Reproductive steroid responses in fish exposed to pulp mill condensates (original) (raw)
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Since 1997, a number of approaches (artificial stream exposures, lab bioassays) have been used to identify waste-stream sources of contaminants at the Irving Pulp & Paper Ltd. (IPP) mill, in Saint John, Canada. These studies have shown that chemical recovery condensates have the greatest potential for reducing circulating and gonadal steroids in mummichog (Fundulus heteroclitus), an endemic fish species. A solid phase extraction technique was developed to isolate the hormonally-active substances from the condensates, and a toxicity identification evaluation (TIE) approach was used to gain a better understanding of the chemical characteristics of the active substances. The extract was fractionated by high performance liquid chromatography and the fractions were used in a seven-day bioassay. Mummichog were exposed in static aquaria with daily renewal to either the whole condensate extract, or one of the six fractions at 1 % v/v. Steroid reductions in recent exposures were not as pronounced as had been observed during studies which investigated the overall potency of the extract at the same concentration. As well, differences in responses were observed between the sexes. A dose-response experiment indicated that greater steroid reductions are elicited at 4% v/v in male mummichog. Therefore, the TIE was continued at 4%v/v, however, no steroid reductions were observed in any of the fractions. Some fractions induced increases in plasma testosterone, which had never been observed previously. Ongoing work is focusing on developing an understanding of the extent to which variability in the fish responses are linked to reproductive state and differences in responses between the sexes, while additional work is concentrating on variability in condensate constituents and the fractionation protocol.
Evidence of Estrogenic Mixture Effects on the Reproductive Performance of Fish
Environmental Science & Technology, 2007
Recent research into the effects of mixtures of estrogenic chemicals has revealed the capacity for similarly acting chemicals to act in combination, according to the principles of concentration addition. This means that, collectively, they may pose a significant environmental risk, even when each component is present at a low and individually ineffective concentration. The aim of this study was to investigate the ecological significance of mixture effects at low-effect concentrations by assessing the combined effect of estrogenic chemicals on the reproductive performance of fish. Pairs of fathead minnows were exposed to five estrogenic chemicals.
Aquatic Toxicology, 2006
The Endocrine Modulators Study Group (EMSG) of the European Chemical Industry has proposed an extended fish early-life stage (ELS) test based on OECD test guideline 210 in combination with a fish pair-breeding reproduction study as a possible alternative for fish full life cycle testing. In this paper the androgen methyldihydrotestosterone (MDHT) was tested in an extended ELS test with fathead minnow supplementary to such a test with the weak estrogen 4-tert-pentylphenol (4TPP). Main endpoints were secondary sexual characteristics (SSC), plasma vitellogenin (VTG) induction and gonadal development. Early blastula embryos were exposed to 0, 0.10, 0.32 and 1.0 g MDHT l −1 for up to 114 days post-hatch (dph). A batch of fish exposed to 1.0 g l −1 was transferred to clean water after 30 or 63 dph for the remainder of the study. Ethinylestradiol (EE2) was included as estrogenic reference substance at 0.01 g l −1 . Exposure to MDHT had no significant effect on hatching success or survival, but significantly increased the condition factor of fish exposed for 63 and 114 dph (up to 150% of the control). At 63 dph MDHT exposure induced appearance of tubercles on the snout (a male SSC) of more than 80% of fish. Compared to the controls, plasma VTG was not detectable or significantly lower in fish exposed to MDHT at 0.10 g/l, but not significantly affected at higher MDHT concentrations. Both lower levels of MDHT significantly inhibited the development of female gonads as of 30 dph. Fish exposed to MDHT at 0.32 and 1.0 g l −1 showed higher incidences of mixed sex gonads (10-25%) and smaller testes or dysplasia of gonadal tissue. Dysplasia was present in 80% of the fish continuously exposed to 1.0 g l −1 up to 114 dph, but reversible when fish were transferred to dilution water. Results indicate that suppression of ovarian development was the most sensitive endpoint for MDHT exposure after 30 dph. Other endpoints (e.g., growth and SSC) required exposure during at least up to 63 dph to yield a significant effect. Androgenic effects on VTG production required even longer exposure, i.e., until sufficient number of females had matured.
