Larval exposure to environmentally relevant mixtures of alkylphenolethoxylates reduces reproductive competence in male fathead minnows

Aquatic Toxicology 79 (2006) 268–277

Larval exposure to environmentally relevant mixtures of alkylphenolethoxylates reduces reproductive competence in male fathead minnows Travis J. Bistodeau a , Larry B. Barber b , Stephen E. Bartell a , Roberto A. Cediel a , Kent J. Grove a , Jacob Klaustermeier a , Janet C. Woodard a , Kathy E. Lee c , Heiko L. Schoenfuss a,∗ a

Department of Biological Sciences, Saint Cloud State University, 720 Fourth Avenue South, St. Cloud, MN 56301, United States b U.S. Geological Survey, 3215 Marine Street, Boulder, CO 80303, United States c U.S. Geological Survey, 2280 Woodale Drive, Mounds View, MN 55112, United States Received 8 April 2006; received in revised form 22 June 2006; accepted 23 June 2006

Abstract The ubiquitous presence of nonylphenolethoxylate/octylphenolethoxylate (NPE/OPE) compounds in aquatic environments adjacent to wastewater treatment plants (WWTP) warrants an assessment of the endocrine disrupting potential of these complex mixtures on aquatic vertebrates. In this study, fathead minnow larvae were exposed for 64 days to a mixture of NPE/OPE, which closely models the NPE/OPE composition of a major metropolitan WWTP effluent. Target exposure concentrations included a total NPE/OPE mixture load of 200% of the WWTP effluent concentration (148 ␮g/L), 100% of the WWTP effluent concentration (74 ␮g/L) and 50% of the WWTP effluent concentration (38 ␮g/L). The NPE/OPE mixture contained 0.2% 4-t-octylphenol, 2.8% 4-nonylphenol, 5.1% 4-nonylphenolmonoethoxylate, 9.3% 4nonylphenoldiethoxylate, 0.9% 4-t-octylphenolmonoethoxylate, 3.1% 4-t-octylphenoldiethoxylate, 33.8% 4-nonylphenolmonoethoxycarboxylate, and 44.8% 4-nonylphenoldiethoxycarboxylate. An additional exposure of 5 ␮g/L 4-nonylphenol (nominal) was conducted. The exposure utilized a flow-through system supplied by ground water and designed to deliver consistent concentrations of applied chemicals. Following exposure, larvae were raised to maturity. Upon sexual maturation, exposed male fish were allowed to compete with control males in a competitive spawning assay. Nest holding ability of control and exposed fish was carefully monitored for 7 days. All male fish were then sacrificed and analyzed for plasma vitellogenin, developmental changes in gonadal tissues, alterations in the development of secondary sexual characters, morphometric changes, and changes to reproductive behavior. When exposed to the 200% NPE/OPE treatment most larvae died within the first 4 weeks of exposure. Both the 100% and 50% NPE/OPE exposures caused a significant decrease in reproductive behavior, as indicated by an inability of many of the previously exposed males to acquire and hold a nest site required for reproduction. In contrast, the 5 ␮g/L 4-nonylphenol exposure resulted in significantly enhanced reproductive behavior compared to that of control males and a majority of the nesting sites were held by previously exposed males. No significant change in the development of gonadal tissues was observed. The 100% NPE/OPE exposure resulted in a significant reduction in the gonadal somatic index and in the prominence of secondary sexual characteristics of exposed larvae. This study indicates that NPE/OPE mixtures have an effect on the reproductive competence of previously exposed male fathead minnows. In addition, 4-nonylphenol concentrations utilized in all exposures were below regulatory guidelines, suggesting that evaluation of 4-nonylphenol alone may not be sufficient for identifying potentially adverse effects of this suite of compounds usually found as mixtures in the aquatic environment. © 2006 Elsevier B.V. All rights reserved. Keywords: Alkylphenolethoxylates; Nonylphenolethoxylates; Octylphenolethoxylate; Fathead minnow; Reproduction; Wastewater treatment plant effluent

1. Introduction The potential for wastewater treatment plant (WWTP) effluent to alter normal endocrine function in aquatic organisms is

∗

Corresponding author. Tel.: +1 320 308 3130; fax: +1 320 308 4166. E-mail address: [email protected] (H.L. Schoenfuss).

