Effects of a Chelating Agent (EDTA) on Rosy Red Minnows, Pimephales promelas, Exposed to Lead Poisoning

Mike Dietzel, Jessica Hart, and Kelsey Roth Biology Department Loras College 1450 Alta Vista St. Dubuque, IA 52001

Abstract: In this experiment we tested the effect of EDTA, a chelating agent, on fish exposed to lead poisoning. EDTA is approved by the FDA to treat lead poisoning in humans, but its effects on fish are unknown. We ran the Kaplan Survival Analysis to analyze whether or not EDTA had a statistically significant effect on the survival of rosy red minnow, Pimephales promelas, exposed to Pb(NO3)2. While our results were inconclusive, the observable characteristics, such as: eating, swimming, and grouping patterns, suggest that the minnows were greatly affected by the lead poisoning and that EDTA helped treat their unusual behaviors.

Introduction Chelation therapy is a treatment that involves administration of a chemical solution, such as ethylenediaminetetraacetic acid, or EDTA. EDTA works by treating lead poisoning by pulling toxins and heavy metals from the bloodstream (Ehrlich 2011). The clinical signs of chronic lead poisoning include lowered food intake, weakness, and weight loss. Mortality directly due to lead poisoning may be secondary to the losses due to non-lethal effects of lead such as reproductive problems, increased susceptibility to disease and infection, behavioral changes, and increased predation due to anemia and weakened muscles (Michigan DNR). The impacts of lead on aquatic ecosystems can be detrimental to all involved. When lead is present in aquatic ecosystems, it bioconcentrates in the skin, bones, kidneys, and liver of fish rather than in muscle and can be especially problematic to people who when they consume the whole fish, whom are potentially exposed to high concentrations of lead. Along with that, lead concentrations that exceed 100 ppb will also affect gill function, but the toxicity of lead to fish also depends in part on the species, with goldfish being relatively resistant due to their ability to excrete lead via their gills (Soloman 2008). Lead can also affect behavior in fish, such as interactions between each other. In fact lead and mercury are the only trace metals that so far have been shown to disrupt nest building, spawning, or courtship behaviors of fish (Scott & Sloman 2004). In addition, Ahmed and Bibi (2010) found that when fish are exposed to high levels of lead they exhibit a wide range of effects such as muscular and neurological degeneration and destruction, growth inhabitation, mortality, reproductive problems, and paralysis. They also found that at concentrations of 5.0 g/l and higher a significant amount of lead was absorbed into

the tissues of the fish. The addition of the chelating agent EDTA starts to significantly decrease levels of lead in the blood after ten treatments (Mikirova et al. 2011). Arefi et al. (2011) found that with a single treatment (1 g every 12 hours) of EDTA for a period of 10 days, there was a significant decrease in lead levels. Knowing what the symptoms of lead poisoning are, the goal of our research is to see whether the effects and symptoms of lead poisoning could be treated through the use of the chelating agent EDTA. Not much research has been done regarding whether chelation therapy will reduce the effects of lead poisoning in fish. For our experiment we examined the behavioral and mortality effects lead nitrate had on Pimephales promelas, Rosy Red minnows, for a period of two weeks and determined whether the chelating agent EDTA reversed the effects. Based off of the research found, we hypothesize that between the two groups of minnows exposed to Pb(NO3)2, the group with the chelating agent, EDTA, treatment will has a significantly greater survival rate. Methods Two fish tanks were prepared with a lead concentration of 15 ppm using lead nitrate. A third tank had the same concentration of nitrate found in the other tanks to be used as a control; this was obtained using calcium nitrate. Calcium nitrate was used because we know that the calcium will not harm the fish and we were determining the effect of nitrate on the fish. We allowed the water to filter through the tanks for two days without introducing any fish. Rosy red minnows, Pimephales promelas, were then introduced to the tanks; 13-15 fish were added per tank. We then began observing the fish for mortality rates, once a day. We began recording observable behaviors of lead poisoning in the fish that were exposed to the lead nitrate such as:

