The Effects of Reclaimed Water on Fathead Minnows

Kevin Daniels

Env 499

11/4/11

The Effects of Reclaimed Water on Fathead Minnows

Introduction

Becauseof many years of drought, the southwest is facing serious water problems. As water becomes more limited in the southwest and countless other places worldwide, the overall use of reclaimed water will undoubtedly increase. Reclaimed water could potentially become a drinking water source as it is in places like Singapore (Guan, 2007). Yet, many questions still remain about the environmental impacts that reclaimed water will have. This is why it is crucial to conduct research to understand the full benefits and consequences of reclaimed water use. The goal of my research is to determine the potential health effects that reclaimed water poses to fish. This information is important because the effects that fish experience from reclaimed water can be linked to the effects that humans might experience from exposure to reclaimed water.

Reclaimed water is wastewater that has been treated and has had contaminants removed. The three main contaminants that are found in wastewater are pathogens, heavy metals, and organic chemicals (Scholz, 2008). Unfortunately, reclaimed water does not have all of the organic chemicals removed. In my research I will be focusing on the organic chemicals left in reclaimed water that are supposed to be removed after waste water is treated. More specifically, I will be researching one class of organic chemicals remaining in reclaimed water, endocrine disruptors. The endocrine system of an organism is involved in the glands and hormones of the body and coordinates bodily functions such as growth and reproduction (Conn, 1998). Understanding howendocrine disruptors in reclaimed water affects the endocrine system will allow us to see if reclaimed water is harmful or not.

In the United States, reclaimed water is not used for human consumption; however it is used extensively in the southwest for irrigation of parks, ball fields, golf courses and cropsResearch indicatesthat reclaimed water has beneficial effects on plant growth (Jiao, 2010). Yet little is known about the fate of organic compounds remaining in reclaimed water and non-target effects of consumers of those plants.

Research has shown that reclaimed water contains endocrine disrupting compounds, and that exposure to reclaimed water affects the reproduction and survival of fish exposed to reclaimed water in a laboratory environment (Scholz, 2008). The results indicated that gonadal development in fish exposed to endocrine disruptors was characterized by sex reversal in males and reduction in Gonad Somatic Index (Scholz, 2008). Other research demonstrated that reclaimed water as a whole is having a harmful effect on fish (De Peyster, 1993). Their experiments exposed caged Fathead Minnows to effluent water, showing differences in the fish’s survival rates, growth rates and swimming performances (De Peyster, 1993). My research is the next step to understanding how fish are affected by reclaimed water because I will compare fish living and breeding in an effluent stream to fish in a reference stream, rather than introducing fish to reclaimed water or estrogen compounds in the laboratory environment.

My research is the next step to understanding how fish are affected by reclaimed water because I will compare fish living and breeding in an effluent stream to fish in a reference stream, rather than introducing fish to reclaimed water or estrogen compounds in the laboratory environment. This will allow us to see if effects found in the laboratory are similar to those in the wild, and thus determine the real-world implications of exposure to effluent in the environment.

Within the city of Flagstaff, there are two reclaimed water plants. The first is Wildcat Hill Wastewater TreatmentPlant, which is located on the east side of Flagstaff. This reclaimed water plant supplies both recreational areas and golf courses with reclaimed water. The second reclaimed water plant is the Rio de Flag Reclamation Plant. Reclaimed water from this plant is used for irrigation on the west side of town and is also used to recharge the Rio de Flag stream. Both of Flagstaff’s water reclamation plants produce class A+ reclaimed water, which is the highest quality reclaimed water that can be produced(City of Flagstaff Official Website). The differences between this water and the reclaimed water being consumed by humans in Singapore are the use of a advanced membrane filtration system that breaks down organic compounds such as estrogen in the water (Guan, 2007). Because water from the wastewater treatment plants in Flagstaff is released directly into streams, the potential impact to wildlife and humans are high if the organic molecules remaining in the water are indeed problematic.

To test the effects of this whole reclaimed water on fish in situ, I will compare fish maturity and sexual development from an effluent stream and a reference stream isolated from human populations. I predict that fish living in reclaimed water will be different internally and externally, including: gonad size, overall body size, and secondary sexual characteristics (i.e. coloration and number of tubercles.) The overall goal of this research is to determine what those differences mean in terms of fish performance and fitness.