0166-445X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2006.06.015

well established and has been attributed to natural and synthetic estrogens as well as estrogen mimics. Exposure to WWTP effluent has been shown to induce vitellogenin in wild male fish (Purdom et al., 1994; Sumpter and Jobling, 1995; Harries et al., 1996, 1997) and to alter normal gonad development including induction of intersex (Harries et al., 1997; Jobling et al., 1998). The quality of gametes produced by intersex fish identified in wild populations also is significantly reduced (Jobling et al.,

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2002). Although the results described above suggest reduced reproductive competence in male fishes exposed to estrogenic compounds, understanding how these compounds affect wild fish populations is critical in our assessment of endocrine disruption. Among the estrogenic compounds found in WWTP effluent, the nonylphenolethoxylates and octylphenolethoxylates (NPE/OPE) are especially prominent due to their concentrations (usually in the ␮g/L range) and their ubiquitous presence in WWTP effluent and surface waters (Ahel and Giger, 1985; Naylor et al., 1992; Bennie et al., 1997; Barber et al., 2000; Kolpin et al., 2002). Mixtures of long-chained and short-chained NPE/OPE are widely used as surfactants in industrial and commercial detergents with the longer chained NPE/OPE (10–20 ethylene oxide, EO, units) having greater surfactant qualities and the shorter chained NPE/OPE (1–4 EO units) being more estrogenic (Jobling and Sumpter, 1993; Jobling et al., 1996). In 1990, alkylphenol polyethoxylate production in the United States exceeded 200 million kg, 60% of which is estimated to be discharged to the aquatic environment via WWTP effluent (Naylor et al., 1992; Talmage, 1994). Concentrations of these compounds in treated wastewater effluent varies widely between effluents and temporally within an effluent (Ahel et al., 1994a; Barber et al., 2000). In river waters, concentrations of NPE/OPE are usually below 1 ␮g/L (Naylor et al., 1992; Ahel et al., 1994b) but can be as high as 4 ␮g/L for nonylphenol (NP), 17 ␮g/L for nonylphenol monoethoxylates (NP1EO), and 45 ␮g/L for nonylphenol monoethoxycarboxylates (NP1EC) in selected rivers (Ahel et al., 1987). Not surprisingly, concentrations of NPOs are also variable but generally higher in sewage effluents with values for NP reported to be in the range of 10–200 ␮g/L by Ahel et al. (1994b). Concentrations for NPEOs and NPECs are also elevated in a ratio to NP that is comparable to that ratio found in river water samples. Octylphenol (OP) is usually found in concentrations below those measured for NP (Barber et al., 2000; Kolpin et al., 2002). In order to develop a mixture of NPE/OPE for the exposure of larval fathead minnow, we elected to use NPE/OPE concentrations reported by Barber et al. (2000) for a major metropolitan wastewater treatment plant on the Upper Mississippi River. Effluent from this plant has a history of eliciting estrogenic responses (vitellogenin induction in male fish) in feral and wild fishes (Folmar et al., 1996, 2001) and has been studied extensively (Schoenfuss et al., 2002). In addition, concentrations for NPE/OPE compounds in this treated wastewater effluent fall within the range of reported values in other studies and in other effluents (see above) thus providing broad applicability of our results. Exposure of fish to NPE/OPE has been shown to alter normal endocrine function (Ankley et al., 1998) including vitellogenin synthesis in male fish (Jobling and Sumpter, 1993; Jobling et al., 1996), inhibition of normal growth of testicular tissues (Jobling et al., 1996), pathological changes to reproductive tissues (Miles-Richardson et al., 1999), and reduction in prominence of secondary sexual characters and decreases in the number of spawning events (Harries et al., 2000). Of particular concern is the degradation of the parent NPE surfactants within sewage treatment works into the shorter chained NPE (Giger et

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al., 1984), which are more estrogenic (Jobling et al., 1996). The final stable degradation product NP is often the only compound utilized in NPE/OPE studies and is the only NPE/OPE currently (2006) under regulatory scrutiny. The U.S. Environmental Protection Agency has set a 4-day average concentration limit not to exceed 6.6 ␮g/L NP as a chronic toxicity water quality criterion (USEPA, 2005). However, the criterion does not address the effects of other NPE/OPE species, such as 4-t-octylphenol (OP), which is a potent estrogen mimic (Jobling et al., 1996), nor the effects of NPE/OPE mixtures usually observed in WWTP effluents (Barber et al., 2000). Larvae may be particularly susceptible to endocrine disruption, because early life cycle development is highly regulated by hormones of the endocrine system (Janz and Weber, 2000). Although the most sensitive period of development appears to be species specific, long term early life-cycle research has demonstrated that exposure to ethinylestradiol, estradiol, and endogenous androgens can cause vitellogenin synthesis (Lange et al., 2001; Van Aerle et al., 2002), altered sex ratios (Nimrod and Benson, 1998), and abnormal gonad tissue development (Van Aerle et al., 2002) during these liable periods. Larval medaka exposures to NP can alter sex ratios and result in intersex fish upon maturation (Gray and Metcalfe, 1997). Similarly, larval medaka exposures to OP can cause detrimental changes in mating behavior and a decrease in fertilization of eggs (Gray et al., 1999). These results indicate that individual NPE/OPE compounds possess the ability to alter endocrine function in fish larval populations, yet their combined action remains unclear. Unfortunately, linking effects observed in laboratory studies to decline in reproductive competence of exposed wild fish has proven difficult. Utilizing reproductive competence, the ability to successfully compete for reproductive resources, may be one way in which to complement laboratory studies to gain a better understanding of the reproductive effects of exposure in the aquatic environment. Reproductive competence is an endpoint that encompasses many endpoints used in endocrine disruption research. The fathead minnow (Pimephales promelas) provides a particularly attractive laboratory model species to test the effects of NPE/OPE exposure on reproductive competence. Previous studies have sought to evaluate estrogenic effects on fathead minnow reproductive performance by quantifying egg production (Harries et al., 2000). However, by evaluating the reproductive competence through male reproductive competition, it is possible to assess the effects of estrogenic compounds on male fathead minnows and to eliminate egg production as a potentially confounding factor. Male fathead minnows practice paternal nest care, which requires the male fish to obtain and defend a nest site, attract a female, and successfully guard the developing larvae until hatching (Unger, 1983). The inability to acquire and vigorously defend a nest site until larvae hatch will prevent most males from reproducing, thus making the nest site an indispensable reproductive resource. The aggressive behaviors employed in nest site defense are generally considered under the control of androgens, whose down regulation in the presence of estrogens (Trudeau et al., 1993) could reduce aggressive behaviors needed to defend a nest site. In this context, secondary sexual

