Recent evidence indicates that some of California’s coastal estuaries are contaminated with the chemical 4-nonylphenol (4-NP). Tissue burdens of 4-NP detected in the intertidal arrow goby (Clevelandia ios) in California are among the highest recorded worldwide, however, it remains unknown whether these fish are impacted by this 4-NP contamination. 4-NP is an established endocrine disrupting compound with estrogenic properties that can alter reproductive function. Furthermore, evidence that estrogens can modulate iono- and osmo-regulatory function in fishes implies that estuarine fishes exposed to 4-NP may also experience an impaired ability to maintain hydromineral balance. In Chapter 1 of this research, the time course of detectable xenoestrogen biomarker responses including gene transcripts encoding vitellogenins (vtgA and vtgC), choriogenins (chgL and chgHm), and estrogen receptors esr1 and esr2a were examined using quantitative real-time reverse transcription PCR (qRT-PCR) in adult male arrow gobies exposed to either 4-NP or E2. Specifically, adult gobies were treated with 4-NP at 10 μg/L (low 4-NP dose), or 4-NP at 100 μg/L (high 4-NP dose), ethanol vehicle (negative control), or 17β-estradiol (E2) at 50 ng/L (positive control) for 21 days. This 21 day exposure period was following by a 21 day depuration period to assess the time pattern of biomarker recovery. Results from these experiments indicated that 4-NP can induce increases in relative mRNA levels encoding vitellogenins, choriogenins, and estrogen receptor esr1 – but not esr2a – in the liver within 72 hrs, and that these transcriptional changes return to pre-exposure levels within 12 days of the termination of 4-NP or E2 exposure. In sum, these findings validate the use of mRNA levels for several estrogen-responsive genes as accurate biomarkers for short-term 4-NP exposure in the arrow goby. In Chapter 2, I evaluated the effects of 4-NP and E2 exposures on the osmoregulatory ability of C. ios. I exposed adult arrow gobies to 4-NP (10 μg/L or 100 μg/L) or E2 (50 ng/L) for 14 days, and then transferred the fish from a 33 ppt salinity (control) environment to either 20 ppt, or 5 ppt conditions. Whole body water content was then measured, and the relative mRNA levels for the ion channels Na+/K+/2Cl--cotransporter1 (nkcc1) and Na+/H+ exchanger-3 (nhe3), and the aquaporin water channel aquaporin-3 (aqp3) were quantified in the gill epithelium by qRT-PCR. Results showed that fish treated with 4-NP exhibited higher whole body water content, suggesting that 4-NP exposure results in excessive water uptake during hypoosmotic challenge. 4-NP treated gobies also exhibited elevated nkcc1 and reduced nhe3 and aqp3 mRNAs in the gill even prior to transfer of fish from the 33 ppt acclimation salinity. At 6 hrs after salinity transfer, transcripts encoding nkcc1 remained elevated in the gill epithelium of 4-NP treated gobies transferred to 20 ppt or maintained at 33 ppt (salinity control), while nhe3 and aqp3 mRNAs were still less abundant in gills of these fish. These findings point to impaired maintenance of water balance in gobies exposed to 4-NP, with those changes in fluid homeostasis possibly mediated in part by changes in gill ionic regulation. Taken as a entirety, the findings provided by this research reinforces accumulating data showing the potential for 4-NP to disrupt reproductive physiology in vertebrates, and points to the possibility that 4-NP may impair the ability of fish to regulate ion and water balance under changing salinity conditions.

Estrogenic contamination of waterways is of world-wide concern due to the adverse effects observed in aquatic biota. Recently, wastes from agricultural activities have been identified as likely sources of steroid estrogens released into the environment. Wastes from dairying activities are of particular concern in New Zealand. This project included development of analytical methods to measure free and conjugated estrogens, measurement of estrogens from the source to receiving environments and an investigation of effluent treatment technologies. The analytical method developed in this study was based on GC-MS measurement of free estrogens (17α-estradiol (17α-E2), 17β-estradiol (17β-E2) and estrone (E1)) and LC-IT-MS measurement of their sulfate-conjugates (17α-E2-3S, 17β-3S, E1-3S) in raw and treated farm dairy shed effluents (DSE). Effluents from farms in the Canterbury and Waikato Regions, two regions where dairy farming is the dominant land-use, were collected and analysed. All effluents demonstrated high concentrations of steroid estrogens, particularly 17α-E2 (median 760 ng/L). Estrogenic activity was also elevated, at up to 500 ng/L 17β-E2 equivalents using the E-Screen, an in vitro cell proliferation bioassay. Comparison to the chemical data indicated that for most samples, the highest proportion of estrogenic activity was derived from steroid estrogens naturally excreted by dairy cows. Conjugated estrogens were measured in several raw effluent samples, at similar concentrations to those of free estrogens, particularly E1. Dairy effluent treatment systems reduced free estrogen concentrations by 63-99% and reduced estrogenic activity by up to 89%. In spite of high removal efficiencies, estrogens remained elevated in the treated effluents that are discharged into waterways. Steroid estrogens and estrogenic activity were detected in streams and groundwater in areas impacted by dairy farming. Although concentrations were generally low, in two streams the concentrations were above levels regarded as safe for aquatic biota (<1 ng/L). The results demonstrate that dairy effluents are indeed a major source of estrogens to the environment and to waterways.