irregular eating and or the lack of eating, irregular tail movements for example swimming backwards or upper part of tailfin moving at a different pace than the bottom, jittery swimming/ jagged movements, running into side of tank or other fish, burrowing, getting stuck, or other unusual behaviors. After two days, the treatment with the chelating agent EDTA, began in one of the lead tanks. The lead tank with the EDTA was given two capsules of EDTA once a day for seven days with food; fish in the other tanks were also given food at the same time. We then continued to watch the mortality rates between the tanks for any observable trends. The experiment ended when the lead group with no chelating agent all died. Results Half of the initial group of minnows from PetSmart died within the first day of our experiment; we were forewarned by the store manager that their new stock of minnows was experiencing many fatalities, so the initial loss of 6 fish in both the lead nitrate and calcium nitrate groups we believe were due to the shipment of frail fish that PetSmart received. The overall trend of figure 1 shows that the minnows treated with Pb(NO3)2 and EDTA (the chelating agent was added after day two) had the greatest survival percentage over the nine day experiment. Of the fifteen initial minnows ten were living after day eight. The second best treatment, in terms of number of minnows surviving the experiment, was the Ca(NO3)2. This treatment group started with thirteen minnows and at the end of the experiment six were living. However, of the seven total that died from the Ca(NO3)2 group, six died within the first twentyfour hours of the experiment. We believe this is due to factors nonrelated to our experiment for these reasons: 1.) It is common knowledge that calcium does not harm minnows and 2.) Our data shows that nitrates didnt have a statistically significant difference on the survival of the

minnows within the Ca(NO3)2 and Pb(NO3)2 treatment groups. The treatment with the worst survival percentage was the fish tank polluted by Pb(NO3)2. All thirteen of these minnows were dead by the end of the nine day experiment. Similarly to the Ca(NO3)2, six minnows were dead when we made our first observations on day one. Regardless of the cause of these fatalities, the remaining seven minnows died by day nine.

Fig. 1. A Kaplan Survival Analysis graph which shows what percentage of the initial minnows, for each treatment group, were surviving after a given number of days. The test was run for a total of nine days. Table 1 Means for Survival Time. The mean life span is given for the minnows of each chemical treatment. The confidence intervals show which treatments have statistically different mean life spans.

Table 2 Overall Comparisons. The degrees of freedom (df), Chi-Square value, and p value (sig.) are given. The critical Chi-Square value for two df at the 95% confidence level is 5.99. Since the Chi-Square value is greater than 5.99 we would expect the p value to be less than .05, as it is, and therefore a statistically significant difference is present between our treatment groups.

Outside of the Kaplan Survival Analysis data, we recorded visual observations which helped us locate characteristics of lead poisoning. The first characteristic noted was that all the dead fish from the lead nitrate group were mutilated in some way. This observation was not seen in the calcium nitrate group. Some of the observable behaviors that we noticed were unusual swimming patterns, such as swimming into the sides of the tank, swimming in one place, and jerky swimming patterns as well. One of the most noticeable behaviors the minnows exhibited was the abnormal movement of their tail fin. The top part of the tail fin would move at an exceedingly fast rate while the bottom part of the tail fin would hardly move. They also had a tendency, especially towards the end to propel themselves backwards because of loss of ability to stay in one spot. Within the lead group, we were unable to determine if the eating of the bodies was post-mortem or cause of death. The behavior of the lead poisoned group was the state in

