The Relationship between Water Flow Rate and Nitrate Concentration in

Southwest Ohio Freshwater

Bryce Linkous
Cody Loveless; Jennifer Lindahl
Will Joesten
Professor Jessa
CHM 145 B
23 April 2016

Abstract:
Based on the volume and flow of a body of water, the higher volume of water, related to a larger
watershed, will show higher concentrations of nitrate versus smaller, slower bodies of water.
The nitrate concentrations of validation samples, NA and NB, calculated by an ion-selective
electrode, were found to be 4.68 and 8.46 mg/L, respectively. The nitrate concentration of the
Great Miami River, Four Mile Creek, and a private pond in Monroe, Ohio were found to be 9.17,
11.8, and 5.16 mg/L, respectively. The larger body of water, the Great Miami River, was not
statistically different from that of the small, private pond; the moderately sized Four Mile Creek
returned the highest concentration of nitrate. Hence, the data observed rejects the original
hypothesis.

Introduction:
According to the Ohio Legislative Service Commission, the Ohio land mass is comprised of 52%
agricultural land.7 With so much of the state covered in crops, farmers go to great lengths to
produce the most bountiful harvest in a climate that can change from wet and cold to warm and
dry in under a month. Such completion has driven farmers in recent decades to widespread
fertilization of their crops. Once such common fertilizer is nitrogen, commonly found in its
nitrate form, NO3-. This commercial fertilizer is the third most common fertilizer used in the
United States behind only potash and phosphate.2 In Ohio, the EPA reports over 300,000,000 kg
of nitrogen were purchased in 2011.1
Nitrogen, though it occurs naturally, can be detrimental to the environment in high
concentrations as it is a potential health threat to humans, and especially infants; furthermore,
nitrogen is a major cause of algae blooms in lakes and rivers.5 These blooms deprive the water
of oxygen as algae dies and decomposes, in turn killing fish and other aquatic organisms. Nitrate
tests, along with tests for E. coli and total Coliform bacteria, are required by the state of Ohio

before water may be deemed suitable for drinking; nitrate concentration must be under 10 ppm,
or 10 mg/L, to be suitable for drinking.8
Ions may be detected in aqueous solutions using an ion-selective electrode, or ISE, specifically
turned to the charge of the ion. The ISE is capable of detecting NO3-, and by measuring a
specific voltage carried by the ions through the water, this may be used to measure the
concentration of nitrate.
Within southwest Ohio are several key water sources, important to local wildlife, recreation, and
commerce. Because southwest Ohio is one of the most populated regions of the state, observing,
and protecting, water in these areas is especially crucial. In this region, as within any local area,
several types of freshwater bodies exist. These range from stagnant and stationary water in lakes
and ponds to fast flowing water of some streams and rivers. As water flow rate correlates to the
size of watershed and change in elevation, some connection must exist between the flow rate and
the level of nitrate present in the water. Thus, the amount of nitrate present in a surface water
source is dependent upon the volume of water flow into that source; therefore, the Great Miami
River, which has the highest volume of water flow, will have the greatest amount of nitrate
present, while the private pond, which has the lowest volume of water flow, will have the least
amount of nitrate present.
The watershed of the Great Miami River includes a large portion of land in southwest Ohio, with
748 square miles draining into the river. This encompasses land from eight counties: Miami,
Hardin, Champaign, Darke, Auglaize, Mercer, Shelby, and Logan. Of the 748 square miles, 79%
is farmland potentially fertilized with nitrates.4 The Ohio EPA reports agriculture as the second
largest detriment to the health of this river. Also, because the Great Miami River flows into the
Ohio River, greater implications of high nitrate levels follow, as the Ohio flows into the
Mississippi and finally into the Gulf of Mexico.
Four Mile Creek, stretching through two southwest Ohio counties, Preble and Butler, receives
water from 322 square miles that is also largely cropland and pasture, though some surrounding
municipalities are responsible for industrial pollutants. A small portion of this rivers watershed
falls within the Indiana border.3 As this river is home to a plethora of fish species and
macroinvertebrates (63 and 344, respectively), high nitrate concentration could pose threats to
the biodiversity of the ecosystem.6
Private water systems are common throughout southwest Ohio as well, including private ponds,
streams, springs, and wells. In order to analyze small, stagnant bodies of water, a private pond in
Monroe, Ohio was used to observe nitrate levels in water in an isolated setting. The pond was
made artificially, and is not regularly treated with chemicals. The watershed of this pond has not
been officially recorded, though little to no water flows into the pond from the surrounding
environment outside of the acreage of the private property. It is known to have a small water
flow/input as the pond was man-made.

