320 VOLUME 114 | NUMBER 3 | March 2006 • Environmental Health Perspectives

Review

The health implications of exposure to

nitrates in drinking water were first reported

in the scientific literature by Comly in 1945

after observing cyanosis in infants in Iowa,

where well water was used in formula preparation (Comly 1987). Since then, most studies on the health effects of nitrates in drinking

water have focused on infants because they

are thought to be the most vulnerable to this

exposure. More recent evaluations of the

health implications of nitrates in drinking

water have examined reproductive and developmental effects (Table 1) (Arbuckle et al.

1986, 1988; Dorsch et al. 1984; Fan and

Steinberg 1996; Fan et al. 1987; Gelperin

et al. 1975; Scragg et al. 1982; Super et al.

1981; Tabacova and Balabaeva 1993;

Tabacova et al. 1997, 1998).

Data are insufficient on the incidence of

methemoglobinemia among infants in the

United States and on the effects that exposure to nitrate levels above the maximum

contaminant limit (MCL) may have on other

populations such as pregnant women. We do

know that people in the United States who

get their water from public water systems are

occasionally exposed to biologically relevant

doses of nitrate in their drinking water [U.S.

Environmental Protection Agency (EPA)

1990]. In addition, a considerable number of

people (~ 16% of the U.S. population) use

private water systems that are usually located

in areas considered more vulnerable to nitrate

contamination (U.S. EPA 2002a).

In this review, we summarize the experimental and epidemiologic studies on nitrates

in drinking water with reference to adverse

reproductive and developmental effects.

Previous reviews by Fan and colleagues (Fan

and Steinberg 1996; Fan et al. 1987) focused

on nitrates in drinking water, methemoglobinemia, and reproductive toxicity. In this

review, we expand on these previous works,

with a focus on maternal exposure and reproductive effects. Because a drinking water

source may play a crucial role in exposure, we

also present a discussion on sources and

occurrence of nitrates in drinking water in the

United States.

Nitrate and Drinking Water

Nitrate occurs naturally in soil containing

nitrogen-fixing bacteria, decaying plants, septic system effluent, and animal manure. Other

sources of nitrate include nitrogenous fertilizers and airborne nitrogen compounds emitted

by industry and automobiles [U.S. Geological

Survey (USGS) 1996c]. Nitrate penetrates

through soil and remains in groundwater for

decades (Spalding and Exner 1993; USGS

1999). Groundwater is the source for > 50%

of drinking water supplies, 96% of private

water supplies, and an estimated 39% of public water supplies (USGS 1996a).

Factors affecting nitrate in drinking

water. People who get water from shallow

wells (< 100 feet below land surface) in areas

with well-drained soils and high nitrogen

inputs (e.g., close proximity to agricultural

areas) have an increased risk of exposure to

nitrate-rich groundwater. Agricultural activities are the largest nonpoint sources of nitrate

contamination of groundwater because

of greater use of nitrogen fertilizer on crops

and the trend toward concentrated animal

farming (Spalding and Exner 1993; USGS

1996b). Private wells are usually shallower

and closer to sources of nitrate contamination, whereas public supply wells are usually

in deeper groundwater aquifers where contamination is less likely (USGS 1996a).

Drinking water regulations. The U.S.

EPA sets the standards for water quality regulations written in the Safe Drinking Water

Act (SDWA) and its subsequent amendments

(SDWA 1974). The SDWA specifies MCL

for contaminants, defined as the concentrations above which adverse human health

effects may occur (U.S. EPA 2002b). The

enforceable MCL applies only to public

drinking water systems and government or

privately run companies supplying water to at

least 25 people or with ≥ 15 service connections (U.S. EPA 2003).

Drinking water standards are usually

set at a fraction of the no observed adverse

effect levels (NOAELs) because potential

health risks are often unknown or hard to

predict. The standards are based on data from

experimental animal studies and available

human health reports. A margin of safety is

usually built into the standard to account for

issues such as extrapolating from animals to

humans. This safety factor (orders of magnitude) is usually higher if potential health

effects are more uncertain [Risk Assessment

Information System (RAIS) 1995]. The

MCL for nitrate in drinking water was set

at 10 mg/L nitrate–nitrogen (NO3–N) or

45 mg/L nitrate (NO3

–), on the basis of

214 methemoglobinemia cases reported to

the American Public Health Association for

which nitrate concentration data were available (Walton 1951). Because the current

MCL was nominally based on human exposure data, no uncertainty or modifying factors

were used, so there is no safety factor built

Address correspondence to D. Manassaram,

CDC/NCEH/EHHE, Health Studies Branch, 4770

Buford Highway NE, MS F-46, Chamblee, GA

30341 USA. Telephone: (770) 488-3438. Fax: (770)

488-3450. E-mail: dmanassaram@cdc.gov

The authors declare they have no competing

financial interests.

Received 16 June 2005; accepted 3 November 2005.