Evidence of feminising effects, including additional or enlarged female organs, have been reported in the (sub)-tropical freshwater prosobranch Marisa cornuarietis exposed to vertebrate estrogen receptor agonists. The primary symptom is an increase in the number of eggs laid, but this is only observed when exposure occurs at relatively low temperatures. This research project exposed temperate freshwater prosobranchs and a pulmonate to 17β-oestradiol (10-200 ngL-l, nominal) in an outdoor mesocosm subject to natural seasons to determine whether similar effects occur in European native temperate freshwater gastropods. Laboratory exposures to 17β-oestradiol (1-100 ngL-l, nominal) and Bisphenol-A (0.2-20 ngL-l, nominal) were also carried out over a range of different temperatures and photoperiods to simulate natural seasons. In the mesocosm exposures, significant increases in reproduction were measured in Viviparus viviparus, Bithynia tentaculata and, if the mortality rate was not significantly increased, Planorbarius corneus. It was observed that increases only occurred after the onset of autumn. In the laboratory, the oviposition rate in P. corneus was constant at 20oC with a 16h photoperiod, but declined significantly at 15oC with a 12h photoperiod, except at 100 ngL-l 17β-oestradiol, when the rate remained constant. There were no similar effects from Bisphenol-A exposure. Small increases in reproduction were observed in all the prosobranch exposures to both compounds, but the interpretation of the data was confounded by several factors (test chemical degradation, high mortality rates and parasitized organisms) and there were no significant differences. In conclusion, there were indications that all of the assessed species were capable of increased reproduction, and in P. corneus this appears to be a perpetuation of summer oviposition rates in autumnal conditions. The consequences of this effect in annual semelparous pulmonates may not be detrimental at the population level, but the long-term fitness of iteroparous prosobranchs that practice ‘restrained reproduction’ in early breeding seasons may be adversely affected.

For the past few years there has been increasing concern surrounding a group of chemicals known as phthalate esters. In mammals, phthalates are known antiandrogens, interfering with the production or activity of testosterone. Phthalates are ubiquitous in the aquatic environment. With recent findings suggesting that antiandrogens may be responsible for much of the endocrine disruption found in wild fish populations, the study of phthalate esters has become integral to determining whether or not these anti-androgenic chemicals are of concern. I investigated whether di-n-butyl phthalate (DBP) was able to cause antiandrogenic endocrine disruption in fish under controlled laboratory conditions. Three experiments were undertaken. In the first study, two generations of fathead minnows were exposed to nominal concentrations of 6 to 100 μg/L for 21 and 150 days, respectively. The second experiment examined the effects of early life-stage exposure to DBP (50, 100 and 200 μg DBP/L) on three-spined sticklebacks. The final experiment examined the effects of DBP on adult male three-spined sticklebacks in a 21-day nesting study (15 and 35 μg DBP/L). DBP had no effect on the fecundity, survival, growth, sex ratio, or gonadal histology of the exposed fish in any of the experiments. Further, it failed to alter the expression of two steroidogenic genes in adult male sticklebacks. In contrast, DBP was often found to significantly alter plasma androgen concentrations in both species, and spiggin concentrations in the three-spined stickleback, most notably causing significantly reduced spiggin concentrations in the adult males exposed to DBP. Ultimately, DBP-exposure did not disrupt the ability of the fish to reproduce successfully, and did not appear to alter reproductive behaviours or the expression of secondary sexual characteristics. In conclusion, while DBP did appear to have some capacity for endocrine disruption in fish, it was unable to interfere with the ability of the fish to develop normally and reproduce successfully. Thus, environmentallyrelevant concentrations of phthalate esters are likely not of particular concern to fish populations.

