Drinking Water Nitrate and Human Health: An Updated Review

International Journal of
Environmental Research
and Public Health
Review
Drinking Water Nitrate and Human Health: An
Updated Review
Mary H. Ward 1, *, Rena R. Jones 1 ID , Jean D. Brender 2 , Theo M. de Kok 3 , Peter J. Weyer 4 ,
Bernard T. Nolan 5 , Cristina M. Villanueva 6,7,8,9 ID and Simone G. van Breda 3
1 Occupational and Environmental Epidemiology Branch, Division of Cancer Epidemiology and Genetics,
National Cancer Institute, 9609 Medical Center Dr. Room 6E138, Rockville, MD 20850, USA;
rena.jones@nih.gov
2 Department of Epidemiology and Biostatistics, Texas A&M University, School of Public Health,
College Station, TX 77843, USA; jdbrender@sph.tamhsc.edu
3 Department of Toxicogenomics, GROW-school for Oncology and Developmental Biology,
Maastricht University Medical Center, P.O Box 616, 6200 MD Maastricht, The Netherlands;
t.dekok@maastrichtuniversity.nl (T.M.d.K.); s.vanbreda@maastrichtuniversity.nl (S.G.v.B.)
4 The Center for Health Effects of Environmental Contamination, The University of Iowa, 455 Van Allen Hall,
Iowa City, IA 52242, USA; peter-weyer@uiowa.edu
5 U.S. Geological Survey, Water Mission Area, National Water Quality Program, 12201 Sunrise Valley Drive,
Reston, VA 20192, USA; btnolan@usgs.gov
6 ISGlobal, 08003 Barcelona, Spain; cvillanueva@isiglobal.org
7 IMIM (Hospital del Mar Medical Research Institute), 08003 Barcelona, Spain
8 Department of Experimental and Health Sciences, Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain
9 CIBER Epidemiología y Salud Pública (CIBERESP), 28029 Madrid, Spain
* Correspondence: wardm@mail.nih.gov

Received: 17 May 2018; Accepted: 14 July 2018; Published: 23 July 2018
Abstract: Nitrate levels in our water resources have increased in many areas of the world largely due
to applications of inorganic fertilizer and animal manure in agricultural areas. The regulatory limit
for nitrate in public drinking water supplies was set to protect against infant methemoglobinemia,
but other health effects were not considered. Risk of specific cancers and birth defects may be
increased when nitrate is ingested under conditions that increase formation of N-nitroso compounds.
We previously reviewed epidemiologic studies before 2005 of nitrate intake from drinking water
and cancer, adverse reproductive outcomes and other health effects. Since that review, more than
30 epidemiologic studies have evaluated drinking water nitrate and these outcomes. The most
common endpoints studied were colorectal cancer, bladder, and breast cancer (three studies each),
and thyroid disease (four studies). Considering all studies, the strongest evidence for a relationship
between drinking water nitrate ingestion and adverse health outcomes (besides methemoglobinemia)
is for colorectal cancer, thyroid disease, and neural tube defects. Many studies observed increased risk
with ingestion of water nitrate levels that were below regulatory limits. Future studies of these and
other health outcomes should include improved exposure assessment and accurate characterization
of individual factors that affect endogenous nitrosation.
Keywords: drinking water; nitrate; cancer; adverse reproductive outcomes; methemoglobinemia;

thyroid disease; endogenous nitrosation; N-nitroso compounds
1. Introduction
Since the mid-1920s, humans have doubled the natural rate at which nitrogen is deposited
onto land through the production and application of nitrogen fertilizers (inorganic and manure),
Int. J. Environ. Res. Public Health 2018, 15, 1557; doi:10.3390/ijerph15071557 www.mdpi.com/journal/ijerph
Int. J. Environ. Res. Public Health 2018, 15, 1557 2 of 31
the combustion of fossil fuels, and replacement of natural vegetation with nitrogen-fixing crops such
as soybeans [1,2]. The major anthropogenic source of nitrogen in the environment is nitrogen fertilizer,
the application of which increased exponentially after the development of the Haber–Bosch process
in the 1920s. Most synthetic fertilizer applications to agricultural land occurred after 1980 [3]. Since
approximately half of all applied nitrogen drains from agricultural fields to contaminate surface and
groundwater, nitrate concentrations in our water resources have also increased [1].
The maximum contaminant level (MCL) for nitrate in public drinking water supplies in the United
States (U.S.) is 10 mg/L as nitrate-nitrogen (NO3 -N). This concentration is approximately equivalent to
the World Health Organization (WHO) guideline of 50 mg/L as NO3 or 11.3 mg/L NO3 -N (multiply
NO3 mg/L by 0.2258). The MCL was set to protect against infant methemoglobinemia; however
other health effects including cancer and adverse reproductive outcomes were not considered [4].
Through endogenous nitrosation, nitrate is a precursor in the formation of N-nitroso compounds
(NOC); most NOC are carcinogens and teratogens. Thus, exposure to NOC formed after ingestion of
nitrate from drinking water and dietary sources may result in cancer, birth defects, or other adverse
health effects. Nitrate is found in many foods, with the highest levels occurring in some green leafy
and root vegetables [5,6]. Average daily intakes from food are in the range of 30–130 mg/day as
NO3 (7–29 mg/day NO3 -N) [5]. Because NOC formation is inhibited by ascorbic acid, polyphenols,
and other compounds present at high levels in most vegetables, dietary nitrate intake may not result
in substantial endogenous NOC formation [5,7].
Studies of health effects related to nitrate exposure from drinking water were previously reviewed
through early 2004 [8]. Further, an International Agency for Research on Cancer (IARC) Working
Group reviewed human, animal, and mechanistic studies of cancer through mid-2006 and concluded
that ingested nitrate and nitrite, under conditions that result in endogenous nitrosation, are probably
carcinogenic [5]. Here, our objective is to provide updated information on human exposure and to
review mechanistic and health effects studies since 2004. We summarize how the additional studies
contribute to the overall evidence for health effects and we discuss what future research may be
most informative.
2. Drinking Water Nitrate Exposures in the United States and Europe

Approximately 45 million people in the U.S. (about 14% of the population) had self-supplied
water at their residence in 2010 [9]. Almost all (98%) were private wells, which are not regulated by the
U.S. Environmental Protection Agency (EPA). The rest of the population was served by public water
supplies, which use groundwater, surface water, or both. The U.S. Geological Survey’s National Water
Quality Assessment (USGS-NAWQA) Project [10] sampled principal groundwater aquifers used as
U.S. public and private drinking water supplies in 1988–2015. Nitrate levels in groundwater under
agricultural land were about three times the national background level of 1 mg/L NO3 -N (Figure 1) [11].
The mixed land use category mostly had nitrate concentrations below background levels reflecting
levels in deeper private and public water supply wells. Based on the NAWQA study, it was estimated
that 2% of public-supply wells and 6% of private wells exceeded the MCL; whereas, in agricultural
areas, 21% of private wells exceeded the MCL [10]. The USGS-NAWQA study also revealed significant
decadal-scale changes in groundwater nitrate concentrations among wells sampled first in 1988–2000
and again in 2001–2010 for agricultural, urban, and mixed land uses [12]. More sampling networks
had increases in median nitrate concentration than had decreases.
A study of U.S. public water supplies (PWS) using data from EPA’s Safe Drinking Water
Information System estimated that the percentage of PWS violating the MCL increased from 0.28 to
0.42% during 1994–2009; most increases were for small to medium PWS (<10,000 population served)
using groundwater [13]. As a result of increasing nitrate levels, some PWS have incurred expensive
upgrades to their treatment systems to comply with the regulatory level [14–16].
Int. J. Environ. Res. Public Health 2018, 15, x FOR PEER REVIEW 3 of 30
Figure 1.
Figure 1. Boxplots
Boxplots of of
nitrate concentrations
nitrate concentrationsin shallow groundwater
in shallow beneath
groundwater agricultural
beneath and urban
agricultural and
land uses, and at depths of private and public drinking water supplies beneath
urban land uses, and at depths of private and public drinking water supplies beneath mixed mixed land use.land
The
number
use. The of sampled
number wells were
of sampled wells 1573 (agricultural
were land), 1054
1573 (agricultural land),(urban), and 3417
1054 (urban), (mixed).
and 3417 The
(mixed).
agricultural
The andand
agricultural urban wells
urban were
wells weresampled
sampledto to
assess land
assess landuse
useeffects,
effects,whereas
whereasthethe mixed
mixed category
wells were
wells weresampled
sampled at depths
at depths of private
of private and supplies.
and public public supplies. Medianofdepths
Median depths wells inof
thewells in the
agricultural,
urban, and mixed categories were 34, 32, and 200 feet, respectively. The height of the upper of
agricultural, urban, and mixed categories were 34, 32, and 200 feet, respectively. The height barthe
is
upper
1.5 barthe
times is 1.5 timesofthe
length length
the of thethe
box, and box, and bound
lower the lowerwas bound was truncated
truncated at thedetection
at the nitrate nitrate detection
limit of
limitmg/L
0.05 of 0.05NOmg/L
3 -N.NO3-N.
In Europe,
In Europe, the
the Nitrates
Nitrates Directive
Directive was
was set in 1991
set in 1991 [17,18]
[17,18] to
to reduce
reduce or or prevent
prevent nitrate
nitrate pollution
pollution
from agriculture. Areas most affected by nitrate pollution are designated as ‘nitrate vulnerable zones’
from agriculture. Areas most affected by nitrate pollution are designated as ‘nitrate vulnerable zones’
and are
and are subject
subjecttotomandatory
mandatoryCodes
Codesof of Good
Good Agricultural
Agricultural Practice
Practice [18].[18].
The The results
results of compliance
of compliance with
with this directive have been reflected in the time trends of nitrate in some countries. For example,
this directive have been reflected in the time trends of nitrate in some countries. For example, nitrate
nitrateinlevels
levels in groundwater
groundwater in Denmark
in Denmark increasedincreased in 1950–1980
in 1950–1980 and decreased
and decreased since thesince
1990sthe 1990s
[19]. [19].
Average
Average nitrate levels in groundwater in most other European countries have been stable at around
nitrate levels in groundwater in most other European countries have been stable at around 17.5 mg/L
17.5 (4
NO mg/L
mg/L NO 3 (4 mg/L NO3-N) across Europe over a 20-year period (1992–2012), with some
NO
3 3 -N) across Europe over a 20-year period (1992–2012), with some differences between
differences between countries both in trends and concentrations. Average concentrations are lowest
countries both in trends and concentrations. Average concentrations are lowest in Finland (around
in Finland (around 1 mg/L NO3 in 1992–2012) and highest in Malta (58.1 mg/L in 2000–2012)[20].
1 mg/L NO3 in 1992–2012) and highest in Malta (58.1 mg/L in 2000–2012) [20]. Average annual nitrate
Average annual nitrate concentrations at river monitoring stations in Europe showed a steady decline
concentrations at river monitoring stations in Europe showed a steady decline from 2.7 NO3 -N in 1992
from 2.7 NO3-N in 1992 to 2.1 mg/L in 2012 [20], with the lowest average levels in Norway (0.2 mg/L
to 2.1 mg/L in 2012 [20], with the lowest average levels in Norway (0.2 mg/L NO3 -N in 2012) and
NO3-N in 2012) and highest in Greece (6.6 mg/L NO3-N in 2012).
highest in Greece (6.6 mg/L NO3 -N in 2012).
Levels in finished public drinking water have been published only for a few European countries.
Levels in finished public drinking water have been published only for a few European countries.
Trends of nitrate in drinking water supplies from 1976 to 2012 in Denmark showed a decline in public
Trends of nitrate in drinking water supplies from 1976 to 2012 in Denmark showed a decline in
supplies but not in private wells [21]. In Spain, median concentrations were 3.5 mg/L NO3 (range:
public supplies but not in private wells [21]. In Spain, median concentrations were 3.5 mg/L NO3
0.4−66.8) in 108 municipalities in 2012 [22], and 4.2 mg/L (range: <1−29) in 11 provinces in 2010 [23].
(range: 0.4−66.8) in 108 municipalities in 2012 [22], and 4.2 mg/L (range: <1−29) in 11 provinces in
Levels in other countries included a median of 0.18 mg/L (range: <0.02−7.9) in Iceland in 2001−2012
2010 [23]. Levels in other countries included a median of 0.18 mg/L (range: <0.02−7.9) in Iceland in
[24], a mean of 16.1 mg/L (range: 0.05−296 mg/L) in Sicily, Italy in 2004−2005 [25] and a range from
2001−2012 [24], a mean of 16.1 mg/L (range: 0.05−296 mg/L) in Sicily, Italy in 2004−2005 [25] and a
undetected to 63.3 mg/L in Deux-Sèvres, France in in 2005−2009 [26].
range from undetected to 63.3 mg/L in Deux-Sèvres, France in in 2005−2009 [26].
Nitrate levels in bottled water have been measured in a few areas of the EU and the U.S. and
Nitrate levels in bottled water have been measured in a few areas of the EU and the U.S. and have
have been found to be below the MCL. In Sicily, the mean level was 15.2 mg/L NO3(range: 1.2−31.8
been found to be below the MCL. In Sicily, the mean level was 15.2 mg/L NO3 (range: 1.2−31.8 mg/L)
mg/L) in 16 brands [25] and in Spain, the median level was 5.2 mg/L NO3 (range: <1.0−29.0 mg/L) in
in 16 brands [25] and in Spain, the median level was 5.2 mg/L NO3 (range: <1.0−29.0 mg/L) in
9 brands [23]. In the U.S., a survey of bottle water sold in 42 Iowa and 32 Texas communities found
9 brands [23]. In the U.S., a survey of bottle water sold in 42 Iowa and 32 Texas communities found
varying but generally low nitrate levels. Nitrate concentrations ranged from below the limit of
detection (0.1 mg/L NO3-N) to 4.9 mg/L NO3-N for U.S. domestic spring water purchased in Texas.
varying but generally low nitrate levels. Nitrate concentrations ranged from below the limit of detection
(0.1 mg/L NO3 -N) to 4.9 mg/L NO3 -N for U.S. domestic spring water purchased in Texas.
There are few published studies of nitrate concentrations in drinking water outside the U.S. and
Europe. Nitrate concentrations in groundwater were reported for Morocco, Niger, Nigeria, Senegal,
India-Pakistan, Japan, Lebanon, Philippines and Turkey with maximum levels in Senegal (median
42.9 mg/L NO3 -N) [5]. In India, nitrate in drinking water supplies is particularly high in rural areas,
where average levels have been reported to be 45.7 mg/L NO3 [27,28] and 66.6 mg/L NO3 [28];
maximum levels in drinking water exceeded 100 mg/L NO3 in several regions [27,29]. Extremely
high levels of nitrate have been reported in The Gaza Strip, where nitrate reached concentrations of
500 mg/L NO3 in some areas, and more than 50% of public-supply wells had nitrate concentrations
above 45 mg/L NO3 [30].
3. Exposure Assessment in Epidemiologic Studies

