A catchment-based study of endocrine disruption in surface waters: multivariate evaluation of the health of a sentinel fish species exposed to sewage treatment works effluent

Summary of the results in the context of EDCAT 5 project aims: 1. By comparing appropriate biomarkers in fish sampled from STW-impacted sites and control sites during the pre-remediation period, to determine whether there was evidence for any effects that might be attributed to the presence of estrogenic (or androgenic, or anti-androgenic/-estrogenic) endocrine disrupting chemicals in the former. This aim was addressed by measuring concentrations of the estrogen-dependent yolk protein precursor vitellogenin, and the androgen-dependent nest glue spiggin in male and female sticklebacks. In addition histological examination of the gonadal structure of fish captured at the impacted and non-impacted sites was employed to seek evidence of overt alterations in reproductive physiology of the fish. For a subset of matched samples from the two rivers, the relative induction of hepatic choriogenin mRNA, a biomarker of estrogen exposure, was measured. Conclusions: Chemistry data provided by EDCAT3&4 showed that estrogenicity of the effluent was low prior to remediation and lower still following installation of the GAC plant. No evidence of overt estrogenic effects was detected in male sticklebacks in the Ray, VTG and ChG levels were similar in males from both rivers. Nor was there any evidence of alterations in spiggin concentrations in the kidneys of males from the Ray compared to the Ock. However, VTG concentrations in female sticklebacks from the Ray were increased following the STW upgrade as were hepatic ChG transcript levels, and kidney spiggin concentrations. No changes in these elements of the reproductive system were observed in females from the Ock across the same time periods. Chemical analysis of the effluent indicated that prior to installation of the GAC plant substantial concentrations of anti-androgenic chemicals were present, together with a wide range of other organics. Concentrations of these were much reduced following the plant upgrade. It is reasonable to suppose that the changes observed in the female reproductive endocrine system following the upgrade were related to the removal of some or all of this complex mixture of chemicals. The absence of effects in males may be related to the balance between exogenous and endogenous signals, or to the specificity of effects exerted by the chemicals present. No intersex fish were detected from either river. A significant bias in favour of females was detected in the stickleback populations in both rivers suggesting a factor associated with life-history of the fish, rather than contaminant burden, was responsible. 2. By comparing appropriate biomarkers in fish sampled from STW-impacted sites and control sites during the pre-remediation period, to determine whether there was evidence for any effects that might be attributed to the presence of “conventionally” toxic chemicals. This was addressed by measurement of the activity of a key Phase I transforming enzyme in the liver of fish, either using direct enzymatic assay (EROD) or by quantifying the levels of expression of the corresponding gene (CYP1A). Conclusions: EROD activity was significantly greater in fish from the Ray than the Ock in two samples collected prior to the installation of the GAC plant (2006, 2007) and this likely reflects the differential contaminant loading in the two rivers. A single sample following the commissioning of the GAC plant (2008) indicated that EROD activity had increased among fish from the Ock while that in fish from the Ray remained unchanged. While a delayed recovery of this biomarker in fish from the Ray may be expected depending on the route of exposure (direct via water or indirect via contaminated food) the reasons for elevated EROD activity in fish from the Ock/Childrey Brook are not immediately evident. Provision of a full data set for Cyp1A expression awaits the repeat of the assay. When this is complete the factors underlying the EROD findings may become clear. 3. To determine whether the adaptive capacity and energetic status of fish varied between the STW-impacted and non-impacted sites. This was addressed by measurement of indicators of stress (whole-body corticosteroid levels), metabolic status (whole-body glucose levels) and anabolic activity (RNA:DNA ratios). Conclusions: The data provide no evidence that the stress response of fish captured in the Ray prior to installation of the GAC plant was modified by exposure to the effluent. However, large variations in whole-body corticosteroid and glucose concentrations in fish from both rivers, with clear trends over time, were closely linked to perturbations in the river flow regime. Whether there was interaction between environmental and chemical factors in determining corticosteroid and glucose status is difficult to discern but it seems likely that variation in these indicators of the stress axis was driven primarily by environmental factors. The RNA:DNA ratios were closely linked with seasonal change in temperature and closely matched observed patterns of weight and length gain in stickleback populations in the two rivers. The longer growth period enjoyed by fish in the Ray was clearly evident. For both rivers, mean anabolic activity was greater during 2008 than 2007 and it seems likely that this is related to adverse effects associated with the periods of extreme flow change observed on both rivers in 2007. 4. To assess whether there were differences in population size and structure between STW-impacted and non-impacted sites. This was addressed by comparison of key somatic measures, in particular frequency distributions for fork length. Conclusions: Because of the extreme patchiness of the distribution of stickleback populations in both rivers accurate abundance estimates were not obtained. However, the catch per unit effort across the life of the project was similar for both rivers. While population size, and age structure (both rivers hosted annual populations), appeared to be similar fish in the Ray were overall larger than those from the Ock, and spawned earlier. The differences in growth and timing of spawning between the rivers were likely to have been associated with the Rodbourne STW effluent. Downstream of the discharge on the Ray water temperatures were consistently 2 – 3oC above those of the Ock. This temperature difference, in combination with the introduction of additional nutrients into the river which is likely to have affected the availaibility of food, probably accounts for the different growth profile among the sticklebacks in the two rivers. However, over and above this difference, there was a significant increase in size of sticklebacks in the Ray between the matched pre- and post-remediation periods in the Ray while no change in size of the fish in the Ock occurred during the same period. Similarly, the RNA:DNA ratio was higher in fish from the Ock during 2007 but greater in fish from the Ray during 2008. Taken together, these observations suggest that there was an improvement in the status of the fish in the Ray following the commissioning of the GAC plant, while the population in the Ock remained relatively stable. It is reasonable to suppose that this may be linked to the reduction of the chemical load entering the Ray at Rodbourne following the installation of the GAC plant. The Ray is “cleaner” now than was the case prior to remediation but remains nutrient rich and several degrees warmer than the Ock, this combination of factors providing fish in the Ray with greater scope for growth relative to populations in the Ock.

