Prenatal Exposure to Nitrate from Drinking Water and Markers of Fetal Growth Restriction: A Population-Based Study of Nearly One Million Danish-Born Children

Background: High levels of nitrate (NO3−) in drinking water cause methemoglobinemia in infants; however, few studies have examined the potential effects of low-level exposure on fetal growth, and the results have been inconsistent. Objectives: We sought to assess the association between maternal exposure to nitrate in drinking water during pregnancy and offspring size at birth in a nationwide study of full-term (≥37 wk gestation) live-born singletons. Methods: We estimated maternal nitrate exposure for 898,206 births in Denmark during 1991–2011 by linkage of individual home address(es) with nitrate data from the national monitoring database. Maternal address during pregnancy, infant size at birth [i.e., birth weight, low birth weight (LBW), body length, and birth head circumference] and covariates were compiled from the Danish Civil Registration System, the Danish Medical Birth Register, and The Integrated Database for Longitudinal Labor Market Research. Linear and logistic models with generalized estimating equations were used to account for multiple births to an individual. Nitrate exposure was modeled using five categories and as a log-transformed continuous variable. Results: There was evidence of a decreasing trend in models for term birth weight using categorical or continuous measures of exposure. Modeling exposure continuously, a difference of −9.71 g (95% confidence interval: −14.60, −4.81) was predicted at 25 mg/L (half the value of the European Union drinking water standard) compared with 0 mg/L NO3−. Body length also decreased as nitrate concentrations increased in categorical and continuous models. There was little evidence of an association between NO3− and head circumference or LBW. Discussion: Although the estimated effects were small, our findings for live singleton births to Danish-born parents suggest that maternal intake of nitrate from drinking water may reduce term birth weight and length, which are markers of intrauterine growth. However, there was little evidence for an association between nitrate and head circumference or LBW. Future studies in other populations and with data on dietary sources of nitrate are encouraged to confirm or refute these findings. https://doi.org/10.1289/EHP7331

