Journal of Exposure Science and Environmental Epidemiology (2008) 18, 129–133
r 2008 Nature Publishing Group All rights reserved 1559-0631/08/$30.00
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Are nitrate levels in groundwater stable over time?
PERRI ZEITZ RUCKARTa, ALDEN K. HENDERSONa, MICHELE LYNBERG BLACKb AND
W. DANA FLANDERSc
a
Agency for Toxic Substances and Disease Registry, Atlanta, Georgia, USA
Centers for Disease Control and Prevention, National Center for Injury Prevention and Control, Atlanta, Georgia, USA
c
Centers for Disease Control and Prevention, National Center for Environmental Health, Atlanta, Georgia, USA
b
Epidemiologists often use a retrospective study design to examine for associations between an exposure and the occurrence of adverse health effects.
Several of these studies used this approach to examine for an association between elevated levels of nitrate in drinking water and related health effects such
as methemoglobinemia, cancer, neural tube effects, or spontaneous abortions. Often, data on exposures that occurred before these health outcomes were
not available. Consequently, researchers use measurements of exposures at the time of the study to represent exposures that occurred before people
developed these conditions. An opportunity to examine the stability of nitrate in water occurred during a survey of private water wells in nine Midwestern
states. In this survey, water samples from 853 homes with drilled wells were collected in May 1994 and in September 1995 and nitrate-nitrogen (nitrate-N)
was measured by the colorimetric cadmium reduction method. Nitrate-N levels from the same well over time were assessed by a mixed-effects analysis of
variance. Analysis showed no significant difference in between the initial level and those measured 16 months later. Furthermore, analysis showed that
most of the variance in the nitrate concentrations in well water was due to well-to-well variation (89%) rather than to residual error (12%). This
observation showed that a single measurement of nitrate in water from drilled wells could represent longer periods of exposure.
Journal of Exposure Science and Environmental Epidemiology (2008) 18, 129–133; doi:10.1038/sj.jes.7500561; published online 11 April 2007
Keywords: nitrate, groundwater, exposure, aquifer.
Introduction
The level of nitrate in groundwater has been increasing over
the past three decades (Mueller et al., 1995; Mueller and
Helsel, 1996). Fertilizers and organic waste account for much
of the increase in the nitrate in groundwater. Soil bacteria
may also increase nitrate levels in groundwater by oxidizing
ammonia into nitrite and then to nitrate ion in oxygen-rich
water (Dorsch et al., 1984). Although the nitrate ion is not
toxic, enzymes in saliva and gastrointestinal secretions can
reduce it to the more toxic nitrite (Swann, 1975). Nitrite is
not only directly toxic, it also reacts with secondary and
tertiary amines and amides to form carcinogenic N-nitroso
compounds.
Elevated nitrate levels in drinking water have been
associated with methemoglobinemia, cancer, neural tube
defects, and spontaneous abortions (Swann, 1975; Scragg
et al., 1982; Maloney et al., 1983; Dorsch et al., 1984; Fan
et al., 1987; CDC, 1994). Methemoglobinemia is the most
widely recognized health outcome associated with ingestion
of nitrate-contaminated water. It is an acute and potentially
1 Address all correspondence to: Dr. Alden Henderson, Agency for Toxic
Substances and Disease Registry, 1600 Clifton Road, MS E-31, Atlanta,
GA 30333, USA. Tel.: þ 1 404 498 1351. Fax: þ 1 404 498 0077.
E-mail: [email protected]
Received 14 March 2006; accepted 4 February 2007; published online 11
April 2007
fatal illness that primarily affects infants. While N-nitroso
compounds have been linked to cancer in animals (Swann,
1975), the evidence of stomach, brain, esophageal, and
nasopharyngeal cancers in humans from these compounds
remains inconclusive (Eichholzer and Gutzwiller, 1998).
Studies by Scragg et al. (1982) and Dorsch et al. (1984)
suggest a possible association between neural tube defects
and elevated nitrate levels. An investigation of a cluster of
women with spontaneous abortions in LaGrange County,
Indiana, from 1991 to 1994 suggested that elevated nitrate
levels in drinking water may be associated with spontaneous
abortions (CDC, 1994).
To prevent adverse health effects from ingesting nitrate in
drinking water, the Environmental Protection Agency (EPA)
established a maximum contaminant level (MCL) of 10 mg/l
for nitrate (nitrate-N) in public water systems (US EPA,
1994). Unlike other MCLs, the nitrate MCL does not
include a 10–100-fold uncertainty factor (Fan and Steinberg,
1996). The nitrate MCL is based on studies of infant
methemoglobinemia and nitrate levels in drinking water.