A Review of Nitrates in Drinking Water: Maternal Exposure and Adverse

Reproductive and Developmental Outcomes

Deana M. Manassaram, Lorraine C. Backer, and Deborah M. Moll

Centers for Disease Control and Prevention, National Center for Environmental Health, Health Studies Branch, Atlanta, Georgia, USA

In this review we present an update on maternal exposure to nitrates in drinking water in relation to

possible adverse reproductive and developmental effects, and also discuss nitrates in drinking water

in the United States. The current standard for nitrates in drinking water is based on retrospective

studies and approximates a level that protects infants from methemoglobinemia, but no safety factor

is built into the standard. The current standard applies only to public water systems. Drinking water

source was related to nitrate exposure (i.e., private systems water was more likely than community

system water to have nitrate levels above the maximum contaminant limit). Animal studies have

found adverse reproductive effects resulting from higher doses of nitrate or nitrite. The epidemiologic evidence of a direct exposure–response relationship between drinking water nitrate level and

adverse reproductive effect is still not clear. However, some reports have suggested an association

between exposure to nitrates in drinking water and spontaneous abortions, intrauterine growth

restriction, and various birth defects. Uncertainties in epidemiologic studies include the lack of individual exposure assessment that would rule out confounding of the exposure with some other cause.

Nitrates may be just one of the contaminants in drinking water contributing to adverse outcomes.

We conclude that the current literature does not provide sufficient evidence of a causal relationship

between exposure to nitrates in drinking water and adverse reproductive effects. Future studies incorporating individual exposure assessment about users of private wells—the population most at risk—

should be considered. Key words: developmental outcomes, drinking water, groundwater, maternal

exposure, nitrate, nitrite, private wells, reproductive health. Environ Health Perspect 114:320–327

(2006). doi:10.1289/ehp.8407 available via http://dx.doi.org/ [Online 3 November 2005]
into the MCL for nitrates in drinking water

(Johnson and Kross 1990; Walton 1951).

The appropriateness of the current MCL

is questioned (Avery 1999). Complicating

matters is the existence of methemoglobinemia in infants without exposure to water that

contains elevated nitrate levels or any apparent exogenous nitrate or nitrite exposure.

These reports suggested that bacterial infection and subsequent overproduction of nitric

oxide, or other substances in drinking water,

such as copper, cause methemoglobinemia in

infants (Avery 1999; Felsot 1998; Hanukoglu

and Danon 1996; Hegesh and Shiloah 1982).

However, case reports persist of methemoglobinemia in infants related to well water

containing nitrate levels above the MCL

(Knobeloch et al. 2000). Private water systems are not regulated, and methemoglobinemia is not a reportable disease, so there is

insufficient data on whether exposure to

nitrate levels above the MCL is a problem

among users of such systems.

Occurrence of nitrates in drinking water.

The National Water Quality Assessment

(NAWQA) program of the USGS assessed

water quality of aquifer systems that cover the

water resources of > 60% of the population in

the contiguous United States. On the basis of

the NAWQA findings, approximately 15% of

Nitrate in drinking water and reproductive health

Environmental Health Perspectives • VOLUME 114 | NUMBER 3 | March 2006 321

Table 1. Summary of epidemiologic studies that evaluated exposure to nitrate in drinking water and reproductive and developmental effects.

Reference No. of subjects Exposure Outcome Results

Brender et al. 2004 184 cases, Drinking water nitrate level, NTDs Increased risk for NTDs

225 controls dietary nitrates and nitrites, shown with nitrosatable drug use

nitrosatable drugs (OR = 2.7; 95% CI, 1.4–5.3), and

nitrate level in water ≥ 3.5 mg/L

(OR = 1.9; 95% CI, 0.8–4.6)

Cedergren et al. 2002 71,978 infants Drinking water source; address Congenital cardiac defects OR = 1.18 (95% CI, 0.97–1.44); 

close to conception or early no excess risk for cardiac defects shown

pregnancy with an increase in nitrate levels

Bukowski et al. 2001 546 infant cases, Drinking water source, mother’s Premature birth, low birth Higher nitrate concentrations

4,098 infant controls address at delivery weight, IUGR associated with prematurity

(OR = 2.37; 95% CI, 1.07–4.80), and

IUGR (OR = 2.56; 95% CI, 1.44–4.45)

Croen et al. 2001 538 infant cases, Mother’s address before and NTDs Moderate (OR = 1.9; 95% CI, 0.73–4.7)

539 infant controls during first trimester, drinking but not significant increased risk of

water source, interview data NTDs with nitrate levels above the

on water consumption MCL, significant increased risk

(OR = 4.0; 95% CI, 1.0–15.4) of

anencephaly associated with nitrate

levels above the MCL

Tabacova et al. 1997 61 pregnancies Public drinking water source, Pregnancy complications Methemoglobin levels significantly higher

NO2 in ambient air; based in women with anemia, toxemia, and

on address during pregnancy threatened abortion/premature delivery

(mean range 2.8–6.6%), compared with

normal pregnancy (mean 1.3%)

Tabacova et al. 1998 51 mother–infant pairs Public drinking water source, Neonatal health status Maternal and cord blood methemoglobin

NO2 in ambient air, nitrate levels higher in cases of abnormal birth

in food; based on address at outcomes (preterm births, low birth

delivery weight). Mother’s methemoglobin was

associated with cord blood

methemoglobin (r = 0.79, p < 0.0001)

Arbuckle et al. 1988 130 infant cases, Household water sample, CNS defects Higher nitrate levels in private well sources.

260 infant controls drinking water source Increased risk of having an infant with a

CNS defect associated with exposure to

nitrate exposure from private well sources

(ROR = 2.30; 95% CI, 0.73–7.29)

Aschengrau et al. 1989 286 cases, Public drinking water source, Spontaneous abortion Any detectable level nitrates was

1,391 controls address at pregnancy outcome associated with decrease in

frequency of spontaneous abortion

(OR = 0.5; 95% CI, 0.2–0.9).

