exposure characteristics are identified.

Data needs relating to both prenatal and childhood exposures, and developmental effects expressed either

prenatally or during childhood, are discussed in detail in the Developmental Toxicity subsection above.

Biomarkers of Exposure and Effect

Exposure. There are no biomarkers of exposure that are specific to nitrate or nitrite. Although nitrate

and nitrite can be detected in blood, saliva, and urine (mostly nitrate), nitrate and nitrite are also produced

endogenously via the nitrate-nitrite-nitric oxide pathway. Sources for nitrate and nitrite levels in the body

may therefore include not only ingested food and drinking water, but also oxidation of nitric oxide

produced endogenously. Similarly, N-nitroso compounds that may be detected in the blood or urine may

indicate exposure to nitrate or nitrite; however, these compounds may also be products of the endogenous

nitrate-nitrite-nitric oxide pathway.

Effect. Biomarkers of effects from exposure to nitrate or nitrite are not specific to nitrate or nitrite.

Blood methemoglobin level has been used as a biomarker of nitrate and nitrite toxicity; however,

methemoglobinemia may be elicited by other substances such as selected drugs, pesticides, industrial and

commercial products, and medical conditions such as pediatric gastrointestinal infection, sepsis, and

sickle cell crisis (ATSDR 2013a). Methemoglobinemia may also be inherited (genetic conditions that

result in decreased activity of enzymes that reduce methemoglobin or the presence of hemoglobin M).

Urinary levels of various N-nitroso compounds have been measured as an index of nitrosation; however,

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N-nitroso compounds can form via endogenous nitrosation and do not require the intake of nitrate or

nitrite.

Absorption, Distribution, Metabolism, and Excretion. No information was located regarding

the pharmacokinetics of nitrate or nitrite following inhalation or dermal exposure. However, numerous

reviews are available regarding the pharmacokinetics of ingested nitrate and nitrite (Bailey et al. 2012;

Bryan and van Grinsven 2013; IARC 2010; JECFA 2003a, 2003b; Lundberg and Govoni 2004; Lundberg

and Weitzberg 2013; Lundberg et al. 2008, 2009; Weitzberg and Lundberg 2013; Weitzberg et al. 2010;

WHO 2011b). Ingestion is the major source of exposure to nitrate and nitrite. The data adequately

describe the pharmacokinetics of nitrate and nitrite; additional studies do not appear necessary.

A PBPK model (Zeilmaker et al. 1996, 2010b) simulates the kinetics of methemoglobin formation

resulting from gastrointestinal absorption of nitrate in adult humans. The model is adequate for this

purpose; however, the model is not considered adequate for the purpose of simulating the kinetics in

infants. Additional information is needed to adapt the model to infants for the purpose of quantitative risk

assessment.

Comparative Toxicokinetics. Significant differences exist regarding the kinetics of the nitrate-

nitrite-nitric oxide pathway in humans and laboratory animals, thus precluding the usefulness of results

from laboratory animals to evaluate the toxicokinetics of nitrate or nitrite in humans.

Methods for Reducing Toxic Effects. Ingestion is the most likely route of overexposure to nitrate

or nitrite. Methods for reducing peak absorption include oral administration of activated charcoal within

a short period following significant ingestion (Seifert 2004) and use of mouthwash containing

chlorhexidine (an active antibacterial), which may decrease the reduction of salivary nitrate to nitrite (van

Maanen et al. 1996b). No information was located regarding methods to reduce the body burden of

nitrate or nitrite. Adequate data are available regarding methods for reducing nitrate- or nitrite-induced

methemoglobinemia (e.g., Barclay 1998; Leikin and Paloucek 2008; Seifert 2004). In several rat studies,

tumorigenicity associated with concurrent exposure to nitrite and various amino compounds was

modulated by coexposure to selected antioxidants such as ascorbic acid, catechol, 3-methoxycatechol,

tert-butylhydroquinone, α-tocopherol, and propyl gallate (Chan and Fong 1977; Mirvish et al. 1976, 1983;

Miyauchi et al. 2002; Mohktar et al. 1988; Yada et al. 2002; Yoshida et al. 1994); thioproline (which may

serve as a nitrite scavenger when nitrosated to nitrosothioproline) (Tahira et al. 1988); or soy bean

(Mokhtar et al. 1988).

150 NITRATE AND NITRITE

3. HEALTH EFFECTS

Children’s Susceptibility. Data needs relating to both prenatal and childhood exposures, and

developmental effects expressed either prenatally or during childhood, are discussed in detail in the

Developmental Toxicity subsection above.

Ingestion of relatively large amounts of nitrate or nitrite can result in methemoglobinemia. The first

6 months of postnatal life is a period of increased susceptibility to methemoglobinemia; possible

contributing factors to this increased susceptibility include a higher pH in the infant stomach, greater

proportion of fetal hemoglobin (which appears to be more readily oxidized to methemoglobin than adult

hemoglobin), and higher concentration of NADH-dependent methemoglobin reductase (an enzyme

involved in the reduction of methemoglobin to hemoglobin). Some investigators have reported

significant associations between nitrate levels in drinking water (or living in areas presumed to have

elevated nitrate levels in drinking water sources) and risk of childhood type 1 diabetes (Dahlquist et al.

1990; Kostraba et al. 1992; Parslow et al. 1997; Virtanen et al. 1994). However, no such relationship was

observed in two other studies (van Maanen et al. 2000; Zhao et al. 2001). Refer to Section 3.2.2.2

(Metabolic Effects) for summaries of these study reports.

Child health data needs relating to exposure are discussed in Section 6.8.1, Identification of Data Needs:

Exposures of Children.

3.12.3 Ongoing Studies

The following ongoing study pertaining to nitrate was identified in National Institutes of Health (NIH)

Research Portfolio Online Reporting Tools (RePORTER 2014): Dr. Paul A Romitti, College of Public

Health, University of Iowa, is evaluating risk of birth defects associated with nitrate in drinking water.

151 NITRATE AND NITRITE

4. CHEMICAL AND PHYSICAL INFORMATION

4.1 CHEMICAL IDENTITY

Information regarding the chemical identity of nitrate and nitrite is provided in Table 4-1 and information

regarding the chemical identity of selected inorganic nitrate and nitrite compounds is provided in

Table 4-2. Information regarding ammonia and urea is provided in Table 4-3.

Inorganic nitrate and nitrite are naturally occurring ionic species that are part of the earth’s nitrogen cycle

(see Figure 5-1). These anions are the products formed via the fixation of nitrogen and oxygen.

Chemical processes, biological processes, and microbial processes in the environment convert nitrogen

compounds to nitrite and nitrate via nitrogen fixation and nitrification. Compounds such as urea are

converted via hydrolysis to ammonia, protonation of ammonia to ammonium (cation), followed by

oxidation of ammonium to form nitrite, and then oxidation to form nitrate. Nitrate and nitrite are not

neutral compounds, but rather the ionic (anionic; negatively charged) portions of compounds, commonly

found in commerce as organic and inorganic salts. As used in this profile, the word “ion” is implied and

not used, unless added for clarity.

Nitrate and nitrite typically exist in the environment as highly water-soluble inorganic salts, often bound

when not solubilized to metal cations such as sodium or potassium. The nitrate ion is the more stable

form as it is chemically unreactive in aqueous solution; however, it may be reduced through biotic

processes with nitrate reductase to the nitrite ion. The nitrite ion is readily oxidized back to the nitrate ion

via Nitrobacter (a genus of proteobacteria), or conversely, the nitrite ion may be reduced to various

compounds (IARC 2010; WHO 2011b).

Under certain conditions, nitrite may be converted to a class of compounds called N-nitrosamines. In

foods, endogenous production of N-nitrosamines occurs when nitrite reacts with secondary amines or

amides. Several factors, including the presence of antioxidants, such as vitamin C, affect the rate of

formation. N-nitrosamines are a class of chemical compounds that have a nitroso (N=O) group bonded to

an amine (-N(R)R’) with a general chemical structure of RN(R’)-N=O (IARC 94).

There is a wide range of both organic and inorganic nitrate and nitrite compounds. Common nitrate and

nitrite salts include potassium nitrate, potassium nitrite, sodium nitrate, sodium nitrite, and ammonium

nitrate; these salts are highly soluble in water, dissociate under environmental conditions, and exist as

152 NITRATE AND NITRITE

4. CHEMICAL AND PHYSICAL INFORMATION

ions (WHO 1978, 2011b). Common inorganic fertilizers that contribute to environmental concentrations

of nitrate and nitrite include ammonia and urea.

4.2 PHYSICAL AND CHEMICAL PROPERTIES

Information regarding the physical and chemical properties of selected inorganic nitrate and nitrite

compounds is provided in Table 4-4 and information regarding the physical and chemical properties of

ammonia and urea is provided in Table 4-5.

153 NITRATE AND NITRITE

4. CHEMICAL AND PHYSICAL INFORMATION

Table 4-1. Chemical Identity of Nitrate and Nitrite Ions

a

Characteristic

Nitrate ion

Nitrite ion

Synonym(s)

Nitrate ion; nitrate(1-); nitrate ion

(NO

3

-

); nitrate ion(1-); nitrato; nitric

acid, ion (1-)

Nitrite ion; nitrite (1-); nitrite anion;

nitrite ion (NO

2

-

); nitrite ion (1-);

nitrogen dioxide(1-); nitrogen

peroxide ion (1-); nitrous acid,

ion (1-)

Registered trade name(s)

No data

Chemical formula

NO

3

-

NO

2

-

Chemical structure

b

O

N

+

O

N

O

Ionic weight

62.005

45.995

Identification numbers:

CAS registry

14797-55-8

14797-65-0

NIOSH RTECS

No data

EPA hazardous waste

No data

DOT/UN/NA/IMDG shipping

c

UN3218; UN1447

No data

HSDB

Not applicable

NCI

No data

EPA Pesticide Chemical

Code

No data

a

All information obtained from IARC (2010), except where noted.

b

HSDB 2007.

c

ChemIDplus 2014.

CAS = Chemical Abstracts Service; DOT/UN/NA/IMDG = Department of Transportation/United Nations/North

America/International Maritime Dangerous Goods Code; EPA = Environmental Protection Agency;

HSDB = Hazardous Substances Data Bank; NCI = National Cancer Institute; NIOSH = National Institute for

Occupational Safety and Health; RTECS = Registry of Toxic Effects of Chemical Substances

154 NITRATE AND NITRITE

4. CHEMICAL AND PHYSICAL INFORMATION

Table 4-2. Chemical Identity of Selected Inorganic Nitrate and Nitrite

Compounds

a

Ammonium

Sodium

Potassium

Characteristic

nitrate

nitrite

nitrate

nitrite

Synonym(s)

Nitric acid,

ammonium salt;

ammonium nitrate

(NH

4

NO

3

); Emulite;

EXP 200; German

saltpeter; Norge

saltpeter; Norway

saltpeter;

Norwegian

saltpeter; Plenco

12203; Varioform I;

ZhVK

Nitric acid,

sodium salt;

Chile saltpeter;

niter; nitric acid

sodium

salt(1:1);

saltpeter; soda

niter; nitrate of

soda

;

cubic

niter; nitratine

Nitrous acid,

sodium salt;

nitrous acid

soda; nitrous

acid sodium

salt (1:1)

Nitric acid,

potassium

salt; niter;

nitre; nitric

acid

potassium salt

(1:1);

saltpeter;

saltpetre;

nitrate of

potash

Nitrous acid,

potassium

salt; Chile

saltpeter;

niter; nitric

acid sodium

salt (1:1);

salpeter; soda

niter

Registered trade

name(s)

No data

Chemical formula

NH

4

NO

3

NaNO

3

NaNO

2

KNO

3

KNO

2

Chemical structure

Trigonal NH

4

+

NO

3

-

Na

+

NO

3

-

Trigonal

Na

+

NO

2

-

Orthorhombic

K

+

NO

3

-

K

+

NO

2

-

N

H

+

N

+

O

Na

+

N

+

O

N

O

Na

+

K

+

N

+

O

N

O

K

+

O

Identification

numbers:

CAS registry

6484-52-2

7631-99-4

7632-00-0

7757-79-1

7758-09-0

NIOSH RTECS

BR9050000

WC5600000

RA1225000

TT3700000

TT3750000

EPA hazardous

No data

waste

DOT/UN/NA/IMDG

UN 2426; UN 0223;

UN 1498;

UN 1500;

UN 1486;

UN 1488;

shipping UN 1942; UN 2067; IMO 5.1 IMO 5.1 IMO 5.1 IMO 5.1

UN 2068; UN 2069;

UN 2070; UN 2071;

UN 2072; UN 0222

IMO 5.1; NA 1942;

IMO 1.1; IMO 9.0

HSDB

475

726

757

1227

1216

NCI

No data

NFPA instability

hazard

b

3

No data

155 NITRATE AND NITRITE

4. CHEMICAL AND PHYSICAL INFORMATION

Table 4-2. Chemical Identity of Selected Inorganic Nitrate and Nitrite

Compounds

a

Ammonium

Sodium

Potassium

Characteristic

nitrate

nitrite

nitrate

nitrite

EPA Pesticide

076101

c

076104

c

076204

c

076103

c

076203

c

Chemical Code

a

All information obtained from IARC 2010 and HSDB 2007, except where noted.

b

NFPA 2002; instability hazard 3 = materials that in themselves are capable of detonation or explosive

decomposition or explosive reaction, but that require a strong initiating source or that must be heated under

confinement before initiation.

c

EPA 2014f.

CAS = Chemical Abstracts Service; DOT/UN/NA/IMDG = Department of Transportation/United Nations/North

America/International Maritime Dangerous Goods Code; EPA = Environmental Protection Agency;

HSDB = Hazardous Substances Data Bank; NCI = National Cancer Institute; NFPA = National Fire Protection

Association; NIOSH = National Institute for Occupational Safety and Health; RTECS = Registry of Toxic Effects of

Chemical Substances

156 NITRATE AND NITRITE

4. CHEMICAL AND PHYSICAL INFORMATION

Table 4-3. Chemical Identity of Ammonia

a

and Urea

b

Characteristic

Ammonia

Urea

Synonym(s)

Anhydrous ammonia, ammonia gas;

Alphahydrate; carbamide; carbonyl

aqua ammonia; liquid ammonia

diamide; carbonyldiamine; isourea

Registered trade name(s)

BCMW; BUSAN 1215

UAL-37; N-Dure; UF-Concentrate-

85; Ureacin-20

Chemical formula

NH

3

CH

4

N

2

O

Chemical structure

H

O

H

2

N

H

NH

2

Ionic weight

17.03

60.06

Identification numbers:

CAS registry

7664-41-7

57-13-6

NIOSH RTECS

No data

EPA hazardous waste

No data

DOT/UN/NA/IMDG shipping

UN 1005; UN 3318; UN 2672; UN

No data

2073

HSDB

162

163

NCI

No data

EPA Pesticide Chemical

005302

085702

Code

a

HSDB 2012.

b

HSDB 2003.

CAS = Chemical Abstracts Service; DOT/UN/NA/IMDG = Department of Transportation/United Nations/North

America/International Maritime Dangerous Goods Code; EPA = Environmental Protection Agency;

HSDB = Hazardous Substances Data Bank; NCI = National Cancer Institute; NIOSH = National Institute for

Occupational Safety and Health; RTECS = Registry of Toxic Effects of Chemical Substances

157 NITRATE AND NITRITE

4. CHEMICAL AND PHYSICAL INFORMATION

Table 4-4. Physical and Chemical Properties of Selected Inorganic Nitrate and

Nitrite Compounds

a

Ammonium

Potassium

Characteristic

nitrate

Sodium nitrate

Sodium nitrite

nitrate

nitrite

CAS

6484-52-2

7631-99-4

7632-00-0

7757-79-1

7758-09-0

Molecular weight

80.043

84.995

68.985

101.103

85.093

Color

White; colorless

White to pale

Colorless

Pale yellow

(pure); gray or yellow

brown (fertilizer

grade)

Physical state

Solid

Melting point

169.7°C

306°C; 308°C

271°C

334°C; 337°C

440°C

Boiling point

Decomposes at

380°C;

320°C;

400°C;

537°C;

~210°C (200– decomposes decomposes decomposes explodes

260°C)

Density:

1.725 g/cm

3

2.26 g/cm

3

2.17 g/cm

3

2.11 g/cm

3

1.915 g/cm

3

at 20°C/4°C

Odor

Odorless

b

No data

Odorless

No data

Odor threshold:

No data

Taste threshold

No data

Solubility:

Water at 25°C

213 g/100 g

91.2

c

g/100 g

84.8 g/100 g

38.3 g/100 g

312 g/100 g

Organic solvent(s)

Acetone,

Ammonia,

Ammonia;

ammonia, hydrazine, ethanol, glycerol; sl sol alcohol

ethanol, ethanol, methanol ethanol

isopropanol, methanol,

methanol

acetone, glycerol

Partition coefficients

Not available

Vapor pressure

Not available

Henry's law

Not available

constant

Flashpoint

Not available

Flames up when

498ºC; May

Not flammable

heated to 540°C explode above

530°C

Flammability limits

Not available

Explosive limits

Not available

>1,000°C

Not available

a

All information obtained from HSDB 2007, unless otherwise noted.

b

Lewis 2002.

c

Lide 2013.

CAS = Chemical Abstracts Service; HSDB = Hazardous Substances Data Bank

158 NITRATE AND NITRITE

4. CHEMICAL AND PHYSICAL INFORMATION

Table 4-5. Physical and Chemical Properties of Ammonia

a

and Urea

b

Characteristic

Ammonia

Urea

CAS

7664-41-7

57-13-6

Molecular weight

17.03

60.06

Color

Colorless

White

Physical state

Gas

Crystal/powder

Melting point

-77.7°C

132.70°C

Boiling point

-33.35 °C at 760 mm Hg

Decomposes

Density:

at 20°C/4°C

0.696 g/L (liquid)

1.3230

Odor

Sharp, pungent, irritating

Slight odor of ammonia; odorless

Odor threshold:

Water: 1.5 mg/L; Air: 5.2 µL/L

No data

Taste threshold

No data

Solubility:

Water

4.82x10

5

mg/L at 24°C

5.45x10

5

mg/L at 25°C

Organic solvent(s)

Alcohol, chloroform, ether

Alcohol; acetic acid; pyrimidine

Partition coefficients

No data

-2.11

Vapor pressure at 25°C

7.51x10

3

mm Hg

1.2x10

-5

mm Hg

Henry's law constant

1.61x10

-5

atm m

3

/mole at 25°C

No data

Flashpoint

No data

Flammability limits

No data

Explosive limits

No data

a

HSDB 2012

b

HSDB 2003

CAS = Chemical Abstracts Service; HSDB = Hazardous Substances Data Bank

159 NITRATE AND NITRITE

4. CHEMICAL AND PHYSICAL INFORMATION

Nitrate is the most oxidized form of nitrogen present in the environment (oxidation state of nitrogen +5).

It accounts for the majority of the total available nitrogen in surface waters (Environment Canada 2012),

perhaps due to its formation by converting the ammonium ion (e.g., from fertilizer and manure) through a

2-step oxidation process, first to nitrite and then to nitrate. In compounds, nitrate and nitrite typically

exist in an oxidation state of 1

-

. Nitrate is the conjugate base of nitric acid (HNO

3

), a strong acid with

pKa of -1.38 at 25°C (Dean 1985). Nitric acid and salts of nitric acid completely dissociate in aqueous

solutions, except for nitrates of mercury and bismuth (Environment Canada 2012; WHO 1978). Nitrite is

the conjugate base of nitrous acid (HNO

2

), a weak acid with a pKa of 3.14 at 25°C (Dean 1985); nitrite

readily decomposes to yield water and dinitrogen trioxide (N

2

O

3

), or nitric acid, nitric oxide (NO), and

water (H

2

O) (WHO 1978, 2011b).

160 NITRATE AND NITRITE

4. CHEMICAL AND PHYSICAL INFORMATION

This page is intentionally blank.