A key element when studying the relationship between
nitrate-contaminated drinking water and adverse acute health
outcomes is the exposure to nitrate levels before the disease
occurrence. Since measurements of nitrate levels may not be
available, information on the stability of nitrate levels in
ground water would be helpful to determine whether
historical data may be used or testing conducted on new
Zeitz et al.
samples. Several studies reported the stability of nitrate in
groundwater. A study in Czechoslovakia showed that nitrate
levels in farming communities doubled from 24 mg/l in 1961–
1965 to 43 mg/l in 1986 as fertilizer use increased eight times
(Benes et al., 1989). A smaller increase (21 mg/l in 1968 to
28 mg/l in 1986) was observed in wells surrounded by
protection zones. The study concluded that climatic conditions were mainly responsible for short-term changes in
nitrate levels in the upper parts of the aquifer whereas
anthropogenic influences (especially farming) were mainly
responsible for long-term increases.
A study in Minnesota showed that nitrate concentrations
at the water table are closely linked to seasonal recharge and
with nitrogen fertilizer applications (Landon et al., 2000).
Nitrate levels in the 65-ha research area ranged from 0.05 to
40 mg/l over a 2-year period. A comparison of nitrate
concentrations from the same irrigation wells in Kansas
taken over 20 years showed a slight increasing nitrate
concentration (Townsend and Young, 2000). This conclusion
was based on results from 32 wells which had concentrations
less than 5 mg/l in the 1970s and no data on rainfall and land
use practices. Nitrate concentrations in 66 shallow groundwater sources in Kentucky showed seasonal variations. This
was attributed to low flow rates in the summer and higher
flow rates in the winter (Montgomery et al., 1997).
The present study examines the stability of nitrate levels in
853-drilled wells in nine upper mid-western states by
comparing nitrate-N levels that were measured three times
over a 17-month period.
Methods
Almost 2.5 million households in the upper Midwestern
states depend on private well water as their source of drinking
water. State health and environmental departments in nine
Midwestern states (Illinois, Iowa, Kansas, Minnesota,
Missouri, Nebraska, North Dakota, South Dakota, and
Wisconsin) and the Centers for Disease Control and
Prevention (CDC) conducted a study to determine the
presence of bacteria and chemicals in water drawn from
private wells (CDC, 1998).
Households were originally selected for inclusion into the
study using a systematic geographical sampling approach. A
10-mile sampling grid of the entire study area was
constructed using ArcInfo, a geographic information system
software package. County health department staff traveled to
each grid intersection in the 10-mile sampling grid and asked
a resident of the household closest to and within three miles
of each intersection to participate in the study. If a resident
consented, a water sample was collected at the point of use
that most often supplied drinking water to household
residents (generally the kitchen tap). Additionally, a short
interview was conducted to obtain information on well
130
Nitrate stability in groundwater
characteristics, potential sources of contamination, the
number of people who drink the well water, and the
occurrence of diarrhea among residents of the household.
To be eligible to participate in the survey, at least one
member of the household had to drink well water, and the
well could not have been chlorinated in the previous 4 days.
If the resident refused to participate or if an eligibility
requirement was not met, the next closest household was
contacted. From May to November 1994, samples were
collected from 5520 domestic wells and tested for total
coliforms, Escherichia coli, nitrate, and atrazine. Nitratenitrogen concentration (mg/l NO3-N and hereby called
nitrate) was measured according to the colorimetric cadmium-reduction method (American Public Health Association, 1992).
In the following year (May–September 1995), a subset of
household wells (n ¼ 1487) that tested positive in 1994 for E.
coli or had elevated nitrate levels above the MCL were tested
for bacteria and nitrate. A random sample of 10% of the
uncontaminated wells was also re-sampled. In this survey,
household respondents were re-interviewed with the same
questionnaire as the 1994 survey. A third well water sample
was collected approximately 2 weeks after the second
sampling for quality assurance/quality control (QA/QC)
purposes. This analysis in the study consists of the 853-drilled
wells that were sampled in all three sampling rounds.
