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

Developmental Outcomes

Author(s): Deana M. Manassaram, Lorraine C. Backer, Deborah M. Moll

Source: Environmental Health Perspectives, Vol. 114, No. 3 (Mar., 2006), pp. 320-327

Published by: Brogan & Partners

Stable URL: http://www.jstor.org/stable/3436672

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Review_ 

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 epidemio? 

logic 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 indi? 

vidual exposure assessment that would rule out confounding ofthe 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] 

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 prepa? 

ration (Comly 1987). Since then, most stud? 

ies 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 devel? 

opmental 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 expo? 

sure 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% ofthe 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 experi? 

mental 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, methemoglo? 

binemia, and reproductive toxicity. In this 

review, we expand on these previous works, 

with a focus on maternal exposure and repro? 

ductive 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 pub? 

lic 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 activi? 

ties 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 contamina? 

tion, whereas public supply wells are usually 

in deeper groundwater aquifers where conta? 

mination is less likely (USGS 1996a). 

Drinking water regulations. The U.S. 

EPA sets the standards for water quality regu? 

lations written in the Safe Drinking Water 

Act (SDWA) and its subsequent amendments 

(SDWA 1974). The SDWA specifies MCL 

for contaminants, defined as the concentra? 

tions 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 connec? 

tions (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 magni? 

tude) 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 avail? 

able (Walton 1951). Because the current 

MCL was nominally based on human expo? 

sure 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. 

320 volume 1141 number 31 March 2006 ? Environmental Health Perspectives 
Nitrate in drinking water and reproductive health 

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 methemoglobine? 

mia in infants without exposure to water that 

contains elevated nitrate levels or any appar? 

ent exogenous nitrate or nitrite exposure. 

These reports suggested that bacterial infec? 

tion 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 methemo? 

globinemia in infants related to well water 

containing nitrate levels above the MCL 

(Knobeloch et al. 2000). Private water sys? 

tems are not regulated, and methemoglobine? 

mia 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 

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 etal. 2004 

Cedergren etal. 2002 

Bukowski etal. 2001 

Croen et al. 2001 

Tabacova etal. 1997 

Tabacova etal. 1998 

Arbuckle etal. 1988 

Aschengrauetal. 1989 

Dorsch etal. 1984 

Scragg etal. 1982 

Super etal. 1981 

Gelperinetal. 1975 

184 cases, 

225 controls 

71,978 infants 

546 infant cases, 

4,098 infant controls 

538 infant cases, 

539 infant controls 

61 pregnancies 

51 mother-infant pairs 

130 infant cases, 

260 infant controls 

286 cases, 

1,391 controls 

218 infant case-control pairs 

699 perinatal deaths 

486 infants 

30,980 infants 

Drinking water nitrate level, 

dietary nitrates and nitrites, 

nitrosatable drugs 

Drinking water source; address 

close to conception or early 

pregnancy 

Drinking water source, mother's 

address at delivery 

Mother's address before and 

during first trimester, drinking 

water source, interview data 

on water consumption 

Public drinking water source, 

N02 in ambient air; based 

on address during pregnancy 

Public drinking water source, 

N02 in ambient air, nitrate. 

in food; based on address at 

delivery 

Household water sample, 

drinking water source 

Public drinking water source, 

address at pregnancy outcome 

Address at delivery, water 

sample from address 

Drinking water source 

Drinking water well, interview 

data on water consumption 

Community drinking water 

source 

NTDs 

Congenital cardiac defects 

Premature birth, low birth 

weight, IUGR 

NTDs 

Pregnancy complications 

Neonatal health status 

CNS defects 

Spontaneous abortion 

Congenital malformations 

Deaths due to congenital 

malformations 

Premature birth, size at birth 

Infant and fetal mortality 

Increased risk for NTDs 

shown with nitrosatable drug use 

(OR = 2.7; 95% Cl, 1.4-5.3), and 

nitrate level in water > 3.5 mg/L 

(OR = 1.9; 95% Cl, 0.8-4.6) 

OR = 1.18 (95% Cl, 0.97-1.44); 

no excess risk for cardiac defects shown 

with an increase in nitrate levels. 