Aquatic Toxicology, 2006
An extended early-life stage test (based on OECD test guideline 210) was developed to allow the evaluation of a weak environmental oestrogen, 4-tert-pentyphenol (4TPP), on sexual differentiation and gonadal development. Fathead minnow (Pimephales promelas) embryos were exposed to three concentrations of 4TPP (56, 180 and 560 g l −1 ) in a flow-through system, at 25 ± 1 • C, for <107 days post-hatch (dph). In addition, some embryos were exposed to 180 g 4TPP l −1 until 30 or 60 dph, after which they were exposed to dilution water only until 107 dph. At 30, 60 and 107 dph fish were evaluated for growth and gonadal development (via histology), and at 107 dph fish were also evaluated for secondary sexual characteristics (SSC), gonadosomatic index (GSI) and plasma vitellogenin (VTG). There were no effects of 4TPP on hatching success or survival, however, there was a delay in the time taken for embryos to hatch (560 g 4TPP l −1 ). No treatment-related effects were observed on fish growth, with the exception of at 107 dph when the condition factor in female fish was reduced in all 4TPP continuous exposure treatments. Plasma VTG was only elevated in female fish exposed to 180 g 4TPP l −1 and inhibition of gonadal growth (GSI) occurred only in females exposed to 560 g 4TPP l −1 . Histological examination of the gonads revealed delays and disruption in male sexual differentiation and development (180 g 4TPP l −1 ) and no testicular tissue was observed in any fish exposed to 560 g 4TPP l −1 . Mixed gonads (predominately testes with a scattering of primary oocytes) were present in fish exposed to all doses of 180 g 4TPP l −1 at 107 dph. Feminisation of the reproductive ducts (formation of an ovarian like cavity) occurred in the testis of all males exposed to 180 g l −1 , regardless of length of 4TPP exposure. Results indicate that the period of 30-60 dph appears to be the sensitive window for disruption of formation of the reproductive duct and this effect is not reversible when the fish are transferred to dilution water. The data also show that this integrative test is suitable for the detection of a weak environmental oestrogen and comparisons of these results with that of a fish full life-cycle, in medaka, indicate that this test could be a suitable surrogate for a fish full life-cycle. (G.H. Panter). through a variety of mechanisms of action and as a result this has given impetus for the development of screening assays and tests for the detection of EACs, notably in aquatic species. In Europe, the EMSG developed an aquatic research programme to assess the effects of EACs on fish. Within this programme robust and sensitive fish screening assays have already been developed for the evaluation of a range of EACs, using the fathead minnow (Pimephales promelas) . However, although such screening assays will identify the potential endocrine activity of chemicals, further chronic testing will be required to provide more detailed information 0166-445X/$ -see front matter
Critical Reviews in Toxicology, 2012
Endocrine disrupting chemicals (EDCs) enter aquatic ecosystems through discharged effluents, mainly from wastewater treatment works and diffuse run off from land, and affect a wide range of aquatic biota, including fish. Evidence for altered physiology in fish as a consequence of endocrine disruption is global, with some of the most widely reported effects on sexual development and function. In recent years research has shown that fish behaviors can also be affected by EDCs which potentially has wide implications for individual fitness and population level outcomes. This review presents a critical assessment on reported effects of EDCs on behavior in fish, focusing on behaviors associated with reproduction. We investigate commonalities and differences in sexual behaviors between fish species most commonly applied in ecotoxicology, drawing out common principles for impacts of EDCs and then reviewing the evidence for, and implications of, disruptions of these behaviors after exposures to EDCs. In an analysis of the reported effects of exposure to the estrogen, ethinylestradiol, we show that life-stage at exposure is a key factor determining behavioral responses of affected populations. EDC-induced changes in behavior occur at similar concentrations as for established biomarker responses (e.g. vitellogenin induction for estrogens), indicating behavior is equally sensitive (and non-invasive) as an indicator of EDC exposure. Adopting behaviors in fish as indicators of chemical exposure and effects, however, still has many technical and interpretation challenges and there is very little information available on how behaviors under laboratory conditions equate with those occurring in wild populations.
Environmental Toxicology and Chemistry, 2002
In ecotoxicological testing, there are few studies that report on reproductive output (egg production) of marine or estuarine fish. Cunner (Tautogolabrus adspersus) were studied as a potential model species to evaluate the impact of pollutants with estrogenic activity on reproduction in estuarine fish populations. Cunner inhabit marine and estuarine areas where contaminant discharges are likely. Baseline values for cunner gonadosomatic index (GSI), hepatosomatic index (HSI), and plasma vitellogenin (VTG) were determined in a field reference site (April 1999-December 1999). Male and female GSI indicated that cunner spawning is synchronized. Female HSI and VTG increased prior to GSI. From our laboratory observations, cunner are suitable for conducting experiments with reproductive endpoints indicative of both exposure (vitellogenin levels) and effects (egg production). However, cunner are not sexually dimorphic and stripping ripe fish is the only method to distinguish sex. In preparation for laboratory exposure studies with cunner, we designed a laboratory experimental holding system to accommodate cunner's reproductive behavior, a vertical spawning run to the water surface. Cunner were successfully acclimated from overwintering torpor to spawning condition in the laboratory by gradually changing the environmental conditions of fish held at winter conditions (4ЊC and 9:15-h light:dark photoperiod) to spawning condition (18ЊC and 15:9-h light:dark photoperiod). Our results show that cunner successfully spawned daily in the laboratory. They produced fertile eggs in our experimental system designed to accommodate cunner's vertical spawning runs, demonstrating that male and female reproductive behavior was synchronized in the laboratory. Our observations indicate that cunner would be a useful model species for evaluating reproductive effects of environmental contaminants in laboratory studies.
