The Effect of Sewage Treatment Plant Effluent on the Reproductive Behavior of Sailfin Mollies (Poecilia latipinna)

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The Effect of Sewage Treatment Plant Effluent on the Reproductive Behavior of Sailfin Mollies (Poecilia latipinna)
Gabrenya, Annamarie
St. Mary, Colette ( Mentor )
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Gainesville, Fla.
University of Florida
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The Effect of Sewage Treatment Plant Effluent on the Reproductive
Behavior of Sailfin Mollies (Poecilia latipinna)

Annamarie Gabrenya


Endocrine disrupting chemicals (EDC) that are commonly found in the environment have been shown to have to

have deleterious effects on the morphology, physiology, and behavior of wildlife. Water effluent of industrial

and municipal sewage treatment plants is a frequent source of EDCs. Estrogens are a common type of EDC found

in domestic sewage treatment plants (STP), which is thought to enter the sewage effluent through human

excrement containing pharmaceutical chemicals such as the synthetic estrogen ethinyl estradiol in birth control

pills. While the effects of estrogens on morphology and physiology of wildlife, such as fish, have been

well documented, the effects on behavior, specifically sexual behavior, are less well known. The present

study explored the effect of sewage effluent on the sexual behavior of sailfin mollies. It was hypothesized

that female sailfin mollies will choose males from sites that do not receive sewage effluent over males from sites

that do receive effluent. It was also hypothesized that males from polluted sites will display decreased

sexual behavior. Sailfin mollies (Poecilia latipinna) were collected from a reference site receiving no sewage

effluent and an influenced site that contained sewage effluent. Female sailfin mollies were presented a male

molly from both sites and behavior indicative of female preference and male courtship were recorded.

Gonopodium length and standard length were also measured for each male fish. Females did not show a

mate preference. Male behavior, the total time and duration the male spent courting the female, was also

not significantly different between the two sites. Gonopodium length did not differ between sites, contrary

to expectations of an elongated gonopodium in influenced males. I believe that the lack of significant differences

in behavior is mainly attributed to the small sample size, supported by the high amount of variation. Future

studies should increase the sample size and expand the study to include evaluation of more sexual behavior

and quantification of estrogens in the sewage effluent.


Sewage treatment plants (STP) release a concoction of chemicals, natural and synthetic into the

aquatic environment. Many of these chemicals are known to be endocrine disrupting chemicals (EDCs).

The endocrine system is responsible for regulating various functions within the body and does so by

secreting hormones that change the actions of particular tissues or organs. EDCs act to disrupt normal

endocrine functions, resulting in potential abnormalities in the morphology, physiology, and behavior of

animals. Aquatic animals, such as fish, are especially at risk because of constant exposure to low concentrations

of EDCs (Jobling et al., 1996) acquired through aquatic respiration, osmoregulation, and maternal transfer

though uptake in the egg yolk (Van der Kraak et al., 2001). Additionally, because many EDCs are lipid soluble,

they can accumulate in animal tissues, and through biomagnification adversely affect species higher in the food

chain (Tyler et al, 1998).

One class of endocrine disruptor that is quite common in our environment is estrogenic hormones and

estrogen mimics. One common estrogenic chemical that is known to negatively affect wildlife, such as fish, is

human contraceptives, or birth control pills. For example, the yolk protein, vitellogenin, has been found in male

fish after exposure to estrogen (Sumpter and Jobling, 1995). Females that take birth control pills excrete

ethinyl estrodiol and its metabolic byproducts which enter the environment via municipal water treatment

plants. Constant entrance of these estrogens in the environment will continuously produce adverse effects

in exposed wildlife.

Traditional STP processing focuses on removal of nitrogen and phosphorus compounds, but often

estrogen compounds are not removed. STP involves three common treatment steps: preliminary clarification,

aerator tank, and end point clarification. Preliminary clarification removes solid materials from the wastewater.

In the second step, bacteria and protozoa further degrade the biological content. The final clarification step

prepares the wastewater for release back into the environment. Common effluent release points are natural

and man-made wetlands, rivers, or even released back into the ground water. While some processes will

remove estrogens from the sewage influent (Ternes, et al 1999), the majority of processes used fail to do so, due

to the great expense. Although studies conducted in Europe have shown a detection of estrogen in STP

discharge measured in concentrations in the lower ng/L (Ternes, et al 1999; Jobling, et al 1998), this range has

been found to have significant effects on gonad morphology. Desbrow et al. (1998) detected 17p-estradiol

in domestic wastewater ranging from ing/L up to50 and 80 ng/L. Higher concentration of sewage effluent is

directly correlated with the greater degree and number of intersexed roach (Rutilus rutilus) individuals (Jobling et

al. 1998).