Dorsch et al. 1984 218 infant case–control pairs Address at delivery, water Congenital malformations The higher the level of nitrate in the water

sample from address the mother consumed, the greater the 

risk estimate for having a child with a 

malformation. Women who consume 

mainly groundwater had a greater risk of 

having a child with a malformation

(relative risk = 2.8; 95% CI, 1.6–4.4)

Scragg et al. 1982 699 perinatal deaths Drinking water source Deaths due to congenital Highest death rate in area served by

malformations drinking water source with higher

nitrate levels

Super et al. 1981 486 infants Drinking water well, interview Premature birth, size at birth No significant association between higher

data on water consumption nitrate levels in well water and incidence

of premature birth or size at birth

Gelperin et al. 1975 30,980 infants Community drinking water Infant and fetal mortality No increase in fetal or infant mortality

source in areas where community water

contained excess nitrates

Abbreviations: CI, confidence interval; CNS, central nervous system; IUGR, intrauterine growth restriction; MCL, maximum contaminant level; NTD, neural tube defect; OR, odds ratio;

ROR, risk odds ratio.
shallow groundwater sampled beneath agricultural and urban areas had nitrate levels above

the MCL. In comparison, < 10% of samples

taken from 100–200 feet deep exceeded the

MCL, and no sample was found to exceed the

MCL in groundwater that was > 200 feet

below the surface (USGS 1999). Other

reports using the NAWQA data showed

nitrate levels > 3 mg/L (report assumed levels

of ≥ 3 mg/L because of contamination) in

28% of samples taken from public and private

wells. More private wells sampled (11%)

exceeded the MCL than did public wells (2%)

(Squillace et al. 2002).

The U.S. EPA National Pesticide Survey

(U.S. EPA 1992), which sampled private

wells in 38 states and public water systems in

50 states, found 1.2% of public water systems

and 2.4% of private wells exceeded the MCL

for nitrate (Spalding and Exner 1993). From

this survey, the U.S. EPA estimated that

> 4 million people, including some 66,000

infants < 1 year of age, could be served by systems that exceed the MCL for nitrate (U.S.

EPA 1992). A survey by the Centers for

Disease Control and Prevention (CDC) of

> 5,500 private wells in nine midwestern

states found nitrate levels above the MCL in

13.4% of wells sampled (CDC 1998). A survey of 3,351 domestic wells found that 9%

had nitrate levels exceeding the MCL, compared with 1% of public wells (USGS 1995).

Although varied levels of nitrate in drinking water sources have been reported from

state-based and national studies, a relationship between levels of nitrate and source of

water is consistent. Specifically, higher levels

of nitrate were found more often in groundwater than in surface water, in private wells

than in public water supplies, in shallow wells

than in deep wells, and in agricultural than in

urban areas. The higher levels of nitrate in

private water systems are of public health concern because users could be exposed to nitrate

levels above the MCL.

Materials and Methods

We conducted a Medline (PubMed 2005)

search to identify publications in English

through January 2004. Key words and phrases

used to search the database for both animal

and human studies included nitrates, nitrites,

drinking water, pregnancy, fetus, birth defects,

spontaneous abortions, prematurity, stillbirth,

health effects, low birth weight, growth, reproductive outcomes, and drinking water contaminants. We also searched for internal

publications from the CDC, USGS, and U.S.

EPA via the agencies’ websites.

Nitrate Toxicity

Nitrate toxicity is related primarily to the in

vivo conversion to nitrite after ingestion

[National Academy of Sciences (NAS) 1977;

Swann 1975]. The health hazards from consuming water with nitrate are related to the

direct toxicity of nitrite—that is, its ability to

directly oxidize hemoglobin, changing it to

methemoglobin, which cannot bind oxygen.

Accumulation of methemoglobin (methemoglobinemia) occurs if this oxidation process

overwhelms the protective reduction capacity

of the cells (Jaffe 1981; Swann 1975). In addition to drinking water, which contributes an

estimated < 3–21% of the average adult intake

of nitrate, other sources include vegetables,

meat, and meat products preserved with

sodium nitrite [Agency for Toxic Substances

and Disease Registry (ATSDR) 2001; Wogan

et al. 1995]. There is also evidence of endogenous nitrate formation from the oxidation of

nitric oxide independently of dietary sources

(Walker 1996). A further concern relating to

the metabolism of dietary nitrate is the potential in vivo formation of N-nitroso compounds

from nitrite (Swann 1975). Nitrite is also a

substrate in the formation of N-nitroso compounds from nitrosatable drugs. Therefore,

higher dietary nitrate intake results in greater

amounts being formed if there is concurrent

exposure to nitrosatable drugs (Brender et al.

2004).

Reproductive Toxicity

Animal studies have shown some indication

that nitrate, nitrite, and N-nitroso compounds

may traverse the placenta and affect the fetus

in utero (Bruning-Fann and Kaneene 1993;

Fan et al. 1987; Gruener et al. 1973). It has

been suggested that the placental membrane is

effective in separating blood circulation

between mother and fetus from the fourth

month of pregnancy, thus preventing methemoglobin molecules from crossing (L’hirondel

and L’hirondel 2002). Others have suggested

that nitrate or the reduced form (nitrite) may

pass to the fetus through a system of active

transport similar to that of iodide, and fetal

nitrate plasma levels may exceed that of the

mother (Hartman 1982). Tarburton and

Metcalf (1985) found that amyl nitrite caused

both adult and cord blood to oxidize from

hemoglobin to methemoglobin in vitro. Cord

blood was oxidized at a 5- to 6-fold greater

rate than was adult blood. Whether nitrite

may have the same effect in vivo and on fetal

blood if it traverses the placenta is uncertain

(Tarburton and Metcalf 1985).

