Humans have altered the nitrogen cycle

dramatically over the last half-century, and as a

result, nitrate is steadily accumulating in our

water resources. Globally, human nitrogen

production has increased rapidly since 1950

and currently exceeds nitrogen fixed by natural

sources by about 30% (Fields 2004). This figure compares with pre-1950 human inputs,

which were a small fraction of the input from

natural sources (Lambert and Driscoll 2003).

Fertilizer is the largest contributor to anthropogenic nitrogen worldwide; other major

sources include animal and human waste,

nitrogen oxides from utilities and automobiles,

and leguminous crops that fix atmospheric

nitrogen (Fields 2004). These organic and

inorganic sources of nitrogen are transformed

to nitrate by mineralization, hydrolysis, and

bacterial nitrification. Under reducing conditions, nitrate can be biologically transformed

to nitrogen gas through denitrification. Nitrate

not taken up by plants or denitrified migrates

to streams and groundwater.

The U.S. Environmental Protection

Agency (EPA) maximum contaminant level

(MCL) for nitrate in drinking water of

10 mg/L nitrate-nitrogen (nitrate-N) (equivalent to 45 mg/L as nitrate) and the World

Health Organization (WHO) guideline (WHO

2004b) of 50 mg/L as nitrate (equivalent to

11 mg/L as nitrate-N) were promulgated to

protect against methemoglobinemia, or “blue

baby syndrome,” to which infants are especially

susceptible. The regulatory level is usually met

for public water supplies, which are routinely

monitored. Much less is known about private

wells, which in the United States are usually

required to be tested only when the well is constructed or when the property is sold. Some

have suggested recently that the regulatory level

for nitrate in drinking water is overly conservative (Avery 1999; L’hirondel and L’hirondel

2002). However, this discussion of the regulatory level has not thoroughly considered studies

of other chronic health effects including cancer,

adverse reproductive outcomes, and diabetes.

Although a causal role for nitrate in these other

health outcomes is not conclusive, recent studies

that indicate possible adverse effects at nitrate

levels below the MCL are of concern (Brender

et al. 2004b; DeRoos et al. 2003; Ward et al.

1996; Weyer et al. 2001).

In recognition of the widespread contamination of drinking-water sources by nitrate

and the potential for health effects in addition

to methemoglobinemia, a symposium titled

“Drinking Water Nitrate and Health: Recent

Findings and Research Needs” took place at

the annual meeting of the International

Society for Environmental Epidemiology

(1–4 August 2004, New York, New York,

USA). Invited experts presented results from

recent unpublished studies and summarized

the state of knowledge on exposure and

health effects of drinking-water nitrate, with a

focus on cancer and adverse reproductive outcomes. This article summarizes the symposium discussions and recommends promising

areas for future research. Specifically, we discuss the epidemiologic evidence for drinkingwater nitrate and risk of specific cancers,

adverse reproductive outcomes, and other

health outcomes in the context of the current

regulatory limit for nitrate in drinking water.

Nitrate Levels in Groundwater

and Water Supplies

Nitrate is the most common chemical contaminant in the world’s groundwater aquifers

(Spalding and Exner 1993). An estimated 42%

of the U.S. population uses groundwater as their

drinking-water supply (Hutson et al. 2004). In

the United States, total nitrogen in streams and

nitrate in groundwater are highest in agricultural areas, followed by urban areas and areas

with mixed land use (Figure 1). The most

recent data indicate that about 22% of domestic

wells in agricultural areas of the United States

exceeded the MCL (U.S. Geological Survey,

unpublished data). In contrast, 3% of public

supply wells in major aquifers (typical sources

for public water supplies) exceed the MCL

(U.S. Geological Survey, unpublished data).

Environmental Health Perspectives • VOLUME 113 | NUMBER 11 | November 2005 1607

Research

Address correspondence to M.H. Ward, Occupational

and Environmental Epidemiology Branch, Division of

Cancer Epidemiology and Genetics, National Cancer

Institute, 6120 Executive Blvd., EPS 8104, Bethesda,

MD 20892 USA. Telephone: (301) 435-4713. Fax:

(301) 402-1819. E-mail: wardm@mail.nih.gov

We thank K. Cantor of the National Cancer Institute

for his review of the manuscript.

The authors declare they have no competing

financial interests.

Received 25 February 2005; accepted 23 June 2005.

Workgroup Report: Drinking-Water Nitrate and Health—Recent Findings and

Research Needs

Mary H. Ward,1 Theo M. deKok,2 Patrick Levallois,3 Jean Brender,4 Gabriel Gulis,5 Bernard T. Nolan,6 and 

James VanDerslice7

1Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Department of Health and

Human Services, Bethesda, Maryland, USA; 2Department of Health Risk Analysis and Toxicology, University of Maastricht, the

Netherlands; 3Institut National de Santé Publique du Québec and Unité de recherche en santé publique, Centre Hospitalier Universitaire

de Québec, Québec, Canada; 4Department of Health Services Research, Texas State University, San Marcos, Texas, USA; 5Department

of Health Promotion Research, Southern Denmark University and Department of Public Health, University of Trnava, Slovakia; 6U.S.