In particular, estrogens and their mimics are thought to influence wildlife species' sexual and reproductive

behavior. This is alarming because impairment of sexual behavior function coupled with possible

morphological defects could deleteriously affect the fitness of the population. The natural estrogen, 17 p-estradiol,

is known to affect hormonal pathways that regulate the development of gametes, sexual phenotype and

sexual behavior (Arcand-Hoy and Benson, 1998). One example of impairment of sexual behavior is differences

in mate choice, which could result from incorrect signaling, production of incorrect cues, or timing of

particular behavior, etc. Experimental evidence has shown that often male sexual behavior, courtship displays

and mating are decreased, impaired or even inhibited in many wildlife species exposed to EDCs (Zala and

Penn 2003).

Reproductive success depends on many factors, including the individual's ability to produce the correct

sexual behavior at the proper time. Many laboratory exposure studies have found that reproductive

success decreases with exposure to one or a concoction of chemicals, but fewer studies have linked the effect

of occurrence of EDCs in the environment to decreased reproductive success (Mills and Chichester, 2005). This

study examines the effect of sewage effluent on reproductive behavior that are explicitly linked with

reproductive success in the sailfin molly.

The sailfin molly (Poecilia latipinna) was chosen as an experimental organism because they exhibit a high rate

and variety of reproductive behavior. The natural reproductive behavior of sailfin mollies has been well

documented. The females exert particular mate choice behavior and the males display well characterized

courtship behavior such as gonoporal nibbling, gonopodial thrusting and sigmoidal curving of the body for the

male. In addition, male fish display a large colorful dorsal and caudal fin that plays a role in female choice.

They also have a highly modified structure formed from anal fin elements called a gonopodium which is used

for fertilization and has been shown to be sensitive to estrogenic chemicals in other species. These fish

are commonly found throughout the southeastern United States, occurring in fresh, brackish and even coastal

salt water in marsh edges, lowland streams, ponds, swamps and roadside ditches. Their ability to survive in

oxygen depleted and less than ideal waters makes sailfin mollies an ideal fish to study in waters receiving

sewage effluent.

The object of this experiment is to determine whether sewage effluent has an effect on the sexual and

reproductive behavior of sailfin mollies. Specifically, this experiment investigates whether male sailfin

mollies, naturally exposed to estrogen containing sewage effluent, display altered mating behavior and

whether females find males from waters containing sewage effluent less attractive. I hypothesize that male

mollies exposed the sewage effluent will display mating behavior less frequently than sailfin mollies not exposed

to effluent, and that female sailfin mollies will choose unexposed males over exposed males.


Experimental Design

To test these hypotheses, a female was presented two males, one from a "reference site" and the other from

an "influenced site." A 20-gallon aquarium was used in the trials by dividing it in half lengthwise with a clear

plastic divider, then dividing one of these halves into three equal parts using opaque dividers. The undivided

half contained the female while the divided half, in each of the left and right compartments, contained either a

fish from the "influenced" or "reference" site. The middle compartment contained no fish, serving as a neutral

zone for females. Male fish were separated using opaque plastic dividers to avoid visual contact and male to

male competition. The dividers did not have holes so chemical cues which may diffuse through the water

where limited, but this permitted better viewing with the video camera.

The experimental trials were performed within 4 days of the female releasing a brood. The experimental

aquarium was prepared no later than a day before the test to roughly match the holding tank conditions with

6ppt salinity. The water was dechlorinated and aerated for at least 24 hours. Males were isolated in their own

tanks 24 hours prior to female exposure. This isolation has been determined to induce natural mating rates

typically found in natural environments for fish of all sizes (Ptacek and Travis 1997 as in Travis 1994). Females

were left alone until the beginning of the trial. Behavioral observations were conducted between 1100 and

1600 hours. The fish were placed in the experimental tank and given 15 minutes to acclimate to their

new environment, after which video recording began and lasted 25-30 minutes. After the experiment

was completed, the female fish was placed back into her aquarium and the males were euthanized in 1%

neutral buffered MS222 and fixed in neutral buffered formalin for later body length and gonopodium measurements.