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characters are an important component of nest care and defense (Smith, 1978; Unger, 1983), which also appear to be under the control of the endocrine system (Miles-Richardson et al., 1999; Harries et al., 2000). Based on previous studies that identified the mixture composition of several NPE/OPE compounds in WWTP effluent (Barber et al., 2000), the study described in this paper assessed the effects of APE/OPE mixtures on male reproductive competence in fathead minnows. The objectives of this study were to (1) determine whether larval exposure to NPE/OPE mixtures adversely affect the reproductive competence of mature male fathead minnows and (2) to determine whether NP accounts for any observed adverse effects. 2. Material and methods In this study, we investigated the effects of NPE/OPE mixtures and NP singularly on the reproductive competence of male fathead minnows exposed as larvae and reared to maturity. In a series of four exposure experiments (carried out between August 2004 and August 2005), larval fathead minnows were exposed from 3 to 67 days post-hatch to environmentally relevant mixtures (Barber et al., 2000) of NPE/OPE (50%, 100%, or 200% of the NPE/OPE mixture concentrations detected in WWTP effluent) or NP singularly (Table 1). Upon completion of the exposures, fish were allowed to mature to 6 months of age before being sorted by sex. Males from the NPE/OPE mixture or NP exposures were then paired individually with control males from the same experiment and allowed to compete for access to females and a nest site for 7 days. After the competitive assay was completed, fish were analyzed for indications of endocrine disruption including elevated plasma vitellogenin, reduced secondary sexual characters, decreased gonad size, and testis histopathology. 2.1. Chemicals and exposure Water samples from the effluent channel of the Metropolitan Environmental Services Wastewater Treatment Plant in St. Paul, MN were analyzed by gas chromatography/mass spectrometry for NP, NP1EO (4-nonylphenolmonoethoxylate), NP2EO (4-nonylphenoldiethoxylate), NP1EC (4-nonylphenolmonoethoxycarboxylate), NP2EC (4-nonylphenoldiethoxy-

Table 1 Fish exposure experiments and nominal dosing concentrations for the nonylphenolethoxylate/octylphenolethoxylate (NPE/OPE) mixture and nonylphenol (NP) only exposures and total NP concentrations utilized in each exposure Experiment name

Total NPE/OPE concentrationa (␮g/L)

Total NP (␮g/L)

200% NPE/OPE 100% NPE/OPE 50% NPE/OPE NP

148 73.9 38.1 5

4.2 2.1 1.1 5.0

a NPE/OPE mixture consists of NP, NP1EO, NP2EO, NP1EC, NP2EC, OP, OP1EO, and OP2EO in a ratio of 2.8:5.1:9.3:33.8:44.8:0.2:0.9:3.1, respectively.