<p>Industrial and domestic sewage effluents have been found to cause reproductive disorders in wild fish, often as a result of the interference of compounds in the effluents with the endocrine system. This thesis describes laboratory-based exposure experiments and a field survey that were conducted with juveniles of the three-spined stickleback, <i>Gasterosteus aculeatus</i>. This small teleost is a common fish in Swedish coastal waters and was chosen as an alternative to non-native test species commonly used in endocrine disruption studies, which allows the comparison of field data with results from laboratory experiments.</p><p>The aim of this thesis was to elucidate 1) if genetic sex determination and differentiation can be disturbed by natural and synthetic steroid hormones and 2) whether this provides an endpoint for the detection of endocrine disruption, 3) to evaluate the applicability of specific estrogen- and androgen-inducible marker proteins in juvenile three-spined sticklebacks, 4) to investigate whether estrogenic and/or androgenic endocrine disrupting activity can be detected in effluents from Swedish pulp mills and domestic sewage treatment plants and 5) whether such activity can be detected in coastal waters receiving these effluents.</p><p>Laboratory exposure experiments found juvenile three-spined sticklebacks to be sensitive to water-borne estrogenic and androgenic steroid substances. Intersex – the co-occurrence of ovarian and testicular tissue in gonads – was induced by 17β-estradiol (E2), 17α-ethinylestradiol (EE2), 17α-methyltestosterone (MT) and 5α-dihydrotestosterone (DHT). The first two weeks after hatching was the phase of highest sensitivity. MT was ambivalent by simultaneously eliciting masculinizing and feminizing effects. When applying a DNA-based method for genetic sex identification, it was found that application of MT only during the first two weeks after hatching caused total and apparently irreversible development of testis in genetic females. E2 caused gonad type reversal from male to female. E2 and EE2 induced vitellogenin - the estrogen-responsive yolk precursor protein, while DHT and MT induced spiggin – the androgen-responsive glue protein of the stickleback.</p><p>None of the effluents from two pulp mills and two domestic sewage treatment plants had any estrogenic or androgenic activity. Juvenile three-spined sticklebacks were collected during four subsequent summers at the Swedish Baltic Sea coast in recipients of effluents from pulp mills and a domestic sewage treatment plant as well as remote reference sites. No sings of endocrine disruption were observed at any site, when studying gonad development or marker proteins, except for a deviation of sex ratios at a reference site.</p><p>The three-spined stickleback – with focus on the juvenile stage – was found to be a sensitive species suitable for the study of estrogenic and androgenic endocrine disruption.</p>

<p>Deposition remains the most common form of waste disposal in many parts of the world. As a terminus of the products consumed in our society a landfill may contain virtually all sorts of man-made chemicals. Despite this, the harmfulness of landfill leachate has not been extensively evaluated in feral organisms in the environment. In a leachate-contaminated lake, Molnbyggen, in Sweden, our studies reported a low percent of sexually mature (SM) female perch (<i>Perca fluviatilis</i>) that had decreased plasma androgen levels, decreased brain aromatase activity, distinctive sores and fin erosion. The impairments were attributed to unidentified compound(s) present in the leachate. In one out of four other investigated leachate-contaminated lakes, the low percent of SM female perch had reduced plasma sex steroid levels and similar sores as perch in Molnbyggen. The biochemical mechanism causing the disorders was investigated in order to establish a connection between the impairments and possible causative compound(s). Plasma levels of progesterone and 17α-hydroxyprogesterone were unaffected. Ovarian 17,20-lyase activity was decreased while levels of biliary steroid conjugates and hepatic testosterone UDP-glucuronosyltransferase activity did not differ between exposed and reference SM fish. Furthermore, the decreased brain aromatase activity seems to be a secondary effect; possibly a result of low substrate availability. Altogether, this suggests a possible disruption in the synthesis of androgens, knowledge that could be used as a tool in biomarker-directed fractionation studies to pinpoint compound(s). Molnbyggen sediment extracts decreased the testosterone and estradiol concentrations in whole-body homogenates of zebrafish (<i>Danio rerio</i>) after a three week exposure period. This suggests that compound(s) with the potency to alter endocrine function are present in the sediment. Although the first steps have been taken towards identifying compound(s) responsible for this kind of reproductive impairments, they still remain unidentified. Measures have to be taken to identify harmful chemicals in our society, to reduce their number, and to minimise their uncontrolled dispersal.</p>