[1]  R. Tjeerdema,et al.  Reduction of vitellogenin synthesis by an aryl hydrocarbon receptor agonist in the white sturgeon (Acipenser Transmontamus) , 2009, Environmental toxicology and chemistry.

[2]  M. Landman,et al.  Androgenic effects of a Canadian bleached kraft pulp and paper effluent as assessed using threespine stickleback (Gasterosteus aculeatus). , 2009, Aquatic toxicology.

[3]  C T Graham,et al.  Implications of climate change for the fishes of the British Isles. , 2009, Journal of fish biology.

[4]  N. Tatarazako,et al.  The effect of in vivo co-exposure to estrone and AhR-ligands on estrogenic effect to vitellogenin production and EROD activity. , 2009, Environmental toxicology and pharmacology.

[5]  G. Callard,et al.  Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment , 2008, BMC Molecular Biology.

[6]  C. Tyler,et al.  Altered sexual development in roach (Rutilus rutilus) exposed to environmental concentrations of the pharmaceutical 17alpha-ethinylestradiol and associated expression dynamics of aromatases and estrogen receptors. , 2008, Toxicological sciences : an official journal of the Society of Toxicology.

[7]  I. Katsiadaki,et al.  The model anti-androgen flutamide suppresses the expression of typical male stickleback reproductive behaviour. , 2008, Aquatic toxicology.

[8]  S. Scholz,et al.  Molecular biomarkers of endocrine disruption in small model fish , 2008, Molecular and Cellular Endocrinology.

[9]  Jonathan R. Harvey Analysis and Interpretation of Freshwater Fisheries Data , 2008 .

[10]  Xueping Chen,et al.  Choriogenin mRNA as a sensitive molecular biomarker for estrogenic chemicals in developing brackish medaka (Oryzias melastigma). , 2008, Ecotoxicology and environmental safety.

[11]  J. Porcher,et al.  Assessment of seasonal variability of biomarkers in three-spined stickleback (Gasterosteus aculeatus L.) from a low contaminated stream: implication for environmental biomonitoring. , 2008, Environment international.

[12]  L. Chícharo,et al.  RNA:DNA Ratio and Other Nucleic Acid Derived Indices in Marine Ecology , 2008, International journal of molecular sciences.

[13]  Ioanna Katsiadaki,et al.  A cDNA microarray for the three‐spined stickleback, Gasterosteus aculeatus L., and analysis of the interactive effects of oestradiol and dibenzanthracene exposures , 2008 .

[14]  J. Soengas,et al.  Acute and prolonged stress responses of brain monoaminergic activity and plasma cortisol levels in rainbow trout are modified by PAHs (naphthalene, beta-naphthoflavone and benzo(a)pyrene) treatment. , 2008, Aquatic toxicology.

[15]  I. Katsiadaki,et al.  Intercalibration exercise using a stickleback endocrine disrupter screening assay , 2008, Environmental toxicology and chemistry.

[16]  Helmut Segner,et al.  Interference of endocrine disrupting chemicals with aromatase CYP19 expression or activity, and consequences for reproduction of teleost fish. , 2008, General and comparative endocrinology.

[17]  Christopher J Martyniuk,et al.  Microarray analysis in the zebrafish (Danio rerio) liver and telencephalon after exposure to low concentration of 17alpha-ethinylestradiol. , 2007, Aquatic toxicology.

