Effects of Sewage Spillage on the Health of the Wekiva River main content.

Effects of Sewage Spillage on the Health of the Wekiva River

Part of the Young Naturalist Awards Curriculum Collection.

by Vrinda, Grade 9, Florida - 2014 YNA Winner


A view of the Wekiva River, part of an essay on water pollution by sewage
The Wekiva River (upstream)Click to enlarge

As little kids, my brother and I would always jump into the cool and crisp waters of the Wekiva River in Seminole County, Florida. The refreshing waters would slap against our faces as we raced against each other. Those were the beautiful days of my childhood that I will cherish for the rest of my life. However, on August 16, 2013, I was heartbroken to find that my childhood waters had been destroyed. My precious Wekiva River had been polluted by a septic pipe leak. This leak had discharged more than 70,000 gallons of raw sewage and about 1.6 million gallons of wastewater (“Issues”). For days, signs warning people not to swim were put up while the government worked hard to clean up the mess. It took almost $500,000 to patch up the pipe, clean up the river, and conduct water tests.

Government officials say that the rain is enough to clear the sewage out of the river. Still, many residents believe that Wekiva River will never be as clean as it used to be. Like many other residents, I wanted to know if I would still be able to safely play in the river. Even though it had been six months since the leak, I wanted to test the health of the river. Also, I wanted to test if the sewage spill had affected the rest of the river. To do this, I decided to test three different sites: at the spill site, a site half a mile downstream, and a site half a mile upstream.

Photo of Wekiva River downstream from spillage site, from student project.
The Wekiva River downstream of spillage site.Click to enlarge


I hypothesized that the site at the spillage would have the lowest water quality, while the site upstream would have the highest water quality. The site downstream would have the second-highest water quality.


After conducting thorough research, I found six of the best factors to measure the water quality:

Nitrate: Nitrate is vital for the growth of plants, but excessive amounts accelerate aquatic plant growth. This acceleration causes rapid oxygen depletion (“Nitrates and Their Effect”).

Phosphate:Like nitrate, too much phosphate quickens plant growth and depletes oxygen. Then the level of dissolved oxygen becomes too low to support aquatic life (Perlman).

Water Temperature: Water quality is a crucial component of a healthy ecosystem. Aquatic organisms have a narrow range of optimal temperatures. If the temperature varies for a long period of time, these organisms can die (“20010137 (Little Wekiva River).”

pH: pH is a measure of how acidic or basic a substance is. The pH of water can easily be changed by chemical pollution (“Friends of the Shenandoah River”).

Dissolved Oxygen:Dissolved oxygen (DO) is the amount of oxygen available for chemical reactions and consumption by aquatic organisms. Most aquatic organisms cannot live in environments with low DO. Low DO is caused by wastewater treatment plants, urban runoff, and other sources of pollution (“An Introduction to Water Quality Monitoring”).

Fecal Coliform: Fecal coliform live in the digestive tracts of animals and are often indicators of poor water quality. Fecal coliform may be present due to nonpoint sources of pollution.

To analyze all six factors, I decided to create a water quality index of my own. In the book Field Manual for Water Quality Monitoring, 142 scientists were asked to graph the level of water quality (0–100) in correlation to the field measurements for each water quality factor (pH, temperature, dissolved oxygen, etc.). I averaged the curves out and created weights for each factor. The numbers were then added up to give me a water quality index. This water quality index takes consideration of all six factors.

However, I wanted to go one step further with this project. I wanted to see what kinds of bacteria were present in the Wekiva River. To do this, I decided to conduct agar plate testing. I used nutrient agar plates because it creates the optimum environment for the bacteria to proliferate. The plates were streaked to allow the bacteria to spread and be isolated. The plates were incubated overnight to allow for bacterial growth, and then I visually estimated the colony count on the plates. This was a semi-quantitative assay, with bacterial count represented as colony-forming units per milliliter (cfu/ml). Finally, an automated computer analyzer was used to classify the type of bacteria growing on the plates.