161 NITRATE AND NITRITE

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL

5.1 PRODUCTION

Table 5-1 lists the production year, number of facilities, the state where each facility is located, and the

range (in pounds) for each domestic manufacturer that reported the production or formulation of nitrate

compounds in 2012 (TRI12 2014). Table 5-2 lists Toxics Release Inventory (TRI) data for sodium

nitrite, a common nitrite salt. Table 5-3 lists the TRI data for ammonia. Manufacturers are required to

report Toxics Release Inventory (TRI) data to satisfy EPA requirements. The TRI data should be used

with caution since only certain types of facilities are required to report (EPA 2005). Facilities that must

report to the TRI include industries in a specific business sector such as manufacturing, mining, or electric

generation, employ ≥10 full-time employees, and manufacture or process 25,000 pounds of a TRI-listed

chemical or use >10,000 pounds of a TRI listed chemical per calendar year. Therefore, there are some

facilities that may be processing or using nitrate and/or nitrite, but are not required to report to TRI

because they do not meet the regulatory criteria. The amounts reported in Tables 5-1, 5-2, and 5-3

represent those reported by all facilities in each state that are required to report to the TRI and represent

the range of minimum to maximum amounts of each chemical present on-site at these facilities during the

year. This is not an exhaustive list.

Nitrate and nitrite are not stable compounds, but rather the ionic portions of compounds such as inorganic

salts. As used in this profile, the word “ion” is implied and not used, unless added for clarity. Nitrate and

nitrite occur naturally in the environment as a part of the nitrogen cycle. Nitrogen fixation is part of the

natural process by which free nitrogen gas (N

2

) is converted to nitrite, then to nitrate, used by plants, and

returned as free N

2

to the atmosphere. This is called the nitrogen cycle, and is shown in Figure 5-1. This

cycle occurs through the global environment (Newton 2005). Nitrogen exists naturally in soils. Topsoils

contain nitrogen, at content levels as high as 2 to 4 tons/hectare (roughly 1.2–2.4 kg/m

3

in the upper

15 cm of soil; topsoil depths can range between 0 and 30 cm [Hill Laboratories 2014]), typically bound to

organic matter and mineral soil material; available forms of nitrogen, including nitrate, are present in soils

at a few kg/hectare (Taylor 2004). Nitrate is also formed naturally as an end product of oxidation of

vegetable debris and animal and human waste, mainly urine disposed of in waste water. This process is

known as nitrification, which is a microbial process that converts ammonia to nitrate and is the principal

source for nitrate in the terrestrial and aquatic environment (Environment Canada 2012). Under aerobic

conditions, the ammonium ion (e.g., from fertilizer or manure, or discharge from municipal and onsite

waste water treatment systems) is converted to nitrite ion via ammonia-oxidizing bacteria (Nolan 1999).

162 NITRATE AND NITRITE

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL

Table 5-1. Facilities that Produce, Process, or Use Nitrate Compounds

Minimum

Maximum

Number of

amount on site amount on site

State

a

facilities

in pounds

b

in pounds

b

Activities and uses

c

AK

2

1,000,000

9,999,999

1, 5, 12, 14

AL

56

0

49,999,999

1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14

AR

26

0

49,999,999

1, 3, 4, 5, 7, 9, 11, 12, 13

AZ

32

0

99,999,999

1, 4, 5, 6, 7, 11, 12

CA

139

0

499,999,999

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14

CO

39

0

10,000,000,000

1, 5, 7, 11, 12, 13, 14

CT

26

0

99,999

1, 3, 5, 7, 8, 10, 12

DC

4

1,000

9,999

7, 8

DE

6

0

9,999,999

1, 5, 7, 13, 14

FL

36

0

9,999,999

1, 3, 4, 5, 6, 7, 9, 11, 12, 13, 14

GA

57

0

99,999,999

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14

GU

1

100,000

999,999

1, 5

HI

7

0

99,999

1, 5, 9

IA

46

0

999,999,999

1, 3, 4, 5, 7, 8, 10, 11, 12, 13

ID

25

0

9,999,999

1, 2, 3, 5, 6, 7, 8, 10, 12, 13, 14

IL

108

0

499,999,999

1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14

IN

62

0

9,999,999

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14

KS

27

0

999,999,999

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13

KY

44

0

999,999

1, 2, 3, 5, 6, 7, 8, 10, 11, 12, 13

LA

48

0

999,999,999

1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14

MA

42

0

9,999,999

1, 3, 5, 6, 7, 11, 12, 13

MD

21

0

999,999

1, 3, 4, 5, 6, 7, 8, 10, 13, 14

ME

12

0

99,999

1, 5, 11, 12

MI

105

0

9,999,999

1, 5, 6, 7, 8, 9, 10, 11, 12, 14

MN

53

0

999,999,999

1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 14

MO

37

0

49,999,999

1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14

MS

28

100

49,999,999

1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12

MT

10

100

999,999

1, 3, 5, 7, 11, 12, 13

NC

41

0

9,999,999

1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13

ND

10

100

999,999

1, 5, 7, 8, 13

NE

25

100

99,999,999

1, 3, 4, 5, 6, 7, 10, 12, 13

NH

7

0

99,999

1, 5, 7, 10, 11, 12

NJ

40

100

999,999

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14

NM

14

0

499,999,999

1, 5, 6, 10, 11, 12

NV

27

0

499,999,999

1, 2, 3, 5, 6, 7, 10, 11, 12, 13, 14

NY

73

0

999,999

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14

OH

111

0

499,999,999

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14

OK

37

100

99,999,999

1, 3, 4, 5, 6, 7, 10, 11, 12, 14

OR

40

0

49,999,999

1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12

163 NITRATE AND NITRITE

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL

Table 5-1. Facilities that Produce, Process, or Use Nitrate Compounds

Minimum

Maximum

Number of

amount on site amount on site

State

a

facilities

in pounds

b

in pounds

b

Activities and uses

c

PA

67

0

499,999,999

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13

PR

7

100

999,999

1, 3, 5, 7, 8, 14

RI

6

100

99,999

1, 2, 3, 5, 6, 12

SC

43

0

999,999

1, 2, 3, 5, 6, 7, 10, 11, 12, 13, 14

SD

8

1,000

9,999,999

1, 5

TN

45

0

49,999,999

1, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14

TX

132

0

49,999,999

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14

UT

38

0

49,999,999

1, 2, 3, 4, 5, 6, 7, 9, 10, 12, 13

VA

42

0

9,999,999

1, 3, 5, 7, 8, 9, 10, 11, 12, 13

VT

6

100

9,999,999

1, 5, 6, 10, 12

WA

46

0

99,999,999

1, 3, 4, 5, 6, 7, 9, 12, 13, 14

WI

127

0

499,999,999

1, 4, 5, 7, 9, 10, 11, 12, 13, 14

WV

18

0

9,999,999

1, 3, 4, 5, 6, 7, 10, 12, 13, 14

WY

5

10,000

99,999,999

1, 3, 4, 6, 7, 11

a

Post office state abbreviations used.

b

Amounts on site reported by facilities in each state.

c

Activities/Uses:

1. Produce

6. Reactant

11. Manufacturing Aid

2. Import 7. Formulation Component 12. Ancillary/Other Uses

3. Onsite use/processing 8. Article Component 13. Manufacturing Impurity

4. Sale/Distribution 9. Repackaging 14. Process Impurity

5. Byproduct

10. Chemical Processing Aid

Source: TRI13 2014 (Data are from 2013)

164 NITRATE AND NITRITE

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL

Table 5-2. Facilities that Produce, Process, or Use Sodium Nitrite

Minimum

Maximum

Number of amount on site amount on site

State

a

facilities

in pounds

b

in pounds

b

Activities and uses

c

AL

7

1,000

999,999

1, 5, 6, 7, 10, 11, 12

AR

8

0

99,999

2, 3, 5, 6, 8, 9, 12

AZ

3

1,000

9,999

12

CA

11

1,000

499,999,999

1, 3, 6, 7, 9, 11

CO

2

1,000

99,999

7, 9, 11

FL

1

10,000

99,999

12

GA

11

1,000

99,999

1, 5, 6, 7, 9, 11

IA

3

100

99,999

6, 10

ID

1

10,000

99,999

11

IL

32

0

999,999

1, 3, 4, 5, 6, 7, 9, 10, 11, 12

IN

20

100

9,999,999

1, 2, 3, 5, 6, 7, 9, 10, 11, 12, 13

KS

2

1,000

9,999

10, 12

KY

10

100

999,999

6, 7, 10, 11, 12

LA

9

1,000

9,999,999

1, 5, 6, 7, 10, 11, 12

MA

5

1,000

99,999

6, 12

MD

1

10,000

99,999

2, 3, 11

MI

43

0

9,999,999

2, 3, 6, 7, 8, 9, 10, 11, 12

MN

6

10,000

999,999

10, 12

MO

13

0

99,999,999

2, 3, 6, 7, 10, 11, 12

MS

6

1,000

99,999

7, 10, 11, 12

NC

4

1,000

99,999

1, 5, 7, 12

NE

4

1,000

99,999

7, 8, 9

NJ

9

1,000

999,999

7, 9, 11, 12

NM

1

1,000

9,999

12

NV

1

10,000

99,999

2, 3, 12

NY

8

0

9,999,999

1, 4, 5, 7, 10, 11, 12

OH

35

100

999,999

1, 2, 3, 5, 6, 7, 10, 11, 12

OK

3

100

999,999

1, 5, 7, 11

OR

2

10,000

99,999

11

PA

15

0

9,999,999

1, 5, 6, 7, 10, 11, 12

RI

1

100

999

1, 5, 12

SC

19

100

9,999,999

1, 2, 3, 5, 6, 7, 8, 10, 11, 12

SD

3

1,000

99,999

1, 5, 7

TN

5

1,000

999,999

2, 3, 4, 7, 8, 9, 10, 11, 12

TX

38

0

9,999,999

1, 5, 6, 7, 8, 9, 10, 11, 12, 13

UT

1

0

VA

4

1,000

999,999

7, 8, 11, 12

WA

1

0

165 NITRATE AND NITRITE

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL

Table 5-2. Facilities that Produce, Process, or Use Sodium Nitrite

Minimum

Maximum

Number of

amount on site amount on site

State

a

facilities

in pounds

b

in pounds

b

Activities and uses

c

WI

13

100

999,999

7, 9, 11, 12

WV

4

1,000

9,999,999

1, 5, 7, 11, 13

a

Post office state abbreviations used.

b

Amounts on site reported by facilities in each state.

c

Activities/Uses:

1. Produce

6. Reactant

11. Manufacturing Aid

2. Import 7. Formulation Component 12. Ancillary/Other Uses

3. Onsite use/processing 8. Article Component 13. Manufacturing Impurity

4. Sale/Distribution 9. Repackaging 14. Process Impurity

5. Byproduct

10. Chemical Processing Aid

Source: TRI13 2014 (Data are from 2013)

166 NITRATE AND NITRITE

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL

Table 5-3. Facilities that Produce, Process, or Use Ammonia

Minimum

Maximum

Number of amount on site amount on site

State

a

facilities

in pounds

b

in pounds

b

Activities and uses

c

AK

6

0

999,999

1, 2, 3, 5, 11, 12

AL

70

0

49,999,999

1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14

AR

49

0

49,999,999

1, 2, 3, 5, 6, 7, 9, 10, 11, 12, 13

AS

1

1,000

9,999

12

AZ

20

0

49,999,999

1, 5, 6, 7, 9, 10, 11, 12

CA

120

0

99,999,999

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14

CO

19

0

9,999,999

1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, 14

CT

15

0

999,999

1, 2, 3, 5, 6, 7, 8, 10, 11, 12

DC

2

10,000

99,999

12

DE

7

1,000

9,999,999

1, 3, 5, 6, 7, 11, 12

FL

64

0

499,999,999

1, 2, 3, 5, 6, 7, 9, 10, 11, 12, 13

GA

81

0

99,999,999

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13

HI

9

0

999,999

1, 3, 5, 6, 7, 9, 10, 11, 12, 13, 14

IA

79

100

999,999,999

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14

ID

19

100

49,999,999

1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13

IL

112

100

99,999,999

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14

IN

65

0

9,999,999

1, 2, 3, 5, 6, 7, 9, 10, 11, 12, 13, 14

KS

37

0

99,999,999

1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14

KY

47

0

49,999,999

1, 2, 3, 5, 6, 7, 9, 10, 11, 12, 13, 14

LA

72

0

499,999,999

1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14

ME

10

0

999,999

1, 2, 3, 5, 6, 7, 8, 10, 11, 12, 13

MI

70

0

9,999,999

1, 2, 3, 5, 6, 7, 8, 10, 11, 12, 13, 14

MN

61

0

49,999,999

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13

MO

46

0

9,999,999

1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13

MS

34

0

99,999,999

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13

MT

10

0

9,999,999

1, 2, 3, 5, 6, 9, 10, 12, 13

NC

86

0

49,999,999

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14

ND

13

0

99,999,999

1, 3, 4, 5, 6, 9, 10, 11, 12

NE

45

100

499,999,999

1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14

NH

9

0

9,999,999

1, 3, 5, 6, 10, 11, 12

NJ

43

0

999,999

1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13

NM

6

0

99,999

1, 3, 5, 6, 11, 12, 13, 14

NV

12

0

9,999,999

1, 2, 3, 5, 6, 7, 9, 12, 13, 14

NY

50

0

999,999

1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 14

OH

115

0

10,000,000,000

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14

OK

24

0

99,999,999

1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14

OR

31

0

9,999,999

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13

PA

95

0

49,999,999

1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14

167 NITRATE AND NITRITE

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL

Table 5-3. Facilities that Produce, Process, or Use Ammonia

Minimum

Maximum

Number of

amount on site amount on site

State

a

facilities

in pounds

b

in pounds

b

Activities and uses

c

PR

10

0

999,999

1, 2, 3, 4, 5, 6, 7, 10, 12

RI

10

1,000

9,999,999

1, 2, 3, 4, 5, 6, 9, 10, 11

SC

54

0

499,999,999

1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14

SD

10

1,000

999,999

1, 2, 5, 7, 10, 11, 13

TN

70

0

999,999

1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13

TX

211

0

99,999,999

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14

UT

27

0

9,999,999

1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12

WA

29

0

9,999,999

1, 2, 3, 5, 6, 7, 9, 10, 11, 12, 13

WI

81

0

49,999,999

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14

WV

34

0

49,999,999

1, 2, 3, 5, 6, 7, 8, 10, 11, 12, 13, 14

WY

14

0

99,999,999

1, 2, 3, 4, 5, 6, 7, 10, 12, 13

a

Post office state abbreviations used.

b

Amounts on site reported by facilities in each state.

c

Activities/Uses:

1. Produce

6. Reactant

11. Manufacturing Aid

2. Import 7. Formulation Component 12. Ancillary/Other Uses

3. Onsite use/processing 8. Article Component 13. Manufacturing Impurity

4. Sale/Distribution 9. Repackaging 14. Process Impurity

5. Byproduct

10. Chemical Processing Aid

Source: TRI13 2014 (Data are from 2013)

168 NITRATE AND NITRITE

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL

Figure 5-1. Simplified Schematic of the Nitrogen Cycle

Adapted from EEA 2010; EPA 2012a; Vitousek et al. 1997

169 NITRATE AND NITRITE

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL

This oxidation process is an intermediate step in the nitrogen cycle, followed by further oxidation of

nitrite to nitrate ion via nitrite-oxidizing bacteria. These two reactions are mediated by aerobic

chemolithotrophs, Nitrosomonas and Nitrobactor, respectively (WHO 1978). Microbial conversion of

nitrate to nitrite (reduction) may also occur, especially after prolonged storage of vegetables that make the

environment anaerobic.

In nature, nitrate can also be found in igneous and volcanic rocks; however, the high solubility of nitrogen

salts makes minerals containing nitrate rare. Major minerals known are saltpeter (KNO

3

) found in India,

and Chile saltpeter (NaNO

3

) found in deserts of northern Chile (Environment Canada 2012; Hammerl and

Klapotke 2006).

Plants and mammals naturally contain nitrate and nitrite (WHO 2011b). Assimilation of nitrite from soils

occurs via reduction of nitrate to nitrite, which is facilitated by various bacteria and catalyzed by nitrate

reductase (WHO 1978). Mammals endogenously produce nitrate and excrete it in their waste products

(WHO 1978, 2011b).

Various industrial process produce nitrate in their waste streams. Specifically, potassium nitrate, calcium

nitrate, silver nitrate, and sodium nitrate used in several industrial applications have waste waters with

high-nitrate concentrations (Environment Canada 2012).

A major source of anthropogenic nitrate and nitrite is artificial fertilizers (WHO 1978). The majority of

nitrate in the environment derived from fertilizers does not solely originate from nitrate-containing

fertilizers; it also comes from ammonium and urea fertilizers. Nitrate from ammonium and urea

fertilizers is produced through biological processes involving hydrolysis of urea to ammonium and

ammonium nitrification (Kissel et al. 2008). Approximately 11.5 million tons of nitrogen are applied

yearly (as of 1994) in the United States as fertilizer in agricultural areas (Nolan et al. 1997). The

Association of American Plant Food Control Officials and The Fertilizer Institute reported that the United

States used 13.5 thousand tons of nitrogen fertilizer in 2012 (TFI 2014). Ammonium, calcium,

potassium, and sodium salts are all used in commercial fertilizers compounds (IARC 2010; WHO 2011b).

The most common nitrite salt, sodium nitrite, is produced commercially via the reaction of nitrogen

oxides with sodium carbonate or sodium hydroxide solution, typically at a pH higher than 8 (Hammerl

and Klapotke 2006). In 2004, global production of sodium nitrate was about 63 kilotons (IARC 2010).

Ammonium nitrate is manufactured through the reaction of nitric acid and ammonium (HSDB 2007).

Global production of ammonium nitrate in 2002 was reported at 13,608 kilotons (IARC 2010). Between

170 NITRATE AND NITRITE

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL

1998 and 1999, 90 kilotons of Canadian fertilizers were nitrate compounds: 82% as ammonium nitrate

and the remaining 18% from calcium nitrate, calcium ammonium nitrate, and potassium nitrate

(Environment Canada 2012).

According to the 2011 SRI Directory of Chemical Producers, there are 15 domestic producers of

ammonium nitrate in the United States, with an annual capacity of 2,290 metric tons (SRI 2011). There

were six producers of sodium nitrate, two producers of sodium nitrite, and one producer of potassium

nitrite; however, no production volumes or capacities were reported for any of these substances (SRI

2011). Production of ammonium nitrate in 2004 by the United States chemical industry was reported as

6,558 thousands of metric tons and preliminary production data reported 6,353 thousands of metric tons

for the year 2005 (HSDB 2007). Production of ammonium nitrate by the U.S. chemical industry in 1994

through 2003 is listed in Table 5-4. U.S. production of sodium nitrate in 1982 was estimated as

4.75x10

7

kg and at least 5.0x10

7

kg in 1977; U.S. production of sodium nitrite in 1977 was reported as at

least 5.0x10

6

kg; U.S. production of potassium nitrate in 1972 and 1975 were reported as 4.23x10

7

and

9.89x10

7

kg, respectively (HSDB 2007).

Production of ammonia by the U.S. chemical industry in 1995 through 2002 is listed in Table 5-5.

According to 2012 Chemical Data Reporting (CDR) data, the total reported production volumes for

ammonia and urea were 1.75 x10

10

kg/year and 1.17 x10

10

, respectively (EPA 2014g) Consumption

patterns indicate that the major use for these chemicals is in the fertilizer industry (HSDB 2003, 2012).

5.2 IMPORT/EXPORT

In 1984, United States imports of ammonium nitrate were 1.14x10

11

g (109,247 metric tons) and exports

in 1975 were reported as 3.18x10

10

(31,298 metric tons) (HSDB 2007). In 1986, U.S. imports of

potassium nitrate were 3.62x10

6

g (3.56 metric tons) and exports in 1975 were reported as negligible

(HSDB 2007). In 1985, U.S. imports of sodium nitrate were 6.44x10

7

g (63.4 metric tons) and exports in

1985 were reported as 4.81x10

6

(4.73 metric tons) (HSDB 2007). In 1984, U.S. imports of sodium nitrite

were 8.14x10

9

g (8,011 metric tons) and exports in 1984 were reported as 4.03x10

11

(396,635 metric tons)

(exports related to general sodium compounds) (HSDB 2007).