Methods:
Note: All glassware and storage/collection bottles were cleaned according to the following
procedure in each portion of the experiment. The glassware was first rinsed with detergent
before rinsing 3 times with warm tap water. The glassware was then rinsed two times with 20
mL of 1% HCl. Each piece of glassware was then rinsed three more times using 30-50 mL
deionized water. Glassware was finally dried with paper towels before continuing.

Preparation of the Stock Solution

One 1 L volumetric flask, 1 L storage bottle, and 100 mL graduated cylinder were first obtained.
Using 0.722 g KNO3 and deionized water, the 100 mg/L stock solution was made into the
volumetric flask. Once the KNO3 had been measured out and poured into the flask, 12 mL of 3.0
M H2SO4 was added to the flask to acidify the solution. Deionized water then was used to dilute
to 1 L. The solution was mixed well within the flask, then in two 25 mL portions, the plastic
storage bottle was rinsed with the stock solution, each time emptying the rinsed solution into the
sink. Finally, the remaining stock solution was transferred from the flask to the bottle entirely.
Preparation of the Standard Solutions
First, standard solutions were prepared using the 100 mg/L stock solution. Five 100 mL
volumetric flasks were obtained and labeled with the appropriate concentration as follows: 1
mg/L, 2 mg/L, 4 mg/L, 6 mg/L, and 10 mg/L. Roughly 100 mL of stock solution was transferred
into a 150 mL beaker. From this beaker, 1 mL, 2 mL, 4 mL, 6 mL, and 10 mL were added to the
flasks, respectively, using a 10 mL Mohr pipet. Each flask was diluted to 100 mL using
deionized water. Into one 100 mL volumetric flask, a 2.0 M solution of (NH4)2SO4 (ammonium
sulfate) was prepared as the ionic strength adjusting (ISA) solution. 26.428 g (NH4)2SO4 was
measured first, then emptied into the flask. The flask was then diluted to the 100 mL mark, and
poured into a 150 mL Erlenmeyer flask.
Testing the Unknown Validation Samples
Validation samples NA and NB were collected into 400 mL beakers. Approximately 350 mL of
each was collected. A 100 mL graduated cylinder was used to measure 100 mL of NA into three
Erlenmeyer flasks and 100 mL of NB into three separate Erlenmeyer flasks. Flasks were labeled
NA and NB appropriately. 2.0 mL of ISA solution was added to all six validation flasks
using a Mohr pipet, as well as the five standard solutions. All flasks were covered and inverted
to mix. 40 mL of each solution was poured into large labeled test tubes.
The LabQuest device was connected and configured with the ion-selective electrode (ISE) was
inserted into the lowest concentration standard solution first. After the reading stabilized (after
about 1 minute), the measurement in mV was recorded. Reading were taken in order of
increasing concentration, then NA samples were measured, then NB. After each sample the ISE
was rinsed with deionized water and blotted dry.

From the standard solution readings a calibration curve was assembled, electrode potential in mV
versus log[NO3-]. Measurements for the validation samples were interpolated based on the
formula of the linear regression and converted to concentration.
Testing the Environmental Samples
Around 250 mL of water was collected into uniform sample bottles from the following sources:
Great Miami River, St. Rt. 73 Trenton access point; Four Mile Creek, upstream from the St. Rt.
73 overpass; and a private pond located in Monroe, Ohio. Samples were collected within 48
hours prior to the experiment being conducted.
Using the same process described above under Preparation of the Standard Solutions, five
standard solutions of equivalent concentrations were prepared to test against the environmental
samples collected. 2.0 mL of ISA was added to each volumetric flask.
In order to filtrate the environmental samples of solid contaminants, a ring stand was configured
with a large plastic funnel and fluted filter paper. Each of the three 250 mL environmental
samples was gravity-filtrated into 400 mL beakers, using a new filter paper for each of the
samples. Approximately 5 mL of ISA (2 mL per 100 mL of sample) was added and stirred into
the beakers using a glass stir rod. 40 mL of each environmental sample was added to three large,
labeled test tubes, for a total of 9 test tubes. 40 mL of the newly prepared standard solutions
were also emptied into large test tubes.
Measurements of the fourteen solutions were taken in similar fashion to the validation samples:
standard solutions first, and from lowest to highest concentration. A second calibration curve
was assembled, electrode potential (mV) versus log[NO3-], and environmental samples
interpolated using the derived regression and concentrations calculated.
Calculations
For all NA and NB samples and environmental, the observed voltage was used to interpolate the
data using each samples respective calibration curve. The electrode potential, in mV, was set
equal to the equation below, then solved for the nitrate concentration [NO3-] in terms of mg/L N
by using the antilog (10(mV-b)/m):
Electrode potential = m log[NO3-] + b
where m = slope of the line; [NO3-] = nitrate concentration (mg/L); b = y-intercept
Dilutions for the making the standard solutions from the stock solution was calculated according
to the following equation:
M1V1 = M2V2
The data was analyzed using the below formulas for mean and standard deviation:
xm = (xi)/n
where xi = all terms in a data set; n = number of terms in the data set