Higher nitrate concentrations 

associated with prematurity 

(OR = 2.37; 95% Cl, 1.07-4.80), and 

IUGR (OR = 2.56; 95% Cl, 1.44-4.45) 

Moderate (OR = 1.9; 95% Cl, 0.73-4.7) 

but not significant increased risk of 

NTDs with nitrate levels above the 

MCL, significant increased risk 

(OR = 4.0; 95% Cl, 1.0-15.4) of 

anencephaly associated with nitrate 

levels above the MCL 

Methemoglobin levels significantly higher 

in women with anemia, toxemia, and 

threatened abortion/premature delivery 

(mean range 2.8-6.6%), compared with 

normal pregnancy (mean 1.3%) 

Maternal and cord blood methemoglobin 

levels higher in cases of abnormal birth 

outcomes (preterm births, low birth 

weight). Mother's methemoglobin was 

associated with cord blood 

methemoglobin (r = 0.79, p< 0.0001) 

Higher nitrate levels in private well sources. 

Increased risk of having an infant with a 

CNS defect associated with exposure to 

nitrate exposure from private well sources 

(ROR = 2.30; 95% Cl, 0.73-7.29) 

Any detectable level nitrates was 

associated with decrease in 

frequency of spontaneous abortion 

(OR = 0.5; 95% Cl, 0.2-0.9). 

The higher the level of nitrate in the water 

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% Cl, 1.6-4.4) 

Highest death rate in area served by 

drinking water source with higher 

nitrate levels 

No significant association between higher 

nitrate levels in well water and incidence 

of premature birth or size at birth 

No increase in fetal or infant mortality 

in areas where community water 

contained excess nitrates 

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

ROR, risk odds ratio. 

Environmental Health Perspectives ? volume 1141 number 31 March 2006 321 
Manassaram et al. 

shallow groundwater sampled beneath agricul? 

tural 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 sys? 

tems 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 sur? 

vey of 3,351 domestic wells found that 9% 

had nitrate levels exceeding the MCL, com? 

pared with 1% of public wells (USGS 1995). 

Although varied levels of nitrate in drink? 

ing water sources have been reported from 

state-based and national studies, a relation? 

ship between levels of nitrate and source of 

water is consistent. Specifically, higher levels 

of nitrate were found more often in ground? 

water 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 con? 

cern 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, repro? 

ductive outcomes, and drinking water con? 

taminants. 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 (methemo? 

globinemia) occurs if this oxidation process 

overwhelms the protective reduction capacity 

ofthe cells Qaffe 1981; Swann 1975). In addi? 

tion to drinking water, which contributes an 

estimated < 3-21% ofthe 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 endoge? 

nous 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 poten? 

tial in vivo formation of A^-nitroso compounds 

from nitrite (Swann 1975). Nitrite is also a 

substrate in the formation of A^-nitroso com? 

pounds 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 A^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 methe? 

moglobin 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 methe? 

moglobin (10.1%) in their blood (Gruener 

etal. 1973). 

Globus and Samuel (1978) orally admin? 

istered 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 signifi? 

cant 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 preg? 

nancy. Other pregnant hamsters were given 

similar doses of sodium nitrate or similar doses 

of dimethylnitrosamine (positive controls). No 

chromosomal changes occurred in the off? 

spring 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 neo? 

plastic changes were observed in embryonic 

cells from hamsters given doses of 250 and 

500 mg/kg sodium nitrite. The effects in preg? 

nant 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 ofthe 12 heifers in the 

untreated pastures, compared with one of eight 

in the treated pastures, aborted their fetuses. 

322 volume 1141 number 3I March 2006 ? Environmental Health Perspectives 
Nitrate in drinking water and reproductive health 

Blood levels taken at weekly intervals showed 

low, fluctuating levels of methemoglobin in 

all the heifers. The study suggested no associ? 

ation between abortion and levels of methe? 

moglobin. However, the report noted that the 

degenerative changes observed in several 

organs of the aborted fetuses were indicative 

of tissue anoxia, symptomatic of methemo? 

globinemia. 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 signifi? 

cant 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 preg? 

nancy until they aborted or gave birth. The 

dosage was adjusted weekly to maintain methe? 

moglobin levels at 20-30% of total hemoglo? 

bin. 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 non? 

pregnant, 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 evi? 

dent 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) exam? 

ined 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, pericon? 

ceptional 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% con? 

fidence interval (CI), 1.4-5.3]. Among those 

who had their water tested for nitrate, drink? 

ing 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 con? 

sumed, 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 preg? 

nancy period that could be geocoded. The 

Registry of Congenital Malformations pro? 

vided information on cardiac defects. 