171 NITRATE AND NITRITE

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL

Table 5-4. Production of Ammonium Nitrate by the U.S. Chemical Industry

Year

Thousands of metric tons

1994

7,771

1995

7,700

1996

7,708

1997

7,804

1998

8,235

1999

6,920

2000

7,237

2001

5,833

2002

6,436

2003

5,733

Source: HSDB 2007

172 NITRATE AND NITRITE

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL

Table 5-5. Production of Ammonia by the U.S. Chemical Industry

Year

Millions of metric tons

1994

64,510

1995

35,600

1999

16.6

2000

15.7

2001

9.5

2002

10.8

Source: HSDB 2012

173 NITRATE AND NITRITE

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL

The U.S. Department of Agriculture (USDA 2013) has compiled annual import/export data on nitrate

fertilizers (ammonium nitrate, potassium nitrate, and sodium nitrate) for years 2000–2012. The volumes

for ammonium nitrate are provided in Table 5-6, followed by a 2012 comparison for all three fertilizers

(Table 5-7).

5.3 USE

The majority of nitrate in commerce is used in common inorganic fertilizers. Ammonia, urea, ammonium

nitrate, sodium nitrate, potassium nitrate, and calcium nitrate are used as commercial fertilizers;

ammonium nitrate and sodium nitrate are also used in munitions and explosives. These chemicals have

uses defined in several other industrial and consumer categories. Nitrate and nitrite salts are used as

preservatives in beverages. Additional uses include oxidizing agents, in instant cold packs and for the

production of nitrous oxide (ammonium nitrate), and for glass making (potassium nitrate) (EPA 2009a;

IARC 2010; Taylor 2004; WHO 2011b). Potassium and ammonium nitrate may also be used in

pyrotechnics, herbicides, and insecticides (HSDB 2007). Sodium nitrite is mainly used in the food

industry as a preservative, in cured meats for preventing botulism (e.g., it inhibits microbial activity of

certain Clostridium species in cheeses), and in the chemical, pharmaceutical, and agricultural industries

(Hammerl and Klapotke 2006; HSDB 2007; WHO 2011b). Sodium nitrite also has therapeutic uses such

as an antidote for cyanide poisoning and as an antifungal topical agent, for example against MRSA strains

(HSDB 2007; Ormerod et al. 2011; Pokorny and Maturana 2006). Due to the bioactivity of NO, an

endogenous metabolite of nitrite produced under hypoxic conditions, sodium nitrite is being used in

medicinal applications, such as for the treatment of pulmonary arterial hypertension (Blood and Power

2015; Lundberg et al. 2008; Rix et al. 2015). Potassium nitrate has been added to some toothpastes for

cavity prevention and to reduce sensitivity, as well as being used as a curing agent and color fixative in

meats (HSDB 2007). In nature, plants utilize nitrate as an essential nutrient (WHO 2011b).

5.4 DISPOSAL

Disposal methods for anthropogenic sources of nitrate and nitrite are general; unused portions of the

material should be recycled for the approved use or returned to the manufacturer or supplier, while leaks

or spills should be resolved wearing appropriate protective equipment and taking care not to create a

flammable or explosive environment. Response to a small liquid spill involves stopping the leak, soaking

up the liquid with vermiculite or sand, and placing it in a non-combustible container. Response to a large

liquid spill on land involves diking, product recovery, treating residue with soda ash and neutralizing it

with HCl, and flushing residue from the area with water. Response to a solid spill involves picking up the

174 NITRATE AND NITRITE

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL

Table 5-6. U.S. Imports and Exports (Metric Tons) of Selected Fertilizers 2000–

2012

Ammonium

Calcium

Potassium

Sodium

nitrate nitrate nitrate nitrate nitrate nitrate nitrate nitrate

Year

exports

imports

exports

imports

exports

imports

exports

imports

2012

335,080

851,196

Not reported

38,550

15,746

159,135

3,348

148,898

2011

314,764

633,974

Not reported

33,998

16,449

114,861

3,286

90,470

2010

317,737

548,976

Not reported

34,490

9,991

76,849

2,429

70,156

2009

195,455

450,664

Not reported

123,168

8,449

73,871

2,536

79,741

2008

188,818

706,955

Not reported

204,552

4,322

132,571

5,783

149,467

2007

194,038

1,107,220

Not reported

187,640

Not reported

135,912

3,139

72,892

2006

127,244

1,150,523

Not reported

156,997

Not reported

149,633

2,827

68,416

2005

82,237

907,618

Not reported

119,448

Not reported

86,961

2,289

66,655

2004

109,972

1,055,949

Not reported

126,498

Not reported

66,381

2,838

62,812

2003

51,856

1,203,985

Not reported

90,989

Not reported

78,754

2,465

85,565

2002

98,218

989,507

Not reported

99,200

Not reported

100,712

2,810

72,568

2001

19,277

925,534

Not reported

127,586

Not reported

50,791

2,199

89,422

2000

21,611

838,035

Not reported

108,269

Not reported

40,941

2,264

96,067

Source: USDA 2013

175 NITRATE AND NITRITE

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL

Table 5-7. U.S. Exports and Imports for Nitrate Fertilizers in 2012 (Short Tons)

Fertilizer

Exports

Imports

Ammonium nitrate

369,362

938,283

Potassium nitrate

17,357

175,416

Sodium nitrate

3,691

164,132

Urea

370,694

7,654,464

Anhydrous ammonia

41,504

6,938,744

Aqua ammonia

6,549

96,517

All fertilizers

a

10,783,383

35,552,395

a

Includes nitrogen, potassium and phosphate fertilizers

Source: USDA 2013

176 NITRATE AND NITRITE

5. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL

material with implements (e.g., shovels, broom, and pan), placing in a non-combustible container, and

loosely capping the container. Spills to water can be treated with activated charcoal. Ultimate disposal of

the chemicals should take into account several factors (the material's impact on air quality; migration

characteristics; effects on animal, aquatic, and plant life) and must take into account compliance with

environmental and public health regulations. Generally, this involves treatment with sodium carbonate,

neutralization with HCl, and disposal of the resulting sludge in a secure landfill. If incineration is used,

processes to remove nitrogen dioxide and nitrogen oxide should be included (HSDB 2007).

177 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

6.1 OVERVIEW

Nitrate and nitrite are ubiquitous in the environment. Specific salts have occasionally been identified in

hazardous waste sites. Ammonium nitrate, sodium nitrate, and sodium nitrite were identified in 7, 3, and

2, of the 1,832 hazardous waste sites, respectively that have been proposed for inclusion on the EPA

National Priorities List (NPL) (ATSDR 2015). However, the number of sites evaluated for these

substances is not known. The frequency of these sites can be seen in Figures 6-1, 6-2, and 6-3.

Nitrate may enter the environment via natural and anthropogenic sources. Nitrate and nitrite occur

naturally in the environments as a part of the earth’s nitrogen cycle. A major source of anthropogenic

nitrate and nitrite is artificial fertilizers, and various industrial processes also produce nitrate in their waste

streams (Environment Canada 2012; WHO 1978). Inorganic fertilizer and nitrification of animal waste

are the principal sources of nitrate in the environment (Environment Canada 2012; Nolan et al. 1997).

However, contributions from human waste must be taken into account as well. Point and non-point

anthropogenic sources that contribute include industrial waste water, mining (explosives) waste water,

agricultural and urban runoff, feedlot discharges, septic system and landfill leachate, lawn fertilizers,

storm sewer overflow, and nitric oxide and nitrogen dioxide from vehicle exhaust (Environment Canada

2012). Additionally, organic forms of nitrogen in the environment from various sources may undergo

ammonification to form inorganic ammonia and ammonium, and nitrification to form nitrate, and have the

potential to be released into surface waters (Environment Canada 2012). Inorganic nitrate and nitrite in

soil and water can be taken up by plants used for human consumption (ATSDR 2013a).

Exposure from drinking water of private wells is a source of concern as elevated concentrations have been

reported in some wells, yet these water sources are not routinely tested, monitored, or regulated since they

are not covered by the Safe Drinking Water Act (SDWA). About 15% of Americans use private wells as

a source of drinking water and an important percentage of them may have a septic system serving their

homes. Additionally, nitrate and nitrite exposure can occur from the ingestion of foods containing high

levels of these chemicals (ATSDR 2013a).

178 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

Figure 6-1. Frequency of NPL Sites with Ammonium Nitrate Contamination

179 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

Figure 6-2. Frequency of NPL Sites with Sodium Nitrate Contamination

180 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

Figure 6-3. Frequency of NPL Sites with Sodium Nitrite Contamination

181 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

6.2 RELEASES TO THE ENVIRONMENT

The Toxics Release Inventory (TRI) data should be used with caution because only certain types of

facilities are required to report (EPA 2005). This is not an exhaustive list. Manufacturing and processing

facilities are required to report information to the TRI only if they employ 10 or more full-time

employees; if their facility is included in Standard Industrial Classification (SIC) Codes 10 (except 1011,

1081, and 1094), 12 (except 1241), 20–39, 4911 (limited to facilities that combust coal and/or oil for the

purpose of generating electricity for distribution in commerce), 4931 (limited to facilities that combust

coal and/or oil for the purpose of generating electricity for distribution in commerce), 4939 (limited to

facilities that combust coal and/or oil for the purpose of generating electricity for distribution in

commerce), 4953 (limited to facilities regulated under RCRA Subtitle C, 42 U.S.C. section 6921 et seq.),

5169, 5171, and 7389 (limited S.C. section 6921 et seq.), 5169, 5171, and 7389 (limited to facilities

primarily engaged in solvents recovery services on a contract or fee basis); and if their facility produces,

imports, or processes ≥25,000 pounds of any TRI chemical or otherwise uses >10,000 pounds of a TRI

chemical in a calendar year (EPA 2005).

Nitrate is released into the environment through both natural and anthropogenic sources. Naturally

occurring nitrate and nitrite are part of the earth’s nitrogen cycle. Anthropogenic sources, including

animal and human organic wastes as well as nitrogen-containing fertilizers, increase the concentrations of

nitrate in the environment. Nitrate and nitrite are present in the environment, in soils and water, and to a

lesser extent, in air, plant materials, and meat products. Concentrations of nitrite in plants and water are

low relative to nitrate concentration due to the fact that nitrite is easily oxidized to nitrate (WHO 1978).

Nitrate is the ion detected in the majority of groundwater and surface water samples because the nitrite

ion is easily oxidized to nitrate in the environment; the nitrate ion is stable and is chemically unreactive

under most environmental conditions (IARC 2010; WHO 2011b).

6.2.1 Air

Estimated releases of 301,654 pounds (~137 metric tons) of nitrate compounds to the atmosphere from

2,110 domestic manufacturing and processing facilities in 2013, accounted for about 0.1% of the

estimated total environmental releases from facilities required to report to the TRI (TRI13 2014). These

releases are summarized in Table 6-1. Estimated releases of 65,201 (~30 metric tons) pounds of sodium

nitrite were released to the atmosphere from 363 domestic manufacturing and

182 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

Table 6-1. Releases to the Environment from Facilities that Produce, Process, or

Use Nitrate Compounds

a

Reported amounts released in pounds per year

b

Total release

On- and off-

State

c

RF

d

Air

e

Water

f

UI

g

Land

h

Other

i

On-site

j

Off-site

k

site

AK

2

0

310,000

0

1,730,003

0

2,040,003

No data

2,040,003

AL

56

5

10,970,520

5,000

589,240

1,003

11,012,324

553,444

11,565,768

AR

26

0

3,776,021

0

14,399

4,217

3,790,167

4,470

3,794,637

AZ

32

3,295

0

39,894

78

43,189

78

43,267

CA

139

7,172

1,967,150

20,684

956,477

65,682

2,831,868

185,297

3,017,165

CO

39

1

1,850,266

0

54,944

50

1,904,854

407

1,905,261

CT

26

58,098

202,953

0

698

54,000

261,051

54,698

315,749

DC

4

0

No data

0

DE

6

0

2,850,359

0

2,850,359

No data

2,850,359

FL

36

479

850,888

23,373,722

264,705

0

24,261,115

228,680

24,489,794

GA

57

511

12,284,962

0

352,756

196,086

12,573,225

261,090

12,834,315

GU

1

0

181,244

0

196

0

181,440

No data

181,440

HI

7

0

439,915

0

439,915

No data

439,915

IA

46

23,005

6,737,465

0

133,387

28

6,886,605

7,280

6,893,885

ID

25

53

2,462,856

0

1,590,643

0

4,021,989

31,564

4,053,553

IL

108

28,202

6,428,670

14,007

454,232

2,346

6,875,892

51,565

6,927,456

IN

62

439

19,965,218

0

3,287,114

12,527

19,965,662

3,299,636

23,265,298

KS

27

38,262

108,068

340,905

98,569

21

585,186

639

585,825

KY

44

990

5,031,265

0

313,935

532

5,305,617

41,104

5,346,721

LA

48

4,502

10,169,890

1,576,528

55,015

0

11,753,676

52,259

11,805,934

MA

42

10

115

25,928

27,091

217,377

145

270,376

270,520

MD

21

0

739,290

0

84,199

35

739,687

83,837

823,524

ME

12

1,209

2,854,965

0

63

0

2,856,209

28

2,856,237

MI

103

10,021

1,714,827

0

228,050

33,121

1,732,091

253,928

1,986,019

MN

53

1,076

1,471,856

0

78,276

250

1,536,094

15,364

1,551,458

MO

37

2,852

1,752,877

0

241,834

5,100

1,977,139

25,524

2,002,663

MS

28

372

6,495,644

0

329

0

6,496,345

No data

6,496,345

MT

10

0

234,169

0

43,891

0

272,860

5,200

278,060

NC

41

1

6,563,023

0

236,361

236,692

6,793,505

242,572

7,036,077

ND

10

0

113,400

0

15,640

0

129,040

No data

129,040

NE

25

187

11,785,649

0

243,650

40

12,022,108

7,418

12,029,526

NH

7

125

0

39

125

39

164

NJ

40

0

5,313,118

0

41,966

376

5,354,858

602

5,355,460

NM

14

55,000

42,240

0

662,620

0

406,331

353,529

759,860

NV

27

6

2,800

0

3,947,279

2

3,729,262

220,825

3,950,087

NY

73

5,337

5,797,905

0

437,314

28,473

5,804,333

464,696

6,269,029

OH

111

1,987

6,081,057

134,614

182,994

64,665

6,219,720

245,598

6,465,318

OK

37

13,010

4,246,811

534,620

180,969

0

4,970,061

5,349

4,975,410

OR

40

1,000

542,093

0

6,833

0

545,071

4,855

549,926

183 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

Table 6-1. Releases to the Environment from Facilities that Produce, Process, or

Use Nitrate Compounds

a

Reported amounts released in pounds per year

b

Total release

On- and off-

State

c

RF

d

Air

e

Water

f

UI

g

Land

h

Other

i

On-site

j

Off-site

k

site

PA

67

9,139

7,212,765

0

66,541

2,428

7,224,885

65,988

7,290,874

PR

7

0

1,465

120

34

1,465

154

1,619

RI

6

0

121

0

20,098

121

20,098

20,219

SC

43

3,001

2,346,088

0

178,745

458

2,376,696

151,596

2,528,293

SD

8

0

2,995,074

2,000

338,782

6

3,280,324

55,538

3,335,862

TN

45

270

2,748,175

470,956

125,258

1,678

2,772,445

573,892

3,346,337

TX

131

788

14,234,564

7,762,004

876,395

7,165

21,822,716

1,058,200

22,880,916

UT

37

329

97,000

0

1,282,016

29

714,559

664,815

1,379,374

VA

42

3,558

10,978,189

0

4,587

1

10,981,823

4,511

10,986,334

VT

6

0

124,890

0

57,395

0

124,890

57,395

182,285

WA

46

8,770

1,190,505

0

981,730

0

1,631,774

549,231

2,181,004

WI

127

591

2,162,376

0

2,339,390

31,174

3,734,315

799,216

4,533,531

WV

18

18,000

2,140,714

0

2,138

0

2,160,673

179

2,160,852

WY

5

0

633

6,569,900

249

0

6,570,782

No data

6,570,782

Total

2,110

301,654

188,570,641

40,832,332

22,848,913

985,811

242,566,590

10,972,761

253,539,351

a

The TRI data should be used with caution since only certain types of facilities are required to report. This is not an exhaustive list.

Data are rounded to nearest whole number.

b

Data in TRI are maximum amounts released by each facility.

c

Post office state abbreviations are used.

d

Number of reporting facilities.

e

The sum of fugitive and point source releases are included in releases to air by a given facility.

f

Surface water discharges, waste water treatment-(metals only), and publicly owned treatment works (POTWs) (metal and metal

compounds).

g

Class I wells, Class II-V wells, and underground injection.

h

Resource Conservation and Recovery Act (RCRA) subtitle C landfills; other onsite landfills, land treatment, surface impoundments,

other land disposal, other landfills.

i

Storage only, solidification/stabilization (metals only), other off-site management, transfers to waste broker for disposal, unknown

j

The sum of all releases of the chemical to air, land, water, and underground injection wells.

k

Total amount of chemical transferred off-site, including to POTWs.

RF = reporting facilities; UI = underground inject

ion

Source: TRI13 2014 (Data are from 2013)

184 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

processing facilities in 2013, accounted for about 0.8% of the estimated total environmental releases from

facilities required to report to the TRI (TRI13 2014). These releases are summarized in Table 6-2.

Estimated releases of 125,680,001 pounds (~57,007 metric tons) of ammonia were released to the

atmosphere from 2,292 domestic manufacturing and processing facilities in 2013, accounted for about

77% of the estimated total environmental releases from facilities required to report to the TRI (TRI13

2014). These releases are summarized in Table 6-3.

6.2.2 Water

Estimated releases of 188,570,641 pounds (~85,534 metric tons) of nitrate compounds to surface water

from 2,110 domestic manufacturing and processing facilities in 2013, accounted for about 74% of the

estimated total environmental releases from facilities required to report to the TRI (TRI13 2014). These

releases are summarized in Table 6-1. Estimated releases of 2,472,668 pounds (~1,122 metric tons) of

sodium nitrite compounds to surface water from 363 domestic manufacturing and processing facilities in

2013, accounted for about 30% of the estimated total environmental releases from facilities required to

report to the TRI (TRI13 2014). These releases are summarized in Table 6-2. Estimated releases of

4,221,440 pounds (~1,914 metric tons) of ammonia to surface water from 2,292 domestic manufacturing

and processing facilities in 2013, accounted for about 2.6% of the estimated total environmental releases

from facilities required to report to the TRI (TRI13 2014). These releases are summarized in Table 6-3.

EPA (2009d) reported that the Mississippi River drains >40% of the area of the contiguous 48 states and

carries roughly 15 times more nitrate than any other river in the country. EPA (2009d) noted that the

nitrate load in the Mississippi rose from 200,000 to 500,000 tons per year in the 1950s and 1960s to an

average of approximately 1,000,000 tons per year during the 1980s and 1990s; the data indicate that the

nitrate load decreased slightly in the early 2000s.