With the implementation of the Safe Drinking Water Act in 1974, more than 40 years of
monitoring data for public water supplies in the U.S. provide a framework of measurements to
support exposure assessments. Historical data for Europe are more limited, but a quadrennial nitrate
reporting requirement was implemented as part of the EU Nitrates Directive [17,18]. In the U.S.,
the frequency of sampling for nitrate in community water systems is stipulated by their sources
(ground versus surface waters) and whether concentrations are below the MCL, and historically, by
the size of the population served and vulnerability to nitrate contamination. Therefore, the exposure
assessment for study participants who report using a public drinking water source may be based on
a variable number of measurements, raising concerns about exposure misclassification. In a study
of bladder cancer risk in Iowa, associations were stronger in sensitivity analyses based on more
comprehensive measurement data [31]. Other studies have restricted analyses to subgroups with
more complete or recent measurements [32–35], with implications for study power and possible
selection biases. Sampling frequency also limits the extent to which temporal variation in exposure
can be represented within a study population, such as the monthly or trimester-based estimates of
exposure most relevant for etiologic investigations of adverse reproductive outcomes. In Denmark,
limited seasonal variation in nitrate monitoring data suggested these data would sufficiently capture
temporal variation for long-term exposure estimates [36]. Studies have often combined regulatory
measurements with questionnaire and ancillary data to better characterize individual variation in
nitrate exposure, such as to capture changes in water supply characteristics over time or a participant’s
duration at a drinking water source [31,33,37,38]. Most case-control studies of drinking water nitrate
and cancer obtained lifetime residence and drinking water source histories, whereas cohort studies
typically have collected only the current water source. Many studies lacked information about study
participants’ water consumption, which may be an important determinant of exposure to drinking
water contaminants [39].
Due to sparse measurement data, exposures for individuals served by private wells are more
difficult to estimate than exposures for those on public water supplies. However, advances in
geographic-based modeling efforts that incorporate available measurements, nitrogen inputs, aquifer
characteristics, and other data hold promise for this purpose. These models include predictor variables
describing land use, nitrogen inputs (fertilizer applications, animal feeding operations), soils, geology,
climate, management practices, and other factors at the scale of interest. Nolan and Hitt [40] and
Messier et al. [41] used nonlinear regression models with terms representing nitrogen inputs at the land
surface, transport in soils and groundwater, and nitrate removal by processes such as denitrification,
to predict groundwater nitrate concentration at the national scale and for North Carolina, respectively.
Predictor variables in the models included N fertilizer and manure, agricultural or forested land
use, soils, and, in Nolan and Hitt [40], water-use practices and major geology. Nolan and Hitt [40]
reported a training R2 values of 0.77 for a model of groundwater used mainly for private supplies and
Messier, Kane, Bolich and Serre [41] reported a cross-validation testing R2 value of 0.33 for a point-level
private well model. These and earlier regression approaches for groundwater nitrate [42–46] relied
on predictor variables describing surficial soils and activities at the land surface, because conditions
at depth in the aquifer typically are unknown. Redox conditions in the aquifer and the time since
water entered the subsurface (i.e., groundwater age) are two of the most important factors affecting
groundwater nitrate, but redox constituents typically are not analyzed, and age is difficult to measure.
Even if a well has sufficient data to estimate these conditions, the data must be available for all wells in
order to predict water quality in unsampled areas. In most of the above studies, well depth was used
as a proxy for age and redox and set to average private or public-supply well depth for prediction.
Recent advances in groundwater nitrate exposure modeling have involved machine-learning
methods such as random forest (RF) and boosted regression trees (BRT), along with improved
characterization of aquifer conditions at the depth of the well screen (the perforated portion of the
well where groundwater intake occurs). Tree-based models do not require data transformation,
can fit nonlinear relations, and automatically incorporate interactions among predictors [47].
Wheeler et al. [48] used RF to estimate private well nitrate levels in Iowa. In addition to land use
and soil variables, predictor variables included aquifer characteristics at the depth of the well screen,
such as total thickness of fine-grained glacial deposits above the well screen, average and minimum
thicknesses of glacial deposits near sampled wells, and horizontal and vertical hydraulic conductivities
near the wells. Well depth, landscape features, nitrogen sources, and aquifer characteristics ranked
highly in the final model, which explained 77% and 38% of the variation in training and hold-out
nitrate data, respectively.
Ransom et al. [49] used BRT to predict nitrate concentration at the depths of private and
public-supply wells for the Central Valley, California. The model used as input estimates of
groundwater age at the depth of the well screen (from MODFLOW/MODPATH models) and
depth-related reducing conditions in the groundwater. These estimates were generated by separate
models and were available throughout the aquifer. Other MODFLOW-based predictor variables
comprised depth to groundwater, and vertical water fluxes and the percent coarse material in
the uppermost part of the aquifer where groundwater flow was simulated by MODFLOW. Redox
variables were top-ranked in the final BRT model, which also included land use-based N leaching
flux, precipitation, soil characteristics, and the MODFLOW-based variables described above. The final
model retained 25 of an initial 145 predictor variables considered, had training and hold-out R2 values
of 0.83 and 0.44 respectively, and was used to produce a 3D visualization of nitrate in the aquifer. These
studies show that modeling advances and improved characterization of aquifer conditions at depth
are increasing our ability to predict nitrate exposure from drinking water supplied by private wells.
4. Nitrate Intake and Endogenous Formation of N-Nitroso Compounds

Drinking water nitrate is readily absorbed in the upper gastrointestinal tract and distributed
in the human body. When it reaches the salivary glands, it is actively transported from blood into
saliva and levels may be up to 20 times higher than in the plasma [50–53]. In the oral cavity 6–7%
of the total nitrate can be reduced to nitrite, predominantly by nitrate-reducing bacteria [52,54,55].
The secreted nitrate as well as the nitrite generated in the oral cavity re-enter the gastrointestinal tract
when swallowed.
Under acidic conditions in the stomach, nitrite can be protonated to nitrous acid (HNO2 ),
and subsequently yield dinitrogen trioxide (N2 O3 ), nitric oxide (NO), and nitrogen dioxide (NO2 ).
Since the discovery of endogenous NO formation, it has become clear that NO is involved in a wide
range of NO-mediated physiological effects. These comprise the regulation of blood pressure and
blood flow by mediating vasodilation [56–58], the maintenance of blood vessel tonus [59], the inhibition
of platelet adhesion and aggregation [60,61], modulation of mitochondrial function [62] and several
other processes [63–66].
On the other hand, various nitrate and nitrite derived metabolites such as nitrous acid
(HNO2 ) are powerful nitrosating agents and known to drive the formation of NOC, which are
suggested to be the causal agents in many of the nitrate-associated adverse health outcomes. NOC
comprise N-nitrosamines and N-nitrosamides, and may be formed when nitrosating agents encounter
N-nitrosatable amino acids, which are also from dietary origin. The nitrosation process depends on
the reaction mechanisms involved, on the concentration of the compounds involved, the pH of the
reaction environment, and further modifying factors, including the presence of catalysts or inhibitors
of N-nitrosation [66–69].
Endogenous nitrosation can also be inhibited, for instance by dietary compounds like vitamin
C, which has the capacity to reduce HNO2 to NO; and alpha-tocopherol or polyphenols, which can
reduce nitrite to NO [54,70–72]. Inhibitory effects on nitrosation have also been described for dietary
flavonoids such as quercetin, ferulic and caffeic acid, betel nut extracts, garlic, coffee, and green tea
polyphenols [73,74]. Earlier studies showed that the intake of 250 mg or 1 g ascorbic acid per day
substantially inhibited N-nitrosodimethylamine (NDMA) excretion in 25 women consuming a fish
meal rich in amines (nitrosatable precursors) for seven days, in combination with drinking water
containing nitrate at the acceptable daily intake (ADI) [75]. In addition, strawberries, garlic juice,
and kale juice were shown to inhibit NDMA excretion in humans [76]. The effect of these fruits
and vegetables is unlikely to be due solely to ascorbic acid. Using the N-nitrosoproline (NPRO) test,
Helser et al. [77] found that ascorbic acid only inhibited nitrosamine formation by 24% compared with
41–63% following ingestion of juices (100 mL) made of green pepper, pineapple, strawberry or carrot
containing an equal total amount of ascorbic acid.
The protective potential of such dietary inhibitors depends not only on the reaction rates of
N-nitrosatable precursors and nitrosation inhibitors, but also on their biokinetics, since an effective
inhibitor needs to follow gastrointestinal circulation kinetics similar to nitrate [78]. It has been argued
that consumption of some vegetables with high nitrate content, can at least partially inhibit the
formation of NOC [79–81]. This might apply for green leafy vegetables such as spinach and rocket
salad, celery or kale [77] as well as other vegetables rich in both nitrate and natural nitrosation
inhibitors. Preliminary data show that daily consumption of one bottle of beetroot juice containing
400 mg nitrate (the minimal amount advised for athletes to increase their sports performances) for one
day and seven days by 29 young individuals results in an increased urinary excretion of apparent total
nitroso compounds (ATNC), an effect that can only be partially inhibited by vitamin C supplements
(1 g per day) [82].
Also, the amount of nitrosatable precursors is a key factor in the formation of NOC. Dietary
intakes of red and processed meat are of particular importance [83–87] as increased consumption
of red meat (600 vs. 60 g/day), but not white meat, was found to cause a three-fold increase in
fecal NOC levels [85]. It was demonstrated that heme iron stimulated endogenous nitrosation [84],
thereby providing a possible explanation for the differences in colon cancer risk between red and white
meat consumption [88]. The link between meat consumption and colon cancer risk is even stronger
for nitrite-preserved processed meat than for fresh meat leading an IARC review to conclude that
processed meat is carcinogenic to humans [89].
In a human feeding study [90], the replacement of nitrite in processed meat products by natural
antioxidants and the impact of drinking water nitrate ingestion is being evaluated in relation to fecal
excretion of NOC, accounting for intakes of meat and dietary vitamin C. A pilot study demonstrated
that fecal excretion of ATNC increased after participants switched from ingesting drinking water with
low nitrate levels to drinking water with nitrate levels at the acceptable daily intake level of 3.7 mg/kg.
The 20 volunteers were assigned to a group consuming either 3.75 g/kg body weight (maximum
300 g per day) red processed meat or fresh (unprocessed) white meat. Comparison of the two dietary
groups showed that the most pronounced effect of drinking water nitrate was observed in the red
processed meat group. No inhibitory effect of vitamin C intake on ATNC levels in feces was found
(unpublished results).
5. Methemoglobinemia
The physiologic processes that can lead to methemoglobinemia in infants under six months
of age have been described in detail previously [8,91]. Ingested nitrate is reduced to nitrite by
bacteria in the mouth and in the infant stomach, which is less acidic than adults. Nitrite binds
to hemoglobin to form methemoglobin, which interferes with the oxygen carrying capacity of the
blood. Methemoglobinemia is a life-threatening condition that occurs when methemoglobin levels
exceed about 10% [8,91]. Risk factors for infant methemoglobinemia include formula made with
water containing high nitrate levels, foods and medications that have high nitrate levels [91,92], and
enteric infections [93]. Methemoglobinemia related to high nitrate levels in drinking water used to
make infant formula was first reported in 1945 [94]. The U.S. EPA limit of 10 mg/L NO3 -N was set
as about one-half the level at which there were no observed cases [95]. The most recent U.S. cases
related to nitrate in drinking water were reported by Knobeloch and colleagues in the late 1990s in
Wisconsin [96] and were not described in our prior review. Nitrate concentrations in the private wells
were about two-times the MCL and bacterial contamination was not a factor. They also summarize
another U.S. case in 1999 related to nitrate contamination of a private well and six infant deaths
attributed to methemoglobinemia in the U.S. between 1979–1999 only one of which was reported in
the literature [96,97]. High incidence of infant methemoglobinemia in eastern Europe has also been
described previously [98,99]. A 2002 WHO report on water and health [100] noted that there were
41 cases in Hungary annually, 2913 cases in Romania from 1985–1996 and 46 cases in Albania in 1996.
Results of several epidemiologic studies conducted before 2005 that examined the relationship
between nitrate in drinking water and levels of methemoglobin or methemoglobinemia in infants have
been described previously [8]. Briefly, nitrate levels >10 mg/L NO3 -N were usually associated with
increased methemoglobin levels but clinical methemoglobinemia was not always present. Since our
last review, a cross-sectional study conducted in Gaza found elevated methemoglobin levels in infants
on supplemental feeding with formula made from well water in an area with the highest mean nitrate
concentration of 195 mg/L NO3 (range: 18–440) compared to an area with lower nitrate concentration
(mean: 119 mg/L NO3 ; range 18–244) [101]. A cross-sectional study in Morocco found a 22% increased
risk of methemoglobinemia in infants in an area with drinking water nitrate >50 mg/L (>11 as NO3 -N)
compared to infants in an area with nitrate levels <50 mg/L nitrate [102]. A retrospective cohort study
in Iowa of persons (aged 1–60 years) consuming private well water with nitrate levels <10 mg/L
NO3 -N found a positive relationship between methemoglobin levels in the blood and the amount of
nitrate ingestion [103]. Among pregnant women in rural Minnesota with drinking water supplies that
were mostly ≤3 mg/L NO3 -N, there was no relationship between water nitrate intake and women’s
methemoglobin levels around 36 weeks’ gestation [104].
6. Adverse Pregnancy Outcomes

Maternal drinking water nitrate intake during pregnancy has been investigated as a risk factor
for a range of pregnancy outcomes, including spontaneous abortion, fetal deaths, prematurity,
intrauterine growth retardation, low birth weight, congenital malformations, and neonatal deaths.
The relation between drinking water nitrate and congenital malformations in offspring has been the
most extensively studied, most likely because of the availability of birth defect surveillance systems
around the world.
Our earlier review focused on studies of drinking water nitrate and adverse pregnancy outcomes
published before 2005 [8]. In that review, we cited several studies on the relation between maternal
exposure to drinking water nitrate and spontaneous abortion including a cluster investigation that
suggested a positive association [105] and a case-control study that found no association [106]. These
studies were published over 20 years ago. In the present review, we were unable to identify any
recently published studies on this outcome. In Table 1, we describe the findings of studies published
since 2004 on the relation between drinking water nitrate and prematurity, low birthweight, and
congenital malformations. We report results for nitrate in the units (mg/L NO3 or NO3 -N) that
were reported in the publications. In a historic cohort study conducted in the Deux-Sèvres district
(France), Migeot et al. [26] linked maternal addresses from birth records to community water system
measurements of nitrate, atrazine, and other pesticides. Exposure to the second tertile of nitrate
(14–27 mg/L NO3 ) without detectable atrazine metabolites was associated with small-for-gestational
age births (Odds Ratio (OR) 1.74, 95% CI 1.1, 2.8), but without a monotonic increase in risk with
exposures. There was no association with nitrate among those with atrazine detected in their drinking
water supplies. Within the same cohort, Albouy-Llaty and colleagues did not observe any association
between higher water nitrate concentrations (with or without the presence of atrazine) and preterm
birth [107].
Stayner and colleagues also investigated the relation between atrazine and nitrate in drinking
water and rates of low birth weight and preterm birth in 46 counties in four Midwestern U.S. states
that were required by EPA to measure nitrate and atrazine monthly due to prior atrazine MCL
violations [108]. The investigators developed county-level population-weighted metrics of average
monthly nitrate concentrations in public drinking water supplies. When analyses were restricted to
counties with less than 20% private well usage (to reduce misclassification due to unknown nitrate
levels), average nitrate concentrations during the pregnancy were associated with increased rates of
very low birth weight (<1.5 kg Rate Ratio (RR)per 1 ppm = 1.17, 95% CI 1.08, 1.25) and very preterm
births (<32 weeks RRper 1 ppm = 1.08, 95% CI 1.02, 1.15) but not with low birth weight or preterm
birth overall.
In record-based prevalence study in Perth Australia, Joyce et al. mapped births to their water
distribution zone and noted positive associations between increasing tertiles of nitrate levels and
prevalence of term premature rupture of membranes (PROM) adjusted for smoking and socioeconomic
status [109]. Nitrate concentrations were low; the upper tertile cut point was 0.350 mg/L and
the maximum concentration was 1.80 mg/L NO3 -N. Preterm PROM was not associated with
nitrate concentrations.
Among studies of drinking water nitrate and congenital malformations, few before 2005 included
birth defects other than central nervous system defects [8]. More recently, Mattix et al. [110] noted
higher rates of abdominal wall defects (AWD) in Indiana compared to U.S. rates for specific years
during the period 1990–2002. They observed a positive correlation between monthly AWD rates and
monthly atrazine concentrations in surface waters but no correlation with nitrate levels. Water quality
data were obtained from the USGS-NAWQA project that monitors agricultural chemicals in streams
and shallow groundwater that are mostly not used as drinking water sources. A case-control study of
gastroschisis (one of the two major types of AWD), in Washington State [111] also used USGS-NAWQA
measurements of nitrate and pesticides in surface water and determined the distance between maternal
residences (zip code centroids) and the closest monitoring site with concentrations above the MCL for
nitrate, nitrite, and atrazine. Gastrochisis was not associated with maternal proximity to surface water
above the MCL for nitrate (>10 mg/L NO3 -N) or nitrite (>1 mg/L NO2 -N) but there was a positive
relationship with proximity to sites with atrazine concentrations above the MCL. In a USA-wide
study, Winchester et al. [112] linked the USGS-NAWQA monthly surface water nitrate and pesticide
concentrations computed for the month of the last menstrual period with monthly rates of 22 types
of birth defects in 1996–2002. Rates of birth defects among women who were estimated to have
conceived during April through July were higher than rates among women conceiving in other months.
In multivariable models that included nitrate, atrazine, and other pesticides, atrazine (but not nitrate
or other pesticides) was associated with several types of anomalies. Nitrate was associated with birth
defects in the category of “other congenital anomalies” (OR 1.18, 95% CI 1.14, 1.21); the authors did
not specify what defects were included in this category. None of these three studies included local
or regional data to support the assumption that surface water nitrate and pesticide concentrations
correlated with drinking water exposures to these contaminants.
Using a more refined exposure assessment than the aforementioned studies, Holtby et al. [113]
conducted a case-control study of congenital anomalies in an agricultural county in Nova Scotia,
Canada. They linked maternal addresses at delivery to municipal water supply median nitrate
concentrations and used kriging of monthly measurements from a network of 140 private wells to
estimate drinking water nitrate concentrations in private wells. They observed no associations between
drinking water nitrate and all birth defects combined for conceptions during 1987–1997. However, the
prevalence of all birth defects occurring during 1998–2006 was associated with drinking water nitrate
concentrations of 1–5.56 mg/L NO3 -N (OR 2.44, 95% CI 1.05, 5.66) and ≥5.56 mg/L (OR 2.25, 95% CI
0.92, 5.52).
None of the studies of congenital anomalies accounted for maternal consumption of bottled
water or the quantity of water consumed during the first trimester, the most critical period of
organ/structural morphogenesis. Attempting to overcome some of these limitations, Brender, Weyer,
and colleagues [38,114] conducted a population-based, case-control study in the states of Iowa and
Texas where they: (1) linked maternal addresses during the first trimester to public water utilities and
respective nitrate measurements; (2) estimated nitrate intake from bottled water based on a survey of
products consumed and measurement of nitrate in the major products; (3) predicted drinking water
nitrate from private wells through modeling (Texas only); and (4) estimated daily nitrate ingestion
from women’s drinking water sources and daily consumption of water. The study populations were
participants of the U.S. National Birth Defects Prevention Study [115]. Compared to the lowest tertile
of nitrate ingestion from drinking water (<0.91 mg/day NO3 ), mothers of babies with spina bifida
were twice as likely (95% CI 1.3, 3.2) to ingest ≥5 mg/day NO3 from drinking water than control
mothers. Mothers of babies with limb deficiencies, cleft palate, and cleft lip were, respectively,
1.8 (95% CI 1.1, 3.1), 1.9 (95% CI 1.2, 3.1), and 1.8 (95% CI 1.1, 3.1) times more likely to ingest
≥5.4 mg/day of water NO3 than controls. Women were also classified by their nitrosatable drug
exposure during the first trimester [116] and by their daily nitrate and nitrite intake based on a
food frequency questionnaire [117]. Higher ingestion of drinking water nitrate did not strengthen
associations between maternal nitrosatable drug exposure and birth defects in offspring [38]. However,
a pattern was observed of stronger associations between nitrosatable drug exposure and selected birth
defects for women in the upper two tertiles of total nitrite ingestion that included contributions from
drinking water nitrate and dietary intakes of nitrate and nitrite compared to women in the lowest
tertile. Higher intake of food nitrate/nitrite was found to also modify the associations of nitrosatable
drug exposure and birth defects in this study [118,119] as well as in an earlier study of neural tube
defects conducted in south Texas [120]. Multiplicative interactions were observed between higher food
nitrate/nitrite and nitrosatable drug exposures for conotruncal heart, limb deficiency, and oral cleft
defects [118].
In summary, five out of six studies, conducted since the 1980s of drinking water nitrate and central
nervous system defects, found positive associations between higher drinking water nitrate exposure
during pregnancy and neural tube defects or central nervous system defects combined [38,120–123].
The sixth study, which did not find a relationship, did not include measures of association, but
compared average drinking water nitrate concentrations between mothers with and without neural
tube defect-affected births, which were comparable [124].
Table 1. Studies of drinking water nitrate a and adverse pregnancy outcomes published January 2005–March 2018.
First Author, Year, Study Design Years of Outcome

