Effects of Suburban Development Runoff on pH, Nitrate and Dissolved Oxygen Levels in

Barton Creek After a Precipitation Event

Julia Stevens
working under Graduate Student Sara Sopczynski and alongside Lilli Johnson
Thursday, May 9, 2013
BIOL 4342: Population Biology and Ecology
Dr. William J. Quinn

Stevens 2
Abstract
As an increase of urbanization in Austin, there is a correlating increase in runoff and pollution
in the surrounding water systems. Four sites along Barton Creek were sampled to compare
dissolved oxygen concentration, pH, and nitrate concentration, one being undisturbed and the
other three in a neighborhood, or disturbed area. On the night of April 2, a storm occurred
resulting in 3.02 inches of precipitation. This shifted the study to focus on nitrate levels before
and after the storm as a result of runoff. Nitrate concentration had a significant increase after
the storm, with significantly higher levels at all three disturbed sites compared to the
undisturbed. There was one significant finding in the average of dissolved oxygen levels,
where the first disturbed site (site two) had significantly lower levels than the undisturbed
site. There was no significant difference in pH throughout the sites. After a rain event, it was
found that high levels of run off in an urbanized area resulted in an increase in nitrate in the
water.
Introduction
The Barton Creek watershed flows directly into the Colorado River, along with other
watersheds in the Austin area. With the increased urbanization of Austin, the nearby streams
collect runoff of nutrient pollutants at an increased rate (Bulgariu et al. 2012). Runoff of
fertilizers, pesticides, and human and animal wastes contribute to the accumulation of nitrate
in surface water (Mahler et al. 2011). Weather also impacts the nutrient levels and runoff.
During wet periods, levels of nitrogen are higher in creeks and rivers due to increased water
runoff (Kaushal et al. 2008).
In studying three sites in a residential community, the use of pesticides becomes relevant. Set
by the World Health Organization, a maximum of 0.1 g/L of nitrate is allowed in
pesticides(Aslan et al. 2005). Pesticides are used in weed and pest prevention. In a residential
community pesticides are used on a per home basis, but the accumulation of these chemicals
add to the runoff into the creek. The World Health Organization also capped the presence of
nitrate in water at 50 mg/L (Aslan et al. 2005).
Incorporating a wastewater treatment plant in the study allows for analysis of water content
surrounding the treatment plant. Wastewater is defined as low-quality water, such as storm

Stevens 3
water, runoff, and secondary-treated water (Bachand et al. 2000). Nitrogen and phosphorus
are released as discharge near the wastewater treatment plant, resulting in eutrophication
(Hong Guo et al. 2010). The wastewater is cycled back to use as irrigation for the Lost Creek
Country Club. A study comparing nitrate levels of romaine lettuce with drinking water and
wastewater resulted in no significant difference in nitrate content between the two (Castro et
al. 2009). However, a local study found that increased nitrate levels in Barton Creek are more
likely to be from land application of treated wastewater than from other sources (Mahler et al.
2011).
To ensure a thorough study of water composition, dissolved oxygen and pH were studied, as
well as nitrate levels. Dissolved oxygen levels, to support an aquatic community, should be
between five to six parts per million (Hiawassee River Watershed Coalition 2007). For an
aquatic organism to obtain nutrients, the water pH level needs to be stable. While the pH
levels might fluctuate, the creek or water system has the ability to regulate back to a stable pH
(Streams 1991). It has been found that low nitrate levels correlate with a higher pH (Rosso et
al. 2012).
An undisturbed creek site was sampled and compared to three disturbed sites, looking at
nitrate concentration, dissolved oxygen levels, and the pH of the water. The null hypothesis is
that there would be no difference in nitrate, dissolved oxygen, and pH when comparing the
undisturbed site to each individual disturbed site. The alternative hypothesis is that the
nitrate concentration would be higher and the dissolved oxygen lower at the disturbed sites
compared to the undisturbed sites. If higher nitrate concentration is higher, then a correlating
lower pH would be found at the higher nitrate community.
Methods
Location
Four sites were chosen to collect data. The first site, considered the undisturbed site, is
located near the Austin Nature Conservancy, northwest of St. Michaels Academy. Sites two,
three, and four are all located down stream from site one within the Lost Pines subdivision
and considered disturbed sites. Site two is adjacent to the Lost Creek County Club golf
course. Site three is downstream of site two, in the midst of a neighborhood. A bridge runs