Nitrate is commonly detected in various surface waters and groundwaters such as shallow, rural domestic

wells. Contamination of water systems is a consequence of inorganic fertilizer use, animal manures,

septic systems, and waste water treatment (ATSDR 2013a; Nolan 1999; WHO 2011b). Ammonium ions

in sludge from waste water treatment plants, as well as effluents from those plants and septic systems, are

rapidly converted to nitrate (WHO 1978). Various industrial process produce nitrate in their waste

streams. For example, potassium nitrate, calcium nitrate,

185 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

Table 6-2. Releases to the Environment from Facilities that Produce, Process, or

Use Sodium Nitrite

a

Reported amounts released in pounds per year

b

Total release

On- and

State

c

RF

d

Air

e

Water

f

UI

g

Land

h

Other

i

On-site

j

Off-site

k

off-site

AL

7

0

360

0

360

AR

7

0

254

0

254

No data

254

AZ

3

0

9,117

0

9,117

No data

9,117

CA

11

7

0

5

0

7

5

12

CO

2

255

0

255

No data

255

FL

1

0

No data

0

GA

11

98

648,256

0

101,148

68

749,502

68

749,570

IA

3

0

2,517

0

2,517

No data

2,517

ID

1

0

No data

0

IL

32

1,637

18,600

0

90,065

15

20,237

90,080

110,317

IN

20

1

1,130,853

0

3,312,017

4,896

1,130,854

3,316,913

4,447,767

KS

2

0

No data

0

KY

10

1,016

0

41,435

36,320

27,291

51,480

78,771

LA

9

0

48,000

1,500,000

107

0

1,548,000

107

1,548,107

MA

5

0

No data

0

MD

1

16

0

103

16

103

119

MI

42

12,992

4

0

181,207

2,490

18,745

177,948

196,693

MN

6

0

194,173

0

194,173

No data

194,173

MO

13

2,871

0

412

3,685

2,871

4,097

6,968

MS

6

8,489

7,895

0

26,296

3

16,384

26,299

42,683

NC

4

0

4,455

0

4,455

No data

4,455

NE

4

0

21,200

0

1,182

1,637

21,467

2,552

24,019

NJ

9

200

68,032

0

2,898

0

68,290

2,840

71,130

NM

1

0

No data

0

NV

1

2

0

33,641

0

33,642

No data

33,642

NY

8

11,807

4,925

0

2,800

220

16,732

3,020

19,752

OH

35

13,067

731

0

77,303

1,134

13,798

78,437

92,235

OK

3

289

9,010

0

17,405

0

9,299

17,405

26,704

OR

2

0

430

0

430

PA

15

250

0

7

0

250

7

257

RI

1

0

No data

0

SC

19

1,530

69,403

0

787

190

70,933

977

71,910

SD

3

6

121

25

5,500

0

5,652

0

5,652

TN

5

239

0

153

0

239

153

392

TX

38

34

199,659

198,969

54,487

0

449,818

3,331

453,149

UT

1

No data

VA

4

0

40

0

40

WA

1

No data

186 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

Table 6-2. Releases to the Environment from Facilities that Produce, Process, or

Use Sodium Nitrite

a

Reported amounts released in pounds per year

b

Total release

On- and

State

c

RF

d

Air

e

Water

f

UI

g

Land

h

Other

i

On-site

j

Off-site

k

off-site

WI

13

10,231

28

0

69,018

0

10,259

69,018

79,277

WV

4

165

44,552

0

3

0

44,717

3

44,720

Total

363

65,201

2,472,668

1,698,994

4,027,823

50,760

4,469,774

3,845,673

8,315,446

a

The TRI data should be used with caution since only certain types of facilities are required to report. This is not an

exhaustive

list. Data are rounded to nearest whole number.

b

Data in TRI are maximum amounts released by each facility.

c

Post office state abbreviations are used.

d

Number of reporting facilities.

e

The sum of fugitive and point source releases are included in releases to air by a given facility.

f

Surface water discharges, waste water treatment-(metals only), and publicly owned treatment works (

POTWs) (metal

and metal compounds).

g

Class I wells, Class II-V wells, and underground injection.

h

Resource Conservation and Recovery Act (RCRA) subtitle C landfills; other onsite landfills, land treatment, surface

impoundments, other land disposal, other landfills.

i

Storage only, solidification/stabilization (metals only), other off-site management, transfers to waste broker for

disposal, unknown

j

The sum of all releases of the chemical to air, land, water, and underground injection wells.

k

Total amount of chemical transferred off-site, including to POTWs.

RF = reporting facilities; UI = underground injection

Source: TRI13 2014 (Data are from 2013)

187 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

Table 6-3. Releases to the Environment from Facilities that Produce, Process, or

Use Ammonia

a

Reported amounts released in pounds per year

b

Total release

On- and off-

State

c

RF

d

Air

e

Water

f

UI

g

Land

h

Other

i

On-site

j

Off-site

k

site

AK

6

23,099

7,012

136

24,075

0

54,323

No data

54,323

AL

70

4,060,001

209,278

9,343

43,646

294

4,287,915

34,647

4,322,563

AR

47

1,837,750

114,277

0

5,499

511

1,956,076

1,961

1,958,037

AS

1

20

0

20

No data

20

AZ

20

336,273

0

722

0

336,989

6

336,995

CA

118

2,801,456

26,830

2,870

165,409

369

2,978,123

18,811

2,996,934

CO

19

269,063

17,833

0

101,218

2,430

385,942

4,602

390,544

CT

15

74,775

155

0

74,930

No data

74,930

DC

2

165

0

165

No data

165

DE

7

58,659

6,071

0

23

0

64,730

23

64,753

FL

63

5,607,959

244,014

464,183

960,078

0

6,343,579

932,655

7,276,234

GA

81

12,615,696

268,976

0

166,494

153

12,967,436

83,883

13,051,319

HI

9

100,496

1,000

1,200

0

102,696

No data

102,696

IA

79

7,333,058

108,999

0

210,562

6,621

7,545,358

113,882

7,659,240

ID

19

2,844,444

27,824

0

167,548

0

3,023,068

16,749

3,039,817

IL

112

3,081,028

110,035

0

69,293

4,620

3,246,667

18,309

3,264,976

IN

64

1,600,854

45,833

707,485

77,195

0

2,423,117

8,250

2,431,367

KS

37

3,056,601

6,490

38,214

49,185

15,483

3,130,592

35,381

3,165,972

KY

47

971,022

55,064

0

48,663

1,845

1,036,520

40,073

1,076,593

LA

72

13,461,749

585,745

4,582,747

326,520

0

18,630,317

326,444

18,956,761

MA

30

178,727

41

0

1,622

0

178,768

1,622

180,390

MD

16

523,890

48,675

0

2

0

572,565

2

572,567

ME

10

753,203

89,718

0

842,921

No data

842,921

MI

70

1,681,959

36,582

9,790

7,181

7,875

1,731,377

12,010

1,743,387

MN

61

1,870,843

49,112

0

35,092

2,270

1,938,690

18,627

1,957,317

MO

46

422,032

228,193

0

44,490

251

654,800

40,166

694,966

MS

34

4,700,259

188,194

0

2,181

0

4,889,803

830

4,890,633

MT

10

449,711

5,420

0

264,118

0

719,205

44

719,249

NC

86

2,661,108

85,427

0

73,410

110

2,781,752

38,302

2,820,054

ND

13

16,199,585

4,476

11,500

474,130

0

16,689,625

66

16,689,691

NE

44

974,508

26,655

0

162,935

4,643

1,021,771

146,970

1,168,741

NH

9

127,211

447

0

2

127,658

2

127,660

NJ

43

527,774

9,790

0

24,984

74

537,605

25,017

562,622

NM

6

98,819

0

2,300

11,561

0

112,680

No data

112,680

NV

12

132,670

560

0

228,759

1

361,989

1

361,990

NY

50

687,039

50,197

0

974

274

737,570

915

738,485

OH

115

6,430,630

100,258

1,715,361

76,575

2,560

8,244,156

81,229

8,325,384

OK

24

5,433,575

18,832

696,880

137,282

0

6,282,692

3,877

6,286,569

OR

31

1,156,034

35,599

0

9,980

0

1,192,997

8,616

1,201,613

188 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

Table 6-3. Releases to the Environment from Facilities that Produce, Process, or

Use Ammonia

a

Reported amounts released in pounds per year

b

Total release

On- and off-

State

c

RF

d

Air

e

Water

f

UI

g

Land

h

Other

i

On-site

j

Off-site

k

site

PA

95

1,739,875

77,649

59

710,403

4,905

1,829,424

703,467

2,532,891

PR

10

366,211

0

1,499

0

366,211

1,499

367,710

RI

10

7,130

0

2,500

7,130

2,500

9,630

SC

54

3,581,123

163,436

0

15,085

60,935

3,747,126

73,453

3,820,579

SD

10

102,302

1,809

1

21,448

0

104,819

20,741

125,560

TN

70

2,512,358

418,374

0

90,697

0

2,932,376

89,053

3,021,430

TX

209

4,853,142

297,133

17,187,730

397,858

468

21,789,218

947,114

22,736,332

UT

27

520,482

1,484

8

1,162,922

120

1,684,865

151

1,685,016

VA

49

4,358,663

105,431

0

30,198

32,318

4,471,823

54,787

4,526,610

VT

2

4,543

5,357

0

76

0

9,900

76

9,976

WA

29

878,866

55,744

0

100,110

3,880

940,195

98,405

1,038,600

WI

81

593,142

45,129

15,241

14,091

0

640,527

27,076

667,603

WV

34

332,238

227,614

137,238

47,192

13

602,602

141,693

744,294

WY

14

686,181

8,666

297,557

2,791

0

995,195

No data

995,195

Total

2,292

125,680,001

4,221,440

25,879,844

6,565,774

155,524

158,328,597

4,173,986

162,502,583

a

The TRI data should be used with caution since only certain types of facilities are required to report. This is not an

exhaustive list. Data are rounded to nearest whole number.

b

Data in TRI are maximum amounts released by each facility.

c

Post office state abbreviations are used.

d

Number of reporting facilities.

e

The sum of fugitive and point source releases are included in releases to air by a given facility.

f

Surface water discharges, waste water treatment-(metals only), and publicly owned treatment works (POTWs) (metal and

metal compounds).

g

Class I wells, Class II-V wells, and underground injection.

h

Resource Conservation and Recovery Act (RCRA) subtitle C landfills; other onsite landfills, land treatment, surface

impoundments, other land disposal, other landfills.

i

Storage only, solidification/stabilization (metals only), other off-site management, transfers to waste broker for disposal,

unknown

j

The sum of all releases of the chemical to air, land, water, and underground injection wells.

k

Total amount of chemical transferred off-site, including to POTWs.

R

F = reporting facilities; UI = underground injection

Source: TRI13 2014 (Data are from 2013)

189 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

silver nitrate, and sodium nitrate used in several industrial applications have waste waters with high-

nitrate concentrations (Environment Canada 2012). Discharges of these waste streams increase the

concentrations of nitrate and nitrite in surface waters. Treatment of these waste streams may only remove

a portion of nitrogen. Factors such as nitrogen loading, population density, soil drainage characteristics,

and woodland to cropland ratios, affect the transport of nitrogen from land to water (Nolan et al. 1997;

Zhang et al. 1998). Increased levels of nitrite in drinking water may also be a consequence of

contamination by boiler fluid additives (ATSDR 2013a). High risk waters for nitrate contamination

include areas having soils with high permeability, high-nitrogen input, and low woodland to cropland

ratios (Nolan et al. 1997; Zhang et al. 1998).

Natural sources of nitrate and nitrite include wet and dry deposition of atmospheric nitric acid and nitrate

ion. These are formed in the atmosphere as a result of nitrogen cycling. Atmospheric deposition is a

-

factor for nitrate concentrations in water systems (Momen et al. 1999). Atmospheric nitrogen (NO

3

,

NO

2

-

, and NH

4

+

), mainly from natural sources but a result of anthropogenic sources as well, have been

estimated to contribute 182 kilotons of inorganic nitrogen per year to Canadian surface waters via wet and

dry deposition (Environment Canada 2012). In the United States, deposition contributes an estimated

3.2 million tons (3,200 kilotons) of nitrogen per year to watersheds (Momen et al. 1999; Nolan 1999;

Nolan et al. 1997). Owens et al. (1994) reported that nitrogen input to a grass pasture from precipitation

was equivalent to 10% of the nitrogen fertilizer applied during a 5-year period. The concentration of

nitrate-nitrogen in the precipitation during 1975–1980 was reported as 1.1 mg/L (ppm), which correlated

to an input of 12.0 kg nitrate-nitrogen/hectare. A 10-year average was also evaluated for the years 1980–

1990, which resulted in an input of 8.9 kg nitrate-nitrogen/hectare (Owens et al. 1994).

Stagnation of nitrate-containing and oxygen-poor drinking water in galvanized steel pipes and

chlorination disinfectant residues can lead to conditions where nitrite is formed via chemical reactions in

the distribution pipes by Nitrosomonas bacteria (WHO 2011b).

A U.S. Geological Survey (USGS) study across the United States showed that 7% of 2,388 domestic

wells and about 3% of 384 public-supply wells were contaminated with nitrate levels above the EPA

drinking water standard of 10 mg/L (10 ppm) (ATSDR 2013a). Between 1994 and 1996, 24 lakes in the

Adirondack Park, United States, were studied to assess the contribution of in-lake processes, atmospheric

deposition, and watershed cover on the lakes’ nitrate concentrations (Momen et al. 1999). Weighted

means for nitrate concentrations as a result of precipitation near the lakes were reported for all seasons

during the study period and ranged from 13.86 to 35.52 µeq/L. Nitrogen concentrations throughout the

190 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

study period ranged from 2.1 to 22 µmol/L. Both atmospheric deposition and average lake depth were

considered strong factors in concentrations of nitrate in lakes. It was concluded that the average lake

depth was the most important factor; greater average depths correlated to higher nitrate concentrations.

This was attributed to decreased contact time with lake sediment, decreasing the potential for removal

processes (Momen et al. 1999).

Concentrations of nitrate in freshwater downstream from an open-pit coal mining operation have been

reported to exceed 44 mg nitrate/L (Nordin and Pommen 1986). This is attributed to high nitrate levels in

waste streams due to explosive residues. Monitoring studies conducted by the USGS indicate that nitrate

and nitrite levels are several times greater in streams and groundwater in areas classified as agricultural

use rather than as urban use, mixed use, or undeveloped land (USGS 2010a, 2010b).

Policies implemented by the European Union (EU) to reduce nitrogen emissions from agricultural point

sources were reviewed by Velthof et al. (2014). The Nitrates Directive (ND) was implemented to protect

water quality across Europe by inhibiting nitrates released by agricultural sources from leaching into

groundwater and surface waters through the use of good farming practices. Although regional differences

in emissions were large throughout the entire EU, nitrate leaching into groundwater and surface waters

was estimated to decrease by 16% in nitrate leaching vulnerable zones over the period of 2000–2008,

primarily as a result of lower nitrogen emissions from fertilizers and manures (Velthof et al. 2014).

Seawater nitrate concentrations that occur naturally due to nitrification processes can be as high as 2.4 mg

nitrate/L (Environment Canada 2012). Assimilation into biological systems can deplete nitrate

concentrations in marine environments, causing seasonal variations in nitrate concentrations. Winter

concentrations off the Canadian Atlantic coast were reported to be 0.54 mg nitrate/L, a magnitude higher

than summer concentrations of <0.03 mg nitrate/L (Environment Canada 2012).

6.2.3 Soil

Estimated releases of 22,848,913 pounds (~10,364 metric tons) of nitrate compounds to soils from

2,110 domestic manufacturing and processing facilities in 2013, accounted for about 9% of the estimated

total environmental releases from facilities required to report to the TRI (TRI13 2014). An additional

40,832,332 pounds (~18,521 metric tons), constituting about 16% of the total environmental emissions,

were released via underground injection (TRI13 2014). These releases are summarized in Table 6-1. An

estimated release of 4,027,823 pounds (~1,826 metric tons) of sodium nitrite were emitted to soils from

191 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

363 domestic manufacturing and processing facilities in 2013, accounted for about 48% of the estimated

total environmental releases from facilities required to report to the TRI (TRI13 2014). An additional

1,698,994 pounds (~771 metric tons), constituting about 20% of the total environmental emissions, were

released via underground injection (TRI13 2014). These releases are summarized in Table 6-2. An

estimated release of 6,565,774 pounds (~2,978 metric tons) of ammonia were emitted to soils from

2,292 domestic manufacturing and processing facilities in 2013, accounted for about 4% of the estimated

total environmental releases from facilities required to report to the TRI (TRI13 2014). An additional

25,879,844 pounds (~11,739 metric tons), constituting about 16% of the total environmental emissions,

were released via underground injection (TRI13 2014). These releases are summarized in Table 6-3.

In 2012, 13.5 million tons of nitrogen was added to soils as fertilizer (TFI 2014). Therefore, it should be

noted that the totals provided here, for nitrate compounds, ammonia, and ammonium nitrite alone, may be

insignificant, yet contribute to and are representative of, nitrogen releases to the environment.

6.3 ENVIRONMENTAL FATE

Nitrate and nitrite occur naturally in water and soils as part of the nitrogen cycle. Plants and mammals

naturally contain nitrate and nitrite (WHO 2011b). Nitrate is the primary source of nitrogen for plants

(EPA 2009a). Assimilation of nitrite from soils occurs via reduction of nitrate to nitrite, which is

facilitated by various bacteria and catalyzed via nitrate reductase (WHO 1978). The most common forms

of nitrogen that plants assimilate include ammonium (NH

4

+

), nitrate (NO

3

-

), and urea ((NH

2

)2CO)

(Cornell University 2009). Transport, partitioning, and transformation are controlled by various

physicochemical properties, degradation, and other loss processes. Mammals endogenously produce

nitrate and excrete them in their waste products (WHO 1978). Anthropogenic and natural sources of

ammonia in the environment, such as fertilizers or animal waste products, are converted to nitrite via

Nitrosomonas bacteria and then to nitrate via Nitrobactor bacteria. These products may be assimilated

into plants and subsequently the atmosphere, or may leach into groundwater when they are present in

excessive amounts (WHO 2011b).

Nitrate is the most oxidized form of nitrogen present in the environment (oxidation state of +5) and

accounts for the majority of the total available nitrogen in surface waters (Environment Canada 2012).

Nitrate is the conjugate base of nitric acid, HNO

3

, a strong acid with pKa of -1.37 (WHO 1978). Nitric

acid and salts of nitric acid completely dissociate in aqueous solutions (Environment Canada 2012; WHO

1978). Nitrite is the conjugate base of nitrous acid, HNO

2

, a weak acid with a pKa of 3.37; nitrite readily

192 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

decomposes to yield water and dinitrogen trioxide or nitric acid, nitric oxide, and water (WHO 1978;

WHO 2011b).

6.3.1 Transport and Partitioning

Nitrate and nitrite are inorganic water-soluble salts with the potential for rapid migration through soils to

surface water and groundwater (Nolan 1999; Taylor 2004; EPA 2009a). Sorption of anions such as

nitrate is insignificant in most soils; therefore, leaching of excess soil nitrate into oceans, lakes, streams,

and groundwater is an important consideration (Taylor 2004). Drainage characteristics of soils are

strongly related to nitrate levels in shallow wells near agricultural areas (Nolan et al. 1997; Zhang et al.

1998). Other factors affecting leaching potential include the texture of the soil, pH, precipitation rates,

tillage, and the types of crops or vegetation that may be planted in the soils.

The mobility of nitrate in a mid-European semi-natural grassland ecosystem as a function of plant

diversity was investigated (Scherer-Lorenzen et al. 2003). The greatest leaching was observed in bare

ground plots as well as plots planted only with legumes. Experiments with plots containing a wider

variety of plant species indicated that total nitrate plant uptake increased and leaching losses decreased

with increasing plant diversity due to greater root biomass within the soils. The leaching of nitrate

decreased in the following order: bare plots > pure legumes > legumes + grasses > legumes + grasses +

herbs (Scherer-Lorenzen et al. 2003). Annual nitrate leaching in an apple orchard was 4.4–5.6 times

greater in plots treated with conventional farming practices (calcium nitrate fertilizer) as compared to

plots treated by organic farming practices, in which nitrogen application was accomplished by loadings of

chicken manure and alfalfa meal (Kramer et al. 2006). Reduced leaching was accompanied by increased

denitrification in the organic treatment areas. Kitchen et al. (2015) investigated groundwater nitrate as a

result of leaching due to agriculture cropping systems over time (1994–2004) and found the greatest

decreases in groundwater nitrate concentration occurred as groundwater moved through an in-field tree

line or through a riparian zone.