Exposure Description Pregnancy Outcome Summary of Findings
Country Regional Description Ascertainment
No association for >26.99 mg/L vs.
Measurements of atrazine
<14.13 mg/L NO3 in community
Albouy-Llaty, 2016 Historic cohort study metabolites and NO3 in community
2005–2010 Preterm birth water systems with or without
France [107] Deux-Sèvres water systems (263 municipalities)
atrazine detections, adjusted for
were linked to birth addresses
neighborhood deprivation
Maternal addresses during the first
≥5 vs. <0.91 mg/day NO3 from
trimester linked to public water
drinking water spina bifida OR = 2.0
utility nitrate measurements; nitrate
Congenital heart defects (95% CI: 1.3, 3.2)
Brender, 2013 Population-based intake from bottled water estimated
Limb deficiencies ≥5.42 vs. <1.0 mg/day NO3 from
Weyer, 2014 case-control study 1997–2005 with survey and laboratory testing;
Neural tube defects water:
USA [38] Iowa and Texas nitrate from private wells predicted
Oral cleft defects limb deficiencies OR = 1.8 (CI: 1.1,
through modeling; nitrate ingestion
3.1); cleft palate OR = 1.9 (CI: 1.2, 3.1)
(NO3 ) estimated from reported water
cleft lip OR = 1.8 (CI: 1.1, 3.1)
consumption
Conceptions in 1987–1997: no
Maternal addresses at delivery
association with nitrate
Population-based linked to municipal water supply
concentrations
Holtby, 2014 case-control study median nitrate (NO3 -N) Congenital malformations
1988–2006 Conceptions in 1998–2006:
Canada [113] Kings County, Nova concentrations; nitrate in rural combined into one group
1–5.56 mg/L NO3 -N (vs. <1 mg/L)
Scotia private wells estimated from historic
OR = 2.44 (CI: 1.05, 5.66); ≥5.56
sampling and kriging
mg/L OR = 2.25 (CI: 0.92, 5.52)
Linked birth residences to 24 water
distribution zones; computed ORs for tertiles (vs. <0.125 mg/L
Premature rupture of
Record-based prevalence average NO3 -N mg/L from historical NO3 -N): 0.125–0.350 mg/L OR = 1.23
Joyce, 2008 membranes at term
study 2002–2004 measurements; independent (CI: 1.03, 1.52); >0.350 mg/L OR =
Australia [109] (PROM) (37 weeks’
Perth sampling conducted for 6 zones as 1.47 (CI: 1.20, 1.79)
gestation or later)
part of exposure validation; also No association with THM levels
evaluated trihalomethanes (THM)
No correlation observed between
Monthly abdominal wall defect rates
nitrate levels in surface water and
Mattix, 2007 Ecologic study linked to monthly surface water Abdominal wall birth
1990–2002 monthly abdominal wall defects
USA [110] Indiana nitrate and atrazine concentrations defects
Positive correlation with atrazine
(USGS-NAWQA monitoring data b )
levels
Table 1. Cont.
First Author, Year, Study Design Years of Outcome

Exposure Description Pregnancy Outcome Summary of Findings
Country Regional Description Ascertainment
ORs for tertiles (vs. <14.13 mg/L
NO3 ) in community water systems
Measurements of atrazine
with no atrazine detections: 14–27
Migeot, 2013 Historic cohort study metabolites and NO3 in community Small-for-gestational age
2005–2009 mg/L OR = 1.74 (CI: 1.10, 2.75); >27
France [26] Deux-Sèvres water systems (263 municipalities) (SGA) births
mg/L OR = OR 1.51 (CI: 0.96, 2.4); no
were linked to birth addresses
association with nitrate when
atrazine was detected
Counties had one or more water
utility in EPA’s atrazine monitoring
Average nitrate not associated with
program; excluded counties with
low birth weight and preterm birth
Ecologic study >20% of population on private wells
Stayner, 2017 Preterm birth Very low birth weight: RR for 1 ppm
46 counties in Indiana, 2004–2008 and >300,000 population. Computed
USA [108] Low birth weight increase in NO3 -N = 1.17 (CI: 1.08,
Iowa, Missouri, and Ohio county-specific monthly weighted
1.25); Very preterm birth RR for 1
averages of NO3 -N in finished
ppm increase = 1.08 (CI: 1.02, 1.15)
drinking water; exposure metric was
average 9 months prior to birth
Calculated distance between
maternal residence and closest Gastroschisis was not associated with
Population-based
Waller, 2010 stream monitoring site with maternal residential proximity to
case-control study 1987–2006 Gastroschisis
USA [111] concentrations >MCL for NO3 -N, surface water with elevated nitrate
Washington State
NO2 -N, or atrazine in surface water (>10 mg/L) or nitrite (>1 mg/L)
(USGS-NAWQA data b )
Rates of combined and specific birth Birth defect category “other
defects (computed by month of last congenital anomalies”: OR for
menstrual period) linked to monthly continuous log nitrate = 1.15 (CI: 1.12,
Winchester, 2009 Ecologic study Birth defects categorized
1996–2002 surface water nitrate concentrations 1.18); adjusted for atrazine and other
USA [112] USA-wide into 22 groups
(USGS-NAWQA data b ); also pesticides: OR = 1.18, CI: 1.14, 1.21);
evaluated atrazine and other No association with other birth
pesticides (combined) defects
Abbreviations: CI, 95% CI confidence interval; OR, odds ratio; RR, rate ratio; USGS-NAWQA, U. S. Geological Survey National Water Quality Assessment; a nitrate units are specified as
reported in publications. NO3 can be converted to NO3 -N by multiplying by 0.2258; b USGS-NAWQA data for 186 streams in 51 hydrological study areas; streams were not drinking
water sources.
7. Cancer
Most early epidemiologic studies of cancer were ecologic studies of stomach cancer mortality
that used exposure estimates concurrent with the time of death. Results were mixed, with some
studies showing positive associations, many showing no association, and a few showing inverse
associations. The results of ecologic studies through 1995 were reviewed by Cantor [125]. Our previous
review included ecologic studies of the brain, esophagus, stomach, kidney, ovary, and non-Hodgkin
lymphoma (NHL) published between 1999 and 2003 that were largely null [8]. We did not include
ecologic studies or mortality case-control studies in this review due to the limitations of these study
designs, especially their inability to assess individual-level exposure and dietary factors that influence
the endogenous formation of NOC.
Since our review of drinking water nitrate and health in 2005 [8], eight case-control studies and
eight analyses in three cohorts have evaluated historical nitrate levels in PWS in relation to several
cancers. Nitrate levels were largely below 10 mg/L NO3 -N. Most of these studies evaluated potential
confounders and factors affecting nitrosation. Table 2 shows the study designs and results of studies
published from 2005 through 2018, including findings from periodic follow-ups of a cohort study
of postmenopausal women in Iowa (USA) [31,37,126–129]. In the first analysis of drinking water
nitrate in the Iowa cohort with follow-up through 1998, Weyer and colleagues [130] reported that
ovarian and bladder cancers were positively associated with the long-term average PWS nitrate
levels prior to enrollment (highest quartile average 1955–1988: >2.46 mg/L NO3 -N). They observed
inverse associations for uterine and rectal cancer, but no associations with cancers of the breast, colon,
rectum, pancreas, kidney, lung, melanoma, non-Hodgkin lymphoma (NHL), or leukemia. Analyses
of PWS nitrate concentrations and cancers of the thyroid, breast, ovary, bladder, and kidney were
published after additional follow-up of the cohort. The exposure assessment was improved by: (a) the
computation of average nitrate levels and years of exposure at or above 5 mg/L NO3 -N, based on time
in residence (vs. one long-term PWS average nitrate estimate used by Weyer and colleagues); and
(b) by estimation of total trihalomethanes (TTHM) and dietary nitrite intake.
Thyroid cancer was evaluated for the first time after follow-up of the cohort through 2004. A total
of 40 cases were identified [37]. Among women with >10 years on PWS with levels exceeding 5 mg/L
NO3 -N for five years or more, thyroid cancer risk was 2.6 times higher than that of women whose
supplies never exceeded 5 mg/L. With follow-up through 2010, the risk of ovarian cancer remained
increased among women in the highest quartile of average nitrate in PWS [129]. Ovarian cancer risk
among private well users was also elevated compared to the lowest PWS nitrate quartile. Associations
were stronger when vitamin C intake was below median levels with a significant interaction for users
of private wells. Overall, breast cancer risk was not associated with water nitrate levels with follow-up
through 2008 [128]. Among women with folate intake ≥ 400 µg/day, risk was increased for those
in the highest average nitrate quintile (Hazard Ratio (HR) = 1.40; 95% CI: = 1.05–1.87) and among
private well users (HR = 1.38; 95% CI: = 1.05–1.82), compared to those with the lowest average nitrate
quintile. There was no association with nitrate exposure among women with lower folate intake.
With follow-up through 2010, there were 130 bladder cancer cases among women who had used
PWS >10 years. Risk remained elevated among women with the highest average nitrate levels and
was 1.6 times higher among women whose drinking water concentration exceeded 5 mg/L NO3 -N for
at least four years [31]. Risk estimates were not changed by adjustment for TTHM, which are suspected
bladder cancer risk factors. Smoking, but not vitamin C intake, modified the association with nitrate
in water; increased risk was apparent only in current smokers (p-interaction <0.03). With follow-up
through 2010, there were 125 kidney cancer cases among women using PWS; risk was increased among
those in the 95th percentile of average nitrate (>5.0 mg/L NO3 -N) compared with the lowest quartile
(HR = 2.2, 95% CI: 1.2–4.2) [127]. There was no positive trend with the average nitrate level and no
increased risk for women using private wells, compared to those with low average nitrate in their
public supply. An investigation of pancreatic cancer in the same population (follow-up through 2011)
found no association with average water nitrate levels in public supplies and no association among
women on private wells [126].
In contrast to the positive findings for bladder cancer among the cohort of Iowa women, a cohort
study of men and women aged 55–69 in the Netherlands with lower nitrate levels in PWS found no
association between water nitrate ingestion (median in top quintile = 2.4 mg/day NO3 -N) and bladder
cancer risk [131]. Dietary intake of vitamins C and E and history of cigarette smoking did not modify
the association. A hospital-based case-control study of bladder cancer in multiple areas of Spain [33]
assessed lifetime water sources and usual intake of tap water. Nitrate levels in PWS were low, with
almost all average levels below 2 mg/L NO3 -N. Risk of bladder cancer was not associated with the
nitrate level in drinking water or with estimated nitrate ingestion from drinking water, and there was
no evidence of interaction with factors affecting endogenous nitrosation.
Several case-control studies conducted in the Midwestern U.S. obtained lifetime histories of
drinking water sources and estimated exposure for PWS users. In contrast to findings of an increased
risk of NHL associated with nitrate levels in Nebraska PWS in an earlier study [132], there was no
association with similar concentrations in public water sources in a case-control study of NHL in
Iowa [35]. A study of renal cell carcinoma in Iowa [34] found no association with the level of nitrate
in PWS, including the number of years that levels exceeded 5 or 10 mg/L NO3 -N. However, higher
nitrate levels in PWS increased risk among subgroups who reported above the median intake of red
meat intake or below the median intake of vitamin C (p-interaction <0.05). A small case-control study
of adenocarcinoma of the stomach and esophagus among men and women in Nebraska [133] estimated
nitrate levels among long-term users of PWS and found no association between average nitrate levels
and risk.
A case-control study of colorectal cancer among rural women in Wisconsin estimated nitrate
levels in private wells using spatial interpolation of nitrate concentrations from a 1994 water quality
survey and found increased risk of proximal colon cancer among women estimated to have nitrate
levels >10 mg/L NO3 -N compared to levels < 0.5 mg/L. Risk of distal colon cancer and rectal cancer
were not associated with nitrate levels [134]. Water nitrate ingestion from public supplies, bottled water,
and private wells and springs over the adult lifetime was estimated in analyses that pooled case-control
studies of colorectal cancer in Spain and Italy [135]. Risk of colorectal cancer was increased among
those with >2.3 mg/day NO3 -N (vs. <1.1 mg/day). There were no interactions with red meat, vitamins
C and E, and fiber except for a borderline interaction (p-interaction = 0.07) for rectum cancer with
fiber intake. A small hospital-based case-control study in Indonesia found that drinking water nitrate
levels above the WHO standard (>11.3 mg/L as NO3 -N) was associated with colorectal cancer [136].
A national registry-based cohort study in Denmark [32] evaluated average nitrate concentrations in
PWS and private wells in relation to colorectal cancer incidence among those whose 35th birthday
occurred during 1978–2011. The average nitrate level was computed over residential water supplies
from age 20 to 35. Increased risks for colon and rectum cancer were observed in association with
average nitrate levels ≥9.25 mg/L NO3 (≥2.1 as NO3 -N) and ≥3.87 mg/L NO3 (>0.87 as NO3 -N),
respectively, with a significant positive trend. Because the study did not interview individuals, it could
not evaluate individual-level risk factors that might influence endogenous nitrosation.
A case-control study of breast cancer in Cape Cod, Massachusetts (US) [137] estimated nitrate
concentrations in PWS over approximately 20 years as an historical proxy for wastewater contamination
and potential exposure to endocrine disruption compounds. Average exposures >1.2 mg/L NO3 -N
(vs. <0.3 mg/L) were not associated with risk. A hospital-based case-control study in Spain found no
association between water nitrate ingestion and pre- and post-menopausal breast cancers [138].
Table 2. Case-control and cohort studies of drinking water nitrate and cancer (January 2004–March 2018) by cancer site.
First Author Study Design, Years Cancer Sites Evaluation of Effect