Animal studies. Fan et al. (1987) reviewed

the experimental data on reproductive toxicity

and reported no evidence of teratogenic effects

but found indication that nitrates and nitrites

may induce abortion in experimental animals

(Fan and Steinberg 1996; Fan et al. 1987;

NAS 1981). These findings include increased

levels of nitrite and methemoglobin in rats and

subsequently in the fetuses when pregnant rats

were given 2.5–50 mg/kg sodium nitrite in

drinking water or through intraperitoneal

injection. The possibility of increased permeability from placental damage was also tested in

this study by giving another group of pregnant

rats sodium nitrite after labor had begun. The

first newborn had a normal methemoglobin

level (1.2%), whereas those born after the

chemical was given had higher levels of methemoglobin (10.1%) in their blood (Gruener

et al. 1973).

Globus and Samuel (1978) orally administered 0.5 mg/day sodium nitrite to pregnant

mice beginning the first day of gestation and

continuing up to the 14th, 16th, or 18th day

of gestation. The parameters used to measure

embryotoxicity (litter size, gross anatomical

defects, weight, number of resorption sites,

proportion of fetal deaths) showed no significant difference between this group of mice

and the control group given distilled water.

The occurrence of skeletal abnormalities was

similar in both groups; however, an increase

in the production of red blood cells was

reported for the offspring of treated mice.

The investigators suggested that sodium

nitrite may itself traverse the placenta, inducing methemoglobinemia in the fetus that

could stimulate erythropoiesis in the hepatic

cells (Globus and Samuel 1978).

Inui et al. (1979) administered doses of

125, 250, and 500 mg/kg sodium nitrite

to hamsters on the 11th or 12th day of pregnancy. Other pregnant hamsters were given

similar doses of sodium nitrate or similar doses

of dimethylnitrosamine (positive controls). No

chromosomal changes occurred in the offspring of animals treated with lower doses of

sodium nitrite, but mutation occurred at the

highest dose of sodium nitrite (500 mg/kg).

The same dose of sodium nitrate did not

produce such effects. Morphologic and neoplastic changes were observed in embryonic

cells from hamsters given doses of 250 and

500 mg/kg sodium nitrite. The effects in pregnant hamsters treated with sodium nitrite

were similar to those seen in the pregnant

hamsters treated with dimethylnitrosamine as

a positive control (Inui et al. 1979).

Cases of aborted fetuses were observed

in pigs pastured in a feedlot of oats and

rape. Serologic testing ruled out infectious

causes for the abortions but found excessive

nitrate. The report indicated that the levels of

nitrate found in the rape (5.52%) and oats

plants (0.53%) were not safe (Case 1957).

After observing spontaneous abortions in cattle grazing in weedy pastures, Sund (1957)

placed 12 pregnant heifers in pastures with

soil known to have high nitrogen levels and

high-nitrate weeds and eight pregnant heifers

in pastures where prior treatment had killed

high-nitrate weeds. Ten of the 12 heifers in the

untreated pastures, compared with one of eight

in the treated pastures, aborted their fetuses.

Manassaram et al.

322 VOLUME 114 | NUMBER 3 | March 2006 • Environmental Health Perspectives
Blood levels taken at weekly intervals showed

low, fluctuating levels of methemoglobin in

all the heifers. The study suggested no association between abortion and levels of methemoglobin. However, the report noted that the

degenerative changes observed in several

organs of the aborted fetuses were indicative

of tissue anoxia, symptomatic of methemoglobinemia. Sund concluded that the nitrates

in weeds either caused or were related to

the incidence of abortion in cattle grazing in

pastures with soil high in available nitrogen

(Sund 1957).

Winter and Hokanson (1964) indicated

that spontaneous abortion may not be a significant effect of chronic nitrate exposure at levels

sufficient to induce methemoglobinemia in

cattle. Fifteen heifers were given sodium nitrate

daily in their feedings from 2 months of pregnancy until they aborted or gave birth. The

dosage was adjusted weekly to maintain methemoglobin levels at 20–30% of total hemoglobin. Two heifers aborted (one abortion was

caused by vibriosis), and two heifers died from

acute nitrate poisoning.

Other adverse reproductive effects, such as

mummified fetuses; lesions on the cervix,

uterus, and placenta; and maternal death were

also reported by earlier experimental studies

reviewed by Fan et al. (1987). A more recent

study examined the effects on embryo growth

and viability in 48 cows between 2 and 8 weeks

of gestation that were feeding on heavily fertilized grass containing high levels of nitrogen

compared with those in control pasture

(Laven et al. 2002). No evidence was found

that embryo survival or growth was affected

from 20 days onward in pregnant cows grazing

in either pasture.

Two groups of cows, pregnant and nonpregnant, were fed either nitrate rations < 400

ppm nitrate in hay for 5 weeks or a ration of

4,000 ppm nitrate in hay for 8 weeks (Page

et al. 1990). Serum progesterone levels were

depressed in cows fed the higher nitrate

rations. The depressed levels were more evident in nonpregnant cows, less in cows in

early pregnancy, and absent in cows in midpregnancy. The investigators concluded that

early reproductive toxicity from chronic

nitrate exposure may be caused by depression

of serum progesterone, but further studies

need to be done to confirm this conclusion.