sx = ((d2)/(n-1))
where d = | xm - xi |; n = number of terms in the data set
Statistical analysis was performed on the validation samples (NA and NB) using the following ttest and %RSD:
| x m | > t * sm
%RSD = (sx/xm) * 100
Statistical analysis was also performed on the environmental samples, three total, one between
each using the following t-test. A Q-test was used to determine the inclusion of distant data
points:
| xm1 xm2 | > t * (sm12 + sm22)1/2
Q = | suspected value closest neighbor value| / range

Results:

Figure 1: calibration curve for validation samples, based on measured voltages of standard
solutions, with linear fit

Figure 1 displays the calibration curve for the first set of trials, measuring the values of NA and
NB. The plot shows the electrode potential measured by the ISE in relation to the logarithm of
nitrate concentration, giving a straight, negative slope. Smaller voltages relate to higher nitrate
concentrations.
Table 1 shows the values interpolated from the regression in Figure 1. Values of NA and NB are
reported below, and compared against the known concentrations of NA and NB.

Table 1: measured concentrations of NA and NB unknown validation samples; given with

average concentration and standard deviation
NA (mg/L)

NB (mg/L)

4.77

8.27

4.98

8.48

4.30

8.63

Mean

4.68

8.46

Standard Deviation

0.348

0.182

The reported concentrations values were 4.72 mg/L, std. deviation 0.48 mg/L, for NA and 7.77
mg/L, std. deviation 0.74 mg/L, for NB. t-tests performed at the 95% confidence interval, t =
4.30, gave the following inequalities:
NA: 0.040 > 1.50
NB: 0.690 > 0.783
The %RSD for NA and NB are given below:
NA: 7.44%
NB: 2.15%

Figure 2: calibration curve for environmental samples, based on measured voltages of standard
solutions, with linear fit

Figure 2 gives the calibration curve used to interpolate the logarithm of the nitrate concentration
of the environmental samples. Based on an equivalent curve of that of the validation sample
curve, therefore the curves represent very similar regressions.
Table 2 displays the values obtained from interpolated the function given by the calibration
curve. Each triplicate measurement and the mean and standard deviation of each water
collection site are given

Table 2: measured concentrations of environmental samples, given with average concentration

and standard deviation
Great Miami River
(mg/L)

Four Mile Creek

(mg/L)

Private Pond
(mg/L)

10.1

11.0

8.41

9.73

12.4

3.37

9.30

12.1

3.69

Mean

9.70

11.8

5.16

Standard Deviation

0.380

0.726

2.82

The Q-test performed on the first data point of the private pond gave the following inequality for
n = 3, Q = 0.97:
0.937 < 0.97

t-tests performed between the environmental concentrations are given by the inequalities below:
GMR vs. FMC: 2.10 > 1.32
GMR vs. PP: 4.54 > 4.57
FMC vs. PP: 6.64 > 4.53

Discussion:
Based on the t-tests performed on the validation samples, the measurements of NA and NB were
not statistically different from the known values. For NA, the inequality 0.040 > 1.50 was
formulated using t = 4.30 at the 95% confidence interval. Because the inequality does not hold
true, the measured concentration of NA is not statistically different from the known
concentration. For NB, the inequality 0.690 > 0.783 was formulated using t = 4.30 at the 95%
confidence interval. Because this inequality also does not hold true, the measured concentration
of NB is not statistically different from the known concentration. The %RSD of NA and NB
suggest the data is lies close to the mean, since the standard deviation is 7.44% of the mean for
NA, and only 2.15% of the mean for NB. This suggests the data is precise. The validation
samples measured were not statistically different from the known values, demonstrating the
accuracy of the measurements made for the environmental samples should follow with the same
level of accuracy, as the same instrumentation was used for both procedures. The high precision
of the data for NA and NB, based on the %RSD calculated, demonstrate the measurements made