Exposure Description Summary of Drinking-Water Findings a,b
(Year) Country Regional Description Included Modification c
Cohort 1986 nitrate level in 364 pumping Highest vs. lowest quintile intake from
Zeegers, 2006 Incidence, 1986–1995 stations, exposure data available for water (≥1.7 mg/day NO3 -N [median 2.4 No interaction with vitamin
Bladder
Netherlands [131] 204 municipal registries 871 cases, 4359 members of the mg/day] vs. <0.20) RR = 1.11 (CI: 0.87–1.41; C, E, smoking
across the Netherlands subcohort p-trend = 0.14)
Nitrate levels in PWS (1979–2010)
Hospital-based
and bottled water (measurements of Highest vs. lowest quartile average level
multi-center case-control
Espejo-Herrera, brands with highest consumption (age 18-interview) (≥2.26 vs. 1.13 mg/L No interaction with vitamin
Incidence, 1998–2001
2015 based on a Spanish survey); analyses Bladder NO3 -N) OR = 1.04 (CI: 0.60–1.81) C, E, red meat, processed
Asturias, Alicante,
Spain [33] limited to those with ≥70% of Years >2.15 mg/L NO3 -N (75th percentile) meat, average THM level
Barcelona, Vallès-Bages,
residential history with nitrate (>20 vs. 0 years) OR = 1.41 (CI: 0.89–2.24)
Tenerife provinces
estimate (531 cases, 556 controls)
Nitrate levels in PWS (1955–1988)
and private well use among women Highest vs. lowest quartile PWS average Interaction with smoking
Population-based cohort >10 years at enrollment residence (≥2.98 vs. <0.47 mg/L NO3 -N) HR = 1.47 (p-interaction = 0.03); HR =
of postmenopausal with nitrate and trihalomethane (CI: 0.91–2.38; p-trend = 0.11) 3.67 (CI: 1.43–9.38) among
Jones, 2016
women ages 55–69 estimates (20,945 women; 170 Bladder Years >5 mg/L (≥4 years vs. 0) HR = 1.61 current smokers/≥2.98 mg/L
USA [31]
Incidence, 1986–2010 bladder cases); no measurements for (CI: 1.05–2.47; p-trend = 0.03) vs. non-smokers/<0.47 mg/L
Iowa private wells Private well users (vs. <0.47 mg/L NO3 -N NO3 -N); No interaction with
Adjusted for total trihalomethanes on PWS) HR = 1.53 (CI: 0.93–2.54) vitamin C, TTHM levels
(TTHM)
Water source during pregnancy and
Private well use versus PWS associated with
first year of child’s life (836 cases,
Pooled case-control increased risk in 2 regions and decreased
1485 controls); nitrate test strip
Mueller, 2004 studies risk in one; No association with nitrate levels
measurements of nitrate and nitrite
USA, Canada, France, Incidence among children Brain, childhood in water supplies Not described
for pregnancy home (except Italy)
Italy, Spain [139] <15 years (USA <20 years) Astrocytomas (excludes bottled water users):
(283 cases, 537 controls; excluding
7 regions of 5 countries ≥1.5 vs. <0.3 mg/L NO2 -N OR = 5.7 (CI:
bottled water users: 207 cases, 400
1.2–27.2)
controls)
Nitrate levels in public water
supplies (PWS) since 1972 was used Average ≥1.2 mg/L NO3 -N vs. <0.3
Case-control as an indicator of wastewater OR = 1.8, (CI: 0.6–5.0); summed annual
Brody, 2006
Incidence, 1988–1995 contamination and potential Breast NO3 -N ≥ 10 vs. 1–< 10 mg/L OR = 0.9, CI: Not described
USA [137]
Cape Cod, Massachusetts mammary carcinogens and 0.6–1.5); number of years >1 mg/L NO3 -N
endocrine disrupting compounds; ≥8 vs. 0 years OR = 0.9 (CI: 0.5–1.5)
excluded women on private wells
Table 2. Cont.

Highest vs. lowest quintile PWS average
(≥3.8 vs. ≤0.32 mg/L NO3 -N) HR = 1.14 (CI: Interaction with folate for
Population-based cohort Nitrate levels in PWS (1955–1988)
0.95–1.36; p-trend = 0.11); Private well (vs. ≤ PWS (p-interaction = 0.06).
of postmenopausal and private well use among women
Inoue-Choi, 2012 0.32 mg/L NO3 -N) HR = 1.14 (CI: 0.97–1.34); Folate ≥400 µg/d: (≥3.8 vs.
women ages 55–69 >10 years at enrollment residence Breast
USA [128] Private well (vs. ≤0.32 mg/L NO3 -N on ≤0.32 mg/L NO3 -N)
Incidence, 1986–2008 (20,147 women; 1751 breast cases); no
PWS) HR = 1.38 (CI: 1.05–1.82); No HR = 1.40 (CI: 1.05–1.87;
Iowa measurements for private wells
association among those with low folate p-trend = 0.04)
<400 µg/day
Nitrate levels in PWS (2004–2010),
bottled water measurements and
Water nitrate intake based on average nitrate
private wells and springs (2013
levels (age 18 to 2 years prior to interview)
Hospital-based measurements in 21 municipalities in No interaction with red meat,
Espejo-Herrera, and water intake (L/day). Post-menopausal
multi-center case-control León, Spain, the area with highest processed meat, vitamin C, E,
2016 Breast women: >2.0 vs. 0.5 mg/day NO3 -N
Incidence, 2008–2013 non-PWS use) smoking for pre- and
Spain [138] OR = 1.32 (0.93–1.86); Premenopausal
Spain (8 provinces) Analyses include women with ≥70% post-menopausal women
women: >1.4 vs. 0.4 mg/day NO3 -N
of period from age 18 to 2 years
OR = 1.14 (0.67–1.94)
before interview (1245 cases, 1520
controls)
Limited to women in rural areas with
Population-based no public water system (475 cases,
All colon cancers: Private wells ≥10.0 mg/L
case-control, women 1447 controls); nitrate levels at
McElroy, 2008 NO3 -N vs. <0.5 OR = 1.52 (CI: 0.95–2.44);
Incidence, 1990–1992 and residence (presumed to be private Colorectal Not described
USA [134] Proximal colon cancer: OR = 2.91 (CI:
1999–2001 wells) estimated by kriging using
1.52–5.56)
Wisconsin data from a 1994 representative
sample of 289 private wells
Nitrate levels in PWS (2004–2010) for
349 water supply zones, bottled
Interaction with fiber for
Multi-center case-control water (measured brands with highest Water nitrate intake based on average nitrate
rectum (p-interaction = 0.07);
study consumption), and private wells and levels (estimated 30 to 2 years prior to
>20 g/day fiber + >1.0 mg/L
Incidence, 2008–2013 springs (measurements in 2013 in 21 interview) and water intake (L/day)
Espejo-Herrera, 2016 NO3 -N vs. <20 g/day + ≤1.0
Spain (9 provinces) and municipalities in León, Spain, the Colorectal Highest vs. lowest exposure quintiles (≥2.3
Spain, Italy [135] mg/L HR = 0.72 (CI:
population-based controls; area with highest non-PWS use) vs. <1.1 mg /day NO3 -N) OR = 1.49
0.52–1.00).
Italy (two provinces) and Analyses include those with nitrate (CI:1.24–1.78); Colon OR = 1.52 (CI:
No interaction with red meat,
hospital-based controls estimates for ≥70% of period 30 1.24–1.86), Rectum OR = 1.62 (CI: 1.23–2.14)
vitamin C, E
years before interview (1869 cases,
3530 controls)
Table 2. Cont.

Nitrate levels in well water collected during the
Hospital-based raining season (Feb-March 2016) and classified Water nitrate > WHO standard vs. below (> 11.3
Fathmawati, 2017 case-control based on >11.3 or ≤11.3 mg/L as NO3 -N and vs. ≤11.3 mg/L NO3 -N) OR = 2.82 (CI:
Colorectal Not described
Indonesia [136] Incidence, 2014–2016 duration of exposure >10 and ≤10 years 1.08–7.40); > 10 years: 4.31 (CI: 11.32–14.10); ≤10
Indonesia (3 provinces) Analyses included participants who reported years: 1.41 (CI: 0.14–13.68)
drinking well water (75 cases, 75 controls)
Annual average nitrate exposure between ages
20–35 among those who lived ≥75% of study
Population-based
Nitrate levels in PWS and private wells among period at homes with a water sample within 1
record-linkage cohort of
Schullehner, 2018 1,742,321 who met exposure assessment criteria year (61% of Danish population). No information on dietary
men and women ages 35 Colorectal
Denmark [32] (5944 colorectal cancer cases, including 3700 Highest vs. lowest exposure quintile (≥2.1 vs. intakes or smoking
and older, 1978–2011
with colon and 2308 with rectal cancer) 0.16 mg/L NO3 -N); Colorectal: HR = 1.16 (CI:
Denmark
1.08–1.25); colon: 1.15 (CI: 1.05–1.26); rectum:
1.17 (CI: 1.04–1.32)
Interaction with red meat
intake (p-interaction = 0.01);
OR = 1.91 (CI 1.04–3.51)
Population-based case Highest vs. lowest quartile PWS average (≥2.8
Nitrate levels in PWS among those with nitrate among 11+ years >5 mg/L
Ward, 2007 control Kidney (renal cell mg/L NO3 -N vs. <0.62) OR = 0.89 (CI 0.57–1.39);
estimates for ≥70% of person-years ≥1960 (201 NO3 -N and red meat ≥1.2
USA [34] Incidence, 1986–1989 carcinomas) Years >5mg/L NO3 -N 11+ vs. 0 OR = 1.03 (CI:
cases, 1244 controls) servings/day. Interaction
Iowa 0.66–1.60)
with vitamin C showed
similar pattern (p-interaction
= 0.13)
Nitrate and TTHM metrics computed for
duration at water source (11+ years)
Population-based cohort Nitrate levels in PWS (1955–1988) and private 95th percentile vs. lowest quartile PWS average
of postmenopausal well use among women >10 years at enrollment (≥5.00 vs. <0.47 mg/L NO3 -N) HR = 2.23 (CI:
Jones, 2017 No interaction with smoking,
women ages 55–69 residence. PWS measurements for nitrate and Kidney 1.19–4.17; p-trend = 0.35)
USA [127] vitamin C
Incidence, 1986–2010 TTHM; no measurements for private wells Years >5 mg/L (≥4 years vs. 0) HR = 1.54 (CI:
Iowa (20,945 women; 163 kidney cases) 0.97–2.44; p-trend = 0.09)
Private well users (vs. <0.47 mg/L NO3 -N in
PWS) HR = 0.96 (CI: 0.59–1.58)
Private wells: >5.0 mg/L NO3 -N vs. ND OR =
Nitrate levels in PWS among those with nitrate
Population-based 0.8 (CI 0.2–2.5)
estimates for ≥70% of person-years ≥1960 (181
Ward, 2006 case-control Non-Hodgkin PWS average: ≥2.9 mg/L NO3 -N vs. <0.63 OR No interaction with vitamin
case, 142 controls); nitrate measurements for
USA [35] Incidence, 1998–2000 lymphoma = 1.2 (CI 0.6–2.2) C, smoking
private well users at time of interviews
Iowa Years ≥5mg/L NO3-N: 10+ vs. 0 OR = 1.4 (CI:
(1998–2000; 54 cases, 44 controls)
0.7–2.9)
Table 2. Cont.

reported duration at water source (11+ years)
No interaction with vitamin
Population-based cohort Nitrate levels in PWS (1955–1988) and private Highest vs. lowest quartile PWS average (≥2.98
C, red meat intake, smoking
of postmenopausal well use among women >10 years at enrollment mg/L vs. <0.47 mg/L NO3 -N) HR = 2.03 (CI =
Inoue-Choi, 2015 for PWS nitrate
women ages 55–69 residence; PWS measurements for nitrate and Ovary 1.22–3.38; p-trend = 0.003)
USA [129] Interaction with private well
Incidence, 1986–2010 TTHM; no measurements for private wells Years >5 mg/L (≥4 years vs. 0) HR = 1.52 (CI:
use and vitamin C intake
Iowa (17,216 women; 190 ovarian cases) 1.00–2.31; p-trend = 0.05)
(p-interaction = 0.01)
Private well users (vs. <0.47 mg/L NO3 -N in
PWS) HR = 1.53 (CI: 0.93–2.54)
Nitrate levels in PWS (1955–1988) and private
reported duration at water source (11+ years)
well use among women >10 years at enrollment
Population-based cohort 95th percentile vs. lowest quartile PWS average
residence; nitrate and TTHM estimates for PWS
of postmenopausal (≥5.69 vs. <0.47 mg/L NO3 -N) HR = 1.16 (CI:
Quist, 2018 (20,945 women; 189 pancreas cases); no No interaction with smoking,
women ages 55–69 Pancreas 0.51–2.64; p-trend = 0.97)
USA [126] measurements for private wells vitamin C
Incidence, 1986–2011 Years >5 mg/L (≥4 years vs. 0) HR = 0.90 (CI:
Adjusted for TTHM (1955–1988), measured
Iowa 0.55–1.48; p-trend = 0.62)
levels in 1980s, prior year levels estimated by
Private well users (vs. <0.47 mg/L NO3 -N) HR
expert)
= 0.92 (CI: 0.55–1.52)
Controls from prior study of Highest vs. lowest quartile PWS average (>4.32
lymphohematopoetic cases and controls vs. <2.45 mg/L NO3 -N): stomach OR = 1.2 (CI
interviewed in 1992–1994; Proxy interviews for 0.5–2.7); esophagus OR = 1.3 (CI: 0.6–3.1);
Population-based case 80%, 76%, 61% of stomach, esophagus, controls, Years >10 mg/L NO3 -N (9+ vs. 0): stomach OR
Stomach and No interaction with vitamin
Ward, 2008 control respectively. = 1.1 (CI: 0.5–2.3); esophagus OR = 1.2 (CI:
esophagus C, processed meat, or red
USA [133] Incidence, 1988–1993 Nitrate levels (1965–1985) in PWS for ≥70% of 0.6–2.7)
(adenocarcinomas) meat for either cancer
Nebraska person-years (79 distal stomach, 84, esophagus, Private well users (>4.5 mg/L NO3 -N vs. <0.5)
321 controls); Private well users sampling at stomach OR = 5.1 (CI: 0.5–52; 4 cases, 13
interview (15 stomach, 22 esophagus, 44 controls); esophagus OR = 0.5 (CI: 0.1–2.9; 8
controls) cases; 13 controls)
Highest vs. lowest quartile PWS average (>2.46
vs. <0.36 mg/L NO3 -N) HR = 2.18 (CI: 0.83–5.76;
Population-based cohort p-trend = 0.02)
Nitrate levels in PWS (1955–1988) and private No interaction with smoking,
of postmenopausal Years >5 mg/L (≥5 years vs. 0) HR = 2.59 (CI:
Ward, 2010 well use among women >10 years at enrollment vitamin C, body mass index,
women ages 55–69 Thyroid 1.09–6.19; p-trend = 0.04);
USA [37] residence (21,977 women; 40 thyroid cases); no education, residence location
Incidence, 1986–2004 Private well (vs. <0.36 mg/L NO3 -N on PWS)
measurements for private wells (farm/rural vs. urban)
Iowa HR = 1.13 (CI: 0.83–3.66)
Dietary nitrate intake quartiles positively
associated with risk (p-trend = 0.05)
ND = not detected; PWS = public water supplies; a nitrate or nitrite levels presented in the publications as mg/L of the ion were converted to mg/L as NO3 -N or NO2 -N; b Odds ratios
(OR) for case-control studies, incidence rate ratios (RR) and hazard ratios (HR) for cohort studies, and 95% confidence intervals (CI); c Factors evaluated are noted. Interaction refers to
reported p ≤ 0.10 from test of heterogeneity.
Animal studies demonstrate that in utero exposure to nitrosamides can cause brain tumors in the
exposed offspring. Water nitrate and nitrite intake during pregnancy was estimated in a multi-center
case-control study of childhood brain tumors in five countries based on the maternal residential water
source [139]. Results for the California and Washington State sites were reported in our previous
review [8,140]. Nitrate/nitrite levels in water supplies were measured using a nitrate test strip method
in four countries including these U.S. sites; most of these measurements occurred many years after
the pregnancy. Measured nitrate concentrations were not associated with risk of childhood brain
tumors. However, higher nitrite levels (>1.5 mg/L NO2 -N) in the drinking water were associated with
increased risk of astrocytomas.
8. Thyroid Disease
Animal studies demonstrate that ingestion of nitrate at high doses can competitively inhibit
iodine uptake and induce hypertrophy of the thyroid gland [141]. An early study of women in the
Netherlands consuming water with nitrate levels at or above the MCL, found increased prevalence
of thyroid hypertrophy [142]. Since the last review, five studies have evaluated nitrate ingestion
from drinking water (the Iowa cohort study also assessed diet) and prevalence of thyroid disease.
A study of school-age children in Slovakia found increased prevalence of subclinical hypothyroidism
among children in an area with high nitrate levels (51–274 mg/L NO3 ) in water supplies compared
with children ingesting water with nitrate ≤50 mg/L (11 mg/L NO3 -N). In Bulgarian villages with
high nitrate levels (75 mg/L NO3 ) and low nitrate levels (8 mg/L), clinical examinations of the
thyroids of pregnant women and school children revealed an approximately four- and three-fold
increased prevalence of goiter, respectively, in the high nitrate village [143,144]. The iodine status of the
populations in both studies was adequate. Self-reported hypothyroidism and hyperthyroidism among
a cohort of post-menopausal women in Iowa was not associated with average nitrate concentrations in
PWS [37]. However, dietary nitrate, the predominant source of intake, was associated with increased
prevalence of hypothyroidism but not hyperthyroidism. Modeled estimates of nitrate concentrations
in private wells among a cohort of Old Order Amish in Pennsylvania (USA) were associated with
increased prevalence of subclinical hypothyroidism as determined by thyroid stimulating hormone
measurements, among women but not men [145].
9. Other Health Effects