Industrial and domestic sewage effluents have been found to cause reproductive disorders in wild fish, often as a result of the interference of compounds in the effluents with the endocrine system. This thesis describes laboratory-based exposure experiments and a field survey that were conducted with juveniles of the three-spined stickleback, Gasterosteus aculeatus. This small teleost is a common fish in Swedish coastal waters and was chosen as an alternative to non-native test species commonly used in endocrine disruption studies, which allows the comparison of field data with results from laboratory experiments. The aim of this thesis was to elucidate 1) if genetic sex determination and differentiation can be disturbed by natural and synthetic steroid hormones and 2) whether this provides an endpoint for the detection of endocrine disruption, 3) to evaluate the applicability of specific estrogen- and androgen-inducible marker proteins in juvenile three-spined sticklebacks, 4) to investigate whether estrogenic and/or androgenic endocrine disrupting activity can be detected in effluents from Swedish pulp mills and domestic sewage treatment plants and 5) whether such activity can be detected in coastal waters receiving these effluents. Laboratory exposure experiments found juvenile three-spined sticklebacks to be sensitive to water-borne estrogenic and androgenic steroid substances. Intersex – the co-occurrence of ovarian and testicular tissue in gonads – was induced by 17β-estradiol (E2), 17α-ethinylestradiol (EE2), 17α-methyltestosterone (MT) and 5α-dihydrotestosterone (DHT). The first two weeks after hatching was the phase of highest sensitivity. MT was ambivalent by simultaneously eliciting masculinizing and feminizing effects. When applying a DNA-based method for genetic sex identification, it was found that application of MT only during the first two weeks after hatching caused total and apparently irreversible development of testis in genetic females. E2 caused gonad type reversal from male to female. E2 and EE2 induced vitellogenin - the estrogen-responsive yolk precursor protein, while DHT and MT induced spiggin – the androgen-responsive glue protein of the stickleback. None of the effluents from two pulp mills and two domestic sewage treatment plants had any estrogenic or androgenic activity. Juvenile three-spined sticklebacks were collected during four subsequent summers at the Swedish Baltic Sea coast in recipients of effluents from pulp mills and a domestic sewage treatment plant as well as remote reference sites. No sings of endocrine disruption were observed at any site, when studying gonad development or marker proteins, except for a deviation of sex ratios at a reference site. The three-spined stickleback – with focus on the juvenile stage – was found to be a sensitive species suitable for the study of estrogenic and androgenic endocrine disruption.

Deposition remains the most common form of waste disposal in many parts of the world. As a terminus of the products consumed in our society a landfill may contain virtually all sorts of man-made chemicals. Despite this, the harmfulness of landfill leachate has not been extensively evaluated in feral organisms in the environment. In a leachate-contaminated lake, Molnbyggen, in Sweden, our studies reported a low percent of sexually mature (SM) female perch (Perca fluviatilis) that had decreased plasma androgen levels, decreased brain aromatase activity, distinctive sores and fin erosion. The impairments were attributed to unidentified compound(s) present in the leachate. In one out of four other investigated leachate-contaminated lakes, the low percent of SM female perch had reduced plasma sex steroid levels and similar sores as perch in Molnbyggen. The biochemical mechanism causing the disorders was investigated in order to establish a connection between the impairments and possible causative compound(s). Plasma levels of progesterone and 17α-hydroxyprogesterone were unaffected. Ovarian 17,20-lyase activity was decreased while levels of biliary steroid conjugates and hepatic testosterone UDP-glucuronosyltransferase activity did not differ between exposed and reference SM fish. Furthermore, the decreased brain aromatase activity seems to be a secondary effect; possibly a result of low substrate availability. Altogether, this suggests a possible disruption in the synthesis of androgens, knowledge that could be used as a tool in biomarker-directed fractionation studies to pinpoint compound(s). Molnbyggen sediment extracts decreased the testosterone and estradiol concentrations in whole-body homogenates of zebrafish (Danio rerio) after a three week exposure period. This suggests that compound(s) with the potency to alter endocrine function are present in the sediment. Although the first steps have been taken towards identifying compound(s) responsible for this kind of reproductive impairments, they still remain unidentified. Measures have to be taken to identify harmful chemicals in our society, to reduce their number, and to minimise their uncontrolled dispersal.