Additional data on the pregnancy, delivery, 

and newborn health were obtained from med? 

ical records and the hospital discharge reg? 

istry. 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 car? 

diac 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% Cl, 

0.97-1.44) (Cedergren et al. 2002). 

A case-control study in California investi? 

gated the potential association between mater? 

nal 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% Cl, 0.76-9.3). This 

association did not change after adjusting for 

other dietary nitrate intake (OR = 1.9; 95% 

Cl, 0.73-4.7). Increased risk with increasing 

levels of nitrates was observed. Risk estimates 

were higher among groundwater users; how? 

ever, other risk factors (e.g., Hispanic ethnic? 

ity, 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 anen? 

cephaly 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% Cl, 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 rng/L compared with < 5 rng/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 

Environmental Health Perspectives ? volume 1141 number 3I March 2006 323 
Manassaram et al 

defects were increased suggests possible multi? 

ple risk factors. The study by Arbuckle et al. 

(1988) reported a protective relationship for 

users of public and spring water sources com? 

pared 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 

ofthe 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 assess? 

ment?whether the women actually drank the 

water?is a limitation in some investigations. 

The presence of other substances, such as pesti? 

cides, toxic metals, and chemicals (e.g., chlori? 

nated solvents and chlorinated disinfection 

by-products), in private and public water sys? 

tems may be correlated with the presence of 

nitrate. Some of these constituents are reported 

as risk factors for congenital malformations 

(Bove etal. 1992,2002). 

Spontaneous abortions. Aschengrau et al. 

(1989) investigated the quality of community 

drinking water and the occurrence of sponta? 

neous abortions among a group of women in 

the Boston, Massachusetts, area. The popula? 

tion, 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 demo? 

graphic and behavioral information, and water 

quality data were obtained from public records 

of routine analyses of public tap water. All lev? 

els of nitrate were below the MCL. A negative 

association was reported for any detectable 

level of nitrate and the occurrence of sponta? 

neous abortions. The report noted that risk 

estimates may have been diluted by the meas? 

urement, recording, and classification of expo? 

sure, because this information was obtained 

indirecdy 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 lllinois 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, consistendy 

having high nitrate (above the MCL), having 

high nitrate in spring only, or not having 

high nitrate levels. The community water sup? 

ply 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 sim? 

ilar in women who experienced spontaneous 

abortions and in those who experienced term 

delivery. The mean values in women before 

spontaneous abortion did not differ signifi? 

cantly 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 methemo? 

globin 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 inges? 

tion 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 preg? 

nancy. Several of these outcomes have been 

addressed in epidemiologic studies of the 

potential effects of nitrate exposure on repro? 

ductive health (Table 1). Hypothetically, the 

oxidation of hemoglobin to methemoglobin, 

which limits the oxygen-carrying capacity ofthe 

blood, may interfere with the course and out? 

come of pregnancy (NAS 1981; Tabacova and 

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

Bukowski et al. (2001) conducted a popu? 

lation-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 data? 

base provided additional data including demo? 

graphics, 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 signifi? 

cant 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 doseresponse 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 rela? 

tionship is fairly consistent and suggestive; how? 

ever, 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 preg? 

nancy. 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 nitro? 

gen in ambient air. The study, which included 

pregnant women at > 24 weeks' gestation, 

analyzed blood methemoglobin levels as 

markers of exposure to nitrogen compounds. 