The sampling rounds were classified into three phases:
Phase 1 are results of sampling of wells done in 1994; Phase
2(a) are results of the first sampling of wells in 1995, and
Phase 2(b) are results of follow-up sampling conducted 2
weeks after Phase 2(a). In this manuscript, we present the (1)
mean nitrate levels, (2) correlation coefficients between the
phases, (3) a categorical analysis of the distribution of waternitrate measurements in Phase 2(a) as a percentage of Phase
1 measurements, and (4) a mixed-effects analysis of variance
procedure. Because nitrate levels of 2 mg/l or less are
considered background levels (USGS, 2001) and the current
standard is 10 mg/l, these levels provided the basis for the
following categories: r2 mg/l (background level), 42 and
o10 mg/l (low exposure), Z10 and o20 mg/l (moderate
exposure), and greater than or equal to 20 mg/l (high
exposure). We include this categorical analysis because nitrate
ranges like these might be used in an epidemiological study to
assess health effects, and these analyses illustrate stability of
this type of classification over time.
Nitrate levels were logarithmically transformed (base 10)
to stabilize the variance of increasing nitrate levels. To include
samples with a nitrate level that was below the level of
detection, the number one was added to each nitrate
measurement before the levels were transformed. The
mixed-effects analysis of variance for all three phases (logtransformed) tested the model: Yij ¼ a þ Ai þ bj þ Eij where
Yij is a measured value in well i at time j, a is a constant, Ai is
a random effect for well i, bj is a fixed time effect, g is a
Journal of Exposure Science and Environmental Epidemiology (2008) 18(2)
Nitrate stability in groundwater
Zeitz et al.
parameter for the fixed factor X, and Eij is an error term.
Time j was treated categorically (to indicate Phase 1, Phase
2a or Phase 2b samples, respectively) and nearly the same
results were obtained when time was treated as a continuous
variable (t ¼ 0, 1, or 1.04; data not shown). The association
among well age, depth, and diameter with nitrate levels was
examined by adding these variables to the model as fixed
effects (represented by X in the model equation above). The
data were analyzed using SAS for Windows Release 6.12
(SAS Institute Inc., 1992).
Results
Of the 5520 wells sampled in 1994, 1471 were sampled in all
three sampling rounds and were tested for nitrate. For this
analysis, all the dug, bored, and driven wells were excluded to
reduce the effect of contamination. Thus the analysis is based
upon the 853-drilled wells. Of the drilled wells sampled in
Phase 1, there were 425 wells (49.8%) testing positive for
total coliforms, and 199 wells (23.3%) testing positive for
E. coli (concentrations of at least 1 per 100 ml of sample),
and 394 wells (46.2%) had nitrate levels above the MCL of
10 mg/l NO3-N. In Phase 2(a), 381 wells (44.7%) tested
positive for total coliforms, 109 wells (12.8%) tested positive
for E. coli, and 316 wells (37.1%) had nitrate levels above the
MCL of 10 mg/l NO3-N. Most of the drilled wells had a
diameter o6 inches (80.9%), were o25 years (64.5%), and
o100 ft deep (55.1%). Statistically significantly higher
nitrate levels were measured in wells that had diameters Z6
inches (Po0.004), were at least 25 years of age (Po0.001),
or were less than 100 ft deep (Po0.0001). There were 57.5%
of the well owners reported using fertilizer within 100 ft of the
well.
Table 1. Descriptive statistics of nitrate levels, 1994 Midwest WellWater Survey.
The mean nitrate levels in Phases 2(a) and 2(b) was
12.0 mg/l and similar to the Phase 1 level of 13.1 mg/l
(Table 1). For the initial Phase 1 sampling, the nitrate levels
ranged from non-detectable levels to 266.0 mg/l, with a
median of 7.1 mg/l; most (99%) of the values were below
103.0 mg/l. In Phase 2(a), the levels ranged from nondetectable levels to 190 mg/l, with a median of 4.2 mg/l; most
(99%) of the values were below 109 mg/l.
The nitrate levels measured in Phases 2(a) and 2(b) were
highly positively correlated in both transformed and nontransformed states (r ¼ 0.92 and 0.95, respectively,
P ¼ 0.0001 in both cases; Table 2). Levels in Phase 1 were
highly positively correlated with levels measured in both
Phase 2(a) and Phase 2(b). The association became stronger
when the levels were logarithmically transformed (r ¼ 0.88,
P ¼ 0.0001 and r ¼ 0.87, P ¼ 0.0001, respectively).