The males selected for testing were +/- 10mm of the female's total length to increase interactions, as studies

have shown that males are not attracted to significantly larger females and vice versa (Ptacek and Travis 1997 as

in Travis 1994). The color of each male was controlled for to avoid coloration preference exhibited by the

female. Previous observations have shown that females prefer males that are more brightly colored over males

that were not brightly colored. Males were not used from Otter Creek to avoid natal population preference by

the female.

Sailfin Molly Mating Behavior

Male sailfin mollies display three prominent mating behavior towards females. In a courtship display, the male

will display an enlarged dorsal fin complemented by a sigmoidal curving of the body and tilting the body towards

the female. A second behavior, gonopodial thrusting, occurs when the male attempts to forcefully inseminate

the female. The third behavior, gonoporal nibbling, occurs when the male tries to make facial contact with

the female's gonopore. In this experiment, because the male and female fish are divided into

separate compartments, only sigmoidal curving can be considered in the behavioral observations of males.

Female choice was be measured by the amount of time she spent with each male.

Measurements and Statistics

Standard length (SL), gonopodium length, and weight were measured for each male fish used in the

experiment. Standard length was measured after the fish was euthanized and gonopodium length was

measured after the fish were preserved in formalin.

The amount of time each female spent with either male or with neither (middle compartment), and the amount

of time each male spent courting the female were recorded from video recording, using the JWatcher program.

Because there is often a relationship between SL and gonopodium length, a Univariate Analysis of

Covariance (ANCOVA) was performed on these measures. However, no relationship was found for either Lake

Alice or Tumblin Creek sites (F=1.345, df=2, p=0.286) possibly due to the fact that body size was controlled for

and thus did not vary. Therefore, independent t-tests were computed to compare male standard length

and gonopodium length separately.

The relationship between total time a female spent with a male from each site and gonopodium (or body) length

was analyzed using a univariate ANCOVA. Specifically we analyzed the main effects of site and gonopodium

(or body) length and their interaction (the interaction provides a test for the assumption of equal slopes).

Slopes were equivalent in both of these comparisons. Similarly, the relationship between total time males from

each site spent courting and gonopodium (or body) length was analyzed using a univariate ANCOVA. Specifically

we analyzed the main effects of site and gonopodium (body) length and their interaction.

The total time the male spends courting the female, the mean duration of male courting per bout, the total time

the female spends with each male, and the mean duration per bout that the female spends with the male

were analyzed using a paired samples, one-tailed t-test. The confidence level was set at 95% and the alpha level

= 0.05 for all statistical tests.

Collection and Care

Fish were collected between July and September 2006 in North Central Florida. Female sailfin mollies were

collected at Otter Creak in Dixie County, Florida. Male sailfin mollies were collected in Tumblin Creek in the city

of Gainesville and also in Lake Alice on the University of Florida campus. Sites classified as "reference" received

no sewage effluent and were not used as dumping grounds for other chemical waste products. Sites classified

as "influenced" received direct sewage effluent.

Fish from Otter Creek and Tumblin creek were collected by seining in several areas along the creek. Fish from

Lake Alice were collected using fish traps randomly placed throughout the stream leading into the main lake

area. Only mature male fish were collected so they would be receptive to mating. Male fish were determined

as mature if the total length was about 25mm. Female sailfin mollies signal their receptivity to mating upon

maturity and also during a two to three day period after releasing a brood (Ptacek and Travis 1997; Farr and

Travis 1986; Travis 1989). Male mollies also display a higher number and duration of courtship displays when

the female is receptive (Farr and Travis 1986). Studies have shown that nonreceptive females do not exercise

any preference given a choice of males and have high rates of apathy (Ptacek and Travis 1997). Therefore,

only gravid females were collected and maintained until releasing her brood and only receptive females were used

in the experiment to increase rates of female preference.

Female fish were kept in 10-gallon aquaria divided into three equal parts by plastic dividers. One female was kept

in each section so I could detect when she gave birth. Male fish were kept separately in 10-gallon aquaria

according to their place of origin. Fish were kept at 6ppt salinity on a 12-121ight dark cycle. They were fed

TetraMin tropical flake food once or twice a day.


Gonopodium length (t=1.388. df=18, p=0.091) and standard length (t=0.338, df=18, p=0.369) did not
differ significantly between LA males and TB males. The total time the female spent with the male was not related
to gonopodium length (F=2.143, df=1, p=0.165 i.e. slope was not different from zero), and did not differ
between the sites (and (F=2.149, df=l, p=0.165, respectively). Total time each male spent courting the female
was also not related to gonopodium length (F=2.090, df=l, p=0.170), and did not differ between the two sites
(and F=0.713, df=l, p=0.413, respectively). Female mollies were not affected by the size of the males in her
choice of mate (F=0.491, df=1, p=0.494). Male molly courting time did not vary with the males' SL (F=1.001,
df=l, p=0.332).