Endocrine disrupting chemicals (EDCs) are a class of environmental toxicants, of both natural and synthetic origin, that interfere with normal endocrine function. Exposure to EDCs during susceptible periods of development, particularly embryogenesis, can result in profound neurological and reproductive deficits. While the impact of developmental exposure to EDCs on reproductive function and behavior has been much studied, the underlying mechanisms responsible for these observed effects are not well understood. The goal of the research detailed in this dissertation is to elucidate the cellular and molecular targets by which a representative class of EDCs, polychlorinated biphenyls (PCBs), disrupts normal reproductive neuroendocrine function. My specific hypothesis is that PCBs cause changes in sexually dimorphic brain regions underlying sex-specific reproductive physiology and behavior through the perturbation of normal developmental apoptosis, with long-term consequences for reproductive success. The studies detailed herein focus on three areas which contribute to an understanding of the effects of PCBs on neuroendocrine reproductive function: (1) the in vitro effects of PCBs on a neuroendocrine cell line, (2) developmental effects of PCBs on the gestationally exposed F1 generation, and (3) the physiological consequences of these developmental alterations for adult reproductive function. In the first section of this dissertation, the neurotoxic and endocrine disrupting effects of PCBs on a representative developing neuroendocrine cell model, the GT1-7 GnRH cell line, are investigated in time- and dose-response experiments. Treatment and dose-dependent effects are observed for GnRH peptide concentrations, cell viability, apoptotic and necrotic cell death, and caspase activation. In general, GnRH peptide levels are suppressed by high doses and longer durations of PCBs, and elevated at low doses and shorter time points. The suppression of GnRH peptide levels was partially reversed in cultures co-treated with the estrogen receptor antagonist ICI 182,780. All PCBs tested reduced viability and increased both apoptotic and necrotic cell death. The second section of this dissertation examines whether prenatal PCB exposure alters normal neuroendocrine development in the F1 generation, including sexual differentiation of the brain. Disruption of hypothalamic development is detectable as early as the day after birth (postnatal day (P) 1), as indicated by abnormal programmed cell death, and alterations in neuroendocrine gene and protein expression. The third section discusses the physiological impact of developmental PCB exposure on reproductive maturation and adult neuroendocrine function. Pubertal onset is advanced and estrous cyclicity irregular in PCB endocrine-disrupted females. Furthermore, sexual differentiation of female neuroendocrine systems is masculinized/defeminized. Collectively, these results suggest that the disrupted sexual differentiation of the POA can be detected as early as the day after birth, effects that may underlie the adult reproductive phenotype. / text

Current municipal wastewater treatment plants (MWWTP) technologies are insufficiently removing emerging contaminants of concern. These emerging contaminants are an issue as many are known endocrine disrupting compounds (EDCs). EDCs are contaminants that can have severe and irreversible impacts on highly conserved endocrine systems that are critical during developmental periods in vertebrates as well as during adult life. Many EDCs have non-monotonic dose-response curves yet they are not often tested at low, environmentally relevant concentrations. EDC research to date has focused heavily on xenoestrogenic compounds whereas thyroid hormone (TH) disruption has been largely overlooked. TH is conserved in all vertebrates and plays crucial roles in neural development, basal metabolism, and thermoregulation. TH is comprised of thyroxine (T4), often known as the transport form of TH, and triiodothyronine (T3), the more bioactive form of TH. A TH spike occurs in the perinatal period of humans, and when disrupted, this spike can cause severe developmental defects. An analogous, but perhaps more overt, TH spike occurs in amphibians. TH is the sole hormone that drives amphibian metamorphosis, thus providing an excellent model for TH action. Our lab has previously developed the cultured tailfin (C-fin) assay, which uses biopsies from premetamorphic Rana catesbeiana tadpole tailfins cultured in the presence of an exogenous chemical of concern to assess perturbations to TH- and stress-responsive gene transcript levels by QPCR. This thesis uses the C-fin assay to assess the efficacy of removal of biological TH- and stress-altering activity in conventional municipal wastewater treatment systems. We first assess the successive levels of a full-scale conventional activated sludge (CAS) MWWTP in its ability to reduce perturbations of mRNA transcript levels of the critical TH receptors alpha (thra) and beta (thrb), and stress responsive gene transcripts superoxide dismutase (sod), catalase (cat) and heat shock protein 30 (hsp30). Secondary treatment of wastewater effluents removes cellular stress perturbations when compared to influents, but thr disruptions remain after conventional secondary wastewater treatment. We then assess three pilot-sized conventional secondary MWWTP configurations run at two operational conditions. The C-fin assay results suggest that the current understanding of operational conditions and the efficiency of complex MWWTP configurations is not clear-cut when assessed by biological endpoints such as the transcript abundance perturbations in the C-fin assay. Finally, the C-fin assay is used to investigate transcript profiles of genes of interest when the tissues are treated with the endogenous hormones T3, T4, and estradiol (E2). Our results indicate that T4 acts as more than solely a T3-prohormone and that gene expression levels in response to the two different forms of TH can be T3 or T4 specific. E2 effects, although implicated in altering TH-mediated responses in other contexts, do not affect TH-responsive gene transcripts in the C-fin. The data presented use the novel C-fin assay to challenge and advance the currently accepted views of TH-action, as well as develop necessary yet practical biological knowledge for management of emerging contaminant release from MWWTPs. / Graduate / 0383 / 0768 / 0307 / polaw@uvic.ca