Associations between nitrate in drinking water and other non-cancer health effects, including
type 1 childhood diabetes (T1D), blood pressure, and acute respiratory tract infections in children were
previously reviewed [8]. Since 2004, a small number of studies have contributed additional mixed
evidence for these associations. Animal studies indicate that NOC may play a role in the pathology
of T1D through damage to pancreatic beta cells [146]. A registry-based study in Finland [147] found
a positive trend in T1D incidence with levels of nitrate in drinking water. In contrast, an ecological
analysis in Italy showed an inverse correlation with water nitrate levels and T1D rates [148]. A small
T1D case-control study in Canada with 57 cases showed no association between T1D and estimated
intake of nitrate from drinking water (highest quartile >2.7 mg/day NO3 -N) [149]. Concentrations of
nitrate in drinking water (median ~2.1 mg/L NO3 -N) were not associated with progression to T1D in
a German nested case-control study of islet autoantibody-positive children, who may be at increased
risk of the disease [150].
In a prospective, population-based cohort study in Wisconsin (USA), increased incidence of
early and late age-related macular degeneration was positively associated with higher nitrate levels
(≥5 mg/L vs. <5 mg/L NO3 -N) in rural private drinking water supplies [151]. The authors suggested
several possible mechanisms, including methemoglobin-induced lipid peroxidation in the retina.
Potential benefits of nitrate ingestion include lowering of blood pressure due to production of
nitric oxide in the acidic stomach and subsequent vasodilation, antithrombotic, and immunoregulatory
effects [152]. Experimental studies in animals and controlled feeding studies in humans have
demonstrated mixed evidence of these effects and on other cardiovascular endpoints such as vascular
hypertrophy, heart failure, and myocardial infarction (e.g., [152–154]). Ingested nitrite from diet has
also been associated with increased blood flow in certain parts of the brain [155]. Epidemiologic
studies of these effects are limited to estimation of dietary exposures or biomarkers that integrate
exposures from nitrate from diet and drinking water. Recent findings in the Framingham Offspring
Study suggested that plasma nitrate was associated with increased overall risk of death that attenuated
when adjusted for glomerular function (HR: 1.16, 95% CI: 1.0–1.35) but no association was observed
for incident cardiovascular disease [156]. No epidemiologic studies have specifically evaluated nitrate
ingested from drinking water in relation to these outcomes. Another potential beneficial effect of
nitrate is protection against bacterial infections via its reduction to nitrite by enteric bacteria. In an
experimental inflammatory bowel disease mouse model, nitrite in drinking water was associated with
both preventive and therapeutic effects [157]. However, there is limited epidemiologic evidence for a
reduced risk of gastrointestinal disease in populations with high drinking water nitrate intake. One
small, cross-sectional study in Iran found no association between nitrate levels in public water supplies
with mean levels of ~5.6 mg/L NO3 -N and gastrointestinal disease [158].
10. Discussion
Since our last review of studies through 2004 [8], more than 30 epidemiologic studies have
evaluated drinking water nitrate and risk of cancer, adverse reproductive outcomes, or thyroid disease.
However, the number of studies of any one outcome was not large and there are still too few studies to
allow firm conclusions about risk. The most common endpoints studied were colorectal cancer, bladder,
and breast cancer (three studies each) and thyroid disease (four studies). Considering all studies to
date, the strongest evidence for a relationship between drinking water nitrate ingestion and adverse
health outcomes (besides methemoglobinemia) is for colorectal cancer, thyroid disease, and neural
tube defects. Four of the five published studies of colorectal cancer found evidence of an increased
risk of colorectal cancer or colon cancer associated with water nitrate levels that were mostly below
the respective regulatory limits [32,134,135,159]. In one of the four positive studies [159], increased
risk was only observed in subgroups likely to have increased nitrosation. Four of the five studies of
thyroid disease found evidence for an increased prevalence of subclinical hypothyroidism with higher
ingestion of drinking water nitrate among children, pregnant women, or women only [37,144,145,160].
Positive associations with drinking water nitrate were observed at nitrate concentrations close to or
above the MCL. The fifth study, a cohort of post-menopausal women in Iowa, had lower drinking
water nitrate exposure but observed a positive association with dietary nitrate [37]. To date, five of six
studies of neural tube defects showed increased risk with exposure to drinking water nitrate below the
MCL. Thus, the evidence continues to accumulate that higher nitrate intake during the pregnancy is a
risk factor for this group of birth defects.
All but one of the 17 cancer studies conducted since 2004 were in the U.S. or Europe, the majority
of which were investigations of nitrate in regulated public drinking water. Thyroid cancer was studied
for the first time [37] with a positive finding that should be evaluated in future studies. Bladder
cancer, a site for which other drinking water contaminants (arsenic, disinfection by-products [DBPs])
are established or suspected risk factors, was not associated with drinking water nitrate in three
of the four studies. Most of the cancer studies since 2004 evaluated effect modification by factors
known to influence endogenous nitrosation, although few observed evidence for these effects. Several
studies of adverse reproductive outcomes since 2004 have indicated a positive association between
maternal prenatal exposure to nitrate concentrations below the MCL and low birth weight and small
for gestational age births. However, most studies did not account for co-exposure to other water
contaminants, nor did they adjust for potential risk factors. The relation between drinking water
nitrate and spontaneous abortion continues to be understudied. Few cases of methemoglobinemia,
the health concern that lead to the regulation of nitrate in public water supplies, have been reported in
the U.S. since the 1990s. However, as described by Knobeloch et al. [96], cases may be underreported
and only a small proportion of cases are thoroughly investigated and described in the literature.
Based on published reports, [100] areas of the world of particular concern include several eastern
European countries, Gaza, and Morocco, where high nitrate concentrations in water supplies have
been linked to high levels of methemoglobin in children. Therefore, continued surveillance and
education of physicians and parents will be important. Biological plausibility exists for relationships
between nitrate ingestion from drinking water and a few other health outcomes including diabetes
and beneficial effects on the cardiovascular system, but there have been only a limited number of
epidemiologic studies.
Assessment of drinking water nitrate exposures in future studies should be improved by obtaining
drinking water sources at home and at work, estimating the amount of water consumed from each
source, and collecting information on water filtration systems that may impact exposure. These efforts
are important for reducing misclassification of exposure. Since our last review, an additional decade
of PWS monitoring data are available in the U.S. and European countries, which has allowed
assessment of exposure over a substantial proportion of participants’ lifetimes in recent studies.
Future studies should estimate exposure to multiple water contaminants as has been done in recent
cancer studies [31,33,127,129]. For instance, nitrate and atrazine frequently occur together in drinking
water in agricultural areas [161] and animal studies have found this mixture to be teratogenic [162].
Regulatory monitoring data for pesticides in PWS has been available for over 20 years in the U.S.;
therefore, it is now feasible to evaluate co-exposure to these contaminants. Additionally, water
supplies in agricultural areas that rely on alluvial aquifers or surface water often have elevated levels
of both DBPs and nitrate. Under this exposure scenario, there is the possibility of formation of
the nitrogenated DBPs including the carcinogenic NDMA, especially if chloramination treatment is
used for disinfection [163,164]. Studies of health effects in countries outside the U.S. and Europe are
also needed.
A comprehensive assessment of nitrate and nitrite from drinking water and dietary sources as
well as estimation of intakes of antioxidants and other inhibitors of endogenous nitrosation including
dietary polyphenols and flavonoids is needed in future studies. Heme iron from red meat, which
increases fecal NOC in human feeding studies, should also be assessed as a potential effect modifier of
risk from nitrate ingestion. More research is needed on the potential interaction of nitrate ingestion and
nitrosatable drugs (those with secondary and tertiary amines or amides). Evidence from several studies
of birth defects [38,118–120] implicates nitrosatable drug intake during pregnancy as a risk factor for
specific congenital anomalies especially in combination with nitrate. Drugs with nitrosatable groups
include many over-the-counter and prescription drugs. Future studies with electronic medical records
and record-linkage studies in countries like Denmark with national pharmacy data may provide
opportunities for evaluation of these exposures.
Populations with the highest exposure to nitrate from their drinking water are those living in
agricultural regions, especially those drinking water from shallow wells near nitrogen sources (e.g.,
crop fields, animal feeding operations). Estimating exposure for private well users is important because
it allows assessment of risk over a greater range of nitrate exposures compared to studies focusing
solely on populations using PWS. Future health studies should focus on these populations, many
of which may have been exposed to elevated nitrate in drinking water from early childhood into
adulthood. A major challenge in conducting studies in these regions is the high prevalence of private
well use with limited nitrate measurement data for exposure assessment. Recent efforts to model
nitrate concentrations in private wells have shown that it is feasible to develop predictive models
where sufficient measurement data are available [41,48,49]. However, predictive models from one area
are not likely to be directly translatable to other geographic regions with different aquifers, soils, and
nitrogen inputs.
Controlled human feeding studies have demonstrated that endogenous nitrosation occurs after
ingestion of drinking water with nitrate concentrations above the MCL of 10 mg/L NO3 -N (~44 mg/L
as NO3 ). However, the extent of NOC formation after ingestion of drinking water with nitrate
concentrations below the MCL has not been well characterized. Increased risks of specific cancers and
central nervous system birth defects in study populations consuming nitrate below the MCL is indirect
evidence that nitrate ingestion at these levels may be a risk factor under some conditions. However,
confounding by other exposures or risk factors can be difficult to rule out in many studies. Controlled
human studies to evaluate endogenous nitrosation at levels below the MCL are needed to understand
interindividual variability and factors that affect endogenous nitrosation at drinking water nitrate
levels below the MCL.
A key step in the endogenous formation of NOC is the reduction of nitrate, which has been
transported from the bloodstream into the saliva, to nitrite by the nitrate-reducing bacteria that are
located primarily in the crypts on the back of the tongue [165–167]. Tools for measuring bacterial
DNA and characterizing the oral microbiome are now available and are currently being incorporated
into epidemiologic studies [168,169]. Buccal cell samples that have been collected in epidemiologic
studies can be used to characterize the oral microbiome and to determine the relative abundance of
the nitrate-reducing bacteria. Studies are needed to characterize the stability of the nitrate-reducing
capacity of the oral microbiome over time and to determine factors that may modify this capacity such
as diet, oral hygiene, and periodontal disease. Interindividual variability in the oral nitrate-reducing
bacteria may play an important role in modifying endogenous NOC formation. The quantification
of an individual’s nitrate-reducing bacteria in future epidemiologic studies is likely to improve our
ability to classify participants by their intrinsic capacity for endogenous nitrosation.
In addition to characterizing the oral microbiome, future epidemiologic studies should incorporate
biomarkers of NOC (e.g., urinary or fecal NOC), markers of genetic damage, and determine genetic
variability in NOC metabolism. As many NOC require α-hydroxylation by CYP2E1 for bioactivation
and for formation of DNA adducts, it is important to investigate the influence of polymorphisms
in the gene encoding for this enzyme. Studies are also needed among populations with medical
conditions that increase nitrosation such as patients with inflammatory bowel disease and periodontal
disease [8]. Because NOC exposures induce characteristic gene expression profiles [170,171],
further studies linking drinking water intake to NOC excretion and gene expression responses are
relevant to our understanding of health risks associated with drinking water nitrate. The field of
‘Exposome-research’ [172,173] generates large numbers of genomics profiles in human population
studies for which dietary exposures and biobank materials are also available. These studies provide
opportunities to measure urinary levels of nitrate and NOC that could be associated with molecular
markers of exposure and disease risk.
Nitrate concentrations in global water supplies are likely to increase in the future due to population
growth, increases in nitrogen fertilizer use, and increasing intensity and concentration of animal
agriculture. Even with increased inputs, mitigation of nitrate concentrations in water resources
is possible through local, national, and global efforts. Examples of the latter are the International
Nitrogen Initiative [174] and the EU Nitrates Directive [17,18], which aim to quantify human effects
on the nitrogen cycle and to validate and promote methods for sustainable nitrogen management.
Evidence for the effectiveness of these efforts, which include the identification of vulnerable areas,