Epidemiologic Studies of

Reproductive Effects of 

Nitrate in Drinking Water

Birth defects. The effects of exposure to

nitrates in drinking water on the incidence of

birth defects have been evaluated in several

epidemiologic studies (Table 1) (Arbuckle

et al. 1988; Cedergren et al. 2002; Croen

et al. 2001; Fan and Steinberg 1996; Fan

et al. 1987). However, the results from

epidemiologic studies addressing this topic are

equivocal.

In a case–control study of MexicanAmerican women, Brender et al. (2004) examined nitrosatable drug exposure and the

occurrence of neural tube defects (NTDs) in

relation to dietary nitrites and nitrates. They

examined 184 cases of NTD-affected pregnancies from the Texas Neural Tube Defects

Projects and 225 women with normal live

births. All participants were interviewed to

obtain detailed dietary information, periconceptional medication use, and drinking water

source. The water sources of 110 women

(43 cases and 67 controls) were also tested for

nitrate. Nitrosatable drug use was reported as

a risk factor for having an NTD-affected

pregnancy [odds ratio (OR) = 2.7; 95% confidence interval (CI), 1.4–5.3]. Among those

who had their water tested for nitrate, drinking water nitrate level ≥ 3.5 mg/L nitrate–N

was associated with having an NTD-affected

pregnancy (OR = 1.9; 95% CI, 0.8–4.9). The

risk estimate increased drastically (OR = 14;

95% CI, 1.7–660) for women who took

nitrosatable drugs and had nitrate levels

≥ 3.5 mg/L nitrate–N in their drinking water

source. The authors concluded that because

the level of nitrate in the water sampled was

relatively low, and women were not asked

about frequency and amount of water consumed, the amount of nitrate in the water

directly contributed to the increased risk

observed among women who used nitrosatable

drugs (Brender et al. 2004).

A study of 71,978 infants born from 1982

through 1996 was conducted in a Swedish

county served by 80 municipal water systems

(Cedergren et al. 2002). The study assessed

the possible association between mothers’ preconception or early pregnancy exposure to

chlorination by-products and nitrate in public

drinking water and incidence of congenital

cardiac defects. The study population was

identified through the Swedish Birth Registry

and was limited to infants whose mothers

used the municipal water system and had

addresses for the preconception or early pregnancy period that could be geocoded. The

Registry of Congenital Malformations provided information on cardiac defects.

Additional data on the pregnancy, delivery,

and newborn health were obtained from medical records and the hospital discharge registry. Exposure assessment was ascertained by

using a geographic information system to link

the study subjects to specific water supplies.

Groundwater as a source of drinking water

was reported as a potential risk factor for cardiac defects (adjusted OR = 1.31; 95% CI,

1.09–1.57). A very small and not statistically

significant excess risk for cardiac defects was

found to be associated with levels ≥ 2 mg/L

nitrate–N compared with those < 2 mg/L

nitrate–N (adjusted OR = 1.18; 95% CI,

0.97–1.44) (Cedergren et al. 2002).

A case–control study in California investigated the potential association between maternal exposure to nitrates in drinking water and

diet before pregnancy and the risk of NTDs

in the mothers’ infants (Croen et al. 2001).

Case infants (538) with NTDs (both live and

stillborn singleton births) born from 1989

through 1991 were selected from California’s

birth defects program. Control infants (539)

were live births with no malformations

selected from each area birth hospital for the

same time period. Exposure assessment was

done through interviews with the mothers,

which included a detailed beverage and dietary

questionnaire, and the state’s community

water systems data for sources serving their

preconception addresses. The authors found

an increased risk for NTDs among babies

born to mothers living in areas where the

drinking water nitrate level was above the

MCL compared with those in areas below the

MCL (OR = 2.7; 95% CI, 0.76–9.3). This

association did not change after adjusting for

other dietary nitrate intake (OR = 1.9; 95%

CI, 0.73–4.7). Increased risk with increasing

levels of nitrates was observed. Risk estimates

were higher among groundwater users; however, other risk factors (e.g., Hispanic ethnicity, young age, low socioeconomic status, and

no vitamin use) for NTDs were also more

common among groundwater users.

In this study, the authors examined risk

separately for anencephaly and spina bifida

(Croen et al. 2001). Increased risk for anencephaly in babies was associated with their

mother living in an area where the nitrate in

drinking water was above the MCL (OR =

4.0; 95% CI, 1.0–15.4). This association was

not substantially altered when adjusted for

dietary intake of nitrate. A doubling in risk for

anencephaly in babies whose mothers lived in

areas where the nitrate level in groundwater

was ≥ 5 mg/L compared with < 5 mg/L was

observed. No increased risk was observed in

users of mixed water (i.e., a combination of

surface water and groundwater). There was no

increased risk for spina bifida with any level of

nitrate exposure or source of drinking water

(Croen et al. 2001).

Fan and Steinberg (1996) summarized the

studies conducted on maternal exposure to

nitrates in drinking water and birth defects by

Scragg et al. (1982) and Dorsch et al. (1984) in

South Australia, Arbuckle et al. (1988) in New

Brunswick, Canada, and Bove et al. (1992) in

New Jersey. The study by Dorsch et al. (1984)

suggested an increased risk of bearing a child

with a congenital malformation among women

whose homes were served by water with a

nitrate concentration > 5 ppm. Although the

effects of nitrate cannot be discounted, the

finding that the risks associated with multiple

Nitrate in drinking water and reproductive health

Environmental Health Perspectives • VOLUME 114 | NUMBER 3 | March 2006 323
defects were increased suggests possible multiple risk factors. The study by Arbuckle et al.

