Kevin Daniels
Env 499
10/10/12
Morphological and Behavioral Differences between a Fish Population from a Wastewater
Effluent Pond Compared to a Reference Lake
Introduction
As water becomes more limited in the southwest and countless other places worldwide, the overall use of reclaimed water will undoubtedly increase. Globally, reclaimed wastewater effluent (WWE) is an important resource for irrigation, recreation, creation of wetlands, and direct recharge of aquifers. 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. In the last decade many studies have found that exposure to WWE affects several aspects of endocrine function in aquatic vertebrates, including their behavior.
The endocrine system of an organism includes glands and hormones which coordinate bodily functions such as growth and reproduction (Conn, 1998). EDSs in WWE disrupt these essential processes, leading to health and reproductive impairments in wildlife species (Colborn, 1993).
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). 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). This study will look at the relationship that WWE and O. ptychocheilus have on fathead minnows when they are exposed to the WWE and O. ptychocheilus at the same time in their natural environment. Fish living and breeding in an infected WWE pond will be compared to fish in a reference lake, rather than introducing fish to reclaimed water/estrogen compounds and O. ptychocheilus in the laboratory environment.
In our previous experiment, we were interested in one class of organic chemicals remaining in the reclaimed water: endocrine disrupting compounds (EDCs). The study compared morphological and sexual characteristics of fathead minnows 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. The purpose was to further define the risk associated with treated WWE. If exposure to WWE disrupts endocrine function, producing altered developmental and sexual characteristics in this fish system, then fathead minnows can act as an indicator species for monitoring environmental contaminants. Our experiment suggests that fathead minnow morphology is impacted by exposure to WWE. Fish had significant morphological differences in body size, gonadal weight, and secondary sex characteristics between the study sites. The morphological shifts may have detrimental consequences on the fish and the environment. Our results suggest that exposure to WWE affects endocrine function in this model fish species, and that these animals may serve as an indicator species for environmental monitoring for potential exposure to environmental contaminants.

Figure 1: Mean Gonad Weight of Rio vs. Raymond Wildlife Area
Figure 2: Mean Overall Weight of Rio vs. Raymond Wildlife Area

Figure 3: Mean number of Tubercles of Rio vs. Raymond Wildlife Area
Figure 4: Mean Gonadosomatic Index of Rio de Flag vs. Raymond Wildlife Area

However, during the examination of the fish, we discovered that the WWE site was infected with the parasite Ornithodiplostomum ptychocheilus . Which lead to our new question of how does exposure to WWE affect the immune function and infection of fathead minnows residing in the WWE? If fish are infected with O. ptychocheilus and exposed to WWE, then the endocrine system of the fish will be altered, resulting in larger overall and gonadal size.
Figure 5: Brain Comparison of Rio de Flag vs. Raymond Wildlife Area
Ornithodiplostomum ptychocheilus is a trematode that develops in the back of the brain but slowly moves toward the front of the brain while developing (Shirakashi, 2005). When O. ptychocheilus is finished developing, it is encysted in the region of the brain that controls the optomotor response (OMR). The OMR links visual stimuli with motor performance. Studies have exposed fathead minnows to O. ptychocheilus and found that uninfected minnows outperformed infected minnows but only weeks 2 and 4 were significant (Shirakashi, 2005).

Infection may affect rates of habitation and possibly learning, as well as reduction of minnows to detect and respond to motion (Shirakashi, 2005).
Cellular damage to tissue from developing O. ptychocheilus is temporary. Therefore, brain tissues are the same between infected and uninfected hosts. However, the magnitude, duration, or consequence of this temporary damage is unknown (Goater, 2005). Alterations in host visual ability is a result of the O. ptychocheilus larva developing, not encystment (Shirakashi, 2005).
This is based on the correlation of larvae development and periods of significant lower optometer response (OMR). O. ptychocheilus has no effect on anti-predator competence, growth, and survival of the fish (Wisenden, 2012), which is significant since the life cycle of O. ptychocheilus uses fish as an intermediate host in order to get to its final destination, a bird.
This study will look at the relationship that WWE and O. ptychocheilus have on fathead minnows when they are exposed to the WWE and O. ptychocheilus at the same time in their natural environment. Fish living and breeding in an infected WWE pond will be compared to fish in a reference lake, rather than introducing fish to reclaimed water/estrogen compounds and O. ptychocheilus in the laboratory environment. This experiment will be exploring the question of how infection with the parasite affects endocrine processes since the goal of the parasite is to keep the fish small in order for its final host (birds) to be able to consume them, while the WWE leads to larger fish?

Materials and Methods
Study sites
To determine if fish are affected by reclaimed water and O. ptychocheilus , we collected
42 adult fish from a WWE pond at 6,922 ft and a reference lake at 7,040 ft. The experimental group of fish will be collected from the Rio de Flag. At the site of collection, the water is nearly
100% WEE 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 Lake Kinnikinnick, which is within 20 miles of the Rio de Flag.
Experiment set up
Minnow traps with a handful of dog food as bait were set during the day and collected the following morning. The traps set at both sites did not catch anything, so a sane was used at both sites to collect the fish. The fish from the WWE pond were placed in two 10 gallon holding tanks with rock filters. The fish from Lake Kinn. were placed in two different 10 gallon holding tanks with rock filters. There were 9 experimental 5 gallon tanks with rocks, plastic vegetation, half of a clay pot, and an air bubbler. The sides of the experimental tanks were blacked out except for the right quarter of the tank to allow the experimenter to visualize fish eating and effectively use the lever. The experimental tanks were divided into four equal sections by taping three equally spaced vertical margins on the back of the tank.
A yellow spatula was modified to resemble a bird’s beak. This beak was hooked up to a leter that allowed the experimenter to stimulate a predator attack by penetrating the spatula beak

into the water when the fish enters the right quarter of the tank. This pulley system was set up to enter the right quarter of the tank, where the food was placed.
Behavioral Studies
The behavior studies were broken down into two parts. The first part was the baseline activity, which was filmed for three minutes during the morning. The baseline activity recorded the number of times a fish crosses a margin in the absence of food and predation. The second part is the post stimulation activity, which was filmed for five minutes during the afternoon. The post stimulation activity recorded the number of times a fish crosses a margin in the presence of food and predation. Each time a fish entered the right quarter of the tank, the beak was released.
Morphological Measurements
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 will be recorded. The brain of the fish was collected for histology. The overall weight and length, gonad weight, and the collect the brain of the fish will be measured.
The sex of the fish was determined by both sexual characteristics and gonads. 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.
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 that will over power the size constriction from O. ptychocheilus. However, it is anticipated that the fish will experience a decrease OMR but will not experience an effect in anti-predator competence. 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.
Timeline
Literature Set-Up Experiment Collection Clean-up Analysis Discussion
2-3 Weeks 1-2 Weeks 2 Weeks 2 Weeks 1 Week 3-4 Weeks 3-4 Weeks

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