carboxylate), OP, OP1EO (4-t-octylphenolmonoethoxylate), OP2EO (4-t-octylphenoldiethoxylate) content (Barber et al., 2000), and indicated a total NPE/OPE concentration of 74–152 ␮g/L. Based on these analyses, a synthetic NPE/OPE mixture of the above compounds, in similar concentrations and proportions as those found in WWTP effluent, was prepared at a total concentration of 74.9 ␮g/␮L (0.2% OP, 2.8% NP, 5.1% NP1EO, 9.3% NP2EO, 0.9% OP1EO, 3.1% OP2EO, 33.8% NP1EC, 44.8% NP2EC) in ethanol. The concentration and composition of this solution was verified prior to the experiments. The APE/OPE chemicals were obtained from Aldrich Chemical (St. Louis, MO), Cambridge Isotope Laboratories (Cambridge, MA), and Schenectady International Inc. (Schenectady, NY). Twenty-five identical 1 mL aliquots of NPE/OPE mixture stock solutions were prepared in 100% ethanol for each of the three exposure concentrations (Table 1) prior to the exposure experiments and stored at 4 ◦ C until use. For all treatments, fresh aqueous dosing solutions were prepared every 3 days by adding one of the 1 mL aliquots of the appropriate concentration stock solution to 4 L of distilled water in an amber glass bottle containing a Teflon stir-bar. The final aqueous NPE/OPE mixture stock solution concentrations were 16.2, 8.1, and 4.1 mg/L for the 200%, 100%, and 50% NPE/OPE treatments. The concentration of the aqueous NP stock solution was 0.54 mg/L. The solvent concentration did not exceed 2.5 ␮g/L ethanol, well below solvent concentrations used in previous experiments (Schoenfuss et al., 2002). After addition of the aliquot, each amber bottle was gently agitated for 10 s, the neck of the bottle was covered with aluminum foil, and the bottle was placed on a magnetic stir plate. A stainless steel tube was used to draw the stock solution into the treatment aquaria. The stock solution was stirred continuously, and mixed with the ground water inflow (0.1 L/min) to the aquaria at a nominal rate of 0.0009 L/min using a ColePalmer Masterflex 7523-40 peristaltic pump (Vernon Hills, IL), thus achieving approximately seven water exchanges each day. Although we did not measure aquarium concentrations of the exposure compounds, previous studies showed that the continuous input of a quantified stock solution through a flow through system, coupled with monitoring of the well water flow rates, produced concentrations approximating the nominal values. 2.2. Exposure animals Fathead minnow larvae (<72 h post-hatch) were purchased from Environmental Testing and Consulting (Superior, WI) and were used across all treatments except for the 100% NPE/OPE exposure, which used larvae from the F1 generation of mature minnows previously purchased from the same supplier and reared at the St. Cloud State University Aquatic Toxicology Laboratory. In each experiment (one non-dosed ground water control, four dosed ground water exposures) approximately 100–125 larvae were placed into an aerated 20 L all-glass aquaria with continuous ground water flow (0.1 L/min), temperature was maintained at 24–25 ◦ C, and photoperiod was held constant at 16 h light:8 h dark. An attempt was made to count equal numbers of animals into the control and treatment aquaria; however, this

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was not tested as the fragility of the larvae prevented their close examination. Exposure commenced on the third day post-hatch and continued for 64 days. Larvae were fed hatched brine shrimp (Artemia franciscana, San Francisco Bay Brand Inc., Newark, CA) and goldfish flake food (Omega Sea, Sitka, AK) ad libitum for 90 days post-hatch. In addition, a small quantity of whole adult Sally’s Frozen Brine Shrimp (San Francisco Bay Brand Inc., Newark, CA) was introduced to the juvenile diet starting at 54 days post-hatch. After 90 days, fish were fed only frozen brine shrimp and flake food. 2.3. Exposure overview

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reliable results were achieved once larvae were approximately 30 days post-hatch. Adult survival numbers are based on the number of fish that were analyzed approximately 200 days post-hatch. 2.4.2. Sex ratio All adult fish were dissected to determine sex. Visually perceived males used in the competitive spawning assays were processed for histology, while the remaining fish (mostly perceived females) were dissected and sexed based on the gross morphological appearance of their reproductive organs. Fish with distinguishable oocytes were counted as female fish.

Experiments were conducted in a facility supplied by nonchlorinated ground water. All exposures utilized a continuous flow-through system, designed to maintain constant concentrations of the exposure chemicals throughout the experiment. The total nominal NPE/OPE mixture concentration in the 50% exposure was 38 ␮g/L, in the 100% exposure was 74 ␮g/L, and in the 200% exposure was 148 ␮g/L. Water chemistry was monitored for ammonia, pH, and temperature. All exposures, with the exception of the 200% NPE/OPE treatments, lasted 64 days. Exposed fish in the 200% NPE/OPE treatment experienced mortality beyond 95% in the first 3 weeks of exposure and the experiment was terminated after 28 days. This exposure was repeated with similar results and the experiment was terminated after 23 days. Following 64 days of exposure, larvae of the remaining three experiments were moved to clean, 30 L aquaria fed with ground water and raised to maturity (approximately 200 days). Although it was not possible to maintain constant fish density for the duration of the experiment, comparable mortality rates in control and treatment aquaria (with the exception of the terminated 200% NPE/OPE experiment) eliminated a density-based intra-experimental bias.

