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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 Accessed: 13/11/2010 14:36

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http://www.jstor.orgReview_

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 reviewwe presentanupdateon maternalexposureto nitratesin drinkingwaterin relationto possible adversereproductiveand developmentaleffects,and also discussnitratesin drinkingwater in the United States. The currentstandardfor nitratesin drinkingwater is basedon retrospective studiesandapproximates alevelthatprotectsinfantsfrommethemoglobinemia,but no safetyfactor is builtinto the standard. The currentstandardappliesonly to publicwatersystems.Drinkingwater

sourcewas relatedto nitrateexposure(i.e., privatesystemswaterwas more likelythan community system water to have nitrate levels above the maximum contaminant limit). Animal studies have found adversereproductiveeffects resultingfrom higher doses of nitrateor nitrite. The epidemio? logic evidence of a direct exposure-response relationshipbetween drinkingwater nitratelevel and adversereproductiveeffect is still not clear. However, some reportshave suggested an association between exposure to nitrates in drinking water and spontaneous abortions, intrauterine growth restriction,andvariousbirthdefects.Uncertaintiesin epidemiologicstudiesincludethe lackof indi? vidualexposureassessmentthatwould ruleout confoundingofthe exposurewith some othercause. Nitrates may bejust one of the contaminantsin drinkingwater contributingto adverseoutcomes. We concludethat the currentliteraturedoes not providesufficientevidenceof a causalrelationship betweenexposureto nitratesin drinkingwaterandadversereproductive effects.Futurestudiesincorporatingindividualexposureassessmentaboutusersofprivatewells?the populationmost at risk? should be considered.Keywords: developmentaloutcomes, drinkingwater, groundwater,maternal
exposure,nitrate,nitrite, privatewells, reproductivehealth. EnvironHealth Perspect114:320-327 (2006). doi:10.1289/ehp.8407 available via http://dx.doi.org/

[Online3 November2005]

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 arebased 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, HealthStudiesBranch, 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 16June 2005;accepted

3November

2005.

320 volume 1141number

31March 2006 ? Environmental

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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

Table1.Summary

ofepidemiologic

studiesthatevaluatedexposureto nitrate indrinking

waterandreproductive

anddevelopmental

effects.
Reference No.ofsubjects Exposure Outcome Results
Brender etal.2004

Cedergren etal. 2002

Bukowski etal.2001

Croen etal.2001

Tabacova etal. 1997

Tabacova etal. 1998

Arbuckle etal. 1988

Aschengrauetal.

1989

Dorsch etal. 1984

Scragg etal. 1982

Super etal. 1981

Gelperinetal.

1975 184cases,

225controls

71,978 infants

546infant cases, 4,098infant

controls

538infant cases, 539infant controls

61pregnancies

51mother-infant

pairs

130infant cases, 260infant controls

286cases, 1,391 controls

218infant case-control

pairs

699perinatal

deaths

486infants

30,980 infants Drinking water nitrate level, dietary nitrates and nitrites, nitrosatable

drugs

Drinking water source; address closetoconception

orearly pregnancy Drinking water source, mother's address atdelivery

Mother's address before and during first trimester, drinking water source, interview data
onwater consumption

Public drinking water source, N02 inambient air; based

onaddress

during pregnancy

Public drinking water source, N02 inambient air, nitrate. infood; based onaddress

at delivery

Household water sample, drinking water source

Public drinking water source, address atpregnancy

outcome

Address atdelivery,

water sample from address

Drinking water source

Drinking water well,interview data onwater consumption
Community

drinking water
source NTDs

Congenital

cardiac defects

Premature birth, lowbirth weight, IUGR

NTDs

Pregnancy complications

Neonatal health status

CNS defects

Spontaneous

abortion

Congenital

malformations

Deaths due tocongenital malformations

Premature birth, sizeatbirth

Infant and fetalmortality Increased risk for NTDs shown with nitrosatable

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

and nitrate level inwater >3.5mg/L (OR =1.9; 95% Cl, 0.8-4.6) OR =1.18 (95% Cl, 0.97-1.44);
noexcessrisk

for cardiac defects shown with anincrease

innitrate 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 notsignificant

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 theMCL Methemoglobin

levels significantly

higher inwomen 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 incasesofabnormal

birth outcomes (preterm births, lowbirth 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 ofhaving aninfant 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 ofspontaneous

abortion (OR =0.5; 95% Cl, 0.2-0.9). The higher thelevel ofnitrate in thewater themother

consumed,

thegreater

the risk estimate for having achild with a malformation.

Women whoconsume mainly groundwater

had agreater risk of having achild with amalformation (relative risk =2.8; 95% Cl,1.6-4.4) Highest death rate inarea served by drinking water source with higher nitrate levels Nosignificant

association

between higher nitrate levels in wellwater and incidence ofpremature

birth orsizeatbirth Noincrease

in fetalorinfant

mortality inareas where community

water contained excessnitrates
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

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31March 2006 321Manassaram et al.

shallow groundwater sampled beneath agricul? tural and urban areashad 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 privatewater systems areof 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 January2004. 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 averageadult 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 vivoformation of A^-nitroso compounds from nitrite (Swann 1975). Nitrite is also a substrate in the formation of A^-nitrosocom? 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 ratsand 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 ratssodium 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 traversethe 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 heifersin the untreatedpastures,compared with one of eight in the treated pastures, aborted their fetuses.