Geological Survey, Reston, Virginia, USA; 7Washington State Department of Health, Olympia, Washington, USA

Human alteration of the nitrogen cycle has resulted in steadily accumulating nitrate in our water

resources. The U.S. maximum contaminant level and World Health Organization guidelines for

nitrate in drinking water were promulgated to protect infants from developing methemoglobinemia, an acute condition. Some scientists have recently suggested that the regulatory limit for

nitrate is overly conservative; however, they have not thoroughly considered chronic health outcomes. In August 2004, a symposium on drinking-water nitrate and health was held at the

International Society for Environmental Epidemiology meeting to evaluate nitrate exposures and

associated health effects in relation to the current regulatory limit. The contribution of drinkingwater nitrate toward endogenous formation of N-nitroso compounds was evaluated with a focus

toward identifying subpopulations with increased rates of nitrosation. Adverse health effects may

be the result of a complex interaction of the amount of nitrate ingested, the concomitant ingestion

of nitrosation cofactors and precursors, and specific medical conditions that increase nitrosation.

Workshop participants concluded that more experimental studies are needed and that a particularly fruitful approach may be to conduct epidemiologic studies among susceptible subgroups with

increased endogenous nitrosation. The few epidemiologic studies that have evaluated intake of

nitrosation precursors and/or nitrosation inhibitors have observed elevated risks for colon cancer

and neural tube defects associated with drinking-water nitrate concentrations below the regulatory

limit. The role of drinking-water nitrate exposure as a risk factor for specific cancers, reproductive

outcomes, and other chronic health effects must be studied more thoroughly before changes to the

regulatory level for nitrate in drinking water can be considered. Key words: adverse reproductive

outcomes, methemoglobinemia, neoplasms, nitrate, nitrite, N-nitroso compounds, water pollution. Environ Health Perspect 113:1607–1614 (2005). doi:10.1289/ehp.8043 available via

http://dx.doi.org/ [Online 23 June 2005]
The exposure picture is similar in the

European Union. Public water supplies are

largely below the WHO guideline; however, in

some countries, private wells in rural areas have

elevated nitrate concentrations reaching

10–15 times the recommended level (European

Environment Agency 2003). Overall, nitrate

levels exceeded the guideline in about one-third

of the groundwater bodies for which data were

available (European Environment Agency

2003). Several eastern European countries

report high levels of nitrate contamination in a

large proportion of private wells; for example,

in Romania, 20% of 2,000 wells had nitrate

levels > 23 mg/L as nitrate-N (Jedrychowski

et al. 1997). Studies from other countries,

including China, Botswana, Turkey, Senegal,

and Mexico, report private well water levels that

exceed the WHO guideline, in some instances

at levels > 68 mg/L nitrate-N (WHO 2004a).

Fertilizer is the main contributing factor in agricultural areas; however, nitrogen from human

waste appears to be the most important source

in urban areas lacking centralized water and

sanitation systems. Although systematic information on nitrate levels in groundwater in

other parts of the world is more limited, empirical modeling approaches have indicated that

users of shallow wells in areas with high nitrogen inputs, well-drained soils, and unconsolidated rocks are most at risk of consuming

high-nitrate groundwater (Nolan et al. 2002).

Methemoglobinemia

Ingested nitrate is reduced to nitrite, which

binds to hemoglobin to form methemoglobin

(MetHb). Methemoglobinemia occurs when

elevated levels of MetHb (exceeding about

10%) interfere with the oxygen-carrying

capacity of the blood. Infants are particularly

susceptible to developing methemoglobinemia

for several reasons, including their increased

capacity to convert nitrate to nitrite and their

lower levels of the enzyme cytochrome b5

reductase, which converts MetHb back to

hemoglobin. Methemoglobinemia in infants

fed formula made with well water with high

nitrate levels was first reported in 1945 by

Comly (1945). The regulatory level for nitrate

in drinking-water supplies was determined after

a survey of infant methemoglobinemia case

reports in the United States indicated that no

cases were observed at drinking-water nitrate

levels < 10 mg/L nitrate-N (Walton 1951).

Because an estimated 22% of domestic wells in

agricultural regions of the United States exceed

the nitrate MCL (U.S. Geological Survey,

unpublished data), it is likely that significant

numbers of infants are given water containing

> 10 mg/L nitrate-N. Nevertheless, few cases of

methemoglobinemia have been reported since

the MCL was promulgated.

The risk of methemoglobinemia among

infants depends on many factors other than

the ingestion of nitrate in drinking water.

Some foods and medications contain high

levels of nitrate (Sanchez-Echaniz et al. 2001).

Enteric infections, potentially caused by fecal

bacteria contamination in wells, may lead to

the endogenous production of nitrite, as evidenced by numerous published reports of

infants with diarrhea and methemoglobinemia but no apparent exposure to exogenous

MetHb-forming agents (Charmandari et al.

2001; Hanukoglu and Danon 1996; Levine

et al. 1998; Wennmalm et al. 1993). The

consumption of antioxidants such as vitamin

C appears to be a protective factor. Finally,

polymorphisms in the activity of cytochrome

b5 reductase may mediate the effect of

ingested nitrate or endogenously produced

nitrite (Gupta et al. 1999).