2.4.3. Vitellogenin analysis After fish were deeply anaesthetized in 1% 2-phenoxyethanol (Sigma, St. Louis, MO), the tails of male fish were severed to harvest blood using a capillary tube. After blood was extracted, it was immediately centrifuged to isolate plasma. Whenever possible, two aliquots of plasma were collected, placed on ice and transferred to a −80 ◦ C freezer until analysis. A commercially available indirect sandwich enzyme-linked immunosorbant assay (ELISA), specific to the vitellogenin protein molecule synthesized by carp (Cyprinus carpio), was utilized in the vitellogenin analysis (Biosense Laboratories, Bergen, Norway). Substantial cross reactivity exists between carp and fathead minnow vitellogenin and resulting antibodies. Using this assay, vitellogenin content of exposed male fish was compared to the vitellogenin content of control male fish within each experiment.

2.4. Endpoints

2.4.5. Histopathology Following removal, gonads were fixed in Bouin’s solution for 24 h (Gabe, 1976). After fixation, tissues were dehydrated in a series of ethanol and toluene baths before being embedded in paraffin. Embedded tissues were sectioned at approximately 1/3 and 2/3 of the length of the testis using a Reichert-Jung cassette microtome (4–6 ␮m sections). Sectioned tissues were stained using a standard hematoxylin and eosin counter stain protocol modified after Gabe (1976). Histological slides were visually inspected by an experienced histologist for the simultaneous occurrence of ovarian and testicular tissues or any other pathological alterations to gonadal tissues.

A series of biological endpoints were measured in all four exposure experiments to determine whether male fathead minnows suffered reduced reproductive competence as a result of larval exposure. The endpoints include larval survival both during the exposure and during growth to maturity, sex ratio of mature fathead minnows, plasma vitellogenin concentrations in adult males, relative weight of the male reproductive organs (gonadal somatic index, GSI), histopathological analysis of the testis, evaluation of secondary sexual characteristics, and assessment of ability to compete for reproductive opportunities. With the exception of larval survival, all endpoints were evaluated in male fish upon sexual maturity. 2.4.1. Survival Digital photographs were taken of each aquarium throughout the exposure period, using a Nikon Coolpix 995 digital camera (Nikon Corp., Melville, NY). Digital images were magnified to 200% and analyzed using Microsoft Picture Manager software (Redmond, WA) to count surviving larvae. An initial test of this method was conducted to ensure repeatability of the count and

2.4.4. Gonadal somatic index Whole body weights were measured for each male fish at the time of analysis. Gonads were excised from each fish and immediately weighed. Gonad and whole body weights were used to calculate the GSI (GSI = gonad weight/whole body weight).

2.4.6. Secondary sexual characters After the gonads were excised from each fish, the remaining carcass was stored in Bouin’s solution until the secondary sexual character analysis could be completed. Upon sexual maturation male fathead minnows darken in color, develop a thickened dorsal pad, and display tubercles just above the mouth (Smith, 1978). These characters were analyzed using a simple, blind scoring system similar to that described by Smith (1978). Each fish was evaluated by three laboratory technicians who received

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previous training in this analysis technique. The prominence of the tubercles was graded on a scale of 0–3. The dorsal pad was evaluated by a similar method and graded on a scale of 0–3. 2.4.7. Competitive spawning assay Once the entire larval population appeared to be mature (approximately 6-month old), males were identified by the appearance of secondary sexual characters. Both the exposed and treated males received small caudal fin clips (a corner of either the superior or inferior portion of the caudal fin was removed with scissors) so that observations could distinguish fish from the two treatments. These fin clips alternated between the top and bottom of the caudal fin and between treatments to create “blind observations”, where the observer was unaware of the fish’s exposure history. In addition, alternating fin clips avoided any bias between control and exposed males as both groups received similar numbers of upper and lower fin clips. One treated male and one control male of comparable size (judged only visually, as it was important for the integrity of the experiment to avoid extended periods of stress for the fish) were then simultaneously placed into a 7 L all-glass aquarium. Each of these spawning aquaria contained two mature females and a nest site, made of a short section of 8 cm diameter stainless steel pipe cut in half. Twice daily (between 8 and 10 a.m. and 2 and 4 p.m., respectively), for the following 7 days, the nest holding male was identified by its respective fin clip. A nest holding male was defined as one that exhibited aggressive behavior towards other fish in the aquarium, while clearly protecting the nest site. This behavior typically includes butting, using the newly formed tubercles, and chasing (Unger, 1983). Based on the number of visually identifiable males in each experiment, seven competitive spawning scenarios were established for the 100% NPE/OPE experiment, 10 for the 50% experiment, and 24 for the NP experiment. For the analysis of nest holding, each nest site was observed twice daily for approximately 2 min to determine whether a male actively defended a nest site and, when applicable, to identify the caudal fin clip position of the nest holding male. Each observation of a male fish defending a nest site was scored as a nest holding event.