Endocrine Toxicants and Reproductive Success in Fish
Human and Ecological Risk Assessment: An International Journal, 2001
There is compelling evidence on a global scale for compromised growth and reproduction, altered development, and abnormal behaviour in feral fish that can be correlated or in some cases causally linked with exposure to endocrine disrupting chemicals (EDCs). Attributing cause and effect relationships for EDCs is a specific challenge for studies with feral fish as many factors including food availability, disease, competition and loss of habitat also affect reproduction and development. Even in cases where there are physiological responses of fish exposed to EDCs (e.g., changes in reproductive hormone titres, vitellogenin levels), the utility of these measures in extrapolating to whole animal reproductive or developmental outcomes is often limited. Although fish differ from other vertebrates in certain aspects of their endocrinology, there is little evidence that fish are more sensitive to the effects of EDCs. Therefore, to address why endocrine disruption seems so widespread in fish, it is necessary to consider aspects of fish physiology and their environment that may increase their exposure to EDCs. Dependence on aquatic respiration, strategies for iono-osmotic regulation, and maternal transfer of contaminants to eggs creates additional avenues by which fish are exposed to EDCs. This paper provides an overview of responses observed in feral fish populations that have been attributed to EDCs and illustrates many of the factors that need consideration in evaluating the risks posed by these chemicals.
A Model Fish System to Test Chemical Effects on Sexual Differentiation and Development
A model system was characterized which may be used as an in vivo screen for effects of chemicals or environmental mixtures on sexual differentiation and development of reproductive organs and gametes. We evaluated the effects of a model environmental estrogen, ethinyl estradiol (EE2), on the d-rR strain of medaka, Oryzias latipes, using a nano-injection exposure. Gonad histopathology indicated that a single injection of 0.5-2.5 ng EE2/egg can cause phenotypic sex-reversal of genetic males to females. Sex-reversals could be detected as early as seven days post-hatch. Sex-reversed males had female-typical duct development and the secondary sex characteristics we measured were generally consistent with phenotype, with the exception of a few EE2-exposed XX and XY females which possessed ambiguous anal fins. Using discriminant analysis, we determined that the presence or absence of the secondary sex characteristic, a dorsal fin notch, was a very reliable indicator of gonadal sex. No inst...
Journal of Applied Toxicology, 2021
Under the Organisation for Economic Cooperation and Development (OECD), the Ministry of the Environment of Japan (MOE) added Japanese medaka (Oryzias latipes) to the test guideline fish short-term reproduction assay (FSTRA) developed by the United States Environmental Protection Agency (US EPA) using fathead minnow (Pimephales promelas). The FSTRA was designed to detect endocrine disrupting effects of chemicals interacting with the hypothalamic-pituitary-gonadal axis (HPG axis) such as agonists or antagonists on the estrogen receptor (Esr) and/or the androgen receptor (AR) and steroidogenesis inhibitors. We conducted the FSTRA with Japanese medaka, in accordance with OECD test guideline number 229 (TG229), for 16 chemicals including four Esr agonists, two Esr antagonists, three AR agonists, two AR antagonists, two steroidogenesis inhibitors, two progesterone receptor agonists, and a negative substance, and evaluated the usability and the validity of the FSTRA (TG229) protocol. In addition, in vitro reporter gene assays (RGAs) using Esr1 and ARβ of Japanese medaka were performed for the 16 chemicals, to support the interpretation of the in vivo effects observed in the FSTRA. In the present study, all the test chemicals, except an antiandrogenic chemical and a weak Esr agonist, significantly reduced the reproductive status of the test fish, that is, fecundity or fertility, at concentrations where no overt toxicity was observed. Moreover, vitellogenin (VTG) induction in males and formation of secondary sex characteristics (SSC), papillary processes on the anal fin, in females was sensitive endpoints to Esr and AR agonistic effects, respectively, and might be indicators of the effect concentrations in long-term exposure. Overall, it is suggested that the in vivo FSTRA supported by in vitro RGA data can adequately detect effects on the test fish, O. latipes, and probably identify the mode of action (MOA) of the chemicals tested.