I hypothesize that the overall size of the fish from the Rio de Flag will be larger. Estrogenic compounds have been shown to be obesogens, which can cause an animal to be bigger and have more fat (Barringer, 2010). If animals exposed to wastewater effluent are bigger, they might eat more or require more space for their territories. Both of these effects could change the community dynamics within the ecosystem and potentially have effects across trophic levels.

By looking at the gonads, we can get an indication of how the fish developed since they were exposed to wastewater effluent during their maturation from egg to adult. Fish are particularly susceptible to levels of hormones during sexual maturity, which might be missed in short-term laboratory studies (Barringer, 2010). Since there may be chemicals in the water that mimic or block these important hormones, the overall development of the fish might be affected due to the exposure of endocrine disruptors during this life cycle (Barringer, 2010).I hypothesize that the gonads will be larger in the female fish and smaller in the males that are from the Rio de Flag because of the additional hormones the fish were receiving from the reclaimed water.

The coloration and tubercle size/number are important secondary sex characteristics. Secondary sex characteristics are distinctive characteristics that separate the males and females of a species. These characteristics are not directly concerned with reproduction, but are correlated with mate selection.I hypothesis that the number of tubercles on the fish will decrease while the coloration of the fish lightens. The features on a male, coloration and tubercles, could be used to attract females and sometimes to defend territories from other males. The number of tubercles or differences in coloration might make these males less attractive to females. This could potentially result in fewer females choosing to mate with these males, decreasing fitness, and potentially decreasing his ability to defend his territory.

I also hypothesize that populations living in reclaimed water will have a higher number of fish than the reference stream. This is due to the endocrine disruptors that are in the reclaimed water. The endocrine disruptors are making the females gonads larger, which may allow for more offspring. The ultimate goal of my research is to see how the reclaimed water is affecting fish. The changes that the fish endure both internally and externally could ultimately affect the fish’s performance and fitness ability.

If fish are being affected from reclaimed water, this begs the question as to how other species will be affected from reclaimed water. It could also either dismantle the production of reclaimed water, or require an advancement of treatment in order to make reclaimed water safe. If reclaimed water production were dismantled, it not only increases the amount of potable water consumption but also decrease the amount of solutions to fight water shortage. The requirement of advanced treatment would require a significant amount of funds not only to do the research but to innovate existing plants to these new technologies.

Materials and Methods

Study sites

To determine if fish are affected by reclaimed water, we collected 75 adult fish from an effluent stream and a reference stream with minimal human impact. The experimental group of fish will be collected from the Rio de Flag. At the site of collection, the water is nearly 100% wastewater effluent from the wastewater treatment plant. The water has gone through the treatment process to remove the solid waste and many of the other contaminants, but the treatment process is not strong enough to remove all of the smaller molecules, many of which are endocrine disrupting (Conn, 1998).

The reference group of fish will be collected from a cattle tank in the Raymond Wildlife Area, about 40 miles east of Flagstaff. This area has remained relatively undisturbed by humans, with not much runoff or disturbance from cows due to a low cattle density. Therefore, we will use the Raymond Area as a "reference" site, although in nature, no ecosystems are completely isolated from all forms of contamination, so we cannot call any site a "control" site. Instead, we chose a site which is not likely to have abundant contamination by anthropogenic chemicals, and we use assume that they are as close to a "control" as possible.

Experimental Procedures

The experiment procedure to be performed was simple. I will have one site that is 100% effluent steam water and the other site will be natural water.The fish will be caught by using a minnow trap that will have a handful of dog food as bait. The traps will be placed in the streams of the sites along the shore and anchored by rocks. After 24-72 hours, the traps will be collected and the fish will be euthanized in MS222, bagged, numbered, and stored in the freezer. We will record the sex of the fish, the overall color of the fish, the color of the dorsal and pectoral fin, and the number and size of tubercles on the fish. We will also record the overall weight and length, gonad weight, and the collection of the pectoral fin of each fish.