Stevens 4
over the creek between sites two and three. Site four is downstream of site three and the Lost
Creek Wastewater Treatment Plant. At each site, water was collected for testing at three
points. Water samples were collected near each bank and in the middle of the creek. These
points were labeled A, B, and C.
Collection
Each of the designated sampling locations (twelve in all) had a designated clean water bottle
for collection. The samples from these bottles were used for pH testing, done on site, and
nitrate testing performed in the lab. The samples were packed in a cooler with ice packs for
transport back to the lab for nitrate testing. When obtaining the sample, the bottle was held
underwater near the surface with the opening facing upstream. When the bottle was filled it
was taken out of the creek and capped. This process is the dipping method (Decker et al.
2013).
Dissolved Oxygen Testing
Dissolved oxygen was measured using the Hach Water Ecology Kit Model AL-36B. Directions
for a high range (1 to 20 mg/L) dissolved oxygen test were provided in the kits instruction
manual. At each of the three points, at each site, a sample of water was collected in the BOD
bottle, supplied by the HACH kit, by holding the bottle underwater, with the opening facing
upstream, for three minutes. Using nail clippers, the pillow packets for dissolved oxygen
reagent power one and two were opened and the contents were added to the bottle. The
bottle was capped with no air bubbles and vigorously shaken. Once the particulates, or floc,
settled half way down the bottle, the bottle was shaken a second time. The sample turned an
orange/brown, indicating the presence of oxygen. As the floc settled half way, the second time,
the pillow packet of dissolved oxygen reagent powder three was opened and the contents
were added. The bottle was capped again, with no air bubbles, and vigorously shaken, for the
third time. If at any point, when the bottle was capped, air bubbles formed, the sample in the
BOD was discarded and the testing procedures started over with the three-minute water
sample collection.
Upon successful completion of adding the three reagents, a sample of the contents in the
bottle was transferred to a small test tube, provided in the kit. The sample in the tube was

Stevens 5
then poured into a small rectangular glass vile. While swirling the vile, Sodium Thiosulfate
Solution was added drop wise into the glass vile until the orange/brown sample turned clear.
Each drop added represents one mg/L of dissolved oxygen.
pH Testing
The testing was performed at location, as pH can alter over time. The materials for testing
were also included in the Hach Water Ecology Kit Model AL-36B. Two test tubes were each
filled with 5 mL of the sample water from the collection bottles. Six drops of Wide Range 4pH
Indicator Solution was added to one of the test tubes and swirled until a uniform color
appeared throughout the sample. The two test tubes were placed in the color comparator, the
treated sample on the right and untreated sample on the left. The comparator was held up to
the light and the treated sample was compared and matched to a color on the color wheel,
seen through the untreated sample. Each color on the color wheel has a designated pH value.
Nitrate Testing
A standard intermediate nitrate solution was developed to create the standard curve, with
which the samples were plotted to determine nitrate levels. In the lab, a 50ml sample from the
chilled collection bottle was poured into a glass container. One ml of Hydrochloric acid was
added. The spectrophotometer was calibrated using a glass cuvette filled with distilled water,
and the computer was blanked. The distilled water was then discarded. A portion of the
combined 50ml sample plus HCL was poured into the glass cuvette. The spectrophotometer
was run at 220nm and 275nm, returning data that provided the wavelength of nitrate
solution. The returned wavelength between 220 and 275 was then adjusted and entered into
the graph calculated by the intermediate nitrate solution to determine the nitrate levels
present in the creek in g /ml.
Results
Dissolved Oxygen Levels
A one-tailed T-test was used to analyze the data collected for dissolved oxygen levels at a 95%
confidence level. Site one, the undisturbed site, was compared to disturbed sites two, three,
and four. The accepted data at each site was averaged. There was a significant difference
between sites one and two, with site two lower than site one. The p-value between sites one

Stevens 6
and two was 0.00003. There was no significant difference between sites one and three, and
sites one and four. See Figure 1 below. The average dissolved oxygen levels at each site
(average of A,B, and C) for each day were also graphed to depict the fluctuations before and
after the storm. Site one had significantly higher dissolved oxygen levels after the storm than
immediately before (April 2). See Figure 2 on the next page.
Figure 1
Dissolved Oxygen Average At Each Site

Dissolved Oxygen Average

12
10
8
6
Dissolved Oxygen Average
4
2
0
Site 1

Site 2

Site 3

Site 4

Sites

Figure1. The average dissolved oxygen levels through the duration of the study depict a
significant difference between sites one and two, with site two having lower dissolved oxygen
level than site one.

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Figure 2

Dissolved Oxygen
Dissolved Oxygen Levels
(ppm)

Site 1

Site 2

Site 3

Site 4

14
12
10
8
6
4
2
0

Date (2013)

Figure 2. Dissolved oxygen levels throughout the study display a significant difference in
dissolved oxygen before and after the storm for site one, with the levels higher after the
storm. Also, accepted sample days can be seen.
pH Levels
A two-tailed T-test, to account for acidity and basicity, was used to analyze the data collected
for pH levels at a 95% confidence level. The pH averages for sites one through four were
between 7.92 and 8.04. Upon comparing site one to sites two, three, and four, there was no
significant difference between any of the comparisons. See Figure 3 on the next page.