324 volume 1141 number 31 March 2006 ? Environmental Health Perspectives 
Nitrate in drinking water and reproductive health 

Personal interviews with the women fiirnished 

medical and lifestyle histories. Only 16% of 

the study group experienced a normal preg? 

nancy. The others experienced complications, 

including anemia (67%), threatened sponta? 

neous abortion/premature labor (33%), and 

toxemia (23%), with some women having 

more than one of these diagnoses. Among 

women with pregnancy pathology, methemo? 

globin levels were 0.1-11.2% of total hemo? 

globin, compared with 0.4-2.8% in women 

with normal pregnancy. The mean methemo? 

globin levels for all categories of pregnancy 

complications were above the physiologic nor? 

mal level of 2%, with the highest mean level 

occurring in the toxemia group. The report 

suggests an increased risk for pregnancy com? 

plications from exposure to nitrogen com? 

pounds. However, the lack of individual 

exposure assessment and the presence of sev? 

eral 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, 13^00 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 asso? 

ciation 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 methemo? 

globin levels in cord blood and adverse birth 

outcome. Overall, the results show that nitrate 

exposure may have a role in adverse reproduc? 

tive effects. However, the small sample size, 

multiple sources of exposure, and the lack of 

individual exposure assessment make the find? 

ings 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 

etal. 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 previ? 

ous premature births, stillbirths, and sponta? 

neous 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 concentra? 

tions 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 drink? 

ing 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 indi? 

cate 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 ofthe 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 evi? 

dence for an association between exposure to 

nitrate and fetal loss, neonatal mortality, mater? 

nal toxicity, and decrease in number of litters 

and live births (Table 2). Epidemiologic evi? 

dence 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 find? 

ings 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 

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

Reference Animal species Exposure Results summary 

Laven etal. 2002 Cattle 

Bruning-Fann et al. 1996 Swine 

U.S. FDA 1972a, 1972b 

Sinha etal. 1971 

Sleight 1967 

Mice, rats, hamsters, 

rabbits 

Guinea Pigs 

Guinea Pigs 

High nitrate (as available nitrogen) 

in pastures 

Nitrate in drinking water 

KN03orKN02, NaN03orNaN02 

oral intubation 

NaN02 subcutaneous injection 

KN03 or KN02 in drinking water 

NaN03, NaN02, or hydroxylamine 

in feeding 

KN03orNaN02capsules 

Grazing in pasture on oats and rape 

high in nitrate 

Grazing in pastures where soil had 

high levels of available nitrogen, 

and weeds had high nitrate levels 

Grazing in high nitrate pasture did not affect embryo survival or growth 

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

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 

No effect on maternal or fetal survival, no incidence of birth defects 

Abortion induced at 60 mg/kg NaN02 

Maternal death at 70 mg/kg NaN02 

High methemoglobin levels in mothers and fetuses 

Decrease in litter size and number of live births, and fetal loss observed in 

those given KN03 

No live births and aborted fetuses observed in those given KN02 

Two of the 15 heifers fed NaN03 died from acute nitrate poisoning, two 

aborted (one due to vibriosis) 

No abortions in heifers given small doses of KN03 or NaN02 

Three heifers died and three aborted after given higher doses of KN03 

Spontaneous abortion and stillbirths in offspring 

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

mothers' blood 

10 of 12 heifers in weedy pastures aborted 

No association between methemoglobin levels and abortion 

Abbreviations: KN02, potassium nitrite; KN03, potassium nitrate; NaN02, sodium nitrite; NaN03, sodium nitrate. 

Environmental Health Perspectives ? volume 1141 number 3I March 2006 325 
Manassaram et al. 

pregnancy or at time of delivery. Although this 

form of exposure assessment provides infor? 

mation 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 out? 

comes (Bove et al. 1992, 2002). Identifying 

which contaminant in a community water 

system is associated with a particular adverse 

reproductive outcome when multiple conta? 

minants are present is difficult. Future studies 

should conduct individual exposure assess? 

ments such as maternal interviews concerning 

water consumption habits in and outside the 

home and other risk factors such as occupa? 

tional exposures or smoking. 

Many studies on birth defects are also lim? 

ited 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 out? 

come. 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 contamina? 

tion should be encouraged to increase monitor? 

ing or surveillance of such systems. Future 

research could include long-term monitoring or 

surveillance of water systems vulnerable to cont? 

amination. This could provide valuable expo? 

sure 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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