[18]  I. Katsiadaki,et al.  Effects of 17α-ethynylestradiol on EROD activity, spiggin and vitellogenin in three-spined stickleback (Gasterosteus aculeatus) , 2007 .

[19]  C. Tyler,et al.  Functional associations between two estrogen receptors, environmental estrogens, and sexual disruption in the roach (Rutilus rutilus). , 2007, Environmental science & technology.

[20]  Jaeweon Cho,et al.  Occurrence and removal of pharmaceuticals and endocrine disruptors in South Korean surface, drinking, and waste waters. , 2007, Water research.

[21]  Daniel L Villeneuve,et al.  Linkage of biochemical responses to population‐level effects: A case study with vitellogenin in the fathead minnow (Pimephales promelas) , 2007, Environmental toxicology and chemistry.

[22]  Gerd Maack,et al.  Gene expression profiles revealing the mechanisms of anti-androgen- and estrogen-induced feminization in fish. , 2007, Aquatic toxicology.

[23]  I. Schultz,et al.  Temporal changes in gene expression in rainbow trout exposed to ethynyl estradiol. , 2007, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[24]  J. Porcher,et al.  Preliminary investigation of multi-biomarker responses in three-spined stickleback (Gasterosteus aculeatus L.) sampled in contaminated streams , 2007, Ecotoxicology.

[25]  T. Marsh,et al.  The summer 2007 floods in England and Wales – a hydrological appraisal , 2007 .

[26]  Charles R Tyler,et al.  Appropriate 'housekeeping' genes for use in expression profiling the effects of environmental estrogens in fish , 2007, BMC Molecular Biology.

[27]  A. Hontela Corticosteroidogenesis and StAR protein of rainbow trout disrupted by human-use pharmaceuticals: data for use in risk assessment. , 2006, Toxicological sciences : an official journal of the Society of Toxicology.

[28]  M. Westerfield,et al.  Whole-body cortisol is an indicator of crowding stress in adult zebrafish, Danio rerio , 2006 .

[29]  Ioanna Katsiadaki,et al.  Use of the Three-Spined Stickleback (Gasterosteus aculeatus) As a Sensitive in Vivo Test for Detection of Environmental Antiandrogens , 2005, Environmental health perspectives.

[30]  Richard J. Williams,et al.  Predicted Exposures to Steroid Estrogens in U.K. Rivers Correlate with Widespread Sexual Disruption in Wild Fish Populations , 2005, Environmental health perspectives.

[31]  J. Sumpter,et al.  Rapid bioconcentration of steroids in the plasma of 2 sticklebacks ( Gasterosteus aculeatus ) exposed to water-3 borne testosterone and 17 ß-estradiol , 2006 .

[32]  V. Meucci,et al.  Effects of 17β-estradiol, 4-nonylphenol and PCB 126 on the estrogenic activity and phase 1 and 2 biotransformation enzymes in male sea bass (Dicentrarchus labrax) , 2005 .

[33]  T. Sutton,et al.  Temporal changes in the relationship between condition indices and proximate composition of juvenile Coregonus artedi , 2005 .

[34]  G. Monod,et al.  Beta-naphthoflavone inhibits the induction of hepatic oestrogen-dependent proteins by 17alpha-ethynylestradiol in mosquitofish (Gambusia holbrooki) , 2005, Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals.

[35]  Ioanna Katsiadaki,et al.  The juvenile three-spined stickleback (Gasterosteus aculeatus L.) as a model organism for endocrine disruption II--kidney hypertrophy, vitellogenin and spiggin induction. , 2004, Aquatic toxicology.

[36]  N. Hiramatsu,et al.  Quantification of serum levels of precursors to vitelline envelope proteins (choriogenins) and vitellogenin in estrogen treated masu salmon, Oncorhynchus masou. , 2004, General and comparative endocrinology.

[37]  E. Caldarone,et al.  RNA–DNA ratio and other nucleic acid-based indicators for growth and condition of marine fishes , 1999, Hydrobiologia.

[38]  R. Verheyen,et al.  Comparison of vitellogenin responses in zebrafish and rainbow trout following exposure to environmental estrogens. , 2003, Ecotoxicology and environmental safety.

[39]  N. Woo,et al.  Aquatic Hypoxia Is an Endocrine Disruptor and Impairs Fish Reproduction , 2003 .

[40]  T. Braunbeck,et al.  Effects of 17a-ethinylestradiol on the expression of three estrogen-responsive genes and cellular ultrastructure of liver and testes in male zebrafish. , 2003, Aquatic toxicology.

