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Title: Endocrine Disruption in the Marine Environment
Author: Allen et al Y
Author: Burnham CEFAS:
Document Type: Monograph
Annotation: Environment Agency Project ID:EAPRJOUT_1622, Representation ID: 560, Object ID: 2730
Previous research on endocrine disruption in the freshwater and marine aquatic environments has shown that some fish species, particularly rainbow trout, roach and flounder are showing signs of exposure to exogenous oestrogens, as indicated by the presence of the female yolk protein vitellogenin (VTG) in blood plasma and intersex testes in male fish. In light of this work the Endocrine Disruption in the Marine Environment (EDMAR) research programme was initiated in 1998. The overall objectives of this three-year research programme were to investigate whether there is evidence for changes in the reproductive health of both marine fish and invertebrates due to endocrine disruption, and, if so, the possible causes. a a To achieve the EDMAR objectives, a total of 12 scientific workpackages were set up to conduct the research required. Broadly, these comprised development of new methods for detecting endocrine disruption in the marine environment, measurement of biological responses in the field and identification of causal substances and sources. Collectively, these would help to build a picture of biological effects in the UK marine environment and possible casual subastances. Each workpackage was the responsibility of one research organisation, although many were dependent on information inputs and some research support from one or more of the other research organisations. Since androgen-specific biomarkers for fish were absent, a suitable technique needed to be developed which could subsequently be applied in the field. Two biomarkers of androgenic exposure in the three spined stickleback, Gasterosteus aculeatus, were developed and validated: induction of the nest building glue protein, spiggin, using an ELISA (Enzyme-Linked Immunosorbent Assay) technique, and increase in kidney epithelial 5 Molecular probes for VTG and zona radiata protein (ZRP) mRNA were developed for the sand goby Pomatoschistus minutus and the viviparous blenny Zoarces viviparus and tested in the laboratory using a reference oestrogen (ethynyloestradiol (EE2)) and and oestrogenc mimic (octylphenol). This technique does not require large volumes of blood plasma, which are difficult to obtain from small specimens. Initial laboratory experiments dosing sand goby demonstrated a similar sensitivity to VTG induction in flounder. a EDMAR was funded by a consortium of UK Government Departments and Agencies - the Department for Environment, Food and Rural Affairs (DEFRA), the Environment Agency (EA) and the Scotland and Northern Ireland Forum for Environmental Research (SNIFFER). The European Chemical Industry Association (CEFIC) also made a contribution. Six major UK laboratories conducted the research: the CEFAS (Centre for Environment, Fisheries and Aquaculture Science) Burnham, Lowestoft and Weymouth laboratories, the Plymouth Environmental Research Centre (PERC), the Centre for Marine and Coastal Studies in Liverpool (CMACS), the Fisheries Research