The female mollies spent on average 36% of the total observation time (t=l, df=9, p=0.172, Fig 1) in the
proximity of the Lake Alice (LA) males and 45% with the Tumblin Creek (TB) males. The LA male mollies spent
an average of 24% and TB male mollies spent 35% of the observation time courting the females (t=1.242,
df=9, p=0.123, Fig. 2). Neither of these differences was significant.








TT female with LA male TT female withTB male
Figure 1. The total time the female spends with each male and standard deviation.


500000 -




TT LA male courting TT TB male courting
Figure 2. The total time the male spends courting the female and standard deviation.


While the results do not show a significant difference in directionality of female choice, there was a tendency for

the female to choose TB males. The male mollies from Tumblin creek, the reference site, also tend to exhibit

more courting behavior and for longer periods of time. These "tendencies" support the hypothesis that females

will choose fish from the non-contaminated site and contaminated males will show decreased courting behavior.

The lack of significance could in fact be due to the small sample size; thus the interpretation of these results are

open for further experimentation and no definite conclusions can be made.

While this experiment was inconclusive, results are consistent with other studies that show the decrease of

sexual behavior when exposed to estrogens. Adult male guppy (Poecilia reticulata) mating behavior, quantified

by duration and number of sigmoidal displays, decreased after the fish were exposed to a natural estrogen or to

a xenoestrogen (Bayley et al, 1999). Baatrup and Junge (2001) exposed the adult male guppy to the

antiandrogens, p,p'-DDE, flutamide and vinclozolin, and found that male sexual behavior, specifically

sigmoidal display and posturing behavior had decreased in duration. On the contrary, similar exposure

experiments during development of the male guppy saw an increase in sexual behavior (Toft and Baatrup,

2003) although the explanation for this converse effect is unclear. Toft and Baatrup (2003) suggested that

as androgens must be converted to estrogens in the brain to regulate sexual behavior, the increase in

sexual behavior could be due to altered neural development or other unknown estrogenic effects. There are no

other studies in the literature resulting in an increase in sexual behavior as known. Variation in the duration

and total time courting is higher in the LA males than in the TB males, suggesting that there is another

mechanism affecting these behavior, possibly timing of exposure. Further experiments are needed to

determine whether this variation is due to insufficient sample size or if there are other biologically

important influences.

Careful selection of the fish by size proved to be successful. The lack of difference between sampled sites in

standard length (SL) and gonopodium length suggests that we were successful in matching the males for body

size. Additionally, the females' choice was not influenced by SL, suggesting that the male pair was

appropriately matched to the females' SL. The lack of a difference in gonopodium length between the two sites

could possibly be a result of only selecting males that appeared to be mature. In mosquitofish (Gambusia

affinis), gonopodium length is set at maturity and does not experience growth even as the body may continue

to grow larger (Turner, 1941; Angus et al, 2005), and it is likely the gonopodium is also set at maturity in

sailfin mollies. The fact that there was no difference in gonopodium length between the influenced site and

the reference site was actually contrary to expectations. Exposure studies of 17p- estradiol and 17a-

ethynlyestradiol during the development of poeciliids, show elongation of the gonopodium (Doyle and Lim,

2002; Angus, et al, 2005; Toft and Baatrup, 2003).

Although sailfin mollies are common within North Florida, the accessible areas for collection were difficult to

find, resulting in lack of site-type replication. Future studies should find more contaminated and

uncontaminated sites to produce replication within the experiment and increase sample size.

This study may be broadened in scope to include assessment of more male reproductive behavior. Of the

three common sexual behavior, only sigmoidal displays were measured, so decreased (or increased) display of

the other two behavior, gonopodial thrusting and gonoporal nibbling, has yet to be assessed in

estrogen contaminated mollies for sailfin mollies. Also quantification of estrogens found in both reference

and influenced sites would shed light on how the fish are physiologically affected.

Given the considerable amount of literature on the adverse effect of EDCs on development and reproduction

of wildlife, it is important to continue to develop biomarkers so that we may quickly evaluate the potency of

EDC concoctions within our water systems.


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of male western mosquitofish (Gambusia affinis). Comparative Biochemistry and Physiology, Part C 140 330 - 339.

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guppy (Poecilia reticulata) Environmental Health Perspectives. Oct. 109 (10): 1063-1070.

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