for the environmental samples should also provide precise measurements. The environmental
concentration data can be considered reliable based on these validation tests.
t-tests performed on the data gave the statistical relationships between the three sets of data. The
nitrate concentration of Four Mile Creek was found to be statistically different from the Great
Miami River (2.10 > 1.315 is true). The concentration of Four Mile Creek was also statistically
different from the private pond (6.64 > 4.53 is true). The Great Miami River was not found to be
statistically different from the private pond (4.54 > 4.57 is not true).
Four Mile Creek, with an average nitrate concentration of 11.8 mg/L N, had the greatest
concentration of nitrate in the water. The next highest was the Great Miami River with 9.70
mg/L N, followed by the private pond with 5.16 mg/L N. The Great Miami River and private
pond nitrate concentrations are however not statistically different.
The hypothesis is rejected that the greatest volume of body of water, in proportion to highest
flow rate, will produce the water with the highest nitrate concentration. The discrepancy
between hypothesis and results may be attributed to several circumstances. As these
measurements were made prior to the planting and fertilization of crops by local farms, nitrogen
fertilizers have not yet been applied, thus have not yet leached into water sources; nitrate
concentrations therefore may be lower now than in another time of the year, such as late summer.
However, this observation, though true, will cause all concentrations to lower simultaneously,
and does not fully explain the discrepancy in data. The assumption was made that because the
Great Miami was included in the largest watershed area by land, the most farm acreage would
fall within its watershed. However, it is more likely the proximity to farmland is the contributing
factor to nitrogen pollution, as a smaller percentage of the Great Miami River runs along
farmland compared against Four Mile Creek. Another contributing factor that must be
considered is dilution. A combination of direct flow through and proximity to farmland and
volume of water follow different relationships. Proximity to farmland will cause greater nitrate
pollution, but in proportion to the volume of water, per liter, there are less milligrams of
nitrogen. In the case of the private pond, the distance from farmland and small size of the
watershed makes the small volume of water irrelevant to the consideration of nitrate
concentration per liter.
The hypothesis may be rewritten to account for such new observations and findings: because of
the lower volume of water proportional to the influx of agricultural nitrate pollutants, Four Mile
Creek, a moderately-flowing creek, will show the highest concentration of nitrate per liter.

Conclusion:
Living in southwest Ohio my whole life and raised in a small farming town, I am very familiar
with the business of agriculture. I have worked with and around farmers my entire life, and even
work as a large-scale landscaping company myself; yet it was not until this experiment that I
realized the impact of farming and greenscaping on the local water ecosystems. Fertilizers are
used much too freely, from fertilizing corn and soybean crops to fertilizing private lawns. I

believe it is important for the population of Ohio, and the nation, to understand that what they
apply on their lawn does not stay on their lawn. Nitrates in the aquatic environment can have
huge impacts on wildlife. In causing destructive algal blooms, nitrates can quickly ruin entire
sections of rivers. The complexity of pollution is difficult to understand, however the impact
must not be forgotten. Algae blooms kill off other aquatic plants by restricting light from
reaching their leaves, in the short term. Long term impacts can include fish kills and loss of
macroinvertebrate population. When the algae overpopulate and begin to die, bacteria consume
oxygen to decompose the algae, draining the water of dissolved oxygen. Water is not easily
reoxygenated, especially if plants die too. Farmers, particularly those involved in large-scale
commercial farming, should be kept informed on the dangers of fertilizing on the environment.

References:
1

Commercial Fertilizer Purchased. United States Environmental Protection Agency, Office of

Water: Washington, DC, 2016. https://www.epa.gov/nutrient-policy-data/commercial
fertilizer-purchased (accessed Apr. 23, 2016).

Environmental Protection Agency. [Online] Agricultural Fertilizer 2014

https://cfpub.epa.gov/roe/indicator.cfm?i=55 (accessed Apr. 23, 2016).

Fourmile Creek Watershed; Ohio Environmental Protection Agency, Division of Surface Water:
Columbus, OH, 2012.
http://www.epa.ohio.gov/dsw/tmdl/GreatMiamiRiver.aspx#116712986-fourmile-creek
(accessed Apr. 10, 2016).

Great Miami River Watershed; Ohio Environmental Protection Agency, Division of Surface
Water: Columbus, OH, 2012. http://www.epa.ohio.gov/dsw/tmdl/GreatMiamiRiver.aspx
(accessed Apr. 10, 2016).

Jessa, Y.; Novak, M.; Danielson, N. General Chemistry Laboratory Manual; Hayden-McNeil,
Plymouth, 2016; pp. 83-95.

Ohio Environmental Protection Agency, Division of Surface Water. [Online] Biological and
Water Quality Study of Fourmile Creek, Indian Creek, and Select Tributaries 2005 20-99.
http://www.epa.ohio.gov/portals/35/documents/FourmileIndianTSD2008.pdf (accessed
Apr. 10, 2016).

Ohio Legislative Service Commission. [Online] 2004, Dec, 15-17 ff.

http://www.lsc.ohio.gov/fiscal/ohiofacts/dec2004/ohiosnaturalresourcesandenergy.pdf =
(accessed Apr. 23, 2016).

Water Quality. Ohio Department of Health. http://www.odh.ohio.gov/odhprograms/eh/water/

PrivateWaterSystems/quality.aspx (accessed April 10, 2016).