establishment of codes of good agricultural practices, and national monitoring and reporting are
indicated by decreasing trends in groundwater nitrate concentrations in some European countries
after the implementation of the EU Nitrates Directive [19]. However, the effect of this initiative
was variable across the EU. In the U.S., nitrogen applications to crop fields are not regulated and
efforts to reduce nitrogen runoff are voluntary. Although strategies such as appropriate timing of
fertilizer applications, diversified crop rotations, planting of cover crops, and reduced tillage can be
effective [175], concentrations in U.S. ground and surface water have continued to increase in most
areas [10]. Climate change is expected to affect nitrogen in aquatic ecosystems and groundwater
through alterations of the hydrological cycle [176]. Climatic factors that affect nitrate in groundwater
include the amount, intensity, and timing of precipitation. Increasing rainfall intensity, especially in
the winter and spring, can lead to increases in nitrogen runoff from agricultural fields and leaching
to groundwater.
11. Conclusions
In summary, most adverse health effects related to drinking water nitrate are likely due to a
combination of high nitrate ingestion and factors that increase endogenous nitrosation. Some of the
recent studies of cancer and some birth defects have been able to identify subgroups of the population
likely to have greater potential for endogenous nitrosation. However, direct methods of assessing
these individuals are needed. New methods for quantifying the nitrate-reducing bacteria in the oral
microbiome and characterizing genetic variation in NOC metabolism hold promise for identifying
high risk groups in epidemiologic studies.
To date, the number of well-designed studies of individual health outcomes is still too few to draw
firm conclusions about risk from drinking water nitrate ingestion. Additional studies that incorporate
improved exposure assessment for populations on PWS, measured or predicted exposure for private
well users, quantification of nitrate-reducing bacteria, and estimates of dietary and other factors
affecting nitrosation are needed. Studies of colorectal cancer, thyroid disease, and central nervous
system birth defects, which show the most consistent associations with water nitrate ingestion, will be
particularly useful for clarifying these risks. Future studies of other health effects with more limited
evidence of increased risk are also needed including cancers of the thyroid, ovary, and kidney, and
the adverse reproductive outcomes of spontaneous abortion, preterm birth, and small for gestational
age births.
Acknowledgments: This work was partly supported by the Intramural Research Program of the National Cancer
Institute, Division of Cancer Epidemiology and Genetics, Occupational and Environmental Epidemiology Branch.
Two authors (TMdK, SvB) acknowledge financial support from the European Commission in the context of the
integrated project PHYTOME financed under the Seventh Framework Programme for Research and Technology
Development of the European Commission (EU-FP7 grant agreement no. 315683), investigating the possible
replacement of nitrite in meat products by natural compounds. CMV notes that ISGlobal is a member of the
CERCA Programme, Generalitat de Catalunya.
Conflicts of Interest: The authors declare no conflict of interest.
References
1. Davidson, E.A.; David, M.B.; Galloway, J.N.; Goodale, C.L.; Haeuber, R.; Harrison, J.A.; Howarth, R.W.;
Jaynes, D.B.; Lowrance, R.R.; Nolan, B.T.; et al. Excess nitrogen in the U.S. environment: Trends, risks, and
solutions. In Issues in Ecology; Ecological Society of America: Washington, DC, USA, 2012.
2. Vitousek, P.M.; Aber, J.D.; Howarth, R.W.; Likens, G.E.; Matson, P.A.; Schindler, D.W.; Schlesinger, W.H.;
Tilman, D. Human alteration of the global nitrogen cycle: Sources and consequences. Ecol. Appl. 1997, 7,
737–750. [CrossRef]
3. Howarth, R.W. Coastal nitrogen pollution: A review of sources and trends globally and regionally.
Harmful Algae 2008, 8, 14–20. [CrossRef]
4. USEPA. Regulated Drinking Water Contaminants: Inorganic Chemicals. Available online: https://www.
epa.gov/ground-water-and-drinking-water/table-regulated-drinking-water-contaminants (accessed on 23
September 2017).
5. International Agency for Research on Cancer (IARC). IARC Monographs on the Evaluation of Carcionogenic
Risks to Humans: Ingested Nitrate and Nitrite and Cyanobacterial Peptide Toxins; IARC: Lyon, France, 2010.
6. National Research Council (NRC). The Health Effects of Nitrate, Nitrite, and N-Nitroso Compounds; NRC:
Washington, DC, USA, 1981.
7. Mirvish, S.S. Role of N-nitroso compounds (NOC) and N-nitrosation in etiology of gastric, esophageal,
nasopharyngeal and bladder cancer and contribution to cancer of known exposures to NOC. Cancer Lett.
1995, 93, 17–48. [CrossRef]
8. Ward, M.H.; deKok, T.M.; Levallois, P.; Brender, J.; Gulis, G.; Nolan, B.T.; VanDerslice, J. Workgroup report:
Drinking-water nitrate and health-recent findings and research needs. Environ. Health Perspect. 2005, 113,
1607–1614. [CrossRef] [PubMed]
9. Maupin, M.A.; Kenny, J.F.; Hutson, S.S.; Lovelace, J.K.; Barber, N.L.; Linsey, K.S. Estimated Use of Water in the
United States in 2010; US Geological Survey: Reston, VA, USA, 2014; p. 56.
10. U.S. Geological Survey. USGS Water Data for the Nation. Available online: https://waterdata.usgs.gov/nwis
(accessed on 1 January 2018).
11. Dubrovsky, N.M.; Burow, K.R.; Clark, G.M.; Gronberg, J.M.; Hamilton, P.A.; Hitt, K.J.; Mueller, D.K.;
Munn, M.D.; Nolan, B.T.; Puckett, L.J.; et al. The Quality of Our Nation’s Waters—Nutrients in the Nation’s
Streams and Groundwater, 1992–2004; U.S. Geological Survey: Reston, VA, USA, 2010; p. 174.
12. Lindsey, B.D.; Rupert, M.G. Methods for Evaluating Temporal Groundwater Quality Data and Results of
Decadal-Scale Changes in Chloride, Dissolved Solids, and Nitrate Concentrations in Groundwater in the United
States, 1988–2010; U.S. Geological Survey Scientific Investigations Report: 2012–5049; U.S. Geological Survey:
Reston, VA, USA, 2012; p. 46.
13. Pennino, M.J.; Compton, J.E.; Leibowitz, S.G. Trends in Drinking Water Nitrate Violations across the United
States. Environ. Sci. Technol. 2017, 51, 13450–13460. [CrossRef] [PubMed]
14. Van Grinsven, H.J.M.; Tiktak, A.; Rougoor, C.W. Evaluation of the Dutch implementation of the nitrates
directive, the water framework directive and the national emission ceilings directive. NJAS-Wagening. J.
Life Sci. 2016, 78, 69–84. [CrossRef]
15. Vock, D.C. Iowa Farmers Won a Water Pollution Lawsuit, But at What Cost? Available online: http://www.
governing.com/topics/transportation-infrastructure/gov-des-moines-water-utility-lawsuit-farmers.html
(accessed on 10 February 2018).
16. Des Moines Water Works. On Earth Day, Des Moines Water Works Reflects on Resources Spent to Manage
Agrotoxins in Source Waters. Available online: http://www.dmww.com/about-us/news-releases/on-earth-
day-des-moines-water-works-reflects-on-resources-spent-to-manage-agrotoxins-in-source-water.aspx
17. European Commission. The Nitrates Directive. Available online: http://ec.europa.eu/environment/water/
water-nitrates/index_en.html (accessed on 10 May 2018).
18. European Union (EU). Council Directive 91/676/EEC of 12 December 1991 Concerning the Protection of Waters
against Pollution Caused by Nitrates from Agricultural Sources; European Union (EU): Brussels, Belgium, 1991.
19. Hansen, B.; Thorling, L.; Dalgaard, T.; Erlandsen, M. Trend Reversal of Nitrate in Danish Groundwater—A
Reflection of Agricultural Practices and Nitrogen Surpluses since 1950. Environ. Sci. Technol. 2011, 45,
20. European Environment Agency (EEA). Groundwater Nitrate. Available online: https://www.eea.europa.eu/
data-and-maps/daviz/groundwater-nitrate#tab-chart_1_filters=%7B%22rowFilters%22%3A%7B%7D%
3B%22columnFilters%22%3A%7B%22pre_config_country%22%3A%5B%22Slovenia%22%5D%7D%7D
21. Schullehner, J.; Hansen, B. Nitrate exposure from drinking water in Denmark over the last 35 years. Environ.
Res. Lett. 2014, 9, 095001. [CrossRef]
22. Vitoria, I.; Maraver, F.; Sanchez-Valverde, F.; Armijo, F. Nitrate concentrations in tap water in Spain. Gac. Sanit.
2015, 29, 217–220. [CrossRef] [PubMed]
23. Espejo-Herrera, N.; Kogevinas, M.; Castano-Vinyals, G.; Aragones, N.; Boldo, E.; Ardanaz, E.; Azpiroz, L.;
Ulibarrena, E.; Tardon, A.; Molina, A.J.; et al. Nitrate and trace elements in municipal and bottled water in
Spain. Gac. Sanit. 2013, 27, 156–160. [CrossRef] [PubMed]
24. Gunnarsdottir, M.J.; Gardarsson, S.M.; Jonsson, G.S.; Bartram, J. Chemical quality and regulatory compliance
of drinking water in Iceland. Int. J. Hyg. Environ. Health 2016, 219, 724–733. [CrossRef] [PubMed]
25. D’Alessandro, W.; Bellomo, S.; Parello, F.; Bonfanti, P.; Brusca, L.; Longo, M.; Maugeri, R. Nitrate, sulphate
and chloride contents in public drinking water supplies in Sicily, Italy. Environ. Monit. Assess. 2012, 184,
26. Migeot, V.; Albouy-Llaty, M.; Carles, C.; Limousi, F.; Strezlec, S.; Dupuis, A.; Rabouan, S. Drinking-water
exposure to a mixture of nitrate and low-dose atrazine metabolites and small-for-gestational age (SGA)
babies: A historic cohort study. Environ. Res. 2013, 122, 58–64. [CrossRef] [PubMed]
27. Taneja, P.; Labhasetwar, P.; Nagarnaik, P.; Ensink, J.H.J. The risk of cancer as a result of elevated levels of
nitrate in drinking water and vegetables in Central India. J. Water Health 2017, 15, 602–614. [CrossRef]
[PubMed]
28. Suthar, S.; Bishnoi, P.; Singh, S.; Mutiyar, P.K.; Nema, A.K.; Patil, N.S. Nitrate contamination in groundwater
of some rural areas of Rajasthan, India. J. Hazard. Mater. 2009, 171, 189–199. [CrossRef] [PubMed]
29. Gupta, I.; Salunkhe, A.; Rohra, N.; Kumar, R. Groundwater quality in Maharashtra, India: Focus on nitrate
pollution. J. Environ. Sci. Eng. 2011, 53, 453–462. [PubMed]
30. Weinthal, E.; Vengosh, A.; Marei, A.; Kloppmann, W. The water crisis in the Gaza strip: Prospects for
resolution. Ground Water 2005, 43, 653–660. [CrossRef] [PubMed]
31. Jones, R.R.; Weyer, P.J.; DellaValle, C.T.; Inoue-Choi, M.; Anderson, K.E.; Cantor, K.P.; Krasner, S.; Robien, K.;
Freeman, L.E.B.; Silverman, D.T.; et al. Nitrate from drinking water and diet and bladder cancer among
postmenopausal women in Iowa. Environ. Health Perspect. 2016, 124, 1751–1758. [CrossRef] [PubMed]
32. Schullehner, J.; Hansen, B.; Thygesen, M.; Pedersen, C.B.; Sigsgaard, T. Nitrate in drinking water and
colorectal cancer risk: A nationwide population-based cohort study. Int. J. Cancer 2018, 1, 73–79. [CrossRef]
[PubMed]
33. Espejo-Herrera, N.; Cantor, K.P.; Malats, N.; Silverman, D.T.; Tardon, A.; Garcia-Closas, R.; Serra, C.;
Kogevinas, M.; Villanueva, C.M. Nitrate in drinking water and bladder cancer risk in Spain. Environ. Res.
2015, 137, 299–307. [CrossRef] [PubMed]
34. Ward, M.H.; Rusiecki, J.A.; Lynch, C.F.; Cantor, K.P. Nitrate in public water supplies and the risk of renal cell
carcinoma. Cancer Causes Control 2007, 18, 1141–1151. [CrossRef] [PubMed]
35. Ward, M.H.; Cerhan, J.R.; Colt, J.S.; Hartge, P. Risk of non-Hodgkin lymphoma and nitrate and nitrite from
drinking water and diet. Epidemiology 2006, 17, 375–382. [CrossRef] [PubMed]
36. Schullehner, J.; Stayner, L.; Hansen, B. Nitrate, Nitrite, and Ammonium Variability in Drinking Water
Distribution Systems. Int. J. Environ. Res. Public Health 2017, 14, 276. [CrossRef] [PubMed]
37. Ward, M.H.; Kilfoy, B.A.; Weyer, P.J.; Anderson, K.E.; Folsom, A.R.; Cerhan, J.R. Nitrate intake and the risk of
thyroid cancer and thyroid disease. Epidemiology 2010, 21, 389–395. [CrossRef] [PubMed]
38. Brender, J.D.; Weyer, P.J.; Romitti, P.A.; Mohanty, B.P.; Shinde, M.U.; Vuong, A.M.; Sharkey, J.R.; Dwivedi, D.;
Horel, S.A.; Kantamneni, J.; et al. Prenatal nitrate intake from drinking water and selected birth defects in
offspring of participants in the national birth defects prevention study. Environ. Health Perspect. 2013, 121,
39. Baris, D.; Waddell, R.; Beane Freeman, L.E.; Schwenn, M.; Colt, J.S.; Ayotte, J.D.; Ward, M.H.; Nuckols, J.;
Schned, A.; Jackson, B.; et al. Elevated Bladder Cancer in Northern New England: The Role of Drinking
Water and Arsenic. J. Natl. Cancer Inst. 2016, 108. [CrossRef] [PubMed]
40. Nolan, B.T.; Hitt, K.J. Vulnerability of shallow groundwater and drinking-water wells to nitrate in the United
States. Environ. Sci. Technol. 2006, 40, 7834–7840. [CrossRef] [PubMed]
41. Messier, K.P.; Kane, E.; Bolich, R.; Serre, M.L. Nitrate variability in groundwater of North Carolina using
monitoring and private well data models. Environ. Sci. Technol. 2014, 48, 10804–10812. [CrossRef] [PubMed]
42. Eckhardt, D.A.V.; Stackelberg, P.E. Relation of ground-water quality to land use on Long Island, New York.
Ground Water 1995, 33, 1019–1033. [CrossRef]
43. Nolan, B.T.; Hitt, K.J.; Ruddy, B.C. Probability of nitrate contamination of recently recharged groundwaters
in the conterminous United States. Environ. Sci. Technol. 2002, 36, 2138–2145. [CrossRef] [PubMed]
44. Rupert, M.G. Probability of Detecting Atrazine/Desethyl-Atrazine and Elevated Concentrations of Nitrate in Ground
Water in Colorado; Water-Resources Investigations Report 02-4269; U.S. Geological Survey: Denver, CO, USA,
2003; p. 35.
45. Tesoriero, A.J.; Voss, F.D. Predicting the probability of elevated nitrate concentrations in the Puget Sound
Basin: Implications for aquifer susceptibility and vulnerability. Ground Water 1997, 35, 1029–1039. [CrossRef]
46. Warner, K.L.; Arnold, T.L. Relations that Affect the Probability and Prediction of Nitrate Concentration in Private
Wells in the Glacial Aquifer System in the United States; U.S. Geological Survey Scientific Investigations Report
2010–5100; U.S. Geological Survey: Reston, VA, USA, 2010; p. 55.
47. Elith, J.; Leathwick, J.R.; Hastie, T. A working guide to boosted regression trees. J. Anim. Ecol. 2008, 77,
48. Wheeler, D.C.; Nolan, B.T.; Flory, A.R.; DellaValle, C.T.; Ward, M.H. Modeling groundwater nitrate
concentrations in private wells in Iowa. Sci. Total Environ. 2015, 536, 481–488. [CrossRef] [PubMed]
49. Ransom, K.M.; Nolan, B.T.; Traum, J.A.; Faunt, C.C.; Bell, A.M.; Gronberg, J.A.M.; Wheeler, D.C.;
Rosecrans, C.Z.; Jurgens, B.; Schwarz, G.E.; et al. A hybrid machine learning model to predict and visualize
nitrate concentration throughout the Central Valley aquifer, California, USA. Sci. Total Environ. 2017, 601–602,
50. Leach, S.A.; Thompson, M.; Hill, M. Bacterially catalyzed N-nitrosation reactions and their relative
importance in the human stomach. Carcinogenesis 1987, 8, 1907–1912. [CrossRef] [PubMed]
51. Lv, J.; Neal, B.; Ehteshami, P.; Ninomiya, T.; Woodward, M.; Rodgers, A.; Wang, H.; MacMahon, S.;
Turnbull, F.; Hillis, G.; et al. Effects of intensive blood pressure lowering on cardiovascular and renal
outcomes: A systematic review and meta-analysis. PLoS Med. 2012, 9, e1001293. [CrossRef] [PubMed]