Studies that have examined the relationship between nitrate levels in drinking water

and MetHb levels in infants have produced

mixed results (U.S. EPA 1991). The few

experimental studies are largely negative; however, most of these studies evaluated low levels

of drinking-water nitrate and included few

infants. Cofactors such as diarrhea and respiratory diseases reportedly increase MetHb levels

(Shearer et al. 1972; Shuval and Gruener

1972). An epidemiologic study in South

Africa (Super et al. 1981) found an increase in

MetHb levels in infants fed water with nitrate

> 20 mg/L nitrate-N; however, clinical methemoglobinemia was rarely found. A protective

effect of vitamin C intake on MetHb was

noted (Super et al. 1981). More recently, a

retrospective, nested case–control study in

Romania found an association between nitrate

exposure from drinking water and clinical

methemoglobinemia, but also some evidence

of an association with diarrheal disease

(Zeman et al. 2002). Gupta and colleagues

(1999) found cytochrome b5 reductase activities to be higher among those consuming

water with high nitrate levels, indicating a

level of adaptation to the consumption of high

nitrate waters.

Recently, the role of nitrate exposure

alone in causing methemoglobinemia has

been questioned (Avery 1999; Fewtrell 2004;

Hanukoglu and Danon 1996). Clearly, we

need to better understand the interaction of

factors that lead to methemoglobinemia to

assess the relative importance of each factor

and to identify the conditions under which

exposure to nitrate in drinking water poses a

risk of methemoglobinemia.

Nitrate Intake and Endogenous

Formation of N-Nitroso

Compounds

Nitrate is a precursor in the formation of

N-nitroso compounds (NOC), a class of genotoxic compounds, most of which are animal

carcinogens. In the human body, nitrate is a

stable, inert compound that cannot be metabolized by human enzymes. However, the

nitrate-reducing activity of commensal bacteria may convert nitrate into nitrite and other

bioactive nitrogen compounds that affect

physiological processes and human health.

After ingestion, nitrate is readily absorbed

from the upper gastrointestinal tract. Up to

25% is actively excreted in saliva, where about

20% is converted to nitrite by bacteria in the

mouth (Spiegelhalter et al. 1976). This conversion can occur at other sites including the

distal small intestine and the colon.

Under acidic conditions in the stomach,

nitrite is protonated to nitrous acid (HNO2),

which in turn spontaneously yields dinitrogen

trioxide (N2O3), nitric oxide (NO), and nitrogen dioxide (NO2). NO is a bioactive compound known to play a role in vasodilatation

and in defense against periodontal bacteria and

other pathogens. N2O3, on the other hand, is a

powerful nitrosating agent capable of donating

NO+ to secondary and tertiary amines to form

potentially carcinogenic N-nitrosamines (Leaf

et al. 1989). Alternatively, HNO2 can be protonated to H2NO2, which reacts with amides to

form N-nitrosamides. At neutral pH, nitrite can

be reduced by bacterial activity to form NO,

which can react with molecular oxygen to form

the nitrosating compounds N2O3 and nitrogen

tetroxide (N2O4). In addition to the acid-catalyzed and bacterial-catalyzed formation of

nitrosating agents, inducible NO synthase

activity of inflammatory cells can also produce

NO (Ohshima and Bartsch 1994). Together,

these three mechanisms of endogenous nitrosation account for an estimated 40–75% of the

total human exposure to NOC (Tricker 1997).

Other sources of human exposure include preformed NOC found in preserved meats and

fish, beer, certain occupational exposures, and

tobacco products (Tricker 1997).

Several studies support a direct relationship between nitrate intake and endogenous

Ward et al.

1608 VOLUME 113 | NUMBER 11 | November 2005 • Environmental Health Perspectives

Figure 1. Interquartile range of total nitrogen in

streams and nitrate-N in groundwater in agricultural, urban, and mixed land use, and undeveloped

areas of the United States. Upper bound of bar represents 90th percentile and lower bound represents

10th percentile. Along the top of the graph are the

number of stream sampling stations and groundwater networks (group of wells in an aquifer).

12

10

8

6

4

2

0

95 6 118 55 57 34 122 96

Undeveloped Agricultural Urban Mixed

Dominant land use

Concentration (mg/L)

Streams

Groundwater
formation of NOC. High intake of drinkingwater nitrate (above the MCL) is associated

with an increased endogenous capacity to

nitrosate proline (Mirvish et al. 1992; Moller

et al. 1989). In addition, populations with

high rates of esophageal and gastric cancer

excrete high levels of N-nitrosoproline

(Kamiyama et al. 1987; Lu et al. 1986).

Nitrate intake at the acceptable daily intake

level (3.67 mg/kg body weight, 0.84 mg/kg as

nitrate-N) results in increased urinary excretion of NOC, particularly in combination

with increased intake of dietary nitrosatable

precursors (Vermeer et al. 1998). However, a

Canadian population exposed to nitrate below

the acceptable daily intake level showed no

relationship between nitrate levels in drinking

water and urinary nitrosamines (Levallois et al.