In determining the sex of the fish, a photo and description of the sexes will be compared. The male fishes have fat pads and tubercles, and the females do not. The males also have a more bulky head and darker colors. The females have lighter colors and a narrower head. Once the qualitative measurements are collected, the gonads will confirm the observation.

The color of the dorsal fin, pectoral fin, and overall color will be based on a color chart. The color charts for the dorsal and pectoral fin are the same. Zero being no color and three being black. The chart for the overall color looked at color and strips. The fish that do not have any color will be a zero, the fish with a black head will be a one, a two will be a black head and faint black strips, and a four will be a fish with black head and dark strips.

The number of tubercles will be collected by counting the number of tubercles. This will be one of the harder tasks to complete because once the fish were frozen; the tubercles were weakened and fell off. So in order to determine the total number of tubercles, the tubercles that had fallen off have to be counted as well. Under a microscope, an area that contained a tubercle after it fell off will have a black circle where the originally was. The size of the tubercles is based on a size scale. The smallest tubercles will be a one and the larger tubercles will be a three.

Before the fish are weighed, the fish will have to be washed and dried in order to get all dirt off of the fish. Once dried, the fish will be placed on the scale and weighed out to the nearest milligram. The length of the fish will be found by placing the fish next to a ruler and collecting to the nearest micrometer.

The weight of the fishes gonads will be collected by cutting open the fish and extracting them. Once they are out of the fish, a more precise scale will be used in order to get the weight of the fish to the nearest nanogram. The pectoral fin will be collected by cutting it off of the fish. This will be done with a pair of scissors and tweezers. Once it is cut off, it will be directly placed in .5 millimeters of a reagent (RNA Later) that stabilizes the RNA in tissue sample, which will preserve the gene expression profile and allow for the analysis of data for future purposes.

Once all of the data is collected, it will be transferred into excel. A T-test will be used to look at the two different sites and to compare the results. If the results show a strong correlation, graphs will be constructed in order to give a visual of the differences. The graphs will contain standard error bars, linear regression lines, and correlation coefficients.

Anticipated Results

The anticipated results of the experiment should indicate a strong relationship between reclaimed water and gonad size, overall body size, and secondary sexual characteristics. If the Rio de Flag will have more females, and males with smaller gonads, it raises the question of what will this mean for fish performance and population dynamics. Furthermore, if there are fewer males and more females, male fish may not have as much competition for mate selection. Therefore, changes in secondary characteristics may not be important. If fish are impacted by reclaimed water as expected, it makes a strong argument that reclaimed water will have impacts on other species in the environment and that reclaimed water may not be safe for human consumption. This evidence could change the way society thinks about reclaimed water and laws could be passed to either dismantle the reclaimed water production or require a safer product.

Work Cited

Barringer, Felicity. "Water use in southwest heads for a day of reckoning." The New York Times. 28 Sept. 2010: A14. Print.

" City of Flagstaff Official Website." City of Flagstaff Official Website. N.p., n.d. Web. 23 Oct. 2011. <

Conn, P. Michael, H. Maurice Goodman, and Jack L. Kostyo. The endocrine system. New York: Published for the American Physiological Society by Oxford University Press, 1998- 2001. Print.

Colborn, T., Saal, F. S. vom, & Soto, A. M. (1993). Developmental Effects of Endocrine-Disrupting Chemicals in Wildlife and Humans. Environmental Health Perspectives, 101, 378-384.

De Peyster A., R. Donohoe, J. D. Slymen, R. J. Froines, W. A. Olivieri, and M. D. Eisenberg. 1993. Aquatic biomonitoring of reclaimed water for potable use: The San Diego health effects study. Journal of toxicology and environmental health 39:121-141.

Guan Y. K. 2007. Sustainable water management: The Singapore Water Story. WC&P International 49:74.

Jiao Z., Z. Huang, W. Wang, B. Yan, L. Peng, and H. Li. 2010. The effect of reclaimed water on soil performance and the microorganism. Journal of Agro-Environment Science 29:319-323.

Scholz S, Kluver N 2008. Effects of endocrine distrupeters on sexual, gonadal development in fish. Karger 3:136-151