52. Spiegelhalder, B.; Eisenbrand, G.; Preussmann, R. Influence of dietary nitrate on nitrite content of human
saliva: Possible relevance to in vivo formation of N-nitroso compounds. Food Cosmet. Toxicol. 1976, 14,
53. Tricker, A.R.; Kalble, T.; Preussmann, R. Increased urinary nitrosamine excretion in patients with urinary
diversions. Carcinogenesis 1989, 10, 2379–2382. [CrossRef] [PubMed]
54. Eisenbrand, G.; Spiegelhalder, B.; Preussmann, R. Nitrate and nitrite in saliva. Oncology 1980, 37, 227–231.
[CrossRef] [PubMed]
55. Eisenbrand, G. The Significance of N-Nitrosation of Drugs; Nicolai, H.V., Eisenbrand, G., Bozler, G., Eds.; Gustav
Fischer Verlag, Stuttgart: New York, NY, USA, 1990; pp. 47–69.
56. Ceccatelli, S.; Lundberg, J.M.; Fahrenkrug, J.; Bredt, D.S.; Snyder, S.H.; Hokfelt, T. Evidence for involvement
of nitric oxide in the regulation of hypothalamic portal blood flow. Neuroscience 1992, 51, 769–772. [CrossRef]
57. Moncada, S.; Palmer, R.M.J.; Higgs, E.A. Nitric oxide: Physiology, pathophysiology, and pharmacology.
Pharmacol. Rev. 1991, 43, 109–142. [PubMed]
58. Rees, D.D.; Palmer, R.M.; Moncada, S. Role of endothelium-derived nitric oxide in the regulation of blood
pressure. Proc. Natl. Acad. Sci. USA 1989, 86, 3375–3378. [CrossRef] [PubMed]
59. Palmer, R.M.; Ferrige, A.G.; Moncada, S. Nitric oxide release accounts for the biological activity of
endothelium-derived relaxing factor. Nature 1987, 327, 524–526. [CrossRef] [PubMed]
60. Radomski, M.W.; Palmer, R.M.; Moncada, S. Endogenous nitric oxide inhibits human platelet adhesion to
vascular endothelium. Lancet 1987, 2, 1057–1058. [CrossRef]
61. Radomski, M.W.; Palmer, R.M.J.; Moncada, S. The Anti-Aggregating Properties of Vascular
Endothelium—Interactions between Prostacyclin and Nitric-Oxide. Br. J. Pharmacol. 1987, 92, 639–646.
[CrossRef] [PubMed]
62. Larsen, F.J.; Schiffer, T.A.; Weitzberg, E.; Lundberg, J.O. Regulation of mitochondrial function and energetics
by reactive nitrogen oxides. Free Radic. Biol. Med. 2012, 53, 1919–1928. [CrossRef] [PubMed]
63. Ceccatelli, S.; Hulting, A.L.; Zhang, X.; Gustafsson, L.; Villar, M.; Hokfelt, T. Nitric oxide synthase in the rat
anterior pituitary gland and the role of nitric oxide in regulation of luteinizing hormone secretion. Proc. Natl.
Acad. Sci. USA 1993, 90, 11292–11296. [CrossRef] [PubMed]
64. Green, S.J.; Scheller, L.F.; Marletta, M.A.; Seguin, M.C.; Klotz, F.W.; Slayter, M.; Nelson, B.J.; Nacy, C.A. Nitric
oxide: Cytokine-regulation of nitric oxide in host resistance to intracellular pathogens. Immunol. Lett. 1994,
43, 87–94. [CrossRef]
65. Langrehr, J.M.; Hoffman, R.A.; Lancaster, J.R.; Simmons, R.L. Nitric oxide—A new endogenous
immunomodulator. Transplantation 1993, 55, 1205–1212. [CrossRef] [PubMed]
66. Wei, X.Q.; Charles, I.G.; Smith, A.; Ure, J.; Feng, G.J.; Huang, F.P.; Xu, D.; Muller, W.; Moncada, S.; Liew, F.Y.
Altered immune responses in mice lacking inducible nitric oxide synthase. Nature 1995, 375, 408–411.
[CrossRef] [PubMed]
67. D’Ischia, M.; Napolitano, A.; Manini, P.; Panzella, L. Secondary Targets of Nitrite-Derived Reactive Nitrogen
Species: Nitrosation/Nitration Pathways, Antioxidant Defense Mechanisms and Toxicological Implications.
Chem. Res. Toxicol. 2011, 24, 2071–2092. [CrossRef] [PubMed]
68. Mirvish, S.S. Formation of N-nitroso compounds: Chemistry, kinetics, and in vivo occurrence.
Toxicol. Appl. Pharmacol. 1975, 31, 325–351. [CrossRef]
69. Ridd, J.H. Nitrosation, diazotisation, and deamination. Q. Rev. 1961, 15, 418–441. [CrossRef]
70. Akuta, T.; Zaki, M.H.; Yoshitake, J.; Okamoto, T.; Akaike, T. Nitrative stress through formation of
8-nitroguanosine: Insights into microbial pathogenesis. Nitric Oxide 2006, 14, 101–108. [CrossRef] [PubMed]
71. Loeppky, R.N.; Bao, Y.T.; Bae, J.Y.; Yu, L.; Shevlin, G. Blocking nitrosamine formation—Understanding the
chemistry of rapid nitrosation. In Nitrosamines and Related N-Nitroso Compounds: Chemistry and Biochemistry;
Loeppky, R.N., Michejda, C.J., Eds.; American Chemical Society: Washington, DC, USA, 1994; Volume 553,
pp. 52–65.
72. Qin, L.Z.; Liu, X.B.; Sun, Q.F.; Fan, Z.P.; Xia, D.S.; Ding, G.; Ong, H.L.; Adams, D.; Gahl, W.A.;
Zheng, C.Y.; et al. Sialin (SLC17A5) functions as a nitrate transporter in the plasma membrane. Proc. Natl.
Acad. Sci. USA 2012, 109, 13434–13439. [CrossRef] [PubMed]
73. Stich, H.F.; Dunn, B.P.; Pignatelli, B.; Ohshima, H.; Bartsch, H. Dietary Phenolics and Betel Nut Extracts as
Modifiers of n Nitrosation in Rat and Man; IARC Scientific Publications: Lyon, France, 1984; pp. 213–222.
74. Vermeer, I.T.; Moonen, E.J.; Dallinga, J.W.; Kleinjans, J.C.; van Maanen, J.M. Effect of ascorbic acid and green
tea on endogenous formation of N-nitrosodimethylamine and N-nitrosopiperidine in humans. Mutat. Res.
1999, 428, 353–361. [CrossRef]
75. Vermeer, I.T.; Pachen, D.M.; Dallinga, J.W.; Kleinjans, J.C.; van Maanen, J.M. Volatile N-nitrosamine formation
after intake of nitrate at the ADI level in combination with an amine-rich diet. Environ. Health Perspect. 1998,
106, 459–463. [PubMed]
76. Chung, M.J.; Lee, S.H.; Sung, N.J. Inhibitory effect of whole strawberries, garlic juice or kale juice on
endogenous formation of N-nitrosodimethylamine in humans. Cancer Lett. 2002, 182, 1–10. [CrossRef]
77. Helser, M.A.; Hotchkiss, J.H.; Roe, D.A. Influence of fruit and vegetable juices on the endogenous formation
of N-nitrosoproline and N-nitrosothiazolidine-4-carboxylic acid in humans on controlled diets. Carcinogenesis
1992, 13, 2277–2280. [CrossRef] [PubMed]
78. Zeilmaker, M.J.; Bakker, M.I.; Schothorst, R.; Slob, W. Risk assessment of N-nitrosodimethylamine formed
endogenously after fish-with-vegetable meals. Toxicol. Sci. 2010, 116, 323–335. [CrossRef] [PubMed]
79. Khandelwal, N.; Abraham, S.K. Intake of anthocyanidins pelargonidin and cyanidin reduces genotoxic stress
in mice induced by diepoxybutane, urethane and endogenous nitrosation. Environ. Toxicol. Pharmacol. 2014,
37, 837–843. [CrossRef] [PubMed]
80. Conforti, F.; Menichini, F. Phenolic Compounds from Plants as Nitric Oxide Production Inhibitors.
Curr. Med. Chem. 2011, 18, 1137–1145. [CrossRef] [PubMed]
81. Abraham, S.K.; Khandelwal, N. Ascorbic acid and dietary polyphenol combinations protect against genotoxic
damage induced in mice by endogenous nitrosation. Mutat. Res. 2013, 757, 167–172. [CrossRef] [PubMed]
82. De Kok, T.M.; (Maastricht, The Netherlands). Unpublished work. 2018.
83. Haorah, J.; Zhou, L.; Wang, X.J.; Xu, G.P.; Mirvish, S.S. Determination of total N-nitroso compounds and their
precursors in frankfurters, fresh meat, dried salted fish, sauces, tobacco, and tobacco smoke particulates.
J. Agric. Food Chem. 2001, 49, 6068–6078. [CrossRef] [PubMed]
84. Cross, A.J.; Pollock, J.R.; Bingham, S.A. Haem, not protein or inorganic iron, is responsible for endogenous
intestinal N-nitrosation arising from red meat. Cancer Res. 2003, 63, 2358–2360. [PubMed]
85. Bingham, S.A.; Pignatelli, B.; Pollock, J.R.A.; Ellul, A.; Malaveille, C.; Gross, G.; Runswick, S.; Cummings, J.H.;
O’Neill, I.K. Does increased endogenous formation of N-nitroso compounds in the human colon explain the
association between red meat and colon cancer? Carcinogenesis 1996, 17, 515–523. [CrossRef] [PubMed]
86. Bingham, S.A.; Hughes, R.; Cross, A.J. Effect of white versus red meat on endogenous N-nitrosation in the
human colon and further evidence of a dose response. J. Nutr. 2002, 132, 3522s–3525s. [CrossRef] [PubMed]
87. Bingham, S.A. High-meat diets and cancer risk. Proc. Nutr. Soc. 1999, 58, 243–248. [CrossRef] [PubMed]
88. Bouvard, V.; Loomis, D.; Guyton, K.Z.; Grosse, Y.; Ghissassi, F.E.; Benbrahim-Tallaa, L.; Guha, N.;
Mattock, H.; Straif, K. International Agency for Research on Cancer Monograph Working, G. Carcinogenicity
of consumption of red and processed meat. Lancet Oncol. 2015, 16, 1599–1600. [CrossRef]
89. International Agency for Research on Cancer (IARC). IARC Monographs on the Evaluation of Carcionogenic
Risks to Humans: Red Meat and Processed Meat; IARC: Lyon, France, 2018.
90. Phytochemicals to Reduce Nitrite in Meat Products (PHYTOME). Available online: www.phytome.eu
(accessed on 3 May 2018).
91. Greer, F.R.; Shannon, M. American Academy of Pediatrics Committee on Nutrition and the Committee on
Environmental Health. Infant methemoglobinemia: The role of dietary nitrate in food and water. Pediatrics
2005, 116, 784–786. [CrossRef] [PubMed]
92. Sanchez-Echaniz, J.; Benito-Fernandez, J.; Mintegui-Raso, S. Methemoglobinemia and consumption of
vegetables in infants. Pediatrics 2001, 107, 1024–1028. [CrossRef] [PubMed]
93. Charmandari, E.; Meadows, N.; Patel, M.; Johnston, A.; Benjamin, N. Plasma nitrate concentrations in
children with infectious and noninfectious diarrhea. J. Pediatr. Gastroenterol. Nutr. 2001, 32, 423–427.
[CrossRef] [PubMed]
94. Comly, H.H. Landmark article 8 September 1945: Cyanosis in infants caused by nitrates in well-water.
By Hunter H. Comly. JAMA 1987, 257, 2788–2792. [CrossRef] [PubMed]
95. Walton, G. Survey of literature relating to infant methemoglobinemia due to nitrate-contaminated water.
Am. J. Public Health Nation’s Health 1951, 41, 986–996. [CrossRef]
96. Knobeloch, L.; Salna, B.; Hogan, A.; Postle, J.; Anderson, H. Blue babies and nitrate-contaminated well water.
Environ. Health Perspect. 2000, 108, 675–678. [CrossRef] [PubMed]
97. Johnson, C.J.; Bonrud, P.A.; Dosch, T.L.; Kilness, A.W.; Senger, K.A.; Busch, D.C.; Meyer, M.R. Fatal outcome
of methemoglobinemia in an infant. JAMA 1987, 257, 2796–2797. [CrossRef] [PubMed]
98. Lutynski, R.; Steczek-Wojdyla, M.; Wojdyla, Z.; Kroch, S. The concentrations of nitrates and nitrites in food
products and environment and the occurrence of acute toxic methemoglobinemias. Prz. Lek. 1996, 53,
351–355. [PubMed]
99. Ayebo, A.; Kross, B.C.; Vlad, M.; Sinca, A. Infant Methemoglobinemia in the Transylvania Region of Romania.
Int. J. Occup. Environ. Health 1997, 3, 20–29. [CrossRef] [PubMed]
100. World Health Organization. Water and Health in Europe; World Health Organization: Geneva,
Switzerland, 2002.
101. Abu Naser, A.A.; Ghbn, N.; Khoudary, R. Relation of nitrate contamination of groundwater with
methaemoglobin level among infants in Gaza. East Mediterr. Health J. 2007, 13, 994–1004. [CrossRef]
[PubMed]
102. Sadeq, M.; Moe, C.L.; Attarassi, B.; Cherkaoui, I.; ElAouad, R.; Idrissi, L. Drinking water nitrate and
prevalence of methemoglobinemia among infants and children aged 1–7 years in Moroccan areas. Int. J. Hyg.
Environ. Health 2008, 211, 546–554. [CrossRef] [PubMed]
103. Zeman, C.; Beltz, L.; Linda, M.; Maddux, J.; Depken, D.; Orr, J.; Theran, P. New Questions and Insights
into Nitrate/Nitrite and Human Health Effects: A Retrospective Cohort Study of Private Well Users’
Immunological and Wellness Status. J. Environ. Health 2011, 74, 8–18. [PubMed]
104. Manassaram, D.M.; Backer, L.C.; Messing, R.; Fleming, L.E.; Luke, B.; Monteilh, C.P. Nitrates in drinking
water and methemoglobin levels in pregnancy: A longitudinal study. Environ. Health 2010, 9, 60. [CrossRef]
[PubMed]
105. Grant, W.; Steele, G.; Isiorho, S.A. Spontaneous abortions possibly related to ingestion of nitrate-contaminated
well water: LaGrange County, Indiana, 1991–1994. Morb. Mortal. Wkly. Rep. 1996, 45, 569–572.
106. Aschengrau, A.; Zierler, S.; Cohen, A. Quality of community drinking water and the occurrence of
spontaneous abortion. Arch Environ. Health 1989, 44, 283–290. [CrossRef] [PubMed]
107. Albouy-Llaty, M.; Limousi, F.; Carles, C.; Dupuis, A.; Rabouan, S.; Migeot, V. Association between Exposure
to Endocrine Disruptors in Drinking Water and Preterm Birth, Taking Neighborhood Deprivation into
Account: A Historic Cohort Study. Int. J. Environ. Res. Public Health 2016, 13, 796. [CrossRef] [PubMed]
108. Stayner, L.T.; Almberg, K.; Jones, R.; Graber, J.; Pedersen, M.; Turyk, M. Atrazine and nitrate in drinking
water and the risk of preterm delivery and low birth weight in four Midwestern states. Environ. Res. 2017,
109. Joyce, S.J.; Cook, A.; Newnham, J.; Brenters, M.; Ferguson, C.; Weinstein, P. Water disinfection by-products
and prelabor rupture of membranes. Am. J. Epidemiol. 2008, 168, 514–521. [CrossRef] [PubMed]
110. Mattix, K.D.; Winchester, P.D.; Scherer, L.R. Incidence of abdominal wait defects is related to surface water
atrazine and nitrate levels. J. Pediatr. Surg. 2007, 42, 947–949. [CrossRef] [PubMed]
111. Waller, S.A.; Paul, K.; Peterson, S.E.; Hitti, J.E. Agricultural-related chemical exposures, season of conception,
and risk of gastroschisis in Washington State. Am. J. Obstet. Gynecol. 2010, 202, e241–e246. [CrossRef]
[PubMed]
112. Winchester, P.D.; Huskins, J.; Ying, J. Agrichemicals in surface water and birth defects in the United States.
Acta Paediatr. 2009, 98, 664–669. [CrossRef] [PubMed]
113. Holtby, C.E.; Guernsey, J.R.; Allen, A.C.; VanLeeuwen, J.A.; Allen, V.M.; Gordon, R.J. A Population-Based
Case-Control Study of Drinking-Water Nitrate and Congenital Anomalies Using Geographic Information
Systems (GIS) to Develop Individual-Level Exposure Estimates. Int. J. Environ. Res. Public Health 2014, 11,
114. Weyer, P.J.; Brender, J.D.; Romitti, P.A.; Kantamneni, J.R.; Crawford, D.; Sharkey, J.R.; Shinde, M.; Horel, S.A.;
Vuong, A.M.; Langlois, P.H. Assessing bottled water nitrate concentrations to evaluate total drinking water
nitrate exposure and risk of birth defects. J. Water Health 2014, 12, 755–762. [CrossRef] [PubMed]
115. Yoon, P.W.; Rasmussen, S.A.; Lynberg, M.C.; Moore, C.A.; Anderka, M.; Carmichael, S.L.; Costa, P.;
Druschel, C.; Hobbs, C.A.; Romitti, P.A.; et al. The National Birth Defects Prevention Study. Public Health Rep.
2001, 116 (Suppl. 1), 32–40. [CrossRef] [PubMed]
116. Brender, J.D.; Kelley, K.E.; Werler, M.M.; Langlois, P.H.; Suarez, L.; Canfield, M.A. National Birth Defects
Prevention Study. Prevalence and Patterns of Nitrosatable Drug Use among U.S. Women during Early
Pregnancy. Birth Defects Res. A 2011, 91, 258–264. [CrossRef] [PubMed]
117. Griesenbeck, J.S.; Brender, J.D.; Sharkey, J.R.; Steck, M.D.; Huber, J.C., Jr.; Rene, A.A.; McDonald, T.J.;
Romitti, P.A.; Canfield, M.A.; Langlois, P.H.; et al. Maternal characteristics associated with the dietary intake
of nitrates, nitrites, and nitrosamines in women of child-bearing age: A cross-sectional study. Environ. Health
2010, 9, 10. [CrossRef] [PubMed]
118. Brender, J.D.; Werler, M.M.; Shinde, M.U.; Vuong, A.M.; Kelley, K.E.; Huber, J.C., Jr.; Sharkey, J.R.;
Griesenbeck, J.S.; Romitti, P.A.; Malik, S.; et al. Nitrosatable drug exposure during the first trimester
of pregnancy and selected congenital malformations. Birth Defects Res. A 2012, 94, 701–713. [CrossRef]
[PubMed]
119. Brender, J.D.; Werler, M.M.; Kelley, K.E.; Vuong, A.M.; Shinde, M.U.; Zheng, Q.; Huber, J.C., Jr.; Sharkey, J.R.;
Griesenbeck, J.S.; Romitti, P.A.; et al. Nitrosatable drug exposure during early pregnancy and neural tube
defects in offspring: National Birth Defects Prevention Study. Am. J. Epidemiol. 2011, 174, 1286–1295.