2000).

Factors that modulate endogenous nitrosation. Although intake of high drinkingwater nitrate is consistently associated with

endogenous nitrosation capacity, intake of

dietary nitrate is less likely to increase nitrosation, because of the presence of nitrosation

inhibitors in vegetables, the major contributors to dietary nitrate intake (Bartsch et al.

1988; National Academy of Sciences 1981).

Dietary compounds that inhibit endogenous

nitrosation include vitamin C, which has the

capacity to reduce HNO2 to NO, and alphatocopherol, which can reduce nitrite to NO.

Several epidemiologic studies reported no

association or inverse associations between

dietary nitrate intake and human cancers

(Boeing 1991; Forman 1987; Ward et al.

1996), which may be because of the antioxidants and nitrosation inhibitors in nitratecontaining foods (Bartsch et al. 1988).

Inhibitory effects on nitrosation have also

been described with betel nut extracts, ferulic

and caffeic acid, garlic, coffee, and green tea

polyphenols (Stich et al. 1984). In addition,

nondietary factors such as the use of mouthwashes containing chlorhexidine can influence the endogenous nitrosating capacity (van

Maanen et al. 1998).

Apart from the level of nitrosating agents,

the level of nitrosatable precursors in the diet,

which come predominantly from meat and

fish, is a crucial factor in endogenous nitrosation. Dietary intakes of red and processed

meat are of particular importance in the formation of fecal NOC (Bingham 1999;

Bingham et al. 1996, 2002; Cross et al. 2003;

Haorah et al. 2001). Higher consumption of

red meat (600 vs. 60 g/day), but not white

meat, resulted in a 3-fold increase in fecal

NOC levels (Bingham et al. 1996). Colon

cancer incidence is most consistently associated with consumption of red meat (beef,

lamb, and pork), but not with poultry and

fish (Bingham 1999). Dietary supplementation of a diet low in red meat with either heme

iron or inorganic iron demonstrated that heme

in particular was able to stimulate endogenous

nitrosation (Cross et al. 2003), thereby providing a possible explanation for the differences in

colon cancer risk between red and white meat

consumption. Additionally, this linkage may

be stronger for processed meat than for fresh

meat because of the higher NOC and NOC

precursor levels in processed meat.

Endogenous nitrosation can also be stimulated by inflammatory and other medical

conditions. For instance, patients with bilharzia have an increased bladder cancer risk

associated with increased urinary levels of

nitrite and volatile nitrosamines, most likely

generated by the reaction of inflammationderived NO with amines present in the urine

(Tricker et al. 1989). Inflammatory bowel

disease is also related to both increased nitrosation and cancer risk (Lashner et al. 1988).

During inflammatory bowel disease, increased

inducible NO synthase activity can produce

excess NO, which is oxidized to nitrogen

oxides and nitrite, which in turn react with

nitrosatable precursors in colonic contents to

produce NOC. Indeed, ulcerative colitis

patients showed increased levels of inducible

NO synthase in the colonic mucosa (Kimura

et al. 1998) and of NO and nitrite in the

colonic lumen (Lundberg et al. 1997;

Roediger et al. 1990). Increased levels of fecal

NOC have been found in patients with

inflammatory bowel disease and in mice with

chemically induced colitis (de Kok et al.

2005; Mirvish et al. 2003).

Health Effects Associated with

Drinking-Water Nitrate

Cancer. NOC are potent animal carcinogens,

inducing tumors at multiple organ sites

including the esophagus, stomach, colon,

bladder, lympatics, and hematopoietic system

(Bogovski and Bogovski 1981). NOC cause

tumors in every animal species tested, and it is

unlikely that humans are unaffected (Lijinsky

1986). The number of well-designed epidemiologic studies with individual exposure data

and information on nitrosation inhibitors and

precursors are few for any single cancer site,

limiting the ability to draw conclusions about

cancer risk.

Most studies have been ecologic in design,

linking incidence or mortality rates to drinking-water nitrate levels at the town or county

level. The early studies focused on stomach

cancer mortality, and most used drinkingwater nitrate measurements concurrent with

the period of cancer mortality. Results were

mixed, with some studies showing positive

associations, many showing no association, and

a few showing inverse associations (Cantor

1997). Recent ecologic studies of stomach cancer in Slovakia, Spain, and Hungary with historical measurements and exposure levels near

or above the MCL have found positive correlations with stomach cancer incidence or mortality (Gulis et al. 2002; Morales-Suarez-Varela

et al. 1995; Sandor et al. 2001). Two studies

included other cancer sites. In Slovakia, incidence of non-Hodgkin lymphoma (NHL) and

colon cancer was significantly elevated among

men and women exposed to public supply

nitrate levels of 4.5–11.3 mg/L nitrate-N

(Gulis et al. 2002); there was no association

with bladder and kidney cancer incidence. In

Spain, there was a positive correlation between

nitrate levels in public supplies and prostate

cancer mortality, but no relation with bladder

and colon cancer (Morales-Suarez-Varela et al.

1995).