Nitrate leaching from croplands with high fertilizer use is a major source of groundwater nitrate

concentrations. In Nebraska, groundwater concentrations of nitrate have been correlated with nitrogen-

containing fertilizer application rates and residual nitrogen in surface soils (Schepers et al. 1991). The

reduction of nitrate concentrations in groundwater through agricultural management practices was

assessed in Nebraska’s central Platte River valley (Exner et al. 2010). Groundwater nitrate concentration

reports were studied from 1986 to 2003. Peak levels, during 1988, in the primary aquifers were

193 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

26.8 mg nitrate-nitrogen/L. A gradual decline was observed with the implementation of fertilizer

management regulations. In 2003, nitrate-nitrogen levels in the aquifer averaged 22.0 mg/L.

Nitrate in soils and surface water are susceptible to denitrification resulting in gaseous losses to the

atmosphere (Taylor 2004). Nitrate in the atmosphere, emitted by denitrification, industrial processes, and

vehicle exhaust, is deposited on land and water in precipitation, gases, and dry particles (Nolan 1999;

Taylor 2004). Atmospheric deposition is a factor for nitrate concentrations in water systems (Momen et

al. 1999; Nolan 1999; Nolan et al. 1997).

6.3.2 Transformation and Degradation

Nitrate and nitrite has the potential to move into various environmental compartments and are subject to

abiotic and biotic degradation processes. Transformation and degradation processes include

denitrification to atmospheric nitrogen and plant uptake (Newton 2005; Nolan 1999). Conversion is

achieved via biotic process carried out by auto- and heterotrophic bacteria (Hammerl and Klapotke 2006).

Under aerobic conditions in aquatic systems, ammonia and nitrite are converted to nitrate via nitrification.

Conversion is achieved through a biotic process carried out by autotrophic nitrifying bacteria. Under

anaerobic conditions in aquatic systems, bacteria convert nitrate to nitrite, which is further reduced to the

gaseous compounds nitric oxide (NO), nitrous oxide (N

2

O), and N

2

(nitrogen). These compounds are

subsequently released to the atmosphere. Results from a study of denitrification in riverbed sediments

found that potential rates for denitrification are limited by environmental conditions such as available

organic carbon and temperature, rather than concentration of nitrate itself (Pfenning and McMahon1997).

Higher rates were demonstrated in experiments with added carbon sources. Additionally, higher rates of

denitrification were measured at 22°C compared to those at 4°C (Pfenning and McMahon1997).

6.3.2.1 Air

Nitrogen compounds are formed in the air by natural phenomena such as lightning (Hord et al. 2011), or

may be discharged into air from industrial processes, motor vehicles, agricultural practices, or emitted by

denitrification processes. Nitrate is present in air primarily as nitric acid and inorganic aerosols, as well

as nitrate radicals and organic gases or aerosols (WHO 2011b). Nitrate in the atmosphere is subject to

wet and dry deposition and are deposited on land via precipitation, gases, and dry particles (Nolan 1999;

Taylor 2004).

194 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

6.3.2.2 Water

In surface waters, assimilation by plants and algae accounts for the majority of nitrate loss. Reducing

conditions of water system including dissolved oxygen (DO) and dissolved organic carbon (DOC), as

well as temperature and pH, influence the extent of bacterially mediated nitrate loss processes in water

systems (Nolan 1999; WHO 2011b). Biologically mediated reduction processes of nitrate and nitrite

were found to be positively related to DO and inversely related to iron, manganese, ammonium, and DOC

concentrations (Nolan 1999).

Rates of denitrification were examined by measuring N

2

O production using riverbed sediment and

groundwater or surface water collected from the South Platte alluvial aquifer in Colorado, an area with

high nitrate levels due to anthropogenic activity such as fertilizer use, farming practices, and septic

drainage and runoff (Pfenning and McMahon 1996). The greatest N

2

O production was observed in

microcosms containing high levels of organic carbon. The type of organic carbon source was also shown

to be correlated with the denitrification rate. Higher N

2

O production rates were observed with acetate-

amended sediments as compared to sediments amended with fulvic acids. Surface water-derived fulvic

acids resulted in higher denitrification rates as compared to groundwater-derived fulvic acids. Reduction

in microbial activity due to temperature gradients was also investigated. Denitrification rates decreased

by nearly 80% in laboratory experiments when the temperature was lowered from 22 to 4°C.

6.3.2.3 Sediment and Soil

In soils, nitrite is oxidized to nitrate and the majority of nitrate is assimilated by plants and algae (WHO

2011b). Under aerobic conditions, residual or excess nitrate is expected to leach into groundwater and is

not expected to undergo considerable degradation and/or denitrification. Under anaerobic conditions

degradation of nitrate into atmospheric nitrogen is an important removal process (WHO 2011b). Poorly

drained soils, which lack oxygen, promote nitrate conversion to gaseous nitrogen (Nolan et al. 1997).

Denitrification rates were examined using soil samples obtained from different areas of an apple orchard

and measuring the rates of gaseous nitrogen and N

2

O production as well as nitrate leaching (Kramer et al.

2006). Specifically denitrification rates were measured at different locations of the orchard treated with

organic farming practices, conventional farming practices for orchards in the state of Washington, and

integrated treatments from horticultural and pest management practices of conventional and organic

farming practices. Nitrogen application in the conventional plots used calcium nitrate based fertilizer,

while the organic plots used chicken manure and alfalfa meal. The integrated plots used equal parts of

195 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

calcium nitrate fertilizer and chicken manure. Nitrogen emissions were higher in the organically treated

plots, as compared to the conventional and integrated plots. Nitrate leaching was much greater (4.4–

5.6 times higher) in the conventional plots as compared to the organically treated plots.

6.4 LEVELS MONITORED OR ESTIMATED IN THE ENVIRONMENT

Reliable evaluation of the potential for human exposure to nitrate and nitrite depends in part on the

reliability of supporting analytical data from environmental samples and biological specimens.

Concentrations of nitrate and nitrite in unpolluted atmospheres and in pristine surface waters are often so

low as to be near the limits of current analytical methods. In reviewing data on nitrate and nitrite levels

monitored or estimated in the environment, it should also be noted that the amount of chemical identified

analytically is not necessarily equivalent to the amount that is bioavailable. The analytical methods

available for monitoring nitrate and nitrite in a variety of environmental media are detailed in Chapter 7.

Nitrate occurs naturally in the environments as a part of the earth’s nitrogen cycle. Elevated levels may

be present due to anthropogenic sources such as fertilizers, and human or animal wastes. High levels of

nitrate in drinking water pose a health risk to infants, children, and pregnant or nursing women (EPA

2009a).

6.4.1 Air

Anthropogenic emissions of nitrogen oxides (NO

x

) are now of the same order of magnitude as natural

emissions (Hammerl and Klapotke 2006). Air pollution is considered a minor source of exposure to

nitrate (WHO 2011b). Nitrate in the atmosphere is generally a result of nitrogen oxides released into the

atmosphere that are oxidized to nitric acid, in turn forming nitrate particles (Matsumoto and Tanaka

1996). Atmospheric levels of particulate nitrate are highly dependent on temperature and the chemical

composition of aerosol and gases in the atmosphere, especially particulate ammonium nitrate and gaseous

nitric acid (Matsumoto and Tanaka 1996). Reported atmospheric nitrate concentrations range from low

concentrations of 0.1–0.4 μg/m

3

up to higher-level concentrations ranging from 1 to 40 μg/m

3

(WHO

1978, 2011b). Concentrations in Netherland air samples have been reported to range from 1 to 14 μg/m

3

.

Indoor nitrate aerosol concentrations of 1.1–5.6 μg/m

3

appear to be related to outdoor concentrations

(WHO 2011b). Zhuang et al. (1999) evaluated the concentrations of fine and coarse particle nitrate in the

atmosphere over Hong Kong. The average daily concentrations for fine and coarse particle nitrate were

found to be 0.583 and 1.663 µg/m

3

, respectively.

196 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

6.4.2 Water

Nitrate and nitrite concentrations in water are typically expressed as either mg nitrate/L (ppm nitrate) and

mg nitrite/L (ppm nitrite), or mg nitrate as nitrogen (nitrate-nitrogen/L) and mg nitrite as nitrogen (nitrite-

nitrogen/L) (IARC 2010). The federal drinking-water standard maximum contaminant level (MCL) for

nitrate is 10 mg nitrate-nitrogen/L and the MCL for nitrite is 1 mg nitrite-nitrogen/L (EPA 2009c; USGS

2010a; WHO 2011b). Inorganic nitrate and nitrite are very soluble in water and occur naturally in

groundwater and surface water as a result of the earth’s nitrogen cycle. Naturally occurring background

levels of nitrate (concentrations expected if there were no effects of human development and

anthropogenic sources) have been estimated as 1.0 and 0.24 mg nitrate-nitrogen/L for groundwater and

streams, respectively, in the United States (USGS 2010a).

A comprehensive report analyzed nutrient levels in 5,101 wells from 51 different study areas (Burow et

al. 2010; USGS 2010a). Monitoring data from 1993 to 2003 indicated that nitrate levels in groundwater

varied widely across the nation, with some of the highest levels observed in the Northeast (particularly

southern Pennsylvania), the Midwest, the state of California, and select regions of the Northwest

(Washington state and Idaho). The report concluded that nitrate levels in deep aquifers were likely to

continue to increase as shallow groundwater with high levels of nitrate gravitate downward (USGS

2010a). The highest levels of nitrate were observed in oxic groundwater (water containing >0.5 ppm DO)

as opposed to anoxic groundwaters and shallow wells in agricultural areas, which tended to have greater

levels than in urban areas (USGS 2010a). Burow et al. (2010) analyzed these data and reported that

nitrate concentrations exceeded the MCL (10 mg nitrate-nitrogen/L) in 437 wells (8%). Levels exceeded

the MCL in 20% of wells classified as agricultural land-use setting, and 3% were above the MCL in wells

classified as urban use. In monitoring data from bank and in-stream wells in the San Joaquin River in

California, collected between 2006 and 2008, the concentration of nitrate exceeded the detection limit

(0.01 mg/L) in 5% of the groundwater samples and the concentrations in surface waters ranged from 1 to

3 mg/L. It was reported that 17 of the 26 nested monitoring wells, along the river bed and river bank, had

no detectable concentrations of nitrate during the monitoring period (USGS 2013a).

Monitoring data obtained from 1991 to 1995 in shallow groundwater of coastal plains in the Albemarle-

Pamlico Drainage Unit, in North Carolina and Virginia, have indicated the presence of increased nitrate

concentrations as a result of agriculture and anthropogenic sources. Shallow groundwater concentrations

are higher at inner coastal sites with well-drained soils compared with outer coastal sites. Areas with

anthropogenic nitrogen sources, such as fertilizer and manure, had aquifer concentrations >3 mg nitrate-

197 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

nitrogen/L. Levels <2 mg nitrate-nitrogen/L were reported in aquifer waters with greater DOC

concentrations. Groundwaters from areas having well-drained soils had a median concentration of

approximately 0.4 mg nitrate-nitrogen/L. Two of the 20 inner coastal wells had levels >10 mg nitrate-

nitrogen/L. Groundwater concentration of nitrate was nearly undetectable in waters underneath poorly

drained soils in the outer coast (median 0.05 mg nitrate-nitrogen/L). The North Carolina Division of

Water Quality (NCDWQ) selected groundwater samples susceptible to contamination; 40% of the

15 wells in the inner coastal plain of the Albemarle-Pamlico Drainage had levels >10 mg nitrate-

nitrogen/L (USGS 2012).

Nitrate and nitrite were the two most detected inorganic chemicals reported in public water systems

(PWSs) in an analysis supporting the U.S. EPA’s second Six-Year Review of National Primary Drinking

Water Regulations. Occurrence data for nitrate from a Six-Year Review-ICR Dataset include

1,052,487 analytical results from 119,537 public water systems (groundwater 114,764; surface water

4,773) across 44 states during the time period from 1998 to 2005 (EPA 2009a). These water systems are

reported to serve a combined population of 229,508,036. Nitrate was detected in approximately 70% of

the water systems (groundwater 69.4%; surface water 81.3%) at a median concentration of 1.8 mg nitrate-

nitrogen/L (groundwater systems 1.6 mg nitrate-nitrogen/L; surface water systems 2.71 mg nitrate-

nitrogen/L). Maximum concentrations detected in groundwater and surface water systems were 99 and

48.5 mg nitrate-nitrogen/L, respectively. Seven states in the review reported at least one detection of

nitrate greater than the MCL of 10 mg nitrate-nitrogen/L in >5% of their systems. Overall, the

2,973 systems with detections exceeding the MCL serve a combined population of 16,777,093 (EPA

2009a). Occurrence data for nitrite from the Six-Year Review-ICR Dataset includes 397,175 analytical

results from 86,313 public water systems (groundwater 82,738; surface water 3,575) across 44 states

during the time period from 1998 to 2005 (EPA 2009a). These water systems are reported to serve a

combined population of 207,984,813. Nitrite was detected in approximately 22% of the water systems

(groundwater 22%; surface water 23%) at a median concentration of 0.02 mg nitrite-nitrogen/L.

Maximum concentrations detected in groundwater and surface water systems were 13 and 8.68 mg nitrite-

nitrogen/L, respectively. Four states in the review reported at least one nitrite detection greater than the

MCL (1 mg nitrite-nitrogen/L) in more than 1% of their systems. Overall, the 635 systems with

detections exceeding the MCL serve a combined population of 10,067,031 (EPA 2009a).

In 1991, 12% of 631 private wells located on farmlands across 18 states in the United States reported

concentrations >10.2 mg nitrate-nitrogen/L (Bruning-Fann et al. 1994; IARC 2010). Additionally, in

1994, levels of nitrate found in drinking waters across nine mid-western U.S. states ranged from 0.01 to

198 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

266 mg nitrate-nitrogen/L, with a mean value of 8.4 mg nitrate-nitrogen/L and 10% of the water supplies

had concentrations >10 mg nitrate-nitrogen/L (CDC 1998). California groundwater is relied on for

drinking water in 70% of its cities. A study in 1987 indicated that ~10% of the sampled California wells

and >7% of the public water systems in Tulare County, California had levels >45 mg nitrate/L (>10 mg

nitrate-nitrogen/L) (Zhang et al. 1998).

Studies regarding nitrate levels in drinking water outside the United States were summarized (IARC

2010). Zhang et al. (1996) reported that several cities in China (with populations between 10,000 and

100,000) had water supplies with concentrations >11.3 mg nitrate-nitrogen/L. Twenty-eight percent of

public wells monitored in India and 13% in Saudi Arabia had reported levels >11.2 mg nitrate-nitrogen/L

as well. In 1990, average concentrations in Canadian municipal drinking waters were reported to range

from 0.1 to 3.3 mg nitrate/L (0.02–0.75 mg nitrate-nitrogen/L) (Environment Canada 2012). It has been

estimated that 2% of the European population receive their drinking water from private wells and an

estimated 2.4 million people are exposed to water supplies containing nitrate concentrations above

guideline levels (Gangolli et al. 1994). Nitrate concentrations in many European countries have been

reported to be gradually increasing over the last few decades. An average annual increase of 0.7 mg

nitrate/L (0.2 mg nitrate-nitrogen/L) has been observed in some rivers of the United Kingdom (WHO

2011b). Nitrate levels in drinking water in Denmark have increased about 400% over the period from

1940 to 1983 (Moller et al. 1989).

Nitrate was reported to be the most frequently detected nutrient in U.S. streams that exceeded its MCL. It

exceeded the MCL in 2% of all samples obtained (566 out of 27,555) and in at least 1 sample of 50 of the

499 streams surveyed from 1992 to 2001 (USGS 2010a). Many of the streams with levels above the

MCL were located in the upper Midwestern Corn Belt where application rates of fertilizer and manure are

high. Nitrite samples from five streams exceeded the nitrite MCL (USGS 2010a). Flow-adjusted nitrate

concentrations decreased in 25% of 166 streams and rivers sampled by the USGS over the period 1993–

2003; however, concentrations increased over this time period in 20 of the 166 sites (12%) surveyed

(USGS 2009). Decreases in levels were attributed to changes in nitrogen use patterns and implementation

of pollution control strategies. Multiple factors such as land use, nitrogen loading from fertilizer, manure

use, and atmospheric deposition affected the trends observed.

Annual trends of nitrate levels at eight sites along the Mississippi River Basin were studied by the USGS

from 1980 to 2010 (USGS 2013b). Flow-normalized nitrate concentrations were generally reported to be

level or increasing at all of the monitoring sites from 1980 to 2000; however, select locations showed

199 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

greater increases or actual decreases in levels since 2000. The greatest increases over the 30-year period

were observed in the upper Mississippi River (Clinton, Iowa) and Missouri River (Herman, Missouri).

Decreasing flow-normalized nitrate levels over the 30-year period were observed at the Iowa River near

Wapello, Iowa and Illinois River at Valley City, Illinois, suggesting that recent land management and

farming practices may be reducing the nitrate fluxes in these areas (USGS 2013b).

Many surface water bodies in the United States have a large percentage of total nitrate load contributed by

base flow (groundwater discharge, release from other watershed storages, and long-term interflow) in

addition to surface runoff. Mean annual base-flow nitrate levels for 148 surface water and shallow

groundwater sites in the United States from 1990 to 2006 were typically reported to be <1 mg nitrate-

nitrogen/L; however, values as high as 8.48, 11.44, 8.29, and 8.25 mg nitrate-nitrogen/L were reported for

Tulpehocken Creek, Pennsylvania; Indian Creek, Illinois; Salt Creek, Illinois; and Clifty Creek, Indiana,

respectively (USGS 2010b). The highest levels typically were reported for agricultural land use sites that

had frequent fertilizer and manure applications and highly permeable underlying bedrock.

Due to assimilation of nitrate by algae and other plant-life, concentrations of nitrate in surface water are

typically lower than that detected in groundwaters (IARC 2010). Tables 1.4 and 1.5 in the IARC report

summarize concentrations of nitrate from various regions around the globe. The global concentrations in

groundwater were reported to range from 0.02 to 110 mg nitrate-nitrogen/L; mean values ranged from

2.2 to 42.9 mg nitrate-nitrogen/L. Global concentrations of nitrate in surface water were reported to range

from 0 to 22 mg nitrate-nitrogen/L; mean values range from 0.1 to 8.3 mg nitrate-nitrogen/L). Nitrate

levels in rainwater as high as 5 mg nitrate/L (1 mg nitrate-nitrogen/L) have been observed in various

industrial areas (WHO 2011b).

6.4.3 Sediment and Soil

Levels of nitrate and nitrite in soil vary considerably as a function of soil properties, temperature,

precipitation rates, nitrogen loadings, farming practices (tillage, crops planted), and seasonal changes. In

well-drained aerobic soils, the conversion of ammonia into nitrate (nitrification) increases the soil-nitrate

content and in anaerobic soils with high organic matter (such as waterlogged soils or wetlands),

denitrification decreases the levels of nitrate and nitrite in soils. Acidic soils tend to have lower levels of

nitrate since the nitrification process ceases at pH levels below 4.5 (USDA 2014). Typical nitrate levels

in humid temperate soils fluctuate from about 20–65 kg-nitrogen/hectare in cropped soils and 25–150 kg-

200 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

nitrogen/hectare in bare soils (USDA 2014). Certain locations near the South Platte River in northeastern

Colorado had nitrate levels exceeding 500 kg-nitrogen/hectare (Shaffer et al. 1995).

6.4.4 Other Environmental Media

Nitrate and nitrite are common food preservatives. Levels of nitrite and nitrate were evaluated through

several processing steps and storage conditions. Nitrite is oxidized to nitrate during storage. Nitrate

remained relatively constant over a 2-week storage period, while nitrite levels declined over the same

time period. The study also observed minimal influence of the nitrate levels formed from the amount of

nitrite added. Additionally, using nitrate alone resulted in the formation of nitrite after thermal

processing. Cooking resulted in losses of both nitrite and nitrate; 50% nitrite and 10–15% nitrate

remained in the prepared sausage analyzed (Perez-Rodriguez et al. 1996).