When the nitrate levels of wells from Phase 1 were categorized as described previously into background, low, moderate,
and high, most of the wells (75.4%, n ¼ 643) remained in the
same category in Phase 2(a) (Table 3). Of the 345 wells that
initially had background nitrate levels, 92.2% (n ¼ 318)
remained in the background category in Phase 2(a); of the
114 wells that were initially low, 74.6.2% (n ¼ 85) remained
low; of the 226 wells that were initially moderate, 55.8%
(n ¼ 126) remained high; and of the 168 wells that were initially
high, 67.9% (n ¼ 114) remained high. When the nitrate levels
did not remain in the same categories, most changes involved a
shift to the adjacent lower exposure category.
The mixed-effects analysis of variance showed that most
(89%) of the variance in the nitrate measurements was due
mainly to well-to-well variation (s2 ¼ 0.33). Eleven percent of
the variance was due to the within-subject residual error
(s2 ¼ 0.040). Similar results were obtained when we compared Phase 1 to Phase 2(a). The association of higher nitrate
levels with well age (P ¼ 0.02), depth (Po0.0001) and larger
Table 2. Correlation coefficients of the nitrate levels in Phases 1, 2(a),
and 2(b), 1994 Midwestern Well-Water Survey.
Correlations for non-log-transformed nitrate levelsa
Non-log transformed nitrate levels (mg/l NO3-N)
Phase
Mean
SD
Phase 1
Phase 2(a)
Phase 2(b)
13.1
12.0
12.0
22.2
21.2
21.3
Max
266
190
202
Min
Median
Mode
0
0
0
7.1
4.2
4.5
0
0
0
Log transformed nitrate levels (mg/l NO3-N)
Mean
0.76
0.71
0.71
SD
0.61
0.60
0.60
Max
2.43
2.28
2.31
Phase 2(a)
Phase 2(b)
1.0
0.74
1.0
0.73
0.95
1.0
Correlations for log transformed nitrate levelsa
Phase 1
Phase 2(a)
a
Phase
Phase 1
Phase 2(a)
Phase 2(b)
Phase 1
Phase 2(a)
Phase 2(b)
Phase 1
Min
0
0
0
Median
0.91
0.72
0.74
Mode
0
0
0
a
The number one was added to the nitrate levels before log transformation
(base 10) to include those samples with a nondetectable nitrate level.
Journal of Exposure Science and Environmental Epidemiology (2008) 18(2)
Phase 1
Phase 2(a)
Phase 2(b)
1.0
0.88
1.0
Phase 2(b)
0.87
0.92
1.0
All correlations significant (P ¼ 0.0001).
a
One was added to the nitrate levels before log transformation (base 10) to
include those samples with a nitrate level of zero.
131
Nitrate stability in groundwater
Zeitz et al.
Table 3. Distribution of nitrate measurements (mg/l NO3-N) in well water from 853 drilled wells by water-nitrate category in Phase 2(a) as a
percentage of Phase 1 measurements, 1994 Midwestern Well- Water Survey.
Phase 2(a) Number of wells and (%)
Phase 1
Background (r2)
Low (42 to o10)
High (Z10 to o20)
Very high (Z20)
Background (r2)
318
19
17
9
(92.2)b
(16.7)
(7.5)
(5.4)
Low (42 to o10)
21
85
55
13
High (Z10 to o20)
(6.1)
(74.6)b
(24.3)
(7.7)
4
9
126
32
(1.2)
(7.9)
(55.8)b
(19.1)
Very high (Z20)
2
1
28
114
(0.6)
(0.9)
(12.4)
(67.9)b
Totala
345
114
226
168
(100.0)
(100.0)
(100.0)
(100.1)
Mean change (SD)
0.44
+0.06
0.5
+7.23
(2.52)
(3.84)
(15.76)
(28.68)
a
Percent may not add up to 100.0 due to rounding.
Percent of wells in Phase 1 whose nitrate category remained unchanged in Phase 2(a). Phases 1 and 2(a) well water samples taken 1 year apart.
b
diameter (Po0.0001) was apparent in the mixed model.
However, well-to-well variation remained substantial
(s2 ¼ 0.30) and residual error small (s2 ¼ 0.04), even after
accounting for these factors.
Discussion
In this study of 853-drilled private water wells in nine midwestern states, we found that intra-well nitrate levels were not
statistically significantly different over a 17-month period.
Most of the variance in the nitrate measurements in the well
water was due to well-to-well variation. Variability owing to
short-term changes, sample collection and laboratory analysis were found to be small because nitrate levels in samples
taken from the same well 2 weeks apart were extremely
similar. The residual error for intra-well measurements is
consistent with the precision in the laboratory/analytical
method used to measure nitrate further indicating a small
degree of short-term variability (American Public Health
Association, 1992).