In the past decade, several case–control

and cohort studies have evaluated historical

nitrate levels in public water supplies (largely

< 10 mg/L nitrate-N) and risk of several cancers (Table 1). Some studies evaluated factors

affecting nitrosation, such as vitamin C

intake. A cohort study of older women in

Iowa (USA) (Weyer et al. 2001) found a

2.8-fold and 1.8-fold risk of bladder and

ovarian cancers, respectively, associated with

the highest quartile (> 2.46 mg/L nitrate-N)

of the long-term average nitrate levels at the

current residence. They observed significant

inverse associations for uterine and rectal cancer and no significant associations for NHL,

leukemia, colon, rectum, pancreas, kidney,

lung, and melanoma. Case–control studies of

bladder (Ward et al. 2003), brain (Ward et al.

2004), colon and rectum (De Roos et al.

2003), and pancreas cancer (Coss et al. 2004)

in Iowa found no association between cancer

risk and average nitrate levels over almost

30 years. Each study evaluated the interaction

between nitrosation inhibitors or NOC

precursors and nitrate intake from drinking

water. For colon cancer, there was a significant positive interaction between 10 or more

years of exposure above 5 mg/L nitrate-N and

both low vitamin C and high meat intake,

factors likely to increase endogenous NOC

formation (De Roos et al. 2003).

A case–control study of NHL in Nebraska

(USA) (Ward et al. 1996) found a significant

positive association between the average nitrate

level in public water supplies over about

40 years and risk among men and women. In

the highest quartile of nitrate (4.0 mg/L

nitrate-N), risk was elevated 2-fold. However,

a recent study of NHL in Iowa with similar

exposure levels found no association (Ward

et al. 2004). A case–control study of NHL in

Minnesota (USA) (Freedman et al. 2000) with

lower levels of nitrate found an inverse association among those with the highest level

(> 1.5 mg/L nitrate-N). Case–control studies

in Nebraska (Ward et al. 2004) and Germany

(Steindorf et al. 1994) found no association

with long-term average nitrate levels in public

Drinking-water nitrate and health

Environmental Health Perspectives • VOLUME 113 | NUMBER 11 | November 2005 1609
water supplies and adult brain cancer. The

Nebraska study found no evidence of an interaction with vitamin C intake. A case–cohort

analysis of stomach cancer within a cohort

study in the Netherlands (van Loon et al.

1998) found no association with quintiles of

water nitrate intake determined from public

supply levels.

Specific NOC are transplacental neurocarcinogens in animal studies. A study of childhood brain cancer measured nitrate levels in

water supplies using dipstick measurements,

often many years after the pregnancy (Mueller

et al. 2001). Measured levels of nitrate and

nitrite were not associated with risk; however,

women in western Washington State, one of

the three study centers, who used private wells

as their drinking-water source during the pregnancy had a significantly increased risk of

brain cancer in their offspring.

Adverse reproductive outcomes. In 1961,

Schmitz described a possible relationship

between high maternal MetHb levels and

spontaneous abortion. Since then, at least 10

studies have examined the association

between drinking-water nitrate and adverse

reproductive outcomes. Table 2 summarizes

these studies by location, study design, determination of water nitrate, and key findings.

Few studies have been published regarding

water nitrate and the outcomes of spontaneous abortions, stillbirths, premature birth,

or intrauterine growth retardation. Results of

these studies have been inconsistent, possibly

indicating no true effect of water nitrate on

reproductive outcomes at the levels evaluated

in these studies. Alternatively, the inconsistencies may be due to the differing periods

over which exposure was assessed, differing

levels of water nitrate across studies, or differences in exposure to other cofactors.

Results of studies evaluating drinkingwater nitrate and congenital malformations in

offspring are also mixed (Table 2). Four studies (Arbuckle et al. 1988; Brender et al. 2004a,

2004b; Croen et al. 2001, Dorsch et al. 1984)

found positive associations between drinkingwater nitrate and congenital malformations,

Ward et al.

1610 VOLUME 113 | NUMBER 11 | November 2005 • Environmental Health Perspectives

Table 1. Analytic epidemiologic studies of drinking-water nitratea and cancer.

Study design

Reference, year, (case–control, cohort) Years of cancer Cancer sites

country Regional description ascertainment Exposure descriptiona included Summary of findings

Coss et al. 2004 Population-based case–control Average nitrate level in public supplies Pancreas No significant associations with

USA Incidence 1960–1987 (highest quartile > 2.8 mg/L); quartiles of average nitrate or

Iowa 1986–1989 Years of exposure ≥ 7.5 and 10 mg/L number of years ≥ 7.5 or 10 mg/L

DeRoos et al. 2003 Population-based case–control Average nitrate level in public supplies Colon No association with average level,

USA Incidence 1960–1987 categorized into four levels Rectum years > 5 and 10 mg/L;

Iowa 1986–1989 (lowest: ≤ 1.0; highest: > 5mg/L); Significantly elevated risk among

Years of exposure > 5 and >10 mg/L subgroups with below median

vitamin C intake or above median

meat intake and 10 or more

years > 5 mg/L

Freedman 2000 Population-based case–control Average nitrate level in public Non-Hodgkin No increase risk with increasing

USA Incidence water supplies 1947–1980 (157 towns) lymphoma exposure level. OR for > 1.5 mg/L

Minnesota excluding four largest categorized into three levels: ≤ 0.5, > 0.5 to (three cases, four controls) was

cities 1980–1982 ≤ 1.5, > 1.5 mg/L 0.3 (95% CI, 0.1–0.9).