Nitrate and nitrite are present in vegetables, fruits, cured meats, fish, dairy products, beers, cereals, and

cereal products (Gangolli et al. 1994). Nitrate content of foodstuffs is typically higher than nitrite content

(ATSDR 2013a; WHO 2011b). Cured meats have concentrations of <2.7–945 mg of nitrate/kg and <0.2–

1.7 mg nitrite/kg (IARC 2010; WHO 2011b). Concentrations of nitrate in vegetables and fruit is strongly

affected by processing of the food, fertilizer use, and growing conditions and range from 30 to 6,000

mg/kg (ppm) (IARC 2010; WHO 2011b). Celery, lettuce, red beetroot, and spinach have high levels of

nitrate (200–>2,500 mg/kg [ppm]). Parsley, leek, endive, Chinese cabbage, and fennel have high levels

of nitrate ranging from 100 to 250 mg/kg (ppm). Cabbage, dill, and turnips have medium levels of nitrate

ranging from 50 to 100 mg/g. Vegetables with low levels of nitrate (20–50 mg/g) include broccoli,

carrots, cauliflower, cucumber, and pumpkin. Very low levels of nitrate (<20 mg/g) are found in

artichokes, asparagus, eggplant, garlic, onions, green beans, mushrooms, peas, peppers, potatoes, summer

squash, sweet potatoes, tomatoes, and watermelons (ATSDR 2013a). Nitrite concentrations are typically

<10 mg/kg (ppm) and rarely reach 100 mg/kg (ppm); however, exceptions include damaged, outdated,

pickled, and fermented foods in which levels may be as high as 400 mg/kg (ppm) (WHO 2011b). Data

for nitrate and nitrite in foodstuffs are not lacking and several papers have been published that summarize

numerous studies, including IARC (94 2010) and Gangolli et al. (1994). Gangolli et al. (1994) cited a

paper reporting the average daily intakes of nitrate and nitrite in the United States to be 106 and

4.1 mg/day, respectively.

Marshall and Trenerry (1996) analyzed several food types purchased from local supermarkets in Australia

for both nitrate and nitrite. Nitrite was not detected in fruit juices in this study. Nitrate and nitrite was

201 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

detected in various cheeses at levels <10 mg/kg (ppm). Canned meat products had small amounts of

nitrite (<10 mg/kg [ppm]), while nitrate levels were higher (10–25 mg/kg [ppm]). Several samples of ice

were also analyzed for the presence of nitrite and nitrate, and all samples were below Australian food

standards (10 mg/L nitrate; 1 mg/L nitrite).

Nitrite and nitrate levels were determined in several whey-containing food products (Oliveira et al. 1995).

In total, 231 samples from powdered modified milk, powdered non-fat milk, a dairy beverage, and

strawberry- and chocolate-flavored instant mixes were evaluated. Mean nitrate levels ranged between

7.3 and 532 mg/kg (ppm). Mean nitrite levels ranged between 1.1 and 2.5 mg/kg (ppm). One serving of

the product with the highest reported nitrate levels (a chocolate-flavored instant mix) was calculated to be

51.9 mg/serving. One serving of the product with the highest reported nitrite levels (powdered non-fat

milk) was calculated to be 0.1 mg/serving.

From 1993 to 1997, nitrate and nitrite levels were monitored in Danish lettuce, leek, potato, beetroot,

Chinese cabbage, and white cabbage, and spinach (Peterson and Stoltze 1999). Seasonal variation was

observed in nitrate levels. Lettuce exhibited higher concentrations in winter as opposed to summer.

Overall nitrite concentrations were low. Average nitrate concentrations were 2,760, 1,783, 198, and

158 mg/kg fresh weight for lettuce, fresh spinach, leeks, and potatoes, respectively. Average nitrite

concentrations were 11, 0.91, 0.80, 0.15, and 0.14 mg/kg fresh weight for fresh spinach, beetroot,

potatoes, leeks, and lettuce, respectively. The average daily intakes, estimated from consumption surveys

of the vegetables in the study, were 40 mg/day nitrate and 0.09 mg/day nitrite.

Table 6-4 contains data on infant foods examined for nitrate and nitrite (Cortesi et al. 2015). Food

samples of animal origin were composed of a variety of sources such as poultry, beef, rabbit, lamb, and

turkey. Foods samples of plant origin were composed of a variety of sources such peas, legumes,

vegetable broths, cream of pumpkin and carrots, and mixed vegetables. Mixed-origin samples were

composed of both plant and animal sources. The highest average concentration of nitrate was found in

foods of plant origin (45.5 mg/kg), while the highest average concentration of nitrite was found in foods

of animal origin (14.82 mg/kg).

Jones et al. (2014) reported nitrate and nitrite concentrations in fresh breast milk, freeze-thawed breast

milk, freeze thawed colostrum, and several commercially available infant formulas. Data are tabulated in

Table 6-5. Fresh breast milk was collected from 11 mothers of term infants and 13 mothers of preterm

infants. Samples of colostrum (milk expressed days 1–3), transition milk (expressed days 4–7), and

202 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

Table 6-4. Concentrations of Nitrate and Nitrite in Infant Food Products

Food type

Nitrate (mg/kg)

Nitrite (mg/kg)

Homogenized samples of animal

0.35–83.2

6.6–48.87

origin

Freeze dried samples of animal

2.01–80.26

1.3–74.74

origin

Homogenized samples of plant

4.82–131.68

2.26–20.71

origin

Freeze dried samples of plant

19.41–85.03

1.34–6.62

origin

Homogenized samples of mixed

3.77–67.31

1.98–80.22

origin

Source: Cortesi et al. 2015

203 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

Table 6-5. Average Concentrations of Nitrate and Nitrite in Human Milk and Infant

Formula

Milk type

Nitrate (µmol/L)

Nitrite (µmol/L)

Fresh breast milk

16

0.1

Freeze-thawed breast milk

20

0.04

Preterm fresh

12

0.07

Preterm freeze-thawed

22

0.03

Term fresh

11.5

0.13

Term freeze-thawed

12.5

0.04

Freeze-thawed colostrum

41

0.16

Infant formula

43

0.29

Freeze-thawed colostrum

44

0.15

Transition milk

Not reported

0.05

Mature milk

Not reported

0.025

Source: Jones et al. 2014

204 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

mature milk (expressed days >7) were analyzed. Concentrations of nitrate and nitrite in the 10 formulas

evaluated were 9–61 and not detected–1.4 µmol/L, respectively.

6.5 GENERAL POPULATION AND OCCUPATIONAL EXPOSURE

The general public is typically exposed to nitrate and nitrite via ingestion of water and foods that contain

these chemicals. Inhalation and dermal exposure may be possible; however, these routes are not as

prominent. Oral exposure to nitrate and nitrite from contaminated drinking water and food is the

prominent route. Nitrate and nitrite overexposure may occur through ingestion of foods containing high

levels of nitrate and nitrite (ATSDR 2013a). Inorganic nitrate and nitrite can be taken up by plants,

especially leafy vegetables such as lettuce and spinach as well as beet root; vegetables account for about

80% of the nitrate in a typical human diet (ATSDR 2013a; Hord 2011; Lundberg et al. 2009; Peterson and

Stoltze 1999). Contaminated foodstuffs from improper storage of commercial and prepared baby foods

have caused overexposure in children (Dusdieker et al. 1994; Greer and Shannon 2005; Sanchez-Echaniz

et al. 2001).

Iammarino et al. (2014) analysed 75 samples of spinach and 75 samples of lettuce, collected from June

2010 to December 2011, for nitrate and nitrite. Spinach had a greater number of detections compared

with the lettuce samples. Mean nitrate concentrations ranged from 155.5 to 2,149.6 mg/kg; mean nitrite

concentrations ranged from 16.3 to 101.6 mg/kg. Four spinach samples and five lettuce samples had

concentrations of nitrate >2,000 mg/kg. Quantifiable concentrations of nitrite were detected in

15 samples of spinach (28.5–197.5 mg/kg) and one sample of lettuce (66.5 mg/kg).

The remainder of the nitrate in a typical diet comes from drinking water (about 21%) and from meat and

meat products (about 6%) in which sodium nitrate is used as a preservative and color-enhancing agent

(ATSDR 2013a; Lundberg et al. 2008; Saito et al. 2000). For bottle-fed infants, the major source of

nitrate exposure is from contaminated drinking water used to dilute formula, especially when the water is

boiled prior to use (ATSDR 2013a; Fewtrell 2004). A review by Jones et al. (2015) reported daily nitrate

ingestion concentrations for adults. An intake of approximately 3 mg/kg/day for adults was based on a

typical adult diet.

The Fourth National Report on Human Exposures to Environmental Chemicals, published and updated by

the Centers for Disease Control and Prevention (CDC 2013), reported the following data from the

National Health and Nutrition Examination Survey (NHANES) 1999–2008. Nitrate levels in the urine

205 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

(see Table 6-6), and urine (creatinine corrected) (see Table 6-7) were evaluated for various ages and

ethnicities. Mean values of nitrate in the urine were 42.7 and 46.3 mg/L for 7,697 members of the general

U.S. population sampled during 2005–2006 and 7,629 members of the general U.S. population sampled

during 2007–2008, respectively. The highest geometric mean (creatinine corrected) during 2001–2002 of

72.0 mg/L was determined from 374 samples from 6–11 year olds; the highest geometric mean (creatinine

corrected) during 2005–2006 of 60.8 mg/L was determined from 1,054 samples from 6–11 year olds; and

the highest geometric mean during 2007–2008 of 70.2 mg/L was determined from 1,143 samples from 6–

11 year olds. Throughout all survey years, females had a higher geometric mean than males. In the

survey years 2007–2008, 3,789 female samples yielded a mean of 51.0 mg/g, while 5,351 male samples

yielded a mean of 44.6 mg/g (CDC 2013).

No information was located regarding absorption of inhaled inorganic nitrate or nitrite in humans or

laboratory animals. Inhalation of inorganic nitrate or nitrite is not a likely exposure route of concern for

the general population, although inhalation of dust from fertilizer products containing nitrate salts is

possible.

Occupational exposure is primarily via inhalation and dermal routes. Industrial workers and farmers may

be exposed via inhalation of dusts. Dusts may also dissolve in sweat on skin, increasing the potential for

dermal exposure.

Vegetable consumption is a considerable source of nitrate, and drinking water with high levels of nitrate

is also a major contributing factor. Several studies have been conducted assessing exposure to drinking

waters with high levels of nitrate. In 1986 until 1987, Moller et al. (1989) studied 294 Danish adults

between the ages of 20 and 64 years who were exposed to various levels of nitrate in their drinking water

and diet. Twenty-one drinking water supplies contained low (0–5 mg nitrate/L [ppm nitrate])

intermediate (35–59 mg nitrate/L [ppm nitrate]) and high (≥60 mg nitrate/L [ppm nitrate]) nitrate levels,

with mean concentrations of 0.3, 46.5, and 84.4 mg nitrate/L (ppm nitrate), respectively. The median

exposures of total dietary nitrate for the low, intermediate, and high water categories were 37, 89, and

123 mg nitrate/day, respectively. Mean nitrate levels detected in the participant’s 24-hour urine samples

for the low, intermediate, and high water concentration categories were reported as 36, 55, and 73 mg

nitrate, respectively. Overall, the dietary contribution was calculated to be 17% from water and 83%

from food for the low group, and increased to approximately 60% from water and 40% from food for the

intermediate and high groups. Fifty-nine Canadian adults between the ages of 20–74 years used tap water

with low (<3 mg nitrate-nitrogen/L) and high (>3 mg nitrate-nitrogen/L) concentrations of nitrate. The

206 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

Table 6-6. Geometric Mean and Selected Percentiles of Urine Concentrations of

Urinary Nitrate (in mg/L) for the U.S. Population from the National Health and

Nutrition Examination Survey (NHANES)

Geometric

Selected percentiles (95% CI)

Survey

years

mean

(95% CI)

50

th

75

th

90

th

95

th

Sample

size

Total

2001–2002

2005–2006

2007–2008

48.2 (46.2–

50.3)

42.7 (39.6–

46.1)

46.3 (44.6–

48.1)

49.0 (46.0–

52.0)

47.8 (44.4–

51.2)

50.3 (48.4–

52.0)

78.0 (73.0–

83.0)

74.6 (69.8–

79.4)

76.0 (72.7–

79.1)

100 (100–

130)

108 (101–

114)

110 (104–

116)

140 (130–

150)

133 (125–

144)

138 (132–

146)

1,617

7,697

7,629

Age group

6–11 years

2001–2002

2005–2006

2007–2008

62.2 (53.8–

71.8)

51.2 (47.4–

55.4)

55.2 (51.7–

58.9)

68.0 (58.0–

79.0)

54.7 (51.9–

58.2)

60.2 (56.1–

64.3)

94.0 (84.0–

100)

79.2 (72.8–

87.2)

84.5 (80.1–

92.5)

130 (100–

160)

113 (101–

128)

117 (107–

135)

150 (120–

380)

141 (120–

158)

149 (132–

189)

374

1,054

1,143

12–19 years

2001–2002

2005–2006

2007–2008

57.4 (53.5–

61.6)

52.5 (48.5–

56.8)

55.5 (51.5–

59.7)

66.0 (60.0–

69.0)

57.5 (52.3–

62.6)

56.8 (51.9–

61.5)

91.0 (86.0–

95.0)

84.2 (79.9–

88.4)

84.1 (76.2–

94.5)

120 (100–

130)

119 (111–

124)

119 (107–

133)

150 (130–

160)

144 (129–

153)

144 (133–

162)

827

2,106

1,135

≥20 years

2001–2002

2005–2006

2007–2008

45.4 (43.3–

47.5)

40.5 (37.4–

43.9)

44.2 (42.5–

45.9)

49.0 (46.0–

52.0)

45.0 (41.3–

48.3)

48.1 (46.0–

49.7)

78.0 (73.0–

83.0)

71.7 (67.1–

77.4)

73.2 (70.1–

76.5)

100 (100–

130)

105 (98.0–

113)

107 (101–

113)

140 (130–

150)

129 (122–

142)

135 (128–

146)

1,617

4,537

5,351

Gender

Males

2001–2002

2005–2006

2007–2008

57.5 (54.6–

60.6)

48.4 (44.6–

52.6)

51.9 (49.9–

54.1)

63.0 (59.0–

67.0)

52.7 (48.3–

57.8)

56.1 (53.8–

58.0)

89.0 (83.0–

94.0)

79.4 (72.8–

86.5)

79.5 (75.7–

83.5)

130 (100–

140)

110 (103–

121)

112 (105–

119)

150 (140–

170)

136 (123–

152)

137 (131–

149)

1,335

3,765

3,839

Females

2001–2002

2005–2006

2007–2008

40.7 (38.4–

43.2)

37.9 (35.1–

40.8)

41.4 (39.3–

43.7)

43.0 (41.0–

48.0)

42.0 (38.2–

46.0)

43.9 (41.5–

46.3)

72.0 (68.0–

76.0)

69.2 (65.5–

73.4)

71.3 (67.5–

74.8)

100 (98.0–

120)

104 (96.6–

110)

108 (99.8–

115)

130 (120–

150)

130 (124–

140)

138 (132–

149)

1,483

3,932

3,790

207 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

Table 6-6. Geometric Mean and Selected Percentiles of Urine Concentrations of

Urinary Nitrate (in mg/L) for the U.S. Population from the National Health and

Nutrition Examination Survey (NHANES)

Geometric

Selected percentiles (95% CI)

Survey

years

mean

(95% CI)

50

th

75

th

90

th

95

th

Sample

size

Race/ethnicity

Mexican

Americans

2001–2002

2005–2006

2007–2008

53.2 (48.7–

58.2)

47.8 (44.7–

51.2)

48.7 (45.0–

52.6)

59.0 (52.0–

66.0)

52.4 (49.9–

56.2)

51.9 (47.4–

56.7)

84.0 (79.0–

91.0)

77.9 (75.1–

83.1)

75.6 (69.7–

81.3)

120 (100–

150)

113 (104–

120)

111 (100–

122)

160 (130–

180)

148 (133–

156)

148 (127–

164)

707

1,972

1,505

Non-Hispanic

blacks

2001–2002

2005–2006

2007–2008

53.8 (47.8–

60.5)

45.9 (42.1–

50.0)

47.5 (45.0–

50.3)

58.0 (51.0–

64.0)

50.4 (46.6–

54.9)

50.3 (48.6–

52.5)

84.0 (77.0–

93.0)

75.0 (68.8–

80.8)

74.7 (71.9–

77.3)

120 (100–

130)

101 (95.3–

110)

105 (97.1–

116)

140 (130–

170)

127 (114–

148)

134 (125–

150)

680

2,078

1,707

Non-

Hispanic

whites

2001–2002

2005–2006

2007–2008

46.3 (44.1–

48.6)

41.2 (37.6–

45.2)

45.0 (42.7–

47.5)

51.0 (47.0–

53.0)

46.2 (41.3–

50.6)

49.2 (46.1–

52.6)

81.0 (78.0–

85.0)

73.3 (67.3–

80.1)

75.5 (70.5–

80.0)

120 (100–

130)

107 (98.2–

115)

108 (101–

116)

140 (130–

150)

129 (122–

142)

134 (128–

140)

1,228

3,056

3,190

CI = confidence interval

Source: CDC 2013

208 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

Table 6-7. Geometric Mean and Selected Percentiles of Urine Concentrations of

Urinary Nitrate (Creatinine Corrected) (in mg/g of creatinine) for the

U.S. Population from the National Health and Nutrition

Examination Survey (NHANES)

Geometric

Selected percentiles (95% CI)

Survey

years

mean

(95% CI)

50

th

75

th

90

th

95

th

Sample

size

Total

2001–2002

2005–2006

2007–2008

49.8 (47.7–

51.9)

42.6 (40.2–

45.1)

47.7 (45.9–

49.7)

46.9 (44.2–

49.6)

42.4 (40.1–

44.7)

46.0 (44.0–

48.3)

63.8 (61.2–

67.7)

59.7 (55.8–

64.1)

66.5 (62.4–

70.5)

90.9 (84.3–

98.8)

85.5 (81.3–

91.3)

98.0 (92.3–

102)

120 (111–

128)

113 (106–

118)

127 (119–

135)

1,616

7,697

7,628

Age group

6–11 years

2001–2002

2005–2006

2007–2008

72.0 (66.1–

78.4)

60.8 (57.4–

64.5)

70.2 (65.7–

74.9)

66.0 (62.6–

70.4)

57.3 (53.6–

60.6)

65.9 (62.4–

69.5)

87.0 (80.2–

97.7)

76.5 (70.9–

82.1)

89.0 (83.2–

96.5)

129 (96.5–

144)

109 (95.0–

123)

128 (112–

152)

144 (130–

235)

134 (121–

164)

173 (140–

216)

374

1,054

1,143

12–19 years

2001–2002

2005–2006

2007–2008

44.8 (43.4–

46.2)

39.8 (37.8–

41.9)

43.4 (41.3–

45.5)

43.8 (42.6–

45.0)

38.1 (36.3–

40.3)

40.5 (38.6–

43.5)

56.2 (52.2–

59.7)

51.8 (47.7–

56.0)

56.0 (52.7–

59.2)

73.2 (65.2–

85.1)

70.6 (63.1–

78.8)

76.6 (69.0–

86.2)

93.4 (79.0–

104)

88.9 (79.4–

103)

98.0 (85.4–

121)

826

2,106

1,134

≥20 years

2001–2002

2005–2006

2007–2008

48.3 (45.9–

50.9)

41.4 (38.9–

43.9)

46.5 (44.6–

48.4)

46.9 (44.2–

49.6)

41.0 (38.8–

43.7)

44.7 (42.5–

47.0)

63.8 (61.2–

67.7)

58.6 (54.8–

63.1)

65.0 (60.7–

69.2)

90.9 (84.3–

98.8)

85.3 (80.2–

91.0)

96.1 (90.0–

102)

120 (111–

128)

111 (105–

116)

125 (117–

132)

1,616

4,537

5,351

Gender

Males

2001–2002

2005–2006

2007–2008

47.6 (44.7–

50.7)

40.1 (37.5–

42.9)

44.6 (42.8–

46.6)

46.1 (43.4–

48.7)