[CrossRef] [PubMed]
120. Brender, J.D.; Olive, J.M.; Felkner, M.; Suarez, L.; Marckwardt, W.; Hendricks, K.A. Dietary nitrites and
nitrates, nitrosatable drugs, and neural tube defects. Epidemiology 2004, 15, 330–336. [CrossRef] [PubMed]
121. Dorsch, M.M.; Scragg, R.K.; McMichael, A.J.; Baghurst, P.A.; Dyer, K.F. Congenital malformations and
maternal drinking water supply in rural South Australia: A case-control study. Am. J. Epidemiol. 1984, 119,
122. Croen, L.A.; Todoroff, K.; Shaw, G.M. Maternal exposure to nitrate from drinking water and diet and risk for
neural tube defects. Am. J. Epidemiol. 2001, 153, 325–331. [CrossRef] [PubMed]
123. Arbuckle, T.E.; Sherman, G.J.; Corey, P.N.; Walters, D.; Lo, B. Water nitrates and CNS birth defects: A
population-based case-control study. Arch Environ. Health 1988, 43, 162–167. [CrossRef] [PubMed]
124. Ericson, A.; Kallen, B.; Lofkvist, E. Environmental factors in the etiology of neural tube defects: A negative
study. Environ. Res. 1988, 45, 38–47. [CrossRef]
125. Cantor, K.P. Drinking water and cancer. Cancer Causes Control 1997, 8, 292–308. [CrossRef] [PubMed]
126. Quist, A.J.L.; Inoue-Choi, M.; Weyer, P.J.; Anderson, K.E.; Cantor, K.P.; Krasner, S.; Freeman, L.E.B.;
Ward, M.H.; Jones, R.R. Ingested nitrate and nitrite, disinfection by-products, and pancreatic cancer risk in
postmenopausal women. Int. J. Cancer 2018, 142, 251–261. [CrossRef] [PubMed]
127. Jones, R.R.; Weyer, P.J.; DellaValle, C.T.; Robien, K.; Cantor, K.P.; Krasner, S.; Freeman, L.E.B.; Ward, M.H.
Ingested nitrate, disinfection by-products, and kidney cancer risk in older women. Epidemiology 2017, 28,
128. Inoue-Choi, M.; Ward, M.H.; Cerhan, J.R.; Weyer, P.J.; Anderson, K.E.; Robien, K. Interaction of nitrate and
folate on the risk of breast cancer among postmenopausal women. Nutr. Cancer 2012, 64, 685–694. [CrossRef]
[PubMed]
129. Inoue-Choi, M.; Jones, R.R.; Anderson, K.E.; Cantor, K.P.; Cerhan, J.R.; Krasner, S.; Robien, K.; Weyer, P.J.;
Ward, M.H. Nitrate and nitrite ingestion and risk of ovarian cancer among postmenopausal women in Iowa.
Int. J. Cancer 2015, 137, 173–182. [CrossRef] [PubMed]
130. Weyer, P.J.; Cerhan, J.R.; Kross, B.C.; Hallberg, G.R.; Kantamneni, J.; Breuer, G.; Jones, M.P.; Zheng, W.;
Lynch, C.F. Municipal drinking water nitrate level and cancer risk in older women: The Iowa Women’s
Health Study. Epidemiology 2001, 12, 327–338. [CrossRef] [PubMed]
131. Zeegers, M.P.; Selen, R.F.; Kleinjans, J.C.; Goldbohm, R.A.; van den Brandt, P.A. Nitrate intake does not
influence bladder cancer risk: The Netherlands cohort study. Environ. Health Perspect. 2006, 114, 1527–1531.
[CrossRef] [PubMed]
132. Ward, M.H.; Mark, S.D.; Cantor, K.P.; Weisenburger, D.D.; Correa-Villasenor, A.; Zahm, S.H. Drinking water
nitrate and the risk of non-Hodgkin’s lymphoma. Epidemiology 1996, 7, 465–471. [CrossRef] [PubMed]
133. Ward, M.H.; Heineman, E.F.; Markin, R.S.; Weisenburger, D.D. Adenocarcinoma of the stomach and
esophagus and drinking water and dietary sources of nitrate and nitrite. Int. J. Occup. Environ. Health 2008,
134. McElroy, J.A.; Trentham-Dietz, A.; Gangnon, R.E.; Hampton, J.M.; Bersch, A.J.; Kanarek, M.S.; Newcomb, P.A.
Nitrogen-nitrate exposure from drinking water and colorectal cancer risk for rural women in Wisconsin,
USA. J. Water Health 2008, 6, 399–409. [CrossRef] [PubMed]
135. Espejo-Herrera, N.; Gracia-Lavedan, E.; Boldo, E.; Aragones, N.; Perez-Gomez, B.; Pollan, M.; Molina, A.J.;
Fernandez, T.; Martin, V.; La Vecchia, C.; et al. Colorectal cancer risk and nitrate exposure through drinking
water and diet. Int. J. Cancer 2016, 139, 334–346. [CrossRef] [PubMed]
136. Fathmawati; Fachiroh, J.; Gravitiani, E.; Sarto; Husodo, A.H. Nitrate in drinking water and risk of colorectal
cancer in Yogyakarta, Indonesia. J. Toxicol. Environ. Health Part A 2017, 80, 120–128. [CrossRef] [PubMed]
137. Brody, J.G.; Aschengrau, A.; McKelvey, W.; Swartz, C.H.; Kennedy, T.; Rudel, R.A. Breast cancer risk and
drinking water contaminated by wastewater: A case control study. Environ. Health-Glob. 2006, 5, 28.
[CrossRef] [PubMed]
138. Espejo-Herrera, N.; Gracia-Lavedan, E.; Pollan, M.; Aragones, N.; Boldo, E.; Perez-Gomez, B.; Altzibar, J.M.;
Amiano, P.; Zabala, A.J.; Ardanaz, E.; et al. Ingested Nitrate and Breast Cancer in the Spanish
Multicase-Control Study on Cancer (MCC-Spain). Environ. Health Perspect. 2016, 124, 1042–1049. [CrossRef]
[PubMed]
139. Mueller, B.A.; Nielsen, S.S.; Preston-Martin, S.; Holly, E.A.; Cordier, S.; Filippini, G.; Peris-Bonet, R.;
Choi, N.W. Household water source and the risk of childhood brain tumours: Results of the SEARCH
International Brain Tumor Study. Int. J. Epidemiol. 2004, 33, 1209–1216. [CrossRef] [PubMed]
140. Mueller, B.A.; Newton, K.; Holly, E.A.; Preston-Martin, S. Residential water source and the risk of childhood
brain tumors. Environ. Health Perspect. 2001, 109, 551–556. [CrossRef] [PubMed]
141. De Groef, B.; Decallonne, B.R.; Van der Geyten, S.; Darras, V.M.; Bouillon, R. Perchlorate versus
other environmental sodium/iodide symporter inhibitors: Potential thyroid-related health effects.
Eur. J. Endocrinol. 2006, 155, 17–25. [CrossRef] [PubMed]
142. Van Maanen, J.M.; Welle, I.J.; Hageman, G.; Dallinga, J.W.; Mertens, P.L.; Kleinjans, J.C. Nitrate contamination
of drinking water: Relationship with HPRT variant frequency in lymphocyte DNA and urinary excretion of
N-nitrosamines. Environ. Health Perspect. 1996, 104, 522–528. [CrossRef] [PubMed]
143. Radikova, Z.; Tajtakova, M.; Kocan, A.; Trnovec, T.; Sebokova, E.; Klimes, I.; Langer, P. Possible effects of
environmental nitrates and toxic organochlorines on human thyroid in highly polluted areas in Slovakia.
Thyroid Off. J. Am. Thyroid Assoc. 2008, 18, 353–362. [CrossRef] [PubMed]
144. Tajtakova, M.; Semanova, Z.; Tomkova, Z.; Szokeova, E.; Majoros, J.; Radikova, Z.; Sebokova, E.; Klimes, I.;
Langer, P. Increased thyroid volume and frequency of thyroid disorders signs in schoolchildren from nitrate
polluted area. Chemosphere 2006, 62, 559–564. [CrossRef] [PubMed]
145. Aschebrook-Kilfoy, B.; Heltshe, S.L.; Nuckols, J.R.; Sabra, M.M.; Shuldiner, A.R.; Mitchell, B.D.; Airola, M.;
Holford, T.R.; Zhang, Y.; Ward, M.H. Modeled nitrate levels in well water supplies and prevalence of
abnormal thyroid conditions among the Old Order Amish in Pennsylvania. Environ. Health 2012, 11, 6.
[CrossRef] [PubMed]
146. Longnecker, M.P.; Daniels, J.L. Environmental contaminants as etiologic factors for diabetes.
Environ. Health Perspect. 2001, 109 (Suppl. 6), 871–876. [CrossRef] [PubMed]
147. Moltchanova, E.; Rytkonen, M.; Kousa, A.; Taskinen, O.; Tuomilehto, J.; Karvonen, M.; Spat Study, G. Finnish
Childhood Diabetes Registry, G. Zinc and nitrate in the ground water and the incidence of Type 1 diabetes in
Finland. Diabet. Med. 2004, 21, 256–261. [CrossRef] [PubMed]
148. Muntoni, S.; Cocco, P.; Muntoni, S.; Aru, G. Nitrate in community water supplies and risk of childhood type
1 diabetes in Sardinia, Italy. Eur. J. Epidemiol. 2006, 21, 245–247. [CrossRef] [PubMed]
149. Benson, V.S.; Vanleeuwen, J.A.; Taylor, J.; Somers, G.S.; McKinney, P.A.; Van Til, L. Type 1 diabetes mellitus
and components in drinking water and diet: A population-based, case-control study in Prince Edward
Island, Canada. J. Am. Coll. Nutr. 2010, 29, 612–624. [CrossRef] [PubMed]
150. Winkler, C.; Mollenhauer, U.; Hummel, S.; Bonifacio, E.; Ziegler, A.G. Exposure to environmental factors in
drinking water: Risk of islet autoimmunity and type 1 diabetes—The BABYDIAB study. Horm. Metab. Res.
2008, 40, 566–571. [CrossRef] [PubMed]
151. Klein, B.E.K.; McElroy, J.A.; Klein, R.; Howard, K.P.; Lee, K.E. Nitrate-nitrogen levels in rural drinking
water: Is there an association with age-related macular degeneration? J. Environ. Sci. Health Part A 2013, 48,
152. Ahluwalia, A.; Gladwin, M.; Coleman, G.D.; Hord, N.; Howard, G.; Kim-Shapiro, D.B.; Lajous, M.; Larsen, F.J.;
Lefer, D.J.; McClure, L.A.; et al. Dietary Nitrate and the Epidemiology of Cardiovascular Disease: Report
From a National Heart, Lung, and Blood Institute Workshop. J. Am. Heart Assoc. 2016, 5, e003402. [CrossRef]
[PubMed]
153. Kapil, V.; Khambata, R.S.; Robertson, A.; Caulfield, M.J.; Ahluwalia, A. Dietary nitrate provides sustained
blood pressure lowering in hypertensive patients: A randomized, phase 2, double-blind, placebo-controlled
study. Hypertension 2015, 65, 320–327. [CrossRef] [PubMed]
154. Omar, S.A.; Webb, A.J.; Lundberg, J.O.; Weitzberg, E. Therapeutic effects of inorganic nitrate and nitrite in
cardiovascular and metabolic diseases. J. Intern. Med. 2016, 279, 315–336. [CrossRef] [PubMed]
155. Presley, T.D.; Morgan, A.R.; Bechtold, E.; Clodfelter, W.; Dove, R.W.; Jennings, J.M.; Kraft, R.A.; King, S.B.;
Laurienti, P.J.; Rejeski, W.J.; et al. Acute effect of a high nitrate diet on brain perfusion in older adults.
Nitric Oxide 2011, 24, 34–42. [CrossRef] [PubMed]
156. Maas, R.; Schwedhelm, E.; Kahl, L.; Li, H.; Benndorf, R.; Luneburg, N.; Forstermann, U.; Boger, R.H.
Simultaneous assessment of endothelial function, nitric oxide synthase activity, nitric oxide-mediated
signaling, and oxidative stress in individuals with and without hypercholesterolemia. Clin. Chem. 2008, 54,
157. Jadert, C.; Phillipson, M.; Holm, L.; Lundberg, J.O.; Borniquel, S. Preventive and therapeutic effects of
nitrite supplementation in experimental inflammatory bowel disease. Redox Biol. 2014, 2, 73–81. [CrossRef]
[PubMed]
158. Khademikia, S.; Rafiee, Z.; Amin, M.M.; Poursafa, P.; Mansourian, M.; Modaberi, A. Association of nitrate,
nitrite, and total organic carbon (TOC) in drinking water and gastrointestinal disease. J. Environ. Public Health
2013, 2013, 603468. [CrossRef] [PubMed]
159. De Roos, A.J.; Ward, M.H.; Lynch, C.F.; Cantor, K.P. Nitrate in public water supplies and the risk of colon
and rectum cancers. Epidemiology 2003, 14, 640–649. [CrossRef] [PubMed]
160. Gatseva, P.D.; Argirova, M.D. High-nitrate levels in drinking water may be a risk factor for thyroid
dysfunction in children and pregnant women living in rural Bulgarian areas. Int. J. Hyg. Environ. Health
2008, 211, 555–559. [CrossRef] [PubMed]
161. Toccalino, P.L.; Norman, J.E.; Scott, J.C. Chemical mixtures in untreated water from public-supply wells in
the U.S.—Occurrence, composition, and potential toxicity. Sci. Total Environ. 2012, 431, 262–270. [CrossRef]
[PubMed]
162. Joshi, N.; Rhoades, M.G.; Bennett, G.D.; Wells, S.M.; Mirvish, S.S.; Breitbach, M.J.; Shea, P.J. Developmental
abnormalities in chicken embryos exposed to N-nitrosoatrazine. J. Toxicol. Environ. Health Part A 2013, 76,
163. Mitch, W.A.; Sharp, J.O.; Rhoades Trussell, R.; Valentine, R.L.; Alvarez-Cohen, L.; DSedlak, D.L.
N-Nitrosodimethylamine (NDMA) as a Drinking Water Contaminant: A Review. Environ. Eng. Sci. 2003, 20,
164. Krasner, S.W. The formation and control of emerging disinfection by-products of health concern. Philos. Trans.
2009, 367, 4077–4095. [CrossRef] [PubMed]
165. Hezel, M.P.; Weitzberg, E. The oral microbiome and nitric oxide homoeostasis. Oral Dis. 2015, 21, 7–16.
[CrossRef] [PubMed]
166. Hyde, E.R.; Andrade, F.; Vaksman, Z.; Parthasarathy, K.; Jiang, H.; Parthasarathy, D.K.; Torregrossa, A.C.;
Tribble, G.; Kaplan, H.B.; Petrosino, J.F.; et al. Metagenomic analysis of nitrate-reducing bacteria in the oral
cavity: Implications for nitric oxide homeostasis. PLoS ONE 2014, 9, e88645. [CrossRef] [PubMed]
167. Burleigh, M.C.; Liddle, L.; Monaghan, C.; Muggeridge, D.J.; Sculthorpe, N.; Butcher, J.P.; Henriquez, F.L.;
Allen, J.D.; Easton, C. Salivary nitrite production is elevated in individuals with a higher abundance of oral
nitrate-reducing bacteria. Free Radic. Biol. Med. 2018, 120, 80–88. [CrossRef] [PubMed]
168. Vogtmann, E.; Chen, J.; Amir, A.; Shi, J.; Abnet, C.C.; Nelson, H.; Knight, R.; Chia, N.; Sinha, R. Comparison
of Collection Methods for Fecal Samples in Microbiome Studies. Am. J. Epidemiol. 2017, 185, 115–123.
[CrossRef] [PubMed]
169. Sinha, R.; Abu-Ali, G.; Vogtmann, E.; Fodor, A.A.; Ren, B.; Amir, A.; Schwager, E.; Crabtree, J.; Ma, S.; The
Microbiome Quality Control Project Consortium; et al. Assessment of variation in microbial community
amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium. Nat. Biotechnol. 2017,
35, 1077–1086. [PubMed]
170. Hebels, D.G.; Jennen, D.G.; van Herwijnen, M.H.; Moonen, E.J.; Pedersen, M.; Knudsen, L.E.; Kleinjans, J.C.;
de Kok, T.M. Whole-genome gene expression modifications associated with nitrosamine exposure and
micronucleus frequency in human blood cells. Mutagenesis 2011, 26, 753–761. [CrossRef] [PubMed]
171. Hebels, D.G.; Jennen, D.G.; Kleinjans, J.C.; de Kok, T.M. Molecular signatures of N-nitroso compounds in
Caco-2 cells: Implications for colon carcinogenesis. Toxicol. Sci. 2009, 108, 290–300. [CrossRef] [PubMed]
172. Vineis, P.; Chadeau-Hyam, M.; Gmuender, H.; Gulliver, J.; Herceg, Z.; Kleinjans, J.; Kogevinas, M.;
Kyrtopoulos, S.; Nieuwenhuijsen, M.; Phillips, D.H.; et al. The exposome in practice: Design of the
EXPOsOMICS project. Int. J. Hyg. Environ. Health 2017, 220, 142–151. [CrossRef] [PubMed]
173. Hebels, D.G.; Georgiadis, P.; Keun, H.C.; Athersuch, T.J.; Vineis, P.; Vermeulen, R.; Portengen, L.;
Bergdahl, I.A.; Hallmans, G.; Palli, D.; et al. Performance in omics analyses of blood samples in
long-term storage: Opportunities for the exploitation of existing biobanks in environmental health research.
Environ. Health Perspect. 2013, 121, 480–487. [CrossRef] [PubMed]
174. International Nitrogen Initiative. Available online: http://www.initrogen.org/ (accessed on 22 April 2018).
175. Dinnes, D.L.; Karlen, D.L.; Jaynes, D.B.; Kaspar, T.C.; Hatfield, J.L.; Colvin, T.S.; Cambardella, C.A. Nitrogen
management strategies to reduce nitrate leaching in tile-drained midwestern soils. Agron. J. 2002, 94, 153–171.
[CrossRef]
176. Baron, J.S.; Hall, E.K.; Nolan, B.T.; Finlay, J.C.; Bernhardt, E.S.; Harrison, J.A.; Chan, F.; Boyer, E.W.
The interactive effects of excess reactive nitrogen and climate change on aquatic ecosystems and water
resources of the United States. Biogeochemistry 2013, 114, 71–92. [CrossRef]
© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Keywords: drinking water; nitrate; cancer; adverse reproductive outcomes; methemoglobinemia;

2. Drinking Water Nitrate Exposures in the United States and Europe

3. Exposure Assessment in Epidemiologic Studies

4. Nitrate Intake and Endogenous Formation of N-Nitroso Compounds

6. Adverse Pregnancy Outcomes

First Author, Year, Study Design Years of Outcome

First Author, Year, Study Design Years of Outcome

First Author Study Design, Years Cancer Sites Evaluation of Effect

First Author Study Design, Years Cancer Sites Evaluation of Effect

First Author Study Design, Years Cancer Sites Evaluation of Effect

First Author Study Design, Years Cancer Sites Evaluation of Effect

9. Other Health Effects

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