Mueller et al. 2001 Population-based case–control Water source (private well, public supply) Childhood brain No overall association with water

USA 19 counties in San Francisco, during pregnancy; dipstick measurements source. Well use in western

California, area and western of nitrate and nitrite for those still living Washington State increased risk

Washington State 1984–1990 at residence during pregnancy (OR = 2.6; 95% CI, 1.3–5.2); well use

in Los Angeles inversely associated

with risk (OR = 0.2; 95% CI, 0.1–0.8)

Steindorf et al. 1994 Population-based case–control Nitrate levels in municipal supplies after Brain No association with average nitrate

Germany Incidence 1970 (highest quartile: > 5.7 mg/L) level

Rhein-Neckar-Odenwald area 1987–1988

Van Loon et al. 1998 Prospective cohort Nitrate intake from public supplies in Stomach No association with quintiles of

Netherlands Incidence 1986 and intake of tap water (quintiles; water nitrate intake (highest

1986–1992 mean level in highest quintile: 3.7 mg/day) quintile: RR = 0.88)

Ward et al. 1996 Population-based case–control Average nitrate level in public water supplies Non-Hodgkin Significant positive trend with

USA Incidence 1945–early 1980s categorized into quartiles lymphoma increasing quartiles: OR highest

66 counties in eastern Nebraska 1983–1986 (lowest: < 1.6; highest: ≥ 4.0 mg/L); quartile = 2.0 (95% CI, 1.1–3.6)

Ever exposure ≥ 10 mg/L

Ward et al. 2003 Population-based case–control Average nitrate level in public water supplies Bladder Inverse association with quartiles

USA Incidence 1960–1987 (highest quartile men: 3.1 mg/L; of average level among men; no

Iowa 1986–1989 women: 2.4 mg/L); association among women. Similar

Years of exposure ≥ 10 mg/L results for years ≥ 10 mg/L

Ward et al. 2004 Population-based case–control Average nitrate level in public water supplies Brain (gliomas) No association with quartiles of the

USA Incidence 1988–1993 1960–1986 average nitrate level

66 counties of eastern Nebraska

Weyer et al. 2001 Prospective cohort Average nitrate level (1955–1988) in public Non-Hodgkin Positive associations with average

USA Incidence water supplies for residence at enrollment lymphoma, nitrate level for bladder (highest

Iowa (highest quartile: > 2.46 mg/L) leukemia, quartile OR = 2.83) and ovary

1986–1998 colon, rectum, (OR = 1.84) and inverse associations

pancreas, kidney, for uterus (highest quartile

bladder, breast, OR = 0.55) and rectal cancer

ovary, uterine (OR = 0.47)

corpus, lung

and bronchus,

melanoma

OR, odds ratio.

aNitrate levels presented in the original publications as mg/L nitrate were converted to mg/L nitrate-N. 
particularly malformations of the central nervous system, and specifically neural tube

defects (NTDs). In each of these studies,

water nitrate levels associated with increased

risk of these defects were below the MCL,

although the 95% confidence intervals (CIs)

for some of the risk estimates were consistent

with unity and varied by the source of water

(groundwater, mixed, or surface). Two of

these studies (Brender et al. 2004b; Croen

et al. 2001) also examined dietary intake of

nitrates and nitrates and NTDs and found

Drinking-water nitrate and health

Environmental Health Perspectives • VOLUME 113 | NUMBER 11 | November 2005 1611

Table 2. Studies of the relation between drinking-water nitratea and reproductive outcomes.

Reference, Measurement of Reproductive

study population, study design water nitrate outcome Reported findings

Aschengrau et al. 1989 Matched maternal SBs through OR of 0.5 for SB with exposure

Massachusetts (USA) residents residence at pregnancy 27 weeks of gestation to water nitrate levels of

Hospital case–control study outcome to results of 0.1–5.5 mg/L relative to

tap water samples nondetectable levels

Grant et al. 1996 Wells tested for nitrates SBs Water nitrate above U.S. EPA

Indiana (USA) after cluster reported MCL for women with SBs

Cluster investigation

Aschengrau et al. 1993 Matched maternal Congenital anomalies, Neither stillbirths nor congenital

Massachusetts (USA) residents residence during stillbirths, neonatal anomalies associated with

Hospital case–control study pregnancy or deaths detectable levels of water

outcome to results nitrate (0.2–4.5 mg/L); small

of tap water samples positive association between

water nitrates and neonatal deaths.