(1988) reported a protective relationship for

users of public and spring water sources compared with private sources, but the ORs were

not statistically significant. Five water samples

exceeded the Canadian MCL (44 ppm) for

nitrate. Bove et al. (1992) reported a positive

association with water contamination and

NTDs but cautioned that the study did not

provide sufficient evidence of causation for any

of the contaminants in question.

These studies assessed exposure to nitrates in

drinking water on the basis of the source of the

water. The lack of individual exposure assessment—whether the women actually drank the

water—is a limitation in some investigations.

The presence of other substances, such as pesticides, toxic metals, and chemicals (e.g., chlorinated solvents and chlorinated disinfection

by-products), in private and public water systems may be correlated with the presence of

nitrate. Some of these constituents are reported

as risk factors for congenital malformations

(Bove et al. 1992, 2002).

Spontaneous abortions. Aschengrau et al.

(1989) investigated the quality of community

drinking water and the occurrence of spontaneous abortions among a group of women in

the Boston, Massachusetts, area. The population, selected from 1976 through 1978, consisted of women who lived in Massachusetts

during their pregnancy, lived in a town with a

public water supply, and were admitted to a

specific community hospital. Cases were

286 women who had a miscarriage during

their first 27 weeks of pregnancy, and controls

were 1,391 women who had live births. The

women were interviewed to obtain demographic and behavioral information, and water

quality data were obtained from public records

of routine analyses of public tap water. All levels of nitrate were below the MCL. A negative

association was reported for any detectable

level of nitrate and the occurrence of spontaneous abortions. The report noted that risk

estimates may have been diluted by the measurement, recording, and classification of exposure, because this information was obtained

indirectly from public water supply records of

the communities where the women lived at

the time the spontaneous abortions occurred

(Aschengrau et al. 1989).

Gelperin et al. (1975) evaluated data from

1959 through 1966 on infant and fetal deaths

in 16 Illinois communities, nine of which had

nitrate levels ranging from 43 ppm to 123 ppm

(NO3) in their water supply. Communities

were grouped into three categories, consistently

having high nitrate (above the MCL), having

high nitrate in spring only, or not having

high nitrate levels. The community water supply data provided nitrate levels. No significant

increase in fetal deaths was found in areas

consistently reporting nitrate levels above the

MCL in their water compared with other areas.

Skrivan (1971) measured methemoglobin

levels in the blood of pregnant women over a

2-year period to evaluate whether the observed

mean values of methemoglobin would be similar in women who experienced spontaneous

abortions and in those who experienced term

delivery. The mean values in women before

spontaneous abortion did not differ significantly from the mean values among women

with term deliveries (Skrivan 1971).

An earlier evaluation of methemoglobin

levels in pregnancy reported a relationship

between methemoglobinemia and miscarriages

in humans (Schmitz 1961). The study tested

methemoglobin levels in 25 women in their

first trimester of pregnancy. Higher levels were

observed in women who spontaneously aborted

or who threatened abortion in the first

trimester. The report noted nitrates and nitrites

as the most common methemoglobin inducers

and concluded that high maternal methemoglobin levels are possibly related to miscarriages.

Case reports. A report on a cluster of

spontaneous abortions in LaGrange, Indiana,

cited nitrate-contaminated water from private

wells as the possible cause (CDC 1996). The

cases included a 35-year-old woman who

experienced four consecutive miscarriages and

a 37-year-old and a 20-year-old who each

experienced one miscarriage. All three women

lived within 1 mile of each other and were in

the first trimester of pregnancy at the time of

the miscarriages. Testing of the wells serving

the homes of the women found nitrate to be

the only elevated contaminant. The wells had

nitrate levels over the MCL, with reported

levels of 19.0 mg/L, 26 mg/L, and 19.2 mg/L

nitrate–N for the three women, respectively.

Although these incidents of spontaneous

abortion may have been related to the ingestion of nitrate contaminated drinking water,

other possible explanations such as genetic

defects in the fetuses and cluster by chance

could not be ruled out (CDC 1996).

Other reproductive effects. Besides birth

defects and prenatal mortality, reproductive

toxicity includes less readily observed effects

that may be influenced by chronic low-level

exposure to a toxic substance. These effects

include sterility, intrauterine growth restriction,

premature birth, and complications of pregnancy. Several of these outcomes have been

addressed in epidemiologic studies of the

potential effects of nitrate exposure on reproductive health (Table 1). Hypothetically, the

oxidation of hemoglobin to methemoglobin,

which limits the oxygen-carrying capacity of the

blood, may interfere with the course and outcome of pregnancy (NAS 1981; Tabacova and

Balabaeva 1993; Tabacova et al. 1997, 1998).

Bukowski et al. (2001) conducted a population-based case–control study on singleton

births that occurred from 1991 through 1994

to mothers who resided in Prince Edward

Island, Canada, and who used municipal and

private water systems. The study examined

the potential impact of groundwater nitrate

exposure on prematurity and intrauterine

growth restriction (IUGR). The study included

210 cases of IUGR, 336 cases of premature

births, and 4,098 controls that were identified

through a Reproductive Care Program (RCP)

database. The authors developed a nitrate

level exposure map using data on public and

private wells collected from 1990 through

1993. Premature birth was defined as birth at

< 37 weeks of gestation, IUGR was defined as

birth weight < 2,500 g at termed birth, and

controls were defined as “normal” live births

with no other diagnoses. The nitrate data were

subdivided into six exposure categories on the

basis of increasing nitrate–N levels, with the

highest exposure having levels ≥ 5 mg/L. The

study subject was assigned to an exposure

group on the basis of the mother’s residential

address at the time of delivery. The RCP database provided additional data including demographics, lifestyle, health, and reproductive

history (Bukowski et al. 2001).