322 volume 1141number

3 IMarch 2006 ? Environmental

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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 heiferswere given sodium nitrate daily in their feedings from 2 months of preg? nancy until they aborted or gave birth. The dosagewas adjustedweekly 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 acutenitratepoisoning. Other adversereproductive 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 viabilityin 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 eitherpasture. 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 casesof 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 (Brenderet 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 areasbelow 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 riskfor 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 surfacewater and groundwater). There was no increased riskfor 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 Scragget 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

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3 IMarch 2006 323Manassaram 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 studiesassessedexposureto nitratesin drinking wateron the basisof 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 presenceof 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 first27 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 datawere obtained from public records of routine analysesof 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 nitratelevels 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 nitratelevels. 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 theirwatercomparedwith other areas. Skrivan (1971) measured methemoglobin levels in the blood of pregnant women over a 2-year period to evaluatewhether 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 trimesterof pregnancy. Higher levels were observedin women who spontaneously aborted

or who threatened abortion in the first trimester.The reportnoted nitratesand nitrites

as the most common methemoglobin inducers and concluded that high maternal methemo? globin levelsarepossibly relatedto 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,intrauterinegrowth 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-carryingcapacityofthe blood, may interfere with the course and out?

come of pregnancy (NAS 1981; Tabacova and Balabaeva1993; 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 addressat 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 isfairlyconsistent and suggestive;how?
ever, assessing exposure to nitrate based on ecologic classificationmakes it difficult to interpret the findings exclusively in terms of water nitrateexposure (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 areaknown 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 1141number

31March 2006 ? Environmental

Health PerspectivesNitrate 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 (Tabacovaet al. 1997). Tabacova et al. (1998) also evaluated mother-infant pairsin an areaof Bulgariawith chronic exposure to pollution by nitrogen compounds, including drinking water nitrate levels of 8-54 mg/L nitrate-N in municipal watersystem, 13^00 mg/L nitrate-N in wells, and nitrogen oxides in ambient air. They examined methemoglobin levels as markersfor
exposure to nitrogen compounds, measures of oxidative stress,and the health status of infants at birth. Fifty-one mother-infant pairs were recruitedfrom 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 casesof normal birth outcomes. The authors also reportedan association between methemo? globin levels in cord blood and adverse birth outcome. Overall, the resultsshow 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 associatedwith 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 rarelyexceeds this limit. The lack of data on unregulated systems, however, is an important issue. The availabledata on the

occurrences of nitrates in drinking water indi? cate that users of private water systems are most at riskfor exposure to nitratelevels 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 nitrateand fetalloss, neonatal mortality, mater? nal toxicity, and decrease in number of litters and live births (Table 2). Epidemiologic evi? dence for increasedriskfor adversereproductive 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 forfurtherstudies. In most studies presented in this review,
exposure to nitrates in drinking water was assessedprimarilythrough waterqualitydatafor watersystems servingwomen's addressesduring

Table2.Reproductive

anddevelopmental

effects inanimal

studiesthatevaluatedexposureto nitrate ornitrite.
Reference Animal species Exposure Results summary Laven etal. 2002 Cattle
Bruning-Fann

etal.1996 Swine

U.S. FDA 1972a, 1972b

Sinha etal. 1971

Sleight 1967 Mice, rats, hamsters, rabbits Guinea Pigs

Guinea Pigs High

nitrate (asavailable

nitrogen) inpastures Nitrate indrinking water

KN03orKN02,

NaN03orNaN02 oral intubation NaN02 subcutaneous

injection

KN03 orKN02 indrinking water

NaN03, NaN02, orhydroxylamine in feeding KN03orNaN02capsules
Grazing inpasture onoatsand

rape high innitrate
Grazing inpastures

where soilhad high levels ofavailable

nitrogen, and weedshad

high nitrate levels Grazing

inhigh nitrate pasture did not affect embryo survival orgrowth Higher milk and plasma urea levels incowsgrazing

inhigh nitrate pastures Nitrate detected in53% ofsampled

wells,median

concentration

of2.1ppm Noassociation

between thenitrate

level indrinking water and litter size, number ofstillbirths,

deaths, orother known illnesses Noeffectonmaternal

or fetalsurvival,

noincidence

ofbirth defects

Abortion induced at60mg/kg

NaN02 Maternal death at70mg/kg

NaN02 High methemoglobin

levels inmothers and fetuses Decrease inlitter sizeand number oflive births, and fetallossobserved

in thosegiven

KN03 Nolive births and aborted fetusesobserved

inthosegiven

KN02 Two ofthe15heifers

fedNaN03

died from acute nitrate poisoning, two aborted (one due tovibriosis) Noabortions

inheifers given small dosesofKN03

orNaN02 Three heifers died and three aborted after given higher dosesofKN03 Spontaneous

abortion and stillbirths inoffspring Negative testfor infectious diseasebut

high levels ofnitrate found inthe mothers' blood 10of12heifers

inweedy pastures aborted Noassociation

between methemoglobin

levels and abortion

Abbreviations:

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

Environmental

HealthPerspectives ? volume 1141number

3 IMarch 2006 325Manassaram et al.

pregnancyor 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 arepresent 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 maternalinterview. 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 aremore feasible and would provide knowledge about the potential effects of nitratesin drinking water on this out?

come. Spontaneous abortion may be a more sensitive indicator of adverse reproductive effects from relatively low levels of drinking watercontamination. States with large numbers of private wells where groundwateris vulnerableto contamina? tion should be encouraged to increasemonitor? ing or surveillance of such systems. Future researchcould include long-term monitoring or surveillanceofwatersystemsvulnerableto cont? amination. This could provide valuable expo?
sure assessmentinformation to conduct studies

on drinking water contaminants such as nitrates.A discussion on the appropriatenessof the current MCL is beyond the scope of this review. However, future studies with improved exposure assessment (including other dietary
sourcesand medications), adequatesample size, and evaluating endogenous nitrate exposure could help to determine whether nitrates in drinkingwater at the currentMCL increasethe riskforreproductiveand developmental effects.

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