which dead fish were found: the bodies of the fish were usually mutilated and or beheaded. This characteristic was never observed in action although the bodies were discovered in this manner; none of the dead fish bodies from the calcium nitrate group were physically disturbed. Discussion The statistical data from our experiment supports our hypothesis that between the two groups of minnows exposed to Pb(NO3)2, the group with the chelating agent, EDTA, treatment had a significantly greater survival rate than the group without the chelating agent. Similar results were found in a study conducted on young hearing gulls, 14 days after some of the chicks were injected with lead there was a significant difference in the percent of survival when comparing lead-treated groups to control chicks (Burger and Gochfeild, 1994). In the same study by Burger and Gochfeild (1994) there were also accounts of behavioral differences between the lead-treated and control groups such as lead-treated chicks having lower levels of begging for food and lower activity levels. In our study we also observed the Pb(NO3)2 exposed groups of minnows to eat less than the Ca(NO3)2 exposed group have lowed levels of activity. In the two groups of minnows exposed to Pb(NO3)2 there was an observation of the minnows eating the minnows that had died, by ingesting the bodies of the dead the minnows would have ingested lead increasing their exposure to the Pb(NO3)2 which would increase the rate in which the lead poisoning would occur and could also impact its severity. In a previous study that is similar to ours, they found that behavioral effects due to lead were also treated with the help of the chelating agent DMSA, but it did not totally reverse the effects of lead poisoning in fish (Weber et al. 1997).

During our study of the effects of EDTA on the survival of minnows exposed to lead poisoning, we considered new ideas to test through future research. First, we noticed that all the minnows were not showing the same characteristics of lead poisoning. Some were very agitated, wouldnt eat, and had abnormal swimming patterns while others showed no sign of lead poisoning. Future experimentation could test if the sex or the species of the minnow changes the vulnerability to lead poisoning or the effectiveness of EDTA. Other research could be done to determine if there is a more effective dosage of EDTA to increase the survival rate of the minnows. Also, the long term effects of EDTA on minnows are unknown. If the chelating agent has negative effects on the minnows reproductive success or offspring viability could be studied in future research as well.

References: Ahmed, M.S., and Bibi, S. (2010). Uptake and bioaccumulation of water borne lead (Pb) in the fingerlings of a freshwater cyprindid, Catala catala L. Journal of Plant and Animal Science, 20(3), 201-207. Arefi, M., Taghadosinejad, F., Sadeghniiat-Haghighi, K., Salamati, P., Godarz, F., & Saadiyani, E. (2011). Effectiveness of ethylene diamine tetra acetic acid treatment in patients with lead poisoning referred to Baharloo hospital, Tehran. Journal of Isfahan Medical School, 29(143). Burger, J., and Gochfeild, M. (1994). Behavioral impairments of lead-injected young herring gulls in nature. Fundamental and Applied Toxicology, 23. 553-561. Ehrlich, S. D. (2011). EDTA. University of Maryland Medical Center. Retrieved October 17, 2012, from http://www.umm.edu/altmed/articles/ethylenediaminetetraacetic-acid000302.htm. Michigan DNR. (2012). DNR - lead poisoning. Michigan.gov. Retrieved October 17, 2012, from http://www.michigan.gov/dnr/0,1607,7-153-10370_12150_12220-26676--,00.html. Mikirova, N, Cascriara, J., Hunninghake, R., and Riordan, N. (2011). EDTA chelation therapy in the treatment of toxic metals. Riordan Clinic, Medistem Inc. Retrieved October 21, 2012, from http://www.riordanclinic.org/research/articles/SDD-2011-v01n02p81.pdf Scott, G.R. & Sloman, K.A. (2004). The effects of environmental pollutants on complex fish behavior: integrating behavioral and physiological indicators of toxicity. Aquatic Toxicology 68, 369-392.

Soloman, F. (2008). Impacts of metals on aquatic ecosystems and human health. Mining.com Magazine. Retrieved from http://akmininginfo.files.wordpress.com/2012/09/solomon_2008_impacts_of_metals.pdf Weber, D. N., Dingel, W. M., Panos, J. J., & Steinpreis, R. E. (1997). Alterations in neurobehavioral responses in fishes exposed to lead and lead-chelating agents. American zoologist, 37(4), 354-362.