Environmental Effects of Industrial Run-off on Daphnia magna

Part of the Young Naturalist Awards Curriculum Collection.

by Mauree, Grade 11, Iowa - 2005 YNA Winner

Good summer camps never truly end at the closing ceremony. At least this is what I learned last year. While attending a summer camp at the University of Iowa, I met and spoke with Kristina Venzke, who works with the Iowa Army Ammunition Plant's Former Worker Program. The program was developed to help former workers of the Iowa Army Ammunition Plant (IAAAP) get compensation for their exposure to the IAAAP and its contamination. This led me to probe deeper into this issue. Even before I left, I was researching the history of the plant and how the issue concerning contamination from the IAAAP developed.

A sign on a chainlink fence that reads, "U.S. Army Restricted Area."
IAAAP restricted area

It began more than 60 years ago, in 1941, when the IAAAP was built for the purpose of producing munitions and, later, nuclear weaponry. The IAAAP, located in Middletown, Iowa, created nuclear weaponry until 1975, when this part of the plant was moved to the Pantex weapons facility near Amarillo, Texas. Then, in 1988, an environmental survey stated that the IAAAP was a "horror story" of improper hazardous waste disposal (Mansfield). In 1989, the IAAAP was placed on the Environmental Protection Agency (EPA) Superfund list. Cleanup of the contamination left from years of weapons production began in 1992. In 1993, as a result of the EPA's water tests, it was found that the water supply of many homes near the IAAAP was contaminated with RDX (1,3,5 - trinitro - 1,3,5 - triazine). After this contamination was found, these homes were provided with a different water supply.

Up until the 1990's the public was unaware of the fact that the IAAAP had produced nuclear weapons, or that there could be possible health or environmental impacts due to exposure to these explosives. This situation was brought to the public's attention in 1997 when Robert Anderson, a former security guard at the IAAAP, wrote Senator Tom Harkin about his diagnosis of non-Hodgkin's lymphoma. He believed that he had gotten this disease because of his exposure to explosives during his employment at the IAAAP. It was not long before more reports of cancer surfaced from both present and former workers of the IAAAP. In 1998 an employee revealed that the IAAAP actually did produce nuclear weapons. Even then, the Department of Energy (DOE) did not acknowledge the truth of this report until later. Over the course of the next few years, endless tests were conducted, not only on employees but also on local waterways, for possible contamination. Contamination by TNT (2,4,6 - Trinitrotoluene) and RDX were detected, along with high phosphate levels. Radiation levels were within the legal limits. Currently, remediation of the plant and its waterways is underway, and contamination levels have been reduced significantly, which leads to the beginnings of my research on the IAAAP and its effect on local waterways.

Figure 1: Map of study area

The IAAAP covers 19,127 acres of land. Within the IAAAP are three creeks: Brush Creek, Spring Creek, and Long Creek. Two of the creeks, Spring Creek and Long Creek, run through the plant; however, Brush Creek originates on the IAAAP. To begin my research, I contacted Rodger Allison, an installation restoration project manager at the IAAAP. He led me to information about the plant and the findings from the remediation project. After examining the books of information, I decided to talk with some people living in the area about their water supplies. It was incredible how much information I discovered talking to the locals, information that I could not find documented in the IAAAP books. I found out from a local farmer that Brush Creek, a creek that the remediation project had determined was the most contaminated, had at one time run a red, rusty color. After accumulating this useful firsthand information, I began my experiments