39.5 (36.7–

42.8)

42.8 (40.7–

45.1)

61.3 (58.0–

64.5)

55.3 (51.6–

59.5)

60.6 (58.1–

64.4)

86.7 (77.1–

97.3)

77.2 (70.7–

83.0)

85.6 (81.1–

91.3)

114 (97.3–

125)

95.9 (89.6–

102)

111 (101–

121)

1,335

3,765

3,839

Females

2001–2002

2005–2006

2007–2008

51.9 (49.9–

54.1)

45.1 (42.8–

47.6)

51.0 (48.9–

53.2)

51.2 (48.4–

53.0)

45.0 (42.4–

47.4)

50.0 (47.5–

52.7)

69.1 (66.7–

71.2)

64.4 (60.0–

69.6)

72.3 (67.8–

76.8)

100 (91.7–

111)

96.8 (87.8–

105)

107 (101–

116)

129 (118–

140)

128 (117–

134)

146 (129–

163)

1,481

3,932

3,789

209 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

Table 6-7. Geometric Mean and Selected Percentiles of Urine Concentrations of

Urinary Nitrate (Creatinine Corrected) (in mg/g of creatinine) for the

U.S. Population from the National Health and Nutrition

Examination Survey (NHANES)

Geometric

Selected percentiles (95% CI)

Survey

years

mean

(95% CI)

50

th

75

th

90

th

95

th

Sample

size

Race/ethnicity

Mexican

Americans

2001–2002

2005–2006

2007–2008

50.9 (45.7–

56.8)

44.6 (42.6–

46.7)

48.7 (45.1–

52.7)

48.1 (44.9–

51.4)

44.0 (42.2–

45.3)

47.0 (43.5–

50.5)

67.4 (60.3–

77.6)

60.4 (58.1–

62.9)

64.8 (59.7–

69.9)

97.3 (85.7–

117)

89.9 (82.0–

95.4)

93.5 (88.9–

101)

135 (100–

161)

120 (111–

128)

128 (108–

156)

707

1,972

1,505

Non-Hispanic

blacks

2001–2002

2005–2006

2007–2008

38.7 (36.3–

41.3)

32.9 (30.9–

35.0)

35.9 (34.2–

37.7)

38.0 (34.6–

41.3)

31.9 (29.8–

34.1)

34.8 (33.3–

36.6)

53.5 (50.3–

57.4)

45.4 (41.5–

49.6)

49.0 (44.8–

53.6)

70.3 (64.9–

79.3)

64.0 (60.1–

68.0)

69.1 (63.5–

77.4)

91.7 (78.9–

100)

81.2 (75.3–

89.6)

87.8 (78.1–

97.4)

679

2,078

1,706

Non-

Hispanic

whites

2001–2002

2005–2006

2007–2008

51.4 (49.4–

53.4)

43.7 (40.8–

46.8)

49.0 (46.7–

51.4)

49.1 (46.9–

51.5)

43.9 (40.8–

46.8)

47.4 (44.9–

50.5)

66.9 (64.2–

69.4)

61.4 (56.5–

66.2)

68.2 (63.3–

73.1)

95.2 (87.7–

100)

85.5 (80.6–

91.9)

98.3 (91.2–

105)

124 (115–

132)

110 (102–

116)

126 (118–

135)

1,227

3,056

3,190

CI = confidence interval

Source: CDC 2013

210 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

mean urinary nitrate excretion of the participants who consumed drinking water with low-nitrate levels

was 15.0 mg nitrate-nitrogen/day, while the mean value for the participants who consumed drinking water

with higher nitrate levels was 22 mg nitrate-nitrogen/day (Levellois et al. 2000). Higher correlations for

total nitrate intake and urinary excretion were found with dietary nitrate intake as opposed to water nitrate

intake.

Various scenarios for nitrate and nitrite intake have been considered and it has been found that dietary

intake contributes the majority of exposure occurrences. Approximately 89–99% of an adult’s daily

intake of both nitrate and nitrite is from their food when an average to high vegetable diet is consumed

with average water intake. The daily intake contribution from food decreases to 33–56% for nitrate and

7.7–14% for nitrite when average to high consumption of water with nitrate levels (50 mg nitrate/L [ppm

nitrate]) is considered (IARC94_2010). Gangolli et al. (1994) also reported that 88–96% of the average

dietary intake of nitrate comes from food sources (85% of which is attributed to vegetables), while 4–12%

comes from drinking waters. Exposure to dietary nitrate may increase exposure to nitrite due to

endogenous production. Ingested nitrate is readily absorbed from the upper gastrointestinal tract into the

blood and is mainly excreted in the urine (Gangolli et al. 1994). Portions of blood nitrate are transported

to human saliva where it is mostly metabolized to nitrite; approximately 5% of dietary nitrate is

metabolized to nitrite (Gangolli et al. 1994). Gangolli et al. (1994) estimated that the human adult intakes

of nitrate are 2.4 mg nitrate/kg (ppm nitrate) body weight/day from food and 0.33–4.1 mg nitrate/kg body

weight/day from water; the human adult intakes of nitrite are 0.04–0.07 mg nitrite/kg body weight/day

from food and <0.002–0.07 mg nitrite/kg body weight/day from water. These estimates do not account

for the endogenous production of nitrite. Worldwide dietary exposures were estimated from data

collected in the 1997 Total Diet Study in the United Kingdom and additional dietary studies.

Representative exposure estimates for nitrate and nitrite were reported as 58–218 and 0.7–1.6 mg

nitrite/day, respectively (IARC 2010).

Estimated daily intake values of nitrate and nitrite have been calculated based on data from the United

Kingdom and the United States. An individual with an average water intake (1.4 L/day) and average food

consumption or a high vegetable diet is estimated to consume levels 52–80 or 140–220 mg nitrate/day,

respectively. An individual with an average water intake (1.4 L/day) and average food consumption or a

high vegetable diet is estimated to consume levels 0.74 or 2.2 mg nitrite/day, respectively (IARC 2010).

211 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

Dermal exposure to inorganic nitrate or nitrite is not a likely route of concern for the general population,

although absorption following dermal exposure to dust from fertilizer products containing nitrate salts is

possible.

6.6 EXPOSURES OF CHILDREN

This section focuses on exposures from conception to maturity at 18 years in humans. Differences from

adults in susceptibility to hazardous substances are discussed in Section 3.7, Children’s Susceptibility.

Children are not small adults. A child’s exposure may differ from an adult’s exposure in many ways.

Children drink more fluids, eat more food, breathe more air per kilogram of body weight, and have a

larger skin surface in proportion to their body volume than adults. A child’s diet often differs from that of

adults. The developing human’s source of nutrition changes with age: from placental nourishment to

breast milk or formula to the diet of older children who eat more of certain types of foods than adults. A

child’s behavior and lifestyle also influence exposure. Children crawl on the floor, put things in their

mouths, sometimes eat inappropriate things (such as dirt or paint chips), and may spend more time

outdoors. Children also are generally closer to the ground and have not yet developed the adult capacity

to judge and take actions to avoid hazards (NRC 1993).

Children will be exposed to nitrate and nitrite through ingestion of food and drinking water. Nitrate is

commonly detected in various surface waters and groundwaters. High nitrate concentrations in drinking

water are commonly found in privately owned wells, with shallow depths and permeable soils. It has

been estimated that about 15 million families in the United States use private well drinking water. Based

on monitoring data and birthrates from 2000, it was estimated that 40,000 infants <6 months old would be

living in households using drinking water with nitrate levels that exceed the federal standard (10 mg

nitrate-nitrogen/L) (Fewtrell 2004). Additionally, boiling water from private wells may concentrate

nitrate in the water, which may lead to higher exposure of children whose infant foods are prepared using

water that is boiled first (Fewtrell 2004). Gangolli et al. (1994) estimated that the infant intake of nitrate

and nitrite from food is negligible, while the infant intakes of nitrate and nitrite from water are 1.7–8.3 mg

nitrate/kg body weight/day and <0.02 mg nitrite/kg body weight/day, respectively. A review by Jones et

al. (2015) reported dietary nitrate and nitrite concentrations for newborn infants. An intake of

approximately 0.15 mg/kg/day for infants was based on a mean of reported concentrations in breast milk

and formula. Ingestion based on breast milk intake of approximately 150 mL/kg/day, was reported as

0.12 mL/kg/day for nitrate and 0.0007 mL/kg/day for nitrite.

212 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

Infants may have higher exposures as compared to adults if the water source used for formula has high

levels of nitrates and nitrites. Human breast milk has been shown to contain, although not concentrate,

nitrate and is not considered a significant source of infant exposure (IARC 2010).

6.7 POPULATIONS WITH POTENTIALLY HIGH EXPOSURES

Populations using well water in agricultural areas may be exposed to greater levels of nitrate and nitrite as

compared to populations living in urban areas since groundwater in agricultural communities typically

has greater levels of nitrate and nitrite than urban water (Burow et al. 2010). Furthermore, workers who

are employed in occupations where fertilizer use is common (e.g., farming, greenhouse operations) may

be exposed to nitrate and nitrite through dermal routes and inhalation of dust particles.

6.8 ADEQUACY OF THE DATABASE

Section 104(i)(5) of CERCLA, as amended, directs the Administrator of ATSDR (in consultation with the

Administrator of EPA and agencies and programs of the Public Health Service) to assess whether

adequate information on the health effects of nitrate and nitrite is available. Where adequate information

is not available, ATSDR, in conjunction with NTP, is required to assure the initiation of a program of

research designed to determine the health effects (and techniques for developing methods to determine

such health effects) of nitrate and nitrite.

The following categories of possible data needs have been identified by a joint team of scientists from

ATSDR, NTP, and EPA. They are defined as substance-specific informational needs that if met would

reduce the uncertainties of human health assessment. This definition should not be interpreted to mean

that all data needs discussed in this section must be filled. In the future, the identified data needs will be

evaluated and prioritized, and a substance-specific research agenda will be proposed.

6.8.1 Identification of Data Needs

Physical and Chemical Properties. The physical and chemical properties of nitrate salts are

discussed in Chapter 4. These salts are highly soluble in water and dissociate under environmental

conditions and exist as ions (WHO 1978, 2011b). No data needs are identified.

213 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

Production, Import/Export, Use, Release, and Disposal. According to the Emergency

Planning and Community Right-to-Know Act of 1986, 42 U.S.C. Section 11023, industries are required

to submit substance release and off-site transfer information to the EPA. The TRI, which contains this

information for 2012, became available in November of 2013. This database is updated yearly and should

provide a list of industrial production facilities and emissions. Import/export data are available for

ammonium nitrate (USDA 2013). Data for the other compounds assessed in this profile would be useful.

Environmental Fate. The transport and fate of nitrate and nitrite compounds have been studied

(Kramer et al. 2006; Pfenning and McMahon 1996; WHO 2011b). These substances are highly mobile in

soils. Transformation and degradation processes include denitrification to atmospheric nitrogen and plant

uptake (Newton 2005; Nolan 1999). Conversion is achieved via biotic process carried out by auto- and

heterotrophic bacteria (Hammerl and Klapotke 2006). Under aerobic conditions in aquatic systems,

ammonia and nitrite are converted to nitrate via nitrification. Conversion is achieved through a biotic

process carried out by autotrophic nitrifying bacteria. Under anaerobic conditions in aquatic systems,

bacteria convert nitrate to nitrite, which is further reduced to the gaseous compounds nitric oxide (NO),

nitrous oxide (N

2

O), and N

2

(nitrogen). No data needs are identified.

Bioavailability from Environmental Media. Nitrate and nitrite are readily absorbed following

ingestion from water or food sources.

Data assessing absorption from intake of food sources and water containing nitrate and nitrite has been

studied (Gangolli et al. 1994; Kortboyer et al. 1997b). Several reports have indicated the correlation of

methemoglobinemia in adults and children and elevated nitrite levels in the blood (CDC 1997, 2002;

Gautami et al. 1995; Gowans 1990; Greenberg et al. 1945; Sevier and Berbatis 1976; Ten Brink et al.

1982). Adequate data for intake of nitrate and nitrite from drinking water and food are available (ATSDR

2013a; Gangolli et al. 1994; Hord 2011; JECFA 2003c; Lundberg et al. 2009; Peterson and Stoltze 1999).

Data are lacking for absorption from the lungs and skin. Further data may be useful to establish whether

uptake via inhalation or dermal contact of dust is a notable source of exposure, since this may occur

during application of fertilizers containing these chemicals.

Food Chain Bioaccumulation. Nitrate ion and nitrite ion are both a natural part of the earth’s

nitrogen cycle. Plants and mammals naturally contain nitrate and nitrite (WHO 2011b). Assimilation of

nitrite from soils occurs via reduction of nitrate to nitrite, which is facilitated by various bacteria and

catalyzed by nitrate reductase (WHO 1978). Data are available to indicate that nitrate and nitrite may be

214 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

concentrated in several plants and waters intended for human consumption (JECFA 2003c; Peterson and

Stoltze 1999; Zhang et al. 1996, 2003). No data needs are identified.

Exposure Levels in Environmental Media. Reliable monitoring data for the levels of nitrate and

nitrite in contaminated media at hazardous waste sites are needed so that the information obtained on

levels of nitrate and nitrite in the environment can be used in combination with the known body burden of

nitrate and nitrite to assess the potential risk of adverse health effects in populations living in the vicinity

of hazardous waste sites.

Exposure Levels in Humans. Humans are exposed to nitrate and nitrite primarily through the

ingestion of drinking water and consumption of food. Estimated intakes are available (Gangolli et al.

1994; IARC 2010). Biomonitoring data for nitrate levels in urinary samples have been reported (CDC

2013). Continued monitoring of nitrate and nitrite levels in humans is needed.

This information is necessary for assessing the need to conduct health studies on these populations.

Exposures of Children. Children are exposed to nitrate and nitrite by the same exposure routes as

adults (e.g., ingestion of food and water). Data from the NHANES survey discussed in Section 6.5

indicated that higher urinary nitrate levels were typically observed in children as compared to adults.

Continued monitoring of nitrate and nitrite levels in children is needed.

Child health data needs relating to susceptibility are discussed in Section 3.12.2, Identification of Data

Needs: Children’s Susceptibility.

Exposure Registries. No exposure registries for nitrate or nitrite were located. This substance is not

currently one of the compounds for which a sub-registry has been established in the National Exposure

Registry. The substance will be considered in the future when chemical selection is made for sub-

registries to be established. The information that is amassed in the National Exposure Registry facilitates

the epidemiological research needed to assess adverse health outcomes that may be related to exposure to

this substance.

215 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

6.8.2 Ongoing Studies

No ongoing environmental fate studies for nitrate or nitrite were identified using NIH RePORTER or the

Defense Technical Information Center (DTIC) online database. Nitrate and nitrite levels and trends are

monitored in major watersheds and drinking water by organizations such as the USGS and USDA. These

reports are typically available from their websites.

216 NITRATE AND NITRITE

6. POTENTIAL FOR HUMAN EXPOSURE

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217 NITRATE AND NITRITE

7. ANALYTICAL METHODS

The purpose of this chapter is to describe the analytical methods that are available for detecting,

measuring, and/or monitoring nitrate and nitrite, and other biomarkers of exposure and effect to nitrate

and nitrite. The intent is not to provide an exhaustive list of analytical methods. Rather, the intention is

to identify well-established methods that are used as the standard methods of analysis. Many of the

analytical methods used for environmental samples are the methods approved by federal agencies and

organizations such as EPA and the National Institute for Occupational Safety and Health (NIOSH). Other

methods presented in this chapter are those that are approved by groups such as the Association of

Official Analytical Chemists (AOAC) and the American Public Health Association (APHA).

Additionally, analytical methods are included that modify previously used methods to obtain lower

detection limits and/or to improve accuracy and precision.

7.1 BIOLOGICAL MATERIALS

Several methods are available for the analysis of nitrate and nitrite in biological media; details of selected

methods are provided in Table 7-1.

Following the ingestion of nitrate and nitrite, they are readily absorbed from the upper gastrointestinal

tract into the blood and readily excreted in human urine as nitrate. This process is essentially complete at

18 hours following ingestion; minor urinary products of nitrate and nitrite metabolism include ammonia

and urea (Gangolli et al. 1994). Portions of blood nitrate are transported to human saliva where it is

mostly metabolized to nitrite. In human blood and tissues, nitrite is typically oxidized to nitrate.

Concentrations of nitrate in urine and saliva fluctuate; therefore, in order to evaluate exposure more

precisely, a 24-hour collection of urine is recommended. Analysis is achieved via hydrazine reduction

(IARC 2010; Levallois et al. 2000).

Levels of nitrate and nitrite in plasma, urine, and saliva can be measured by gas chromatography/mass

spectrometry (GC/MS) (Bondonno et al. 2012; Tsikas 2005). Frozen samples are treated with

tetraoctylammonium bromide and derivatizing reagent pentafluorobenzyl bromide in acetone solutions at

elevated temperature. Acetone is removed by evaporation under a nitrogen atmosphere and the remaining

aqueous phase is extracted with an isooctane/toluene solution and analyzed by GC/MS (m/z = 62 for

nitrate and 46 for nitrite). Sample procedures must involve precautionary steps to minimize the

218 NITRATE AND NITRITE

7. ANALYTICAL METHODS

Table 7-1. Analytical Methods for Determining Nitrate and Nitrite in Biological

Materials

Sample

Analytical

Sample

Percent

matrix

Preparation method

method

detection limit

recovery

Reference

Urine

Hydrazine reduction

GC-MS

5 ng/L

Not reported

Levallois et al.

2000

Plasma,

Sample derivitization

GC-MS

Not reported

Bondonna et al.

urine, or with tetraoctyl- 2012

saliva ammonium bromide

and pentafluorobenzyl

bromide

Plasma

Deproteinization;

HPLC-UV

0.3–20 µM

Not reported

Hibbs et al.

(serum) Griess reaction spectrometry; (nitrite) 1992; Sun et al.

GC-MS

4–

81 µM (nitrate)

2003; Tsikas

2005

Urine

Griess

GC-MS

690 µmol/

Not reported

Hibbs et al. 1992

24 hours

Urine

International standard

IC-MS/MS

500 µg/L (nitrate)

95

Valentín-Blasini

dilution

et al. 2007

Urine

24-Hour specimen

Colorimetric

Not reported

Tietz 1970

(metabolite- collected, preserve (Berthelot

ammonia) with HCl and

reaction)

refrigerate

Urine

24-Hour specimen

Indophenol

Not reported

Huizenga et

(metabolite-

analyzed immediately, reaction

al. 1994)

ammonia)

or stored up to 8

weeks at -20 °C

Whole blood

Deprotonized using

ace

tonitrile followed by

purification

HPLC/direct

conductivity

detection

0.4 µmol/L

(nitrite)

Yan et al. 2016

GC-MS = gas chromatography-mass spectrometry; HPLC = high-performance liquid chromatography;

IC-MS/MS = ion chromatography-mass spectrometry/mass spectrometry; UV = ultraviolet

219 NITRATE AND NITRITE

7. ANALYTICAL METHODS

endogenous contribution of analytes in the laboratory chemicals and materials (glassware, filtration

equipment, etc.) used for sample collection and work up (Tietz 1970; Tsikas 2005). Chemical

interferences in samples resulting in reduction of nitrate to nitrite, or conversely, the oxidation of nitrite to

nitrate during analysis should be evaluated and accounted for. Microbial conversion via hydrolysis may

cause an increase in values (Tietz 1970). Preparation of blood samples must involve procedures that limit

the oxidation of nitrite by oxyhaemoglobin and loss due to methods requiring acidification or

derivatization. Possible interferences in ammonia quanitification and to the Griess assay, such as

anticoagulants, must also be factored (Huizenga 1994; Tsikas 2005).