3. Results 3.1. Survival Larval survival remained relatively high throughout the exposure period, with the exception of the 200% NPE/OPE exposures (Fig. 1A). After 28 days in the first 200% NPE/OPE exposures, 98% of the larvae had died. This exposure was duplicated shortly thereafter, and a similar trend was observed. Among the remaining three experiments, survival did not differ by more than 8% between each of the exposures and the corresponding control populations (Fig. 1B–D) although there was greater mortality in both the control and treatment of the 100% NPE/OPE experiment (Fig. 1B) than in the 50% NPE/OPE or NP experiments. Over 75% of juveniles in all treatments that had survived exposure reached sexual maturity. Water temperature, pH, ammonia, and dissolved oxygen concentrations were measured weekly throughout the experiments in all treatments and were found to be nearly constant; temperature = 25 ± 0.09 ◦ C (mean ± standard error), pH 7.7 ± 0.02, ammonia = 0.05 ± 0.005 mg/L, and dissolved oxygen = 4.5 ± 0.11 mg/L. 3.2. Sex ratio Sex ratios were approximately 1:1 (male:female) among all treatments and experiments (data not shown), except for the NP only exposure where the sex ratio favored male fish at a 5:3 ratio. It is noteworthy that up to half the males in each exposure aquarium did not display clear secondary sexual characters and could only be sexed in the course of the dissections. As a result, the number of competitive spawning scenarios was lower than would be predicted by the overall mortality in each experiment. 3.3. Vitellogenin analysis Across all treatments, vitellogenin induction above the detection limit was less than 10% for the male fathead minnows in the competitive spawning scenarios (data not shown). The small number of vitellogenin induced males precluded meaningful statistical analysis.

2.5. Statistical analysis

3.4. Gonadal somatic index

All data were assessed for normality prior to any additional analysis using the Prism 4.01 statistical package (GraphPad Software Inc., Oxnard, CA). Data were analyzed using a Student t-test. The nest holding abilities of the exposed and control males in the competitive spawning assays were assessed using a Fisher’s Exact Test (contingency table). Reliability among secondary sexual character scoring was evaluated using a Cronbach’s Alpha test (SPSS for Windows, Release 11.0.0, Standard Version, SPSS Inc., 1989–2001, Chicago, IL, or SYSSTAT 11 for Windows, version 11.00.01 SYSTAT Software Inc., 2004, Richmond, CA). If found reliable, scores from all three observers were averaged and analyzed using a Student t-test. A probability of <0.05 was set as level of significance for all comparisons.

The mean GSI of males exposed as larvae to the 100% NPE/OPE treatment was significantly (p = 0.05 Student ttest, n = 13) smaller than the mean GSI of concurrently reared control males. The 100% NPE/OPE exposure GSI was 0.01 ± 0.002 (mean ± standard error) whereas the control GSI was 0.02 ± 0.002 (Fig. 2A). In the 50% NPE/OPE and the NP only treatments, no significant difference in mean GSI was detected between exposed and control larvae (Fig. 2B and C). 3.5. Histological analysis All of the male fish that were analyzed possessed fully developed and mature gonads. The testicular tissues of all males,

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Fig. 1. Survival rates past 200 days post-hatch for (A) 200% nonylphenolethoxylate/octylphenolethoxylate (NPE/OPE) exposure experiments (squares indicate first replicate, triangles second replicate), (B) 100% NPE/OPE exposure experiment, (C) 50% NPE/OPE exposure experiment, and (D) 4-nonylphenol (NP) only exposure experiment. Solid symbols indicate controls, open symbols indicate respective treatment.

regardless of exposure history, appeared normal. No simultaneous occurrence of ovarian and testicular tissues was noted. Gonadal tissues were free of pathological alterations across all treatments.

(Fig. 3B and E) and the NP only treated males (Fig. 3C and F) did not differ significantly from the corresponding controls.

3.6. Secondary sexual characters

In all three experiments where fish survived to maturity, the competitive spawning assay demonstrated significant changes in the exposed fish’s ability to acquire or defend a nest site from control fish. Fish exposed to the 100% NPE/OPE treatment possessed a reduced ability to hold and defend nest sites (exposed = 23 sites held, control = 48 sites held, n = 71, p = 0.015, Fisher’s Exact Test, Fig. 4A). Similarly, a reduced ability to hold and defend nest sites, was observed for fish exposed to the 50% NPE/OPE treatment (exposed = 56 sites held, control = 79 sites held, n = 135, p = 0.0073, Fisher’s Exact Test, Fig. 4B). In contrast to the 100% and 50% NPE/OPE