Super et al. 1981 Water sample taken Spontaneous No association between water

South West Africa from well used at premature labor from high nitrate regions and

Cross-sectional study time of home visit Size of infant at birth prematurity or size of infant

Bukowski et al. 2001 Residential postal code IUGR Dose–response relation between

Prince Edward Island, Canada at time of delivery Premature birth nitrate level and ORs for IUGR

Case–control study linked to nitrate and prematurity

level exposure map

Scragg et al. 1982 Address at delivery Congenital Elevated OR for any

Dorsch et al. 1984 linked to sources malformations congenital malformation (2.8);

South Australia of water and data malformations of the CNS (3.5);

Case–control study on nitrates musculoskeletal system (2.9) if

primarily drank groundwater.

Elevated ORs for congenital

malformations associated with

nitrate levels ≥ 5 mg/L relative to

nitrate levels < 5 mg/L

Arbuckle et al. 1988 Collected and Congenital OR of 2.3 for CNS malformations

New Brunswick, Canada analyzed a water malformations of with exposure to nitrate 26 mg/L

Case–control study sample at maternal the CNS relative to baseline of 0.1 mg/L

residence at time

of index birth

Ericson et al. 1988 Earliest known maternal NTDs Average water nitrate similar

All deliveries in Sweden address linked to water between cases and controls

Case-control study nitrate results

Croen et al. 2001 Linked periconceptional NTDs Exposure to water

California (USA) addresses to water nitrates > 45 mg/L

Case–control study companies and associated with

databases anencephaly (OR 4.0)

but not with spina bifida;

increased risks for anencephaly

at water nitrate levels below

U.S. EPA MCL among

groundwater drinkers only;

dietary nitrate and nitrite not

associated with NTDs

Cedergren et al. 2002 Linked periconceptional Any congenital Weak association (OR 1.2)

Ostergotland County, Sweden or early pregnancy cardiac defect between water nitrate

Retrospective cohort study address to water supplies ≥ 2 mg/L and cardiac

using a geographic malformations

information system

Brender et al. 2004a Usual periconceptional NTDs OR of 1.9 if water

Brender et al. 2004b drinking-water source nitrates ≥ 3.52 mg/L;

Texas (USA) tested for nitrates increased water nitrate

Counties along Texas–Mexico border associated with

Case–control study spina bifida (OR 7.8)

but not with anencephaly

(OR 1.0); slightly inverse relation

between dietary nitrite, total nitrite

intake and NTDs

Abbreviations: CNS, central nervous system; IUGR, intrauterine growth retardation; SB, spontaneous abortion.

aNitrate units are mg/L as nitrate.
minimal or no effect on risk. In a study of

nitrosatable drug exposure and risk of NTDs

(Brender et al. 2004b), drinking-water nitrates

and dietary nitrites/total nitrites substantially

modified the risk associated with this drug

exposure during the periconceptional period;

higher levels of nitrates in food or water significantly increased the risk of NTDs if women

were exposed to such drugs.

Other health outcomes. Animal studies suggest that nitrate at high doses can competitively

inhibit iodine uptake and induce hypertrophic

changes in the thyroid (Bloomfield et al.

1961). In a human biomonitoring study in the

Netherlands, consumption of water with

nitrate levels at or above the MCL was associated with thyroid hypertrophy (van Maanen

et al. 1994) and genotoxic effects (van Maanen

et al. 1996). Animal studies provide evidence

that NOC can damage the pancreatic beta cells

(Longnecker and Daniels 2001). Three epidemiologic studies (Kostraba et al. 1992;

Parslow et al. 1997; van Maanen et al. 2000)

that were ecologic in design found a positive

correlation between drinking-water nitrate levels below the MCL and the incidence of type I

childhood diabetes, although the association

observed by van Maanen was not statistically

significant. Other studies have found associations between water nitrate exposure and

increased blood pressure (Pomeranz et al.

2000) and acute respiratory tract infections in

children (Gupta et al. 2000).

Recommendations for Future

Research

Experimental/human biomonitoring studies.

Endogenous nitrosation in humans has been

demonstrated in relation to drinking-water

nitrate ingestion at levels above the MCL.

However, further studies are needed to determine the extent of endogenous nitrosation at

intermediate drinking-water nitrate levels

(5–10 mg/L as nitrate-N) and to clarify the

role of nitrate from water versus food sources.

Furthermore, the role of precursors and modulators of NOC formation should be more

fully investigated. These future studies should

be conducted among healthy individuals as

well as individuals with medical conditions

that increase endogenous nitrosation.

In view of the complex kinetics of NOC

formation and the organ specificity of several

of these compounds (Hodgson et al. 1980;

Suzuki et al. 1999), more studies are needed

to evaluate the relationship between nitrate

intake and formation, metabolism, and excretion of NOC. Ideally, a physiologically based

pharmacokinetic model should be developed

as previously recommended (National

Research Council 1995) to predict exposure

to NOC from all sources of nitrate exposure

(exogenous and endogenous), nitrite intake,

the transformation of nitrate into nitrite, and

antioxidant intake. However, this will require

additional data on the formation of individual

NOC as well as their respective toxicologic

characteristics. The results of these investigations will reveal the value of different markers

of NOC exposure in future epidemiologic

studies. Future studies linking NOC exposure

to early markers of effect or to the actual disease will clarify the role of endogenous nitrosation and NOC exposure as etiologic factors.