The Griess assay is one of the first methods used to measure levels of nitrate and nitrite in biological and

environmental samples. The method involves reduction of nitrate to nitrite followed by a diazotization

reaction and then measuring the absorbance of the diazo chromophore in the visible spectrum. To

determine the levels of nitrate and nitrite separately, the procedure is first carried out without the

preliminary reduction step in order to quantify the level of nitrite solely. This assay was originally

performed using sulfanilic acid, which forms a diazonium cation with nitrite under acidic conditions

followed by coupling with α-naphthylamine to form a diazo compound, which contains a strong

absorption band at about 540 nm (Tsikas 2005). Other methods include diazotizing with sulfanilamide

and coupling with N-(1-naphthyl)-ethylenediamine dihydrochloride to form the diazo compound (EPA

1993). GC/MS methods were shown to provide superior quantification of nitrate and nitrite in human

plasma and urine samples when compared to the Griess assays (Tsikas 2005)

Reverse-phase high performance liquid chromatography (HPLC) by means of ion pairing in the mobile

phase without derivitization followed by ultraviolet (UV) detection around 210 nm has also been used to

detect nitrate in urine samples (Tsikas 2005). Urinary nitrate levels can be measure using ion

chromatography-tandem mass spectrometry (IC-MS/MS) by means of internal standard dilution

(Valentín-Blasini et al. 2007).

The level of methemoglobin in the blood is often the biomarker for assessing nitrate exposure

(Manassaram et al. 2010). Methemoglobin can be measured in blood collected via finger stick samples.

Samples are analyzed with portable AVOXimeter 4000 whole-blood oximeter devices. The device

measures total hemoglobin, and further characterizes percentages of oxyhemoglobin, carboxyhemoglobin,

and methemoglobin. The accuracy and precision of the method were reported as ±0.5 and ±0.7%,

respectively. Refer to ATSDR (Agency for Toxic Substance and Disease Registry 2013b) for discussion

of other nitrate and nitrite laboratory tests.

220 NITRATE AND NITRITE

7. ANALYTICAL METHODS

Yan et al. (2016) developed a simple method for the quantitative determination of nitrite in whole blood

samples employing ion chromatography and electrochemical detection. The blood sample is prepared by

adding acetonitrile followed by purification using mini-cartridges to remove interfering compounds. The

detection limit for the method is reported as 0.4 µmol/L.

7.2 ENVIRONMENTAL SAMPLES

Methods are available for determining the level of nitrate and nitrite in a variety of environmental

matrices. A summary of representative methods is shown in Table 7-2.

Ion chromatography and spectrometry methods are the most common analytical techniques employed for

the detection and quantification of nitrate and nitrite in environmental samples; detection limits range

from 0.01 to 1 mg/L (ppm) (IARC 2010; WHO 2011b). Samples must be analyzed as soon as is

reasonably possible in order to minimize any changes in the sample due to microbial transformations.

Sample preservation using chemicals and or deep freezing methods have been reported; however,

interference with the analysis can occur in certain methods (Mulvaney 1996).

Methods based on the Griess assay are available for the determination of nitrate and nitrite in potable

water, raw water and wastewater (EPA 1993; WHO 2011b). The limit of detection for the International

Organization for Standardization ISO method 6777/1 lies within the range of 0.005–0.01 mg/L (ppm)

(WHO 2011b). A continuous-flow spectrometric method (ISO method 7890-1) for the determination of

nitrite, nitrate or the sum of both in various types of water is suitable at concentrations ranging from

0.05 to 5 mg/L (ppm) for nitrite and from 1 to 100 mg/L (ppm) for nitrite and nitrate, both in the

undiluted sample (WHO 2011b).

NIOSH method 7903 employs ion chromatography for the determination of nitric acid in air (NIOSH

1994a). Method 7903 is an analytical technique for determining inorganic acids by measuring the total

concentration of airborne anions. Particulate nitrate has been successfully detected and quantified in

atmospheric samples via ion chromatographic techniques and NO

x

chemiluminescent analyzers (Small et

al. 1975; Yoshizumi et al. 1985)

221 NITRATE AND NITRITE

7. ANALYTICAL METHODS

Table 7-2. Analytical Methods for Determining Nitrate and Nitrite in

Environmental Samples

Sample

Analytical

Sample

Percent

matrix

a

Preparation method

method

detection limit

recovery

Reference

Air/water

Nitrite prepared using

(nitrate, Griess-Ilosvay reaction;

nitrite) nitrate prepared using

hydrazine reduction;

aqueous extracts from

aerosol filters are

analyzed without

pretreatment

Air (nitrate)

Personal air sampled at

0.2–0.5 L/minute for

total sample size of 3–

100 L using silica gel

sample tube; boil

sorbent from sample

tube in bicarbonate/

carbonate buffer for

10 minutes

Air (nitrite)

Ambient air is sampled

at 0.025 L/minute for 3-

L

air sample using glass

sorbent tubes with glass

wool retainers; add

adsorbing solution; add

solution of hydrogen

peroxide, sulfanilamide

and NEDA to extracted

sample, set for

10 minutes

Air (nitrite)

Ambient air is sampled

at 0.025 L/minute for 3-

L

air sample using

diffusive sampler tubes

with three

triethanolamine screens;

add adsorbing solution;

add solution of hydrogen

peroxide, sulfanilamide

and NEDA to extracted

sample, set for

10 minutes

Water

Drinking water or river

(nitrate) water samples are

prepared with

lanthanum (III) chloride

and placed into the cell

UV spectrometry

0.07 ppm

Not

(nitrite); reported

0.2 ppm

(nitrate)

Ion

0.7 μg/sample

Not

chromatography/ reported

conductivity

detector;

NIOSH 7903

Visible

1 μg/sample

Not

absorption reported

spectro-

photometry;

NIOSH 6014

Visible

0.01 μg/sample

Not

absorption reported

spectro-

photometry;

NIOSH 6700

Voltammetry/

20 µg/L

Not

static mercury reported

drop electrode

Oms et al. 1995

NIOSH 1994a

NIOSH 1994b

NIOSH 1998

Markusova et al.

1996

222 NITRATE AND NITRITE

7. ANALYTICAL METHODS

Table 7-2. Analytical Methods for Determining Nitrate and Nitrite in

Environmental Samples

Sample

Analytical

Sample

Percent

matrix

a

Preparation method

method

detection limit

recovery

Reference

Drinking

Clean samples can be

IC/CD

Not reported

93–114

IARC 2010

water used directly (nitrite-N)

83–113

(nitrate-N)

Soil (nitrate)

Soil samples are added

Voltammetry/

20 µg/L

Not

Markusova et al.

to Gohler solutions static mercury reported 1996

followed by drop electrode

neutralization before

analysis

Soil (nitrite,

Extraction of field

UV-Vis

Not reported

Not

Mulvaney 1996

nitrate) samples with 2 M KCL; (λ=540 nm) reported

nitrate reduction;

diazoitization and

coupling with resulting

dye formation

Foods and

Blended/pureed food

Capillary ion

Not reported

>73

Marshall and

juices samples are mixed with electrophoresis (nitrate); Trenerry 1996

(nitrate, water and filtered >88

nitrite)

(nitrite)

Food (nitrate,

Direct injection

HePI-MS

Not reported

Not

Pavlov and

nitrite)

reported

Attygalle 2013

Fish and

Fish muscle

FIA spectrometry

0.01 µg/mL

97.8–

Monser et al. 2002

water homogenized, digested (nitrite); 102.1

(nitrate, with perchloric acid and 0.025 µg/mL (nitrite);

nitrite) centrifuged; nitrate (nitrate) 98.5–

reduction to nitrite using 101.6

copperised cadmium (nitrate)

redactor; reaction with

phosphomolybdenum

blue complex and

ammonium chloride

Water and

Food: homogenization

Potentiometry

0.0037 µ/mL

98.9–

Wardak and

vegetables with deionized water with solid contact

(nitrate)

105.9 Grabarczky 2016

(nitrate)

and heated at 80°C;

ISE (nitrate)

cooled and diluted with

deionized water

Water: direct analysis

Cured meat

Reaction with

Colorimetry;

Not reported

Not

IARC 2010

(nitrite) sulfanilamide followed absorbance reported

by reaction with NEDA

540 nm

Milk and milk

Suspension in buffer

FIA

0.5 mg/kg

Not

IARC 2010

products solution; centrifuge and spectrometry; (nitrate) reported

(nitrate, reduce with cadmium; absorbance 1.0 mg/kg

nitrite) react with sulfanilamide 540 nm (nitrite)

followed by react

ion with

NEDA

223 NITRATE AND NITRITE

7. ANALYTICAL METHODS

Table 7-2. Analytical Methods for Determining Nitrate and Nitrite in

Environmental Samples

Sample

Analytical

Sample

Percent

matrix

a

Preparation method

method

detection limit

recovery

Reference

Dairy

Extract cheese slurry

Spectro-

≥1 µg/g nitrate

Not

IARC 2010

products and using ZnSO

4

and NaOH;

photometry; reported

cheese reduce in Jones absorbance at

(nitrate, reductor using zinc and 522 nm

nitrite)

CdSO

4

Fried bacon

Grind frozen sample;

GC

Not reported

Not

IARC 2010

(N-nitros- vacuum distill with reported

amines) NaOH and mineral oil;

extract and dry with

DCM and anhydrous

NaSO

4

; concentrate

DCM = dichloromethane; FIA = flow injection analysis; GC = gas chromatography; HePI-MS = helium-plasma

ionization-mass spectrometry; IC/CD = ion chromatography/conductivity detector; ISE = ion-selective electrodes;

NEDA = N-1-naphthylethylenediamine dihydrochloride; NIOSH = National Institute for Occupational Safety and

Health; UV = ultraviolet absorbance detection

224 NITRATE AND NITRITE

7. ANALYTICAL METHODS

A sequential injection method coupled with spectrophotometry has been developed for the detection of

nitrate and nitrite in environmental samples such as atmospheric aerosol filter extracts and waste water

samples (Oms et al. 1995). The method is advantageous due to the small volumes of sample and reagents

required for analysis. The detection limits were reported as 0.07 ppm for nitrite and 0.2 ppm for nitrate.

Nitrite is analyzed using the Griess-Ilosvay reaction; nitrate is reduced to nitrite using hydrazine sulphate.

Markusova et al. (1996) developed a sensitive voltammetric method that can determine nitrate levels in

drinking water, river water, or soil extracts three orders of magnitude lower than the allowed levels of

nitrate in drinking water. The method employs a multi-purpose electrochemical analyzer and a

voltammetric cell. Water samples are prepared with lanthanum (III) chloride and placed into the cell; soil

samples are added to Gohler solutions followed by neutralization before analysis. The reported limit of

detection is 20 µg nitrate/L (20 ppb) (5 µg NO

3

—

N/L).

The most commonly used method for soil and soil extract analysis of nitrite is a modified Griess-Ilosvay

colorimetric method using a continuous flow analyzer. Nitrites react with primary aromatic amines to

form a diazonium salt which is then coupled with an aromatic compound; the resulting complex has a

characteristic absorbance band in the UV-Vis spectrum. The concentration of nitrite is proportional to the

color intensity of the resulting azo compound measured using a spectrophotometer or colorimeter. This

technique is also used for sensitive analysis of nitrate following reduction to nitrite. Cadmium reduction

to nitrite is achieved in a column of copperized cadmium with an ammonium chloride (NH4Cl) matrix at

pH between 5 and 10. Other various reducing agents have been reported. Analysis for nitrate must

account for initial concentrations of nitrite in the sample prior to reduction. Maximum accuracy is seen

when absorbance is measured at wavelengths of 540 nm; however, wavelengths between 510 and 550 nm

are acceptable (Mulvaney 1996).

Pavlov and Attygalle (2013) developed an analytical method with minimal sample preparation employing

helium-plasma ionization-mass spectrometry. Nitrate was successfully identified and quantified using

this solvent-less ambient pressure mass spectrometry technique in various foodstuffs. Samples of fruit

juice and meat pieces (i.e., tomato and celery juice, hot dog and beef) can be placed onto glass slides and

analyzed directly without any modification. Quantification of nitrate in such complex matrices is

suggested to be determined with accuracy by spiking with known quantities of radiolabeled nitrate. The

method detection limit for determining the nitrate concentration is in the range of 20 ng/sample and

depends on the specific sample matrix.

225 NITRATE AND NITRITE

7. ANALYTICAL METHODS

Capillary ion electrophoresis has been successfully employed for the determination of nitrite and nitrate in

foods and juices (Marshall and Trenerry 1996). The authors tested the procedure using cheese, cabbage,

fruit juices, and meats. Percent recovery for three processed meat samples ranged from 88 to 118% for

nitrite and from 73 to 106% for nitrate.

7.3 ADEQUACY OF THE DATABASE

Section 104(i)(5) of CERCLA, as amended, directs the Administrator of ATSDR (in consultation with the

Administrator of EPA and agencies and programs of the Public Health Service) to assess whether

adequate information on the health effects of nitrate and nitrite is available. Where adequate information

is not available, ATSDR, in conjunction with NTP, is required to assure the initiation of a program of

research designed to determine the health effects (and techniques for developing methods to determine

such health effects) of nitrate and nitrite.

The following categories of possible data needs have been identified by a joint team of scientists from

ATSDR, NTP, and EPA. They are defined as substance-specific informational needs that if met would

reduce the uncertainties of human health assessment. This definition should not be interpreted to mean

that all data needs discussed in this section must be filled. In the future, the identified data needs will be

evaluated and prioritized, and a substance-specific research agenda will be proposed.

7.3.1 Identification of Data Needs

Methods for Determining Biomarkers of Exposure and Effect.

Exposure. Nitrate and nitrite may be converted to many other compounds in the body, such as N-nitroso

compounds, including nitrosamines. Approximately 25% of absorbed nitrate is secreted to saliva and

about 20% of this is reduced to nitrite. Nitrite is converted to nitric oxide by the acidic environment on

the stomach. Methods exist for the measurement of nitroso compounds and nitrite in plasma and salivary

nitrite (Bondonno et al. 2012). Nitrate in the diet may contribute to nitric oxide levels in the body, and

increases in these levels can be a biomarker of exposure. Ammonia is a minor urinary product of nitrite

and nitrate in which analytical methods are available (Huizenga et al. 1994; Tietz 1970). N-Methyl-

nicotinamide has also been shown to be a potential biomarker of exposure to nitrate and nitrite and there

are methods to measure this (Jansen et al. 1995). No data needs were identified.

226 NITRATE AND NITRITE

7. ANALYTICAL METHODS

Effect. Methemoglobinemia caused by the presence of higher-than-normal levels of methemoglobin is a

biomarker of effect for exposure to high levels of nitrate; however, this effect is not unique for nitrate and

nitrite since other substances may also cause this condition (Bruning-Fann and Kaneene 1993). Methods

are available to measure methemoglobin in the blood (Manassaram et al. 2010).

Methods for Determining Parent Compounds and Degradation Products in Environmental

Media. Methods are available for determining nitrate and nitrite levels in environmental samples such

as air (NIOSH 1994a; Small et al. 1975; Yoshizumi et al. 1985) and water (EPA 1993; Markusova et al.

1996; WHO 2011b).

7.3.2 Ongoing Studies

No ongoing analytical methodologies for nitrate or nitrite were identified using the NIH RePORTER

version 6.1.0 or the DTIC online database.

227 NITRATE AND NITRITE

8. REGULATIONS, ADVISORIES, AND GUIDELINES

MRLs are substance-specific estimates that are intended to serve as screening levels. They are used by

ATSDR health assessors and other responders to identify contaminants and potential health effects that

may be of concern at hazardous waste sites.

MRLs of 4 mg nitrate/kg/day have been derived for acute-, intermediate, and chronic-duration oral

exposure (≤14 days) to nitrate. The MRLs are based on a no-adverse-effect concentration (NOAEC) of

10 mg nitrate-nitrogen/L (44 mg nitrate/L) in drinking water used to prepare formula for infants

<6 months of age (Walton 1951). A NOAEL of 4.33 mg nitrate/kg/day at the NOAEC of 44 mg nitrate/L

was calculated based on estimates of 0.525 L/day for water intake (Kahn and Stralka 2009) and 5.33 kg

for body weight (EPA 2008) of an infant from birth to <3 months of age. A total uncertainty factor of 1

was applied because the point of departure is a NOAEL for nitrate-induced effects on methemoglobin in a

sensitive human subpopulation (i.e., <3-month-old infants, which in many cases may have been at

increased risk of methemoglobinemia due to microbial contamination and associated gastrointestinal

infection). Following ingestion of relatively large amounts of nitrate by healthy normal individuals,

blood methemoglobin levels increase rapidly, followed by a return to normal within several hours

following intake. Repeated ingestion for intermediate- or chronic-duration time periods would be

expected to result in changes in methemoglobin levels similar to those elicited from a single exposure.

Therefore, the acute-, intermediate- and chronic-duration oral MRL values are equivalent. Refer to

Appendix A for additional information regarding derivation of oral MRLs for nitrate.

MRLs of 0.1 mg nitrite/kg/day have been derived for acute-, intermediate, and chronic-duration oral

exposure (≤14 days) to nitrite. The ingestion of nitrate results in the formation of nitrite, which is the

moiety responsible for methemoglobinemia. In adults, approximately 5% of an oral dose of nitrate is

reduced to nitrite in the saliva, most of which is absorbed into the blood in the small intestine. Based on

the assumption of 100% absorption of ingested nitrite, an oral dose of 0.2 mg nitrite/kg/day by an adult

would be expected to result in a nitrite blood level similar to that achieved following ingestion of nitrate

at the oral MRL dose of 4 mg nitrate/kg/day (i.e., 0.2 mg nitrite/kg/day is 5% of an oral dose of nitrate at

the MRL of 4 mg nitrate/kg/day). A modifying factor of 2 was applied to the point of departure (0.2 mg

nitrite/kg/day ÷ 2 = 0.1 mg nitrite/kg/day) because young infants exhibit increased susceptibility to

methemoglobinemia following nitrate ingestion; the modifying factor assumes that the effective

methemoglobin level from a given intake of nitrate by an infant is up to twice that of an adult. Following

ingestion of relatively large amounts of nitrate by healthy normal individuals, blood methemoglobin

228 NITRATE AND NITRITE

8. REGULATIONS, ADVISORIES, AND GUIDELINES

levels increase rapidly, followed by a return to normal within several hours following intake. Repeated

ingestion for intermediate- or chronic-duration time periods would be expected to result in changes in

methemoglobin levels similar to those elicited from a single exposure. Therefore, the acute-,

intermediate-, and chronic-duration oral MRL values are equivalent. Refer to Appendix A for additional

information regarding derivation of oral MRLs for nitrite.

EPA (IRIS 2002) derived an oral reference dose (RfD) of 1.6 mg nitrate-nitrogen/kg/day (i.e., 1.6 mg

nitrogen from nitrate; ~7 mg nitrate/kg/day) based on a NOAEL of 1.6 mg nitrate-nitrogen/kg/day and a

LOAEL of 1.8–3.2 mg nitrate-nitrogen/kg/day (7.92–14.08 mg nitrate/kg/day) for early clinical signs of

methemoglobinemia in excess of 10% among formula-fed infants 0–3 months of age (Bosch et al. 1950;

Walton 1951). An uncertainty factor of 1 was employed because available data defined the NOAEL for

the critical effect in the most sensitive human subpopulation.

EPA (IRIS 2002) derived an RfD of 0.1 mg nitrite-nitrogen/kg/day (~0.33 mg nitrite/kg/day) based on a

NOAEL of 10 mg nitrate-nitrogen/L and a LOAEL of 11–20 mg nitrate-nitrogen/L for early clinical signs

of methemoglobinemia in excess of 10% (Walton 1951). The NOAEL of 10 mg nitrate-nitrogen/L was

converted to an estimated dose of 1 mg nitrate-nitrogen/kg/day based assumptions that a 10-kg child

would ingest 1 L of water/day. EPA applied a modifying factor of 10 to the NOAEL of 1 mg nitrate-

nitrogen/kg/day from the Walton (1951) study to account for the direct toxicity of nitrite, resulting in an

RfD of 0.1 mg nitrite-nitrogen/kg/day. As described in a Drinking Water Criteria Document for

Nitrate/Nitrite (EPA 1990a), the modifying factor of 10 was used to account for an estimated rate of 10%

conversion of ingested nitrate to nitrite in infants compared to an estimated rate of 5% conversion in

adults.