Bukowski et al. (2001) found a significant

relationship between IUGR and higher nitrate

levels. Using the lowest exposure category

(median level of ≤ 1.3 mg/L nitrate–N) as the

comparison group, an adjusted OR showed an

excess risk for higher exposure categories with

median levels 3.1 mg/L (OR = 2.31; 95% CI,

1.47–3.64) and 4.3 mg/L (OR = 2.56; 95%

CI, 1.44–4.45). An increased but not significant risk for the highest exposure group of

5.5 mg/L (OR = 1.34; 95% CI, 0.31–3.99)

was observed, as well as a significant dose–

response association between nitrate exposure

and prematurity. The authors reported excess

risk estimates for the exposure categories with

median levels of 3.1 mg/L (OR = 1.82; 95%

CI, 1.23–2.69), 4.3 mg/L (OR = 2.33; 95%

CI, 1.46–3.68), and 5.5 mg/L (OR = 2.37;

95% CI, 1.07–4.80). The dose–response relationship is fairly consistent and suggestive; however, assessing exposure to nitrate based on

ecologic classification makes it difficult to interpret the findings exclusively in terms of water

nitrate exposure (Bukowski et al. 2001).

In a study in Bulgaria, Tabacova et al.

(1997) investigated the association between

maternal exposure to environmental nitrogen

sources and subsequent complications of pregnancy. The study was done in an area known

to have nitrate levels in drinking water ranging

from 8 to 54 mg/L nitrate–N (depending on

the season and the water supply source), as

well as in the food supply and oxides of nitrogen in ambient air. The study, which included

pregnant women at ≥ 24 weeks’ gestation,

analyzed blood methemoglobin levels as

markers of exposure to nitrogen compounds.

Manassaram et al.

324 VOLUME 114 | NUMBER 3 | March 2006 • Environmental Health Perspectives
Personal interviews with the women furnished

medical and lifestyle histories. Only 16% of

the study group experienced a normal pregnancy. The others experienced complications,

including anemia (67%), threatened spontaneous abortion/premature labor (33%), and

toxemia (23%), with some women having

more than one of these diagnoses. Among

women with pregnancy pathology, methemoglobin levels were 0.1–11.2% of total hemoglobin, compared with 0.4–2.8% in women

with normal pregnancy. The mean methemoglobin levels for all categories of pregnancy

complications were above the physiologic normal level of 2%, with the highest mean level

occurring in the toxemia group. The report

suggests an increased risk for pregnancy complications from exposure to nitrogen compounds. However, the lack of individual

exposure assessment and the presence of several potential sources of exposure prevent

implicating drinking water as a single source

(Tabacova et al. 1997).

Tabacova et al. (1998) also evaluated

mother–infant pairs in an area of Bulgaria with

chronic exposure to pollution by nitrogen

compounds, including drinking water nitrate

levels of 8–54 mg/L nitrate–N in municipal

water system, 13–400 mg/L nitrate–N in wells,

and nitrogen oxides in ambient air. They

examined methemoglobin levels as markers for

exposure to nitrogen compounds, measures of

oxidative stress, and the health status of infants

at birth. Fifty-one mother–infant pairs were

recruited from a local hospital; interviews were

conducted with the mothers, and clinical

records were reviewed for information on

demographics, lifestyle, and medical factors.

Maternal and cord blood were tested for

methemoglobin and markers of oxidative

stress. More than half of maternal blood and

almost half of cord blood had methemoglobin

levels > 2%. The study indicated a strong association between maternal methemoglobin and

cord blood methemoglobin. Maternal and

cord blood methemoglobin levels were higher

in cases of abnormal birth outcomes (preterm

birth, fetal distress, and low birth weight) than

in cases of normal birth outcomes. The authors

also reported an association between methemoglobin levels in cord blood and adverse birth

outcome. Overall, the results show that nitrate

exposure may have a role in adverse reproductive effects. However, the small sample size,

multiple sources of exposure, and the lack of

individual exposure assessment make the findings difficult to interpret in terms of nitrate

exposure from drinking water per se. Other

pollutants potentially associated with abnormal

birth outcomes were not discussed (Tabacova

et al. 1998).

Fan et al. (1987; Fan and Steinberg 1996)

discussed the study by Super et al. (1981) that

evaluated infant morbidity in a rural area of

southwest Africa known to have wells with

high levels of nitrates. Super et al. (1981)

found no association between the incidence

of premature birth or the size of infant at

birth and living in an area with nitrate levels

> 20 mg/L nitrate–N compared with areas

with levels ≤ 20 mg/L. No association was

found between birth weight, current or previous premature births, stillbirths, and spontaneous abortions and nitrate levels > 20 mg/L

nitrate–N. However, increased deaths among

infants previously born to mothers in regions

with nitrate levels > 20 mg/L nitrate–N were

reported.