The prominence of the tubercles of the 100% NPE/OPE exposed males was significantly (p = 0.024 Student t-test, n = 13) reduced. The 100% NPE/OPE exposure tubercle score was 0.9 ± 0.2 (mean ± standard error) whereas the control tubercle score was 2.08 ± 0.4 (Fig. 3A). A similar reduction (p = 0.002 Student t-test, n = 13) was observed for the dorsal pad in these fish with a 100% NPE/OPE exposure dorsal pad score of 1.14 ± 0.2 and a control dorsal pad score of 2.5 ± 0.3 (Fig. 3D). The secondary sexual characteristics of the 50% NPE/OPE

3.7. Competitive spawning assay

Fig. 2. Gonadal somatic index (GSI) for the (A) 100% nonylphenolethoxylate/octylphenolethoxylate (NPE/OPE) exposure experiment, (B) 50% NPE/OPE exposure experiment, and (C) 4-nonylphenol (NP) only exposure experiment. Whiskers indicate data range, the box indicates the 25th and 75th percentile of data, and the center line indicates the median. Only the 100% NPE/OPE exposure was significantly different from control fish (* p < 0.05).

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Fig. 3. Secondary sexual characteristics scores for (A) tubercle results for the 100% nonylphenolethoxylate/octylphenolethoxylate (NPE/OPE) exposure experiment, (B) tubercle results for the 50% NPE/OPE exposure experiment, (C) tubercle results for the 4-nonylphenol (NP) exposure experiment, (D) dorsal pad results for the 100% NPE/OPE exposure experiment, (E) dorsal pad results for the 50% NPE/OPE exposure experiment, and (F) dorsal pad results for the NP only exposure experiment. Whiskers indicate data range, the box indicates the 25th and 75th percentile of data, and the center line indicates the median. Only the 100% NPE/OPE exposure was significantly different from control fish (* p < 0.05).

Fig. 4. Competitive spawning results for (A) the 100% nonylphenolethoxylate/octylphenolethoxylate (NPE/OPE) exposure experiment, (B) the 50% NPE/OPE exposure experiment, and (C) the 4-nonylphenol (NP) only exposure experiment. Bars indicate the percentage of nest sites held by either the control fish or the treated fish in each assay (* p < 0.005).

treatments, the NP only exposed fish were more likely to hold and defend nest sites when competing with control males (exposed = 165 sites held, control = 125 sites held, n = 290, p = 0.0012, Fisher’s Exact Test, Fig. 4C). It is noteworthy that only in the NP experiment a number of males holding nest sites lost control of the site during the competitive spawning assay and were forced out by the competing male (5 of 24, three from control to treatment; two from treatment to control). In all of the other experiments nest sites were acquired by one male within 24 h of the introduction of the males into the spawning scenario and consistently defended. 4. Discussion In this study, we investigated the effects of a mixture containing nonylphenolethoxylates and octylphenolethoxylates at concentrations and proportions similar to those measured in the treated effluent of a major metropolitan WWTP. Exposure of larval fathead minnows to this NPE/OPE mixture during ontogenetically liable periods resulted in significantly altered development and behavior of mature male fish, which may reduce their reproductive competence. Larval exposure to NP alone at

environmentally realistic concentrations also altered the reproductive behavior of males by making them more competitive than their unexposed counterparts. The survival of larvae appeared to be more dependent on the total NPE/OPE concentration of the treatment than the concentration of NP alone in a particular treatment. In the 50% NPE/OPE and NP only treatments, larval survival remained relatively high throughout the entire period, while the high mortality of larvae in the 200% APE treatment, but not in the concurrent control, resulted in the termination of this experiment. This difference in survival correlates with the total NPE/OPE mixture loads, which were highest in the 200% treatment. In contrast, the NP concentration in the 200% NPE/OPE mixture treatment (4.2 ␮g/L) was actually lower than in the NP only treatment (5 ␮g/L), where survival did not differ from the control. The design of this study did not allow us to elucidate the mechanism that may explain the reduction in larval survival at higher NPE/OPE concentrations. Larval survival to the end of the exposure period was slightly lower in all experiments than reported in some other studies (i.e. Siwik et al., 2000), however, those experiments used static renewal and not flow-through systems. We speculate that the flow-through adds an additional