Because many NOC require α-hydroxylation by CYP2E1 for bioactivation and for formation of DNA adducts, it is important to

investigate the influence of polymorphisms in

the gene encoding for this enzyme. One study

found that specific variants in this gene are

linked to increased rectum cancer risk, particularly in subjects with high intake of red and

processed meat, who are exposed to increased

levels of NOC (Le Marchand et al. 2002).

Moreover, gene expression levels of human

CYP2E1 were related to cytotoxicity and DNA

damage by nitrosamines in pancreatic beta-cell

lines, suggesting that such gene environment

interactions are also relevant in type 1 diabetes

(Lees Murdock et al. 2004). These promising

lines of research point to a possible interaction

between drinking-water nitrate exposure and

gene expression of and/or genetic variation in

CYP2E1, which may also influence the risk of

several adverse health outcomes associated with

nitrate exposure.

Epidemiologic studies. Methods must be

developed and validated to improve estimates

of current and historical exposure to nitrate via

food and water, particularly for populations

served by private wells, which are less likely to

be routinely monitored. Future epidemiologic

studies should integrate a) exposure assessment for nitrate intake from drinking water,

nitrate and nitrite intake from the diet, and

amines and amides from dietary and drug

sources, b) endogenous exposure to NOC by

analysis of relevant biological media (e.g.,

saliva, urine, feces), and c) reliable health risk

markers (e.g., biomarkers of genotoxicity) or

diagnosis of actual disease.

Future studies should include populations

with well-characterized long-term exposures,

including those who use private wells, which

can have higher nitrate levels than public supplies. With the increasing availability of public

water supply monitoring data (many U.S. states

have almost 40 years of measurements), further

detailed exposure assessment of populations

using public supplies is also feasible. Drinkingwater contaminants that may occur along with

nitrate, such as agricultural pesticides, should

also be evaluated. Geographic-based modeling

efforts to predict the probability of high nitrate

concentrations in groundwater, using information on nitrogen inputs from agricultural and

urban sources (Nolan et al. 2002), is a promising approach for estimating drinking-water

nitrate exposure for the population using

private wells.

Additional studies of drinking-water

nitrate and cancer are needed to follow up on

the suggestive positive findings to date and to

evaluate other cancer sites in which endogenously formed NOC may play a role. Studies

of reproductive outcomes should address the

exposure period most relevant for the specific

outcome of interest. Maternal residential

mobility between conception and birth may

lead to misclassification of exposure if the

water source at birth is used in studies of

spontaneous abortions and congenital malformations. Studies must be of sufficient size to

allow for examination of specific defects

rather than groups of defects by system,

because combining different defects might

mask associations. More research is needed on

the relation between water nitrate and the

reproductive outcomes of spontaneous abortion, fetal death, premature birth, and

intrauterine growth retardation.

In the design and analysis stage, future epidemiologic studies should consider factors that

modulate endogenous nitrosation, as discussed

above, to be able to evaluate potential interactions of water nitrate intake with these factors, thus providing stronger evidence for or

against an association. In particular, studies of

susceptible populations may be fruitful, and

epidemiologic studies should be designed with

sufficient power to evaluate risk among potentially susceptible subgroups. Such populations

include patients with different forms of chronic

inflammation (such as inflammatory bowel

disease), patients infected with nitrate-reducing

bacteria (such as in periodontal disease), those

with low intake of vitamins and other known

nitrosation inhibitors, or those with a history

of high incidence of potentially NOC-related

diseases. The people of Linxian County in

China, for example, are known for their persistently low intake of several micronutrients and

high risk of esophageal cancer (Blot et al.

1993). Such populations will likely benefit

from preventive measures taken as a result of

these investigations.

Conclusions

Adverse health effects from drinking-water

nitrates are most likely the result of a complex

interaction of the amount of nitrate ingested,

the concomitant ingestion of nitrosating

cofactors and precursors, and medical conditions of the host that may increase nitrosation.

Furthermore, these effects may be attenuated

by inhibitors of endogenous nitrosation such

as vitamin C and alpha-tocopherol. We recommend that future studies take into account

such complexities in understanding the relation between drinking-water nitrates and cancer, adverse reproductive outcomes, and other

health outcomes.

Ward et al.

1612 VOLUME 113 | NUMBER 11 | November 2005 • Environmental Health Perspectives
Several authors (Avery 1999; L’hirondel

and L’hirondel 2002) have questioned the

importance of nitrate in drinking water as a

risk factor for methemoglobinemia and have

suggested that the current nitrate standard

might be safely raised to 15–20 mg/L nitrateN with no increase in methemoglobinemia

cases. A better understanding of the conditions under which nitrate in drinking water

poses a risk of methemoglobinemia is clearly

needed, particularly in light of recent cases of

methemoglobinemia associated with well

water levels between 20 and 30 mg/L nitrateN (Knobeloch et al. 2000). Most importantly,

the role of nitrate as a risk factor for cancer

and adverse reproductive outcomes must be

more thoroughly explored before changes to

nitrate water quality standards are considered.

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