Summary and Discussion

The contamination of groundwater by nitrates

is ubiquitous, but the magnitude of the risk

posed to human health is still unclear. That the

potential toxicity of nitrate in drinking water is

largely based on studies using high concentrations of nitrite is a complication. Another complication is that human studies have provided

little evidence that adverse health effects result

from chronic exposure to low levels of nitrate

in drinking water. Although the current MCL

for nitrates is also in debate, the public drinking water supply rarely exceeds this limit. The

lack of data on unregulated systems, however,

is an important issue. The available data on the

occurrences of nitrates in drinking water indicate that users of private water systems are

most at risk for exposure to nitrate levels above

the MCL. However, a lack of studies focusing

on users of private water systems means that

the extent of the problem is unknown.

Experimental animal studies on nitrate or

nitrite (in the form of sodium or potassium

nitrate/nitrite) and adverse reproductive and

developmental outcomes provide moderate evidence for an association between exposure to

nitrate and fetal loss, neonatal mortality, maternal toxicity, and decrease in number of litters

and live births (Table 2). Epidemiologic evidence for increased risk for adverse reproductive

and developmental outcomes in humans from

exposure to nitrate in drinking water is sparse

and suggestive at best. Nevertheless, the findings of excess birth defects in some studies

(Table 1) suggest the need for further studies.

In most studies presented in this review,

exposure to nitrates in drinking water was

assessed primarily through water quality data for

water systems serving women’s addresses during

Nitrate in drinking water and reproductive health

Environmental Health Perspectives • VOLUME 114 | NUMBER 3 | March 2006 325

Table 2. Reproductive and developmental effects in animal studies that evaluated exposure to nitrate or nitrite.

Reference Animal species Exposure Results summary

Laven et al. 2002 Cattle High nitrate (as available nitrogen) Grazing in high nitrate pasture did not affect embryo survival or growth

in pastures Higher milk and plasma urea levels in cows grazing in high nitrate pastures

Bruning-Fann et al. 1996 Swine Nitrate in drinking water Nitrate detected in 53% of sampled wells, median concentration of 2.1 ppm

No association between the nitrate level in drinking water and litter size,

number of stillbirths, deaths, or other known illnesses

U.S. FDA 1972a, 1972b Mice, rats, hamsters, KNO3 or KNO2, NaNO3 or NaNO2 No effect on maternal or fetal survival, no incidence of birth defects

rabbits oral intubation

Sinha et al. 1971 Guinea Pigs NaNO2 subcutaneous injection Abortion induced at 60 mg/kg NaNO2

Maternal death at 70 mg/kg NaNO2

High methemoglobin levels in mothers and fetuses

Sleight 1967 Guinea Pigs KNO3 or KNO2 in drinking water Decrease in litter size and number of live births, and fetal loss observed in

those given KNO3

No live births and aborted fetuses observed in those given KNO2

Winter 1964 Cattle NaNO3, NaNO2, or hydroxylamine Two of the 15 heifers fed NaNO3 died from acute nitrate poisoning, two

in feeding aborted (one due to vibriosis)

Simon et al. 1959 Cattle KNO3 or NaNO2 capsules No abortions in heifers given small doses of KNO3 or NaNO2

Three heifers died and three aborted after given higher doses of KNO3

Case 1957 Pigs Grazing in pasture on oats and rape Spontaneous abortion and stillbirths in offspring

high in nitrate Negative test for infectious disease but high levels of nitrate found in the

mothers’ blood

Sund et al. 1957 Cattle Grazing in pastures where soil had 10 of 12 heifers in weedy pastures aborted

high levels of available nitrogen, No association between methemoglobin levels and abortion

and weeds had high nitrate levels

Abbreviations: KNO2, potassium nitrite; KNO3, potassium nitrate; NaNO2, sodium nitrite; NaNO3, sodium nitrate.
pregnancy or at time of delivery. Although this

form of exposure assessment provides information in a timely and cost-efficient way,

community-based water quality data provide

only a rough estimate of individual exposure.

This does not account for other issues in

exposure assessment such as other sources of

nitrate exposure, individuals not drinking tap

water, or use of private water systems. The

recent study by Brender et al. (2004) is an

example of the extent of exposure assessment

that should be considered when evaluating

nitrate exposure and reproductive effects.

Drinking water contaminants other than

nitrates have been reported to be associated

with increased risk of adverse pregnancy outcomes (Bove et al. 1992, 2002). Identifying

which contaminant in a community water

system is associated with a particular adverse

reproductive outcome when multiple contaminants are present is difficult. Future studies

should conduct individual exposure assessments such as maternal interviews concerning

water consumption habits in and outside the

home and other risk factors such as occupational exposures or smoking.

Many studies on birth defects are also limited by the time between the end of pregnancy

and the maternal interview. This is difficult for

individual studies to overcome because birth

defects are rare outcomes and prospective

cohort studies may not be feasible to conduct.

Prospective cohort studies on end points such

as spontaneous abortions are more feasible and

would provide knowledge about the potential

effects of nitrates in drinking water on this outcome. Spontaneous abortion may be a more

sensitive indicator of adverse reproductive

effects from relatively low levels of drinking

water contamination.

States with large numbers of private wells

where groundwater is vulnerable to contamination should be encouraged to increase monitoring or surveillance of such systems. Future

research could include long-term monitoring or

surveillance of water systems vulnerable to contamination. This could provide valuable exposure assessment information to conduct studies

on drinking water contaminants such as

nitrates. A discussion on the appropriateness of

the current MCL is beyond the scope of this

review. However, future studies with improved

exposure assessment (including other dietary

sources and medications), adequate sample size,

and evaluating endogenous nitrate exposure

could help to determine whether nitrates in

drinking water at the current MCL increase the

risk for reproductive and developmental effects.

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