. Characteristics of the study population by low birthweight, 1991-2011. Table S2. Difference in the mean birthweight (grams) for NO3concentrations in household drinking water restricted to babies born to mothers who were on public water throughout their pregnancy, and restricted to babies born to mothers whose nitrate levels were never reported above the EU standard of 50 mg/L. Table S3. Difference in mean body length at birth (millimeters) restricted to babies born to mothers who were on public water throughout their pregnancy, and restricted to babies born to mothers whose nitrate levels were never reported above the EU standard of 50 mg/L. Table S4. Difference in mean head circumference (millimeters) restricted to babies born to mothers who were on public water throughout their pregnancy, and restricted to babies born to mothers whose nitrate levels were never reported above the EU standard of 50 mg/L. Table S5. Adjusted odds ratios (aOR) for the association between term low birthweight and household NO3concentration, restricted to babies born to mothers who were on public water throughout their pregnancy, and restricted to babies born to mothers whose nitrate levels were never reported above the EU standard of 50 mg/L. Table S6. Difference in the mean birthweight (g) and body length at birth (mm) and odds of low birthweight for NO3concentrations in household drinking water to babies born during the full cohort  and restricted to the later, lower exposure years (1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011). Table S7. Difference in the mean birthweight (g), birth length (mm), head circumference (mm), and odds ratios (OR) for low birthweight using categorical and continuous variables for NO3concentrations in household drinking water, restricting to those with a recorded value for maternal pre-pregnancy height and weight. Table S8. Difference in the mean birthweight (g), birth length (mm), and head circumference (mm) using categorical and continuous variables for NO3concentrations in household drinking water, adding one additional potential confounder to the base model. Table S9. Adjusted odds ratios for the association between term low birthweight and household NO3concentrations, adding one additional potential confounder to the base model. Table S10. Difference in the mean birthweight (g), birth length (mm), head circumference (mm), and low birthweight for NO3concentrations in household drinking water in two different exposure categorical schemes (five and four categories).
Note: All X 2 tests for difference between strata were significant at p ≤ 0.001 except for the water supply (p = 0.36) and continuous nitrate (p = 0.007). a. The study population: full-term singleton live births in Denmark with a birthweight measurement born January 1, 1991 to December 31, 2011 to Danish-born parents who have at least eight address-linked NO 3 measurements and with non-missing covariates in the base model b. As reported two years prior to birth and standardized to 2009 values c. Maternal height and weight were assessed two years prior to birth and available from 2003 onward only, which reduces the sample size to 3,038 cases and 294,715 non-cases of LBW d. For children born in the period before 1997 smoking was recorded at the first visit with the midwife with no specifications as to the timing. For children born from 1997 onward smoking is during pregnancy. e. As reported two years before birth f. Municipalities in Denmark where the largest town has < 10,000 inhabitants g. Municipalities having a town with between 10,000 and 100,000 inhabitants h. Municipalities having a town with > 100,000 inhabitants i. Public water throughout pregnancy j. Private well at some point in pregnancy k. Public water supply for at least 8 out of the 10 months during pregnancy and unknown water supply for the remaining months l. Available from 1997 onward only, which reduces the sample size to 6,481 cases and 591,873 non-cases of LBW Table S2. Difference in the mean birthweight (grams) for NO 3concentrations in household drinking water restricted to babies born to mothers who were on public water throughout their pregnancy, and restricted to babies born to mothers whose nitrate levels were never reported above the EU standard of 50 mg/L. Note: EU = European Union. CI = Confidence interval. Models were fitted using linear regression with generalized estimating equations in order to control for the non-independence of births from the same mother and were controlled for maternal age, calendar year, sex, gravidity, maternal smoking, maternal education, maternal income, maternal employment status, region, and urbanicity. a. The continuous NO 3 exposure variable was log transformed, ln(x+1) and β (95% CI) shown for exposures x = 25.0 mg/L NO 3 compared to 0 mg/L Table S3. Difference in mean body length at birth (millimeters) restricted to babies born to mothers who were on public water throughout their pregnancy, and restricted to babies born to mothers whose nitrate levels were never reported above the EU standard of 50 mg/L. Note: EU = European Union. CI = Confidence interval. Models were fitted using linear regression with generalized estimating equations in order to control for the non-independence of births from the same mother and were controlled for maternal age, calendar year, sex, gravidity, maternal smoking, maternal education, maternal income, maternal employment status, region, and urbanicity. a. The continuous NO 3 exposure variable was log transformed, ln(x+1) and β (95% CI) shown for exposures x = 25.0 mg/L NO 3 compared to 0 mg/L Table S4. Difference in mean head circumference (millimeters) restricted to babies born to mothers who were on public water throughout their pregnancy, and restricted to babies born to mothers whose nitrate levels were never reported above the EU standard of 50 mg/L.
Base model a Only public Never above 50 mg/L n = 588,981 n = 586,128 n = 584,807  Table S5. Adjusted odds ratios (aOR) for the association between term low birthweight and household NO 3concentration, restricted to babies born to mothers who were on public water throughout their pregnancy, and restricted to babies born to mothers whose nitrate levels were never reported above the EU standard of 50 mg/L. Notes: EU = European Union. CI = Confidence interval. Models were fitted using logistic regression with generalized estimating equations in order to control for the non-independence of births from the same mother and were controlled for maternal age, calendar year, sex, gravidity, maternal smoking, maternal education, maternal income, maternal employment status, region, and urbanicity. a. The continuous NO 3 exposure variable was log transformed, ln(x+1) and aOR (95% CI) shown for exposures x = 25.0 mg/L NO 3 compared to 0 mg/L Table S6. Difference in the mean birthweight (g) and body length at birth (mm) and odds of low birthweight for NO 3concentrations in household drinking water to babies born during the full cohort  and restricted to the later, lower exposure years (1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011) Note: CI = Confidence interval. Models were fitted using linear regression with generalized estimating equations in order to control for the nonindependence of births from the same mother and were controlled for maternal age, calendar year, sex, gravidity, maternal smoking, maternal education, maternal income, maternal employment status, region, and urbanicity. a. Total n is the same as the continuous model n b. The continuous NO 3 exposure variable was log transformed, ln(x+1) and β (95% CI) shown for exposures x = 25.0 mg/L NO 3 compared to 0 mg/L c. The continuous NO 3 exposure variable was log transformed, ln(x+1) and aOR (95% CI) shown for exposures x = 25.0 mg/L NO 3 compared to 0 mg/L Table S7. Difference in the mean birthweight (g), birth length (mm), head circumference (mm), and odds ratios (OR) for low birthweight using categorical and continuous variables for NO 3concentrations in household drinking water, restricting to those with a recorded value for maternal pre-pregnancy height and weight.

Birthweight (g) restricted base model a,b
Birthweight ( Table S8. Difference in the mean birthweight (g), birth length (mm), and head circumference (mm) using categorical and continuous variables for NO 3concentrations in household drinking water, adding one additional potential confounder to the base model.
Categorical NO 3estimation (mg/L) Continuous ≤ 1 > 1 -≤ 2 > 2 -≤ 5 > 5 -≤ 25 > 25 at 25 mg/L NO 3n ∆ (95% CI) p for trend ∆ (95% CI) Birthweight (g) base model a 852,348 Ref ( Note: Models were fitted using linear regression with generalized estimating equations in order to control for the non-independence of births from the same mother. The continuous NO 3 exposure variable was log transformed, ln(x+1) and β (95% CI) shown for exposures x = 25.0 mg/L NO 3 compared to 0 mg/L. a. Base model: Controlled for maternal age, calendar year, sex, gravidity, maternal smoking, maternal education, maternal income, maternal employment status, region, and urbanicity Note: CI = Confidence interval. Models were fitted using linear and logistic regression with generalized estimating equations in order to control for the non-independence of births from the same mother and were controlled for maternal age, calendar year, sex, gravidity, maternal smoking, maternal education, maternal income, maternal employment status, region, and urbanicity. a. n = 852,348 b. n = 848,106 c. n = 588,981 d. Data were available only for births ≥1997