[41]  P. Campbell,et al.  HORMONAL, MORPHOLOGICAL, AND PHYSIOLOGICAL RESPONSES OF YELLOW PERCH (Perca flavescens) TO CHRONIC ENVIRONMENTAL METAL EXPOSURES , 2003, Journal of toxicology and environmental health. Part A.

[42]  I. Katsiadaki,et al.  Detection of environmental androgens: A novel method based on enzyme‐linked immunosorbent assay of spiggin, the stickleback (Gasterosteus aculeatus) glue protein , 2002, Environmental toxicology and chemistry.

[43]  I. Katsiadaki,et al.  The potential of the three-spined stickleback (Gasterosteus aculeatus L.) as a combined biomarker for oestrogens and androgens in European waters. , 2002, Marine environmental research.

[44]  Frank Sacher,et al.  Removal of pharmaceuticals during drinking water treatment. , 2002, Environmental science & technology.

[45]  T. Pottinger,et al.  The three‐spined stickleback as an environmental sentinel: effects of stressors on whole‐body physiological indices , 2002 .

[46]  A. Arukwe,et al.  Molecular cloning of rainbow trout (Oncorhynchus mykiss) eggshell zona radiata protein complementary DNA: mRNA expression in 17beta-estradiol- and nonylphenol-treated fish. , 2002, Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology.

[47]  E. Gorokhova,et al.  Analysis of nucleic acids in Daphnia: development of methods and ontogenetic variations in RNA-DNA content , 2002 .

[48]  A. Hontela,et al.  Cytotoxic and endocrine-disrupting potential of atrazine, diazinon, endosulfan, and mancozeb in adrenocortical steroidogenic cells of rainbow trout exposed in vitro. , 2002, Toxicology and applied pharmacology.

[49]  H. Segner,et al.  Antiestrogenicity of beta-naphthoflavone and PAHs in cultured rainbow trout hepatocytes: evidence for a role of the arylhydrocarbon receptor. , 2000, Aquatic toxicology.

[50]  N. Hiramatsu,et al.  Serum levels of precursors to vitelline envelope proteins (choriogenins) in Sakhalin taimen after treatment with oestrogen and during oocyte growth , 2000 .

[51]  J. Wingfield,et al.  Effects of weather on corticosterone responses in wild free-living passerine birds. , 2000, General and comparative endocrinology.

[52]  T. Pottinger,et al.  Contrasting seasonal modulation of the stress response in male and female rainbow trout , 2000 .

[53]  D. Tillitt,et al.  Ethoxyresorufin-O-deethylase (EROD) Activity in Fish as a Biomarker of Chemical Exposure , 2000, Critical reviews in toxicology.

[54]  Jeane Nicolas Vitellogenesis in fish and the effects of polycyclic aromatic hydrocarbon contaminants , 1999 .

[55]  R. Wootton,et al.  Do random fluctuations in the intervals between feeding affect growth rate in juvenile three‐spined sticklebacks? , 1998 .

[56]  Bertold Hock,et al.  VITELLOGENIN : A BIOMARKER FOR ENDOCRINE DISRUPTORS , 1998 .

[57]  B. Walther,et al.  Oogenesis in Atlantic salmon (Salmo salar L.) occurs by zonagenesis preceding vitellogenesis in vivo and in vitro. , 1998, The Journal of endocrinology.

[58]  A. Arukwe,et al.  Plasma levels of vitellogenin and eggshell zona radiata proteins in 4-nonylphenol and o,p′-DDT treated juvenile Atlantic salmon (Salmo salar) , 1998 .

[59]  Edwin J. Routledge,et al.  Identification of Estrogenic Chemicals in STW Effluent. 2. In Vivo Responses in Trout and Roach , 1998 .

[60]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[61]  A. D. Pickering,et al.  Sexual maturity modifies the responsiveness of the pituitary-interrenal axis to stress in male rainbow trout. , 1995, General and comparative endocrinology.

[62]  T. Pottinger,et al.  Physiological stress in fish during toxicological procedures: a potentially confounding factor , 1995 .

[63]  C. Clemmesen The effect of food availability, age or size on the RNA/DNA ratio of individually measured herring larvae: laboratory calibration , 1994 .

[64]  J. Sumpter,et al.  Estrogenic Effects of Effluents from Sewage Treatment Works , 1994 .

[65]  J. P. Ruyet,et al.  Effect of starvation on RNA, DNA and protein content of laboratory-reared larvae and juveniles of Solea solea , 1991 .

[66]  G. Copp Electrofishing for fish larvae and 0+ juveniles: equipment modifications for increased efficiency with short fishes , 1989 .

[67]  A. D. Pickering,et al.  Poor water quality suppresses the cortisol response of salmonid fish to handling and confinement , 1987 .

[68]  R. Wootton A Functional Biology of Sticklebacks , 1984, Functional Biology Series.