Stevens 8
Figure 3
pH Average At Each Site
9
8

pH Average

7
6
5

pH Average

4
3
2
1
0
Site 1

Site 2

Site 3

Site 4

Sites

Figure 3. The pH values ranged from 7.92 to 8.04. The small standard error represents little
fluctuations in the data.
Nitrate Levels
A one-tailed T-test was used to analyze the accepted data collected for nitrate levels at a 95%
confidence level. Samples collected before the storm were analyzed separately from samples
collected after the storm the night of April 2. First, the average nitrate levels before the storm
were significantly different between site one and two, and site one and four, with sites two
and four having significantly lower nitrate levels than site one. After the storm, sites two,
three, and four all had significantly higher nitrate levels than site one. See Figure 4 on the next
page. The average nitrate levels at each site (average of A, B, and C) for each day was also
graphed to depict length of time after rainfall and nitrate levels. Each disturbed site had
samples collected two times after the storm. Site one was sampled one time after the storm.
Five days after the storm, all three disturbed sites had significantly higher nitrate values than
the data collected prior to the storm. Comparing the average samples after the storm, there is
no significant difference between site one and sites two, three, and four. See Figure 5 on the
next page.

Stevens 9
Figure 4
Nitrate Averages Before and After Storm Event
(Separate)

Amount of Nitrates (g NO3 -N)

28
24
20
16

Before
After

12
8
4
0
Site 1

Site 2

Site 3

Site 4

Sites

Figure 4. The nitrate averages were separated into two categories, before the storm event and
after. Before the storm, sites two and four had significantly lower nitrate levels than site one.
After the storm, all of the disturbed sites, site two, three, and four, had significantly higher
nitrate levels than site one.
Figure 5

Measured Nitrate Levels

Site 1

Site 2

Site 3

Site 4

14
12
Nitrate Levels
(g /mL)

10
8
6
4
2
0
-2
-4
Date (2013)

Figure 5. The measured nitrate levels are depicted throughout the study. The significant rise
in nitrate levels after April 2 and 3 can be seen, as well as the accepted sample days.

Stevens 10
Discussion
In this study, samples were collected between March 23, 2013 and April 7, 2013, however
there were large gaps in collection time. During the study, precipitation was tracked. On
March 23, after collection, there was 0.02 inches of rain. On March 30 and 31, there were trace
amounts of rain. Those were not collection days. On the night of April 2, into the morning of
April 3, there was a total of 3.02 inches of rain. This significant amount of rainfall correlates
with the spike in nitrate levels after the rain. This instigated the before and after rainfall
analysis for nitrate levels. pH and dissolved oxygen did not result in any significant difference
after the rainfall.
The average natural nitrate levels for Barton Creek watershed is found to be at 0.25 mg/L, or
250 g/mL (Mahler et al. 2011). The data in this study measured much lower levels. Site three
reached the highest nitrate content after the storm at 0.00903 mg/L. These extremely low
nitrate levels might be related to the drought encompassing the central Texas region.
The increase in nitrate levels after the storm event brings to light the effects of runoff into the
creek. The runoff from land using fertilizer is linked to higher levels of nitrates within the
watershed (Rosso et al. 2012). Also, it has been found that increased nitrate levels in Barton
Creek are more likely to be from land application of treated wastewater than from other
sources (Mahler et al. 2011). The water from the Lost Creek Wastewater Treatment Plant is
cycled to the Lost Creek Country Club and used for irrigation. The Lost Creek Country Club is
aware of the issue of runoff and uses fertilizer that is highly soluble as well as a treatment
schedule to aim for minimal runoff. There is also a buffer zone between the golf course and
the creek of undisturbed vegetation, this can assist in a decline in nitrate levels that enter the
creek due to runoff (Winter et al. 2005).
With the unexpected storm event, the hypothesis for nitrate levels was divided between
before the storm and after the storm. Before the storm, the study failed to reject the null
hypothesis, as there was no significant difference in the levels of nitrates of site one compared
to sites two, three, and four. After the rain event, the study rejected the null hypothesis, as
sights two, three, and four all had significantly higher levels of nitrate than site one.

Stevens 11
Upon analyzing the dissolved oxygen concentrations in all, for site two, the study supports the
alternative hypothesis that a disturbed site would have lower dissolved oxygen levels than an
undisturbed site. The alternative hypothesis was not supported at sites three or four. The
study also fails to reject the null hypothesis for pH, where the disturbed sites would not have a
pH differing from the undisturbed site.
There are many aspects of this study that could be improved upon. Most importantly, for
future replications, a sample collection schedule should be compiled to assure that each site is
sampled consistently and evenly through the study. There were limitations in determining pH,
as the electronic probe did not work, so the comparator and color wheel were used, which are
less precise. In determining dissolved oxygen levels, completion of the process was dependent
on the sample solution turning clear, there was some human error as to what was clear. It
was learned through the study that glass pipettes and cuvettes should be used, rather than
plastic, as the chemicals used dissolved the plastic, altering the results when preparing the
intermediate nitrate solution and using the spectrophotometer.
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