Mauree taking a sample in the field

Over the past three years, I have tested water quality by using both traditional and non-traditional methods. The traditional method consisted of testing the water for parameters that the EPA also uses. For the non-traditional method I used Daphnia magna. Daphnia magna are freshwater invertebrate crustaceans that are frequently used in water-toxicology research because of their ability to signal stressful levels of pollution; however, methods of usage vary. For instance, the Iowa Hygienic Laboratory uses the LC50 method. This method examines at what concentration levels 50% of the Daphnia magna die. They are also a key link in the lower food chain, being the main source of food for many smaller fish (i.e. minnows), Hydra, and also many small insects (i.e. water skidders). In my previous research, the effects of different water conditions (i.e. seasons and runoff) on Daphnia magna were examined. I examined standard measurements of heart rate and birthrate and documented this data. Along the way I developed a new measurement—reflex. Daphnia magna has a light-sensitive eye, meaning that the eye will track a moving light source. This knowledge helped me to establish a measurement of reflex, providing a measurement of the effect of environmental stress on the nervous system. In my previous research, it was also found that forms of nitrate have detrimental effects on Daphnia magna. They cause high heart rates, low populations, and impaired reflexes. In addition, one form of nitrate, urea, caused the Daphnia magna to form small growths on their midguts, or intestines.

Girl sitting at a desk looking at a small glass bottle containing brownish water.
Mauree with a sample in the lab

For the traditional part of my research this year I tested the water for phosphate, nitrate, ammonium nitrate, pH, dissolved oxygen, and total and free chlorine. I collected water samples at a total of seven locations. At each site I collected four two-gallon containers of water. I collected the water at both the entry and exit points of the creeks. Four different locations were required for Spring Creek. This was because there are three entry sites for this creek and one exit point. Brush Creek had only one collection site because it originates on the IAAAP, and, due to strict security measures after September 11, I was unable to obtain a sample at this entry site. I collected the water samples by lowering a metal bucket down into the creek with a rope. I made sure that no sediment was being brought up from the bottom of the creek because this would contaminate my sample. All of the samples were collected and tested within a six-hour period. I gave each sample a number: Spring Creek entering, 1-3; Spring Creek exiting, 4; Brush Creek exiting, 5; Long Creek entering, 6; and Long Creek exiting, 7. Immediately following collection, I tested the water samples for phosphate, nitrate, ammonium nitrate, pH, dissolved oxygen, and total and free chlorine. I also documented the temperature of the water at each of the test sites. The water tests were conducted in accordance with the standard procedures provided with the HACH water test kit.

For the non-traditional part of my research, I conducted two experiments using Daphnia magna. Before conducting any experiments using Daphnia magna, age controls were set. This was to ensure that the Daphnia magna were all the same age and that some of them would not die from old age at the beginning of the experiment, because this would alter my results. The age controls were set by placing individual adult Daphnia magna in plastic cups filled with spring water. The Daphnia magna were allowed to reproduce, and their offspring were then used in the experiments. The Daphnia magna were fed every week with liquid invertebrate food.

The lab setup

The purpose of the first experiment was to examine their population growth, heart rates, and reflex quality. To begin this experiment, I filled a total of eight plastic aquariums with a designated type of water (Site 1, 2, etc.). The eighth aquarium was for the control, and spring water was used for this type of water. Then I added five age-controlled Daphnia magna to each of the aquariums. Every day I counted the populations and checked the pH and temperature. Every other day I documented the Daphnia magna's heart rate and eye reflex, and also fed the Daphnia magna with the liquid invertebrate food. I monitored their heart rate by examining the Daphnia magna under a microscope and counting the beats per 15 seconds; this was then converted to beats per minute. In order to insure that the microscope's stage would not heat up, causing the Daphnia magna to become stressed, I changed the microscope's light source to a glass rod, bringing in light from the room, which is cool light. I measured their eye reflex by turning off the original light source and moving another light source (a flashlight) around the microscope 180 degrees and giving a rating of: 1) no reflex, 2) an incomplete reflex, and 3) perfect reflex. I then conducted a second trial, using water from the same collection times. Both trials lasted 36 days.


The purpose of my second experiment was to examine how individual Daphnia magna develop in water from each of the test sites. To begin this experiment I set up plastic cups in a grid system. There were four cups per each site number. Each of these cups was then filled with the corresponding type of water. Again, four cups were used for the control, using spring water. To each cup I added an age-controlled Daphnia magna. Every day I documented their heart rate and eye reflex. In addition, I also documented the number of births. Every week I fed the Daphnia magna the liquid invertebrate food. I continued this experiment until all of the Daphnia magna had reached the end of their life cycles. During both of my experiments, my raw data was documented in a notebook and then transferred to a computer for analysis at the end of the experiments.