A girl standing beside a bubbling river wearing plastic gloves and holding a large long-handled scoop. She is covering the scoop with a clear plastic bag to collect the water.
Vrinda collecting samples.Click to enlarge

To test for the six factors (nitrates, phosphates, dissolved oxygen, temperature, pH, and presence or absence of fecal coliform bacteria), I used LaMotte Testing Kits, as recommended to me by Dr. Pramod Arora. These kits used tablets to change the color of the water samples. The color was compared to a key provided by the company to calculate the amount present in the water. To test the six factors, the following materials were used:

  1. LaMotte Water Quality Test Kits®
  2. Sterile one-liter container with cap
  3. Rubber gloves
River map
Figure 1

To conduct the agar plate testing, I went to the microbiology lab at Osceola Regional Medical Center and used the following materials:

  1. Centrifuge
  2. Incubator
  3. Agar plates
  4. Sterile loop
  5. Automated analyzer
A large long-handled scoop is extended into shallow clear water with a sandy bottom. A tuft of tall thin grass in bottom right corner.
Wekiva River (spillage site).Click to enlarge


I tested three different sites along the Wekiva River, as seen in Figure 1. I tested a site upstream of the leak and called it Site A. I tested the site where the leak occurred, Site B. Last but not least, I tested a site downstream of the leak and called it Site C. Like the St. Johns River, the Wekiva River flows from south to north. To validate results, three samples from each site were taken on three different days, and the results were averaged.

Comparing the color of a sample against a reference card to determine concentration
Figure 2

The procedure to test the chemical variables was as following:

  1. In a sterile container, collect a liter of water from each site along the river.
  2. Using the LaMotte Water Quality Test Kits® and the enclosed instructions, test for variables like nitrates, phosphates, dissolved oxygen, pH, temperature change, and the presence or absence of fecal coliform bacteria.
  3. The water samples will change to a different color. To get results, compare the water color to the color key provided in the test kits as seen in Figure 2.

The procedure for the agar plate testing was as follows:

  1. Centrifuge water samples at 1,759 rotations per minute for five minutes.
  2. Decant and discard the top fluid.
  3. Use sterile premade blood agar and MacKonkey medium agar plates.
  4. Use a sterile calibrated disposable loop to collect and streak the sediment across the plates.
  5. Incubate overnight at 35ºC.
  6. Use observation and computer analysis to classify the amount and type of bacteria.

I used distilled water as a control. The independent variable for this experiment was the distance from the sewage spill. The dependent variables were the water quality and the types of cultured bacteria.


LaMotte Testing Results

Graph 1


The semi-quantitative kits gave an approximate concentration of each of the factors in the river. The first variable I tested was pH level. The results can be seen in Graph 1. The optimal pH for a river is 7.4 (“Friends of the Shenandoah River”). Even though Site B had the highest pH, this fact alone does not indicate poor water quality. As a matter of fact, all three pH levels are suitable for the organisms that live in the river.

Graph 2


The next factor that I tested was the dissolved oxygen (DO) content, as seen in Graph 2. The optimal dissolved oxygen content is anything above 6.6 ppm. Site A was above this level, and Site C was just 0.3 ppm less than 6 ppm (“Understanding Water Quality”). Site B, on the other hand, had a lot less DO than an optimal stream should have. 

Graph 3

comparison of nitrate levels in ppm in water samples above, at, and below the spillage site

The third factor tested for was nitrate. More nitrate is found in water that is more polluted. The Virginia Water Quality Standard is 10 ppm of nitrate. In the results shown below, all of the sites had a 10 ppm or less, so they were fine. However, Site B had the highest nitrate concentration, as seen above in Graph 3. Surprisingly, Site A had a greater nitrate concentration than Site C.

Graph 4



The fourth factor tested was phosphate. As the amount of phosphate increases, DO declines and the water quality declines as well. There is no set optimum value for phosphate yet, but the results in Graph 4 show that Site B had the most phosphate. Site C had the second most, and Site A had the least (Perlman). 

Graph 5

A bar chart showing temperature at three sites and the control, distilled water.

The next factor tested was temperature. For this particular variable, the actual temperature isn’t as significant as the change in temperature (“Chapter 5 (Part A): Habitat Assessment and Physical Parameters”). The greater the temperature change, the greater decline there is in water quality. There was a big temperature change between Site B and the other sites, as seen in Graph 5. There was not much temperature change between Site A and Site C.

Table 1: Presence/ Absence of Fecal Coliform Bacteria

  Presence/Absence Time to Test Positive
Site A (Upstream Absent -
Site B (Spillage Site) Present 28 hours
Site C (Downstream) Present 39 hours
Distilled Water Absent -

 The last factor I tested was the presence or absence of fecal coliform bacteria, as seen in Table 1. If fecal coliform bacteria are present, that is a strong indicator that there is some kind of contamination in the river (Murphy). Since LaMotte Test Kits® provide a semi-quantitative analysis, the exact number of bacteria could not be measured. However, the time it takes for the color of the test to change can be taken into account. The faster it changes from orange to yellow, the more fecal coliform bacteria it has. The sample from Site B changed to light yellow faster than the sample from Site C, as can be seen in Figure 3. Therefore, Site B had the most bacteria, while Site C had the second-most bacteria. Site A remained orange for more than 48 hours (see Figure 4) and did not have any fecal coliform bacteria. 

Figure 3 left, Figure 4 right

Water quality cannot be assessed by looking at only one of these factors. Instead, all these factors should be looked as a whole to get a more accurate understanding of the river’s health. I created a water quality index from 0–100 to measure the health of the river. To calculate this index, I used my data to find the “Q-value,” a numerical value that corresponds to the curves of the graphs created by National Sanitation Foundation scientists (“National Sanitation Foundation Water Quality Index”). The Q-value was multiplied by the weight factors I created. Finally, all the products were added up, and the results are shown below in Table 2.

Table 2: Water Quality

Site Tested Water Quality Index Stream Health
Site A (Upstream 75 Good
Site B (Spillage Site) 55 Medium
Site C (Downstream) 70 Medium-Good


90–100: Excellent

70–90: Good

50–70: Medium

25–50: Bad

0–25: Very Bad


According to this water quality index, Site A (upstream) had the healthiest river water. Site B (sewage leak site) had the poorest water quality and just barely managed to be deemed a “medium” river. Site C (downstream) was the second healthiest river and could be deemed as either “medium” or “good.”

Agar Plate Testing Results

The agar plates showed that the least amount of bacterial growth was found in the sample from Site A (upstream), seen in Figure 5. The most growth was found in the sample from Site B (site of leak), as seen in Figure 6. Finally, the second-most bacterial growth was found in a sample from Site C (downstream), as shown in Figure 7. The results from the computer analysis are shown in Table 3.

Agar Plates
Site A (left), Site B (middle), Site C (right)

Table 3: Computer Analysis

Site Tested Bacteria Colony Count (CFU/mL)
Site A

Aeromonas hydrophila

Raoultella ornithinolytica



Site B

Aeromonas hydrophila

Raultella ornithinolytica

Klebsiella pneumoniae




Site C

Aeromonas hydrophila

Raoultella ornithinolytica

Klebsiella pneumoniae




At all three sites, there was a bacterium called Aeromonas hydrophila present. This bacterium is present in nearly all freshwater environments but can cause cellulitis, myonecrosis, and ecthyma gangrenosum when in contact with an open wound (Martin).

Another bacterium found in all three sites was Raoultella ornithinolytica. This gram-negative aerobic bacterium can cause redness and flushing when in contact with human skin (Solak). However, more damage is done to fish. Raoultella ornithinolytica has the ability to convert histidine to histamine in fish and can cause histamine fish poisoning (Solak).

A final bacterium called Klebsiella pneumoniae was found at Sites B and C. This is a naturally occurring bacteria found in the gut of humans or other animals. This bacterium can cause devastating effects if inhaled from contaminated waters. It can cause inflammation and cell death in the lungs (“Klebsiella pneumoniae”). This infection is generally found in people with a weak immune system. However, this bacterium can cause infections through open wounds even if someone has a healthy immune system. It can cause infections like pneumonia, bloodstream infections, surgical site infections, and meningitis (Umeh). Klebsiella pneumoniae was only found at Site B and Site C. It was found the most at Site B, the site of the leak, and was also found downstream of the leak, at Site C.


After conducting this experiment, my hypothesis was proven correct. The site at the leak had the worst water quality and also contained very harmful bacteria. The site downstream from the leak had the second-worst water quality and contained some harmful bacteria, but not as much as the leakage point. The site upstream had the best water quality and did not contain bacteria that are as harmful as the other two sites.

However, there were some limitations to this project. Though there was tremendous supporting evidence, there was no proof that the septic tank leak caused the contamination. Fertilizers, storm runoff, or other environmental factors could have caused the contamination. Another limitation was that the LaMotte Testing Kits® only gave me a semi-quantitative measure of the factors. More thorough and accurate analysis would have to be done to reproduce and validate my results.

This experiment opened my eyes to how much the septic tank leak contaminated the Wekiva River. Even though the county has spent a lot of time and effort trying to clean up the river, there is still harmful pollution floating through my childhood waters. People with open wounds or weak immune systems should definitely not swim in these contaminated waters. In the near future, I will be writing to the government Lake Watch program about the deteriorating health of the stream. For future projects, I hope to create a scrubber bacteria using the principles of competitive inhibition to eliminate the harmful bacteria residing in the Wekiva River.

Works Cited

“20010137 (Little Wekiva River).” St. Johns River Water Management District. Floridaswater.com, n.d. Web. 15 Jan. 2014.

“An Introduction to Water Quality Monitoring.” U.S. Environmental Protection Agency, n.d., Web. 7 Jan. 2014.

“Chapter 5 (Part A): Habitat Assessment And Physicochemical Parameters.” U.S. Environmental Protection Agency, 6 March 2012. Web. 15 Jan. 2014.

Friends of the Shenandoah River, n.d. Web. 15 Jan. 2014.

“Issues.” St. Johns Riverkeeper, n.d. Web. 13 Jan. 2014.

Klebsiella pneumoniae in Healthcare Settings.” Centers for Disease Control and Prevention, 27 Aug. 2012. Web. 26 Feb. 2014.

Martin, Josh. “Aeromonas hydrophila.” Missouri University of Science and Technology, n.d. Web. 27 Feb. 2014.

Mitchell, Mark K. and William Stapp. Field Manual for Water Quality Monitoring: An Environmental Education Program for Schools. Dubuque, Iowa: Kendall Hunt Publishing, 2013.

Murphy, Sheila. “General Information on Fecal Coliform.” City of Boulder/USGS Water Quality Monitoring, 23 April 2007. Web. 7 Jan. 2014.

“National Sanitation Foundation Water Quality Index.” Boulder Area Sustainability Information Network, 27 Dec. 2005. Web. 15 Jan. 2014.

“Nitrates and Their Effect on Water Quality: A Quick Study.” Wheatley River Improvement Group, n.d. Web. 25 Feb. 2014.

Perlman, Howard. “Phosphorus and Water.” USGS Water Science School, n.d. Web. 27 Feb. 2014.

Perlman, Howard. “Water Properties and Measurements.” USGS Water Science School, n.d. Web. 5 Jan. 2014.

Solak, Yalcin, et al. “A rare human infection of Raoultella ornithinolytica in a diabetic foot lesion.” Annals of Saudi Medicine 31.1, Jan-Feb. 2011: 93-94. U.S. National Library of Medicine. Web. 27 Feb. 2014.

Umeh, Obiamiwe. “Klebsiella Infections.” Medscape, n.d.. Web. 27 Feb. 2014.

“Understanding Water Quality.” Water on the Web, n.d. Web. 15 Jan. 2014.

“Water Resource Characterization DSS—Phosphorus.” Watershedss, North Carolina State University Water Quality Group, n.d. Web. 27 Feb. 2014.

“Wekiva River.” St. Johns River Water Management District. Floridaswater.com, 2 Jan. 2013. Web. 15 Jan. 2014.