The information included here about variability and
stability of nitrate levels is important for design, analysis,
and interpretation of epidemiologic studies intended to
address possible health effects of nitrates. Design and analysis
of such studies depend on the variability of the true exposure.
Devine and Smith (1998), for example, have shown that the
sample size is inversely proportional to the variance of
the exposure . In other words, if other factors (average
disease risk in the population, and the dose-response) remain
constant, a smaller sample size will be required if the
population variance of the exposure is larger.
Interpretation of epidemiologic studies also depends on
variability of the exposure, particularly as it relates to
measurement error. For example, the magnitude of measurement error relative to overall variability is a key determinant
of bias owing to measurement error in logistic regression.
With a classical error structure, the regression coefficient
estimated in the presence of measurement error is biased
towards 0 by a factor that depends on the ratio s12/(s12 þ s22),
132
where s12 is the variance of the exposure and s22 is the
variance due to error. In this study, we emphasize that we do
not have true repeat measurements so that we do not have a
true estimate of s22. However, our residual error estimate is
likely a conservative overestimate of the variance owing to
measurement error, since it should include components
owing to changes in nitrate levels over time, variability
between wells in the pattern of change over time, as well as
measurement error. Nevertheless, our results are somewhat
reassuring for the design of epidemiologic studies since our
overestimate of measurement error is small relative to the
overall variability of nitrate levels between wells. The
variability from well-to-well may reflect differences in land
and fertilizer use near the well head, individual characteristics
of the well such as depth and aquifer geology, and perhaps
maintenance of the well.
This study is unique because it evaluated the change in
nitrate levels in the representative wells across a large and
diverse geographic area. In addition, the same well was
measured over time and we used a statistical approach that
evaluated changes within and between wells. Previous studies
were conducted with fewer wells, in smaller areas, and
compared aggregate results (McDonald and Splinter, 1982;
Hallberg et al., 1984; Benes et al., 1989).
Our study observed that nitrate levels were stable during
the study period and that shallower wells were more likely to
have higher nitrate levels. A study in Iowa examined longterm trends in nitrate concentrations in water supplies
(McDonald and Splinter, 1982). In that study, the average
nitrate concentrations in 4597 samples from municipal wells
of all depths grouped together were stable over a 30-year
period. However, the authors concluded that nitrate
concentrations in well water were dependent on time, well
depth, climate, and surface runoff (McDonald and Splinter,
1982). In a second study in Iowa, nitrate levels in 24 of 40
wells increased an average of 14 mg/l from 1975 to 1983
(Hallberg et al., 1984). The authors concluded that the
increase in nitrates in unprotected shallow groundwater
supplies was related to increased application of chemical
fertilizers. Our study could not assess the impact of fertilizer
Journal of Exposure Science and Environmental Epidemiology (2008) 18(2)
Nitrate stability in groundwater
application on nitrate levels because only qualitative
information (such as commercial fertilizer ever used) was
obtained and information on quantity and change over time
was not collected.
About half of the wells were o100 ft deep and more that
one-third were older than 25 years and a fifth had a diameter
greater than 6 inches. These well features were associated
with elevated nitrate levels in our water samples, a finding
which is consistent with findings of other studies that have
shown that well depth, diameter, age, and construction type
are important indicators of well water contamination
(Johnson and Kross, 1990; Kross et al., 1993; US EPA,
1994).
Although most nitrate levels remained stable (76% overall
agreement between water nitrate categories in Phases 1 and
2(a); Table 3), some nitrate levels may have decreased by
about 5%. This small decline could have been owing to
regression to the mean, a statistical phenomenon, or it could
represent an actual gradual decline in nitrate levels. It is
possible that in the absence of continued sources of nitrate
contamination, nitrate levels may naturally decay, and
groundwater may be diluted with uncontaminated water.
However, further work may be warranted to determine
which of these factors explain the slight decline.
The limitations of this study include the use of selfreported data on well characteristics, land use, and fertilizer
use. However, there was good agreement of reported well
type, well depth, and well diameter between Phases 1 and
2(a).
In this study area, nitrate levels appear to be stable over a
period of 17 months. This stability, a change of o0.5 mg/l,
shows that a nitrate level may be representative of the nitrate
concentration in well water for at least 17 months. This
information is important in assessing exposure to nitrates and
useful for conducting epidemiologic studies on the acute
effects of elevated nitrate levels on adverse health outcomes.
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