Table 1: Conventional Test Results

After conducting my experiments, I found that Spring Creek's entry sites had lower levels of ammonium nitrate, free and total chlorine, and phosphate than the creek's exit site (see Table 1 above). Also, the levels of nitrate remained steady at two of the three entry sites. Brush Creek's levels of nitrate, chlorine and phosphate were comparable to the levels in the other creeks, but the concentration of nitrate and the pH level at Brush Creek were the highest of the sites tested. Long Creek's entry site had lower levels of dissolved oxygen and free chlorine than the exit site; however, the entry site was higher in phosphate and nitrate. All of the tested parameters were below the EPA's maximum values.

Graph tracking the growth in the number of Daphnia magna (freshwater invertebrate crustaceans) in a 7 sites and 1 control site over a number of 36 days.
Figure 2: Trial One Population
Trial 2 population, 5-day moving average: chart showing growth in Daphnia magna populations over 36 days across 7 sites with 1 control.
Figure 3: Trial Two Population
Figure 4: Trial One Heart Rate
Figure Five: a line chart showing the Trial Two Heart Rate, a five-day moving average graph for seven sites plus the control.
Figure 5: Trial Two Heart Rate
Figures 2-5: Click on any graph to enlarge.

Looking at the Daphnia magna, I found that population levels in the water from the creeks' exit sites were distinctly lower. This was more evident when a five-day moving average was applied to the data (see Figure 2 and Figure 3). It brought out the population changes and made them more recognizable. At Long Creek, the population level in water from the entry site was close to that in the exit-site water, but was still greater. I also applied a five-day moving average to the heart rate data, which shows that the Daphnia magna had significantly higher heart rates in the exiting creek water compared to the entering creek water (see Figure 4 and Figure 5). I found that in all of the creeks, the birthrates of Daphnia magna were lower in the exiting creeks than in the entering creeks (see Figure 6). I constructed a correlation chart showing how the parameters tested with the Daphnia magna correlated to the parameters tested in the water itself (see Table 2). It was found that there were some direct correlations between birthrate and total chlorine, as well as between heart rate and phosphate. I also found indirect correlations between population growth and phosphate, and heart rate and population growth.

Figure 6: Birthrate
Table 2
Figure 7: Impaired Reflexes
Figure 6, Chart 2, Figure 7: Click on any graph to enlarge.

More investigation is warranted after completing my research. Not only was it found through my research that Daphnia magna were affected neurologically (see Figure 7) by the Brush Creek water, and in three cases by water from Long Creek, but after attending public forums I also discovered another area of concern—the human health issue. After more research, I found that several people who live near Brush Creek and used well water have cancer. I wish to continue to investigate this aspect of the problems at the IAAAP. In addition, I would like to retest the creek waters, adding more sites upstream to see what other pollutants are contributing to the water quality. I was very surprised to see a lower concentration of nitrates in the water downstream from the IAAAP, since most munitions are forms of nitrate. Retesting the water will show if anything upstream is affecting these results. In addition, while retesting the water I would like to take some type of volumetric measurement to see whether the higher dissolved oxygen levels were indeed caused by contaminates from the IAAAP. While the results obtained through the traditional method agree with those using Daphnia magna, it is interesting to note that the Daphnia magna show the effects of contaminated water on aquatic life more precisely than conventional testing methods. It was alarming to see how much the lower food chain, represented by the Daphnia magna, was affected by water quality, even though the traditional water-testing methods showed that all the contaminants within the water were well within legal limits. 

Photo of Daphnia magna

I feel that it is important to use the  Daphnia magna  in conjunction with traditional means of water-quality testing because they can show how certain levels of chemicals, even if they are well within the legal limits, can affect the lower food chain. It is also important that we keep in mind that the IAAAP did not intentionally place their employees and the environment in harm's way. They were employing the best technology available in the 1960s to protect both people and the environment. Now, after many more years of research, we have found that we must go an extra step to help protect not only the environment, but also ourselves, its inhabitants.



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