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stress on larval fathead minnows (i.e. introduction of turbulence, air bubbles) that might diminish the survival of the test animals. The nominal exposure concentrations used in this study were selected based upon concentrations found in the effluent of a modern secondary treatment facility on the Upper Mississippi River. At only twice the total concentration of NPE/OPE compounds measured at this site, larval survival was severely impaired. This result raises concerns about larval survival at other WWTP with higher NPE/OPE effluent loads (Barber et al., 2000). Furthermore, NPE/OPE loads in effluent are likely to experience seasonal variations, which might affect fish populations at critical junctures such as growth and reproduction. The unresolved question of temporal fluctuation in NPE/OPE output by WWTPs warrant further investigations into the prevalence of treated wastewater effluents with NPE/OPE outputs comparable to the one used as reference in this study. Although the ultimate goal of many toxicological studies is to understand the effects of compounds on the reproductive fitness and, by extension, population health of exposed wildlife, experimental constraints and necessities dictate the use of proxies to estimate these ultimate effects. In this study, we applied many of the biomarkers frequently used as proxies in endocrine disruption studies to gauge the reproductive consequences of exposure. However, as is frequently the case in studies in which organisms are exposed to environmentally relevant concentrations, the initial analysis of individual biomarkers can appear inconclusive. The GSI results from this study indicated that the control fish had significantly larger gonads than fish exposed to 100% of the NPE/OPE load they would encounter in the treated effluent. In addition, significant differences in secondary sexual characters between treated larvae and control larvae were detected in the 100% NPE/OPE mixture. The coupling of less prominent secondary sexual characters with reduced gonad size has been noted previously (Smith, 1978), and indicates the biological integrity of our results as well as the potential for reproductive consequences of the larval exposure. Although, male fathead minnows in the 50% NPE/OPE mixture treatment did not display reduced GSI or secondary sexual characteristics, they also suffered from reduced reproductive competence. The ability to compete successfully for reproductive resources encompasses many traditional biomarkers of endocrine disruption as it is the culmination of the entire life history of an animal. This competitive spawning assay was not designed, however, to measure reproductive success as this would entail accounting for the fertility of female fathead minnows, which varies widely (Denny, 1987). Reproductive competence did not correlate with plasma vitellogenin concentrations in mature male fish indicating that either vitellogenin was not induced in larval fish or was cleared from the blood stream during the 4 months between the larval exposure and the adult analysis. In contrast to the competitive spawning results identified in the 100% and 50% NPE/OPE mixture treatments, the competitive spawning results for the NP only treatment demonstrated a positive effect on the exposed males in direct competition with control males. In addition to the greater estrogenicity of

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the NPE/OPE mixture, the potency of mixture treatments might also be explained by its surfactant properties. The 200%, 100%, and 50% NPE/OPE mixture treatments contained a combination of the short-chain alkylphenolethoxylates (NP1EO, NP2EO, NP1EC, NP2EC, OP1EO, OP2EO) in addition to NP and OP, two of the most estrogenic alkylphenol polyethoxylates (Jobling et al., 1993; Jobling et al., 1996). Although less estrogenic than NP and OP, the short-chained alkylphenol polyethoxylates have surfactant properties that might facilitate the uptake of NP and OP. It is possible that the NP only exposure lacked the surfactant properties of the NPE/OPE mixture exposures necessary to facilitate delivery of NP, and as a result, less of the compound is able to reach the circulatory system of the organisms. The competitive spawning assay simulates a component of the selective pressures faced by wild fish during reproductive activity in that it allows for direct competition between males of differing exposure histories for the same reproductive resources (nest sites and females). As many species of fish migrate between environments, including spawning sites and wintering localities, interaction between fish of varying exposure histories is a likely occurrence. Although seldom explicitly acknowledged, the use of secondary sexual characters and reproductive behavior in previous studies (Bayley et al., 1999) imply the possibility that fish of varying exposure histories interact with each other. A gradual decrease in secondary sexual characters or reproductive behavior across a homogeneous cohort of fish would otherwise be of little consequence to the entire fish population. However, it remains to be investigated whether the decline in reproductive competence among a subset of fish in a population adversely affects the health of the entire population. In addition, any altered behavior due to anthropogenic chemicals could diminish the ability to avoid predation, thus indirectly reducing the reproductive success of the fish population. The nominal concentration of 5 ␮g/L utilized in the NP only experiment was selected to approximate the chronic toxicity ambient water quality criterion set by the EPA (USEPA, 2005). In this study, the results indicated that NP exposure at 5 ␮g/L has little effect upon larval survival or GSI, but enhanced competitive behavior. We have seen similar stimulation of estrogen mimic exposed adult male fish in previous experiments, and speculate that exposure to small concentrations of exogenous estrogen mimics might provide a competitive advantage to mature male fish maintained in male-only groups where estrogen release into the ambient water by ovulating female fathead minnows is absent. However, in this study, exposure far preceded the competitive spawning scenarios and fish were in mixed sex groups up to maturation. The long lasting effect of the exposure (albeit a stimulating effect in this experiment) suggests more fundamental modulations in the endocrine system of exposed fishes. In contrast to the stimulatory effect of NP exposure, larvae exposed to the 100% NPE/OPE treatment experienced a marked decline in their reproductive competence as measured by their ability to acquire and defend a nest site. This finding implies that the sole regulatory focus on NP may not be sufficient to assess the effects of NPE/OPE compounds, which are found in mixtures in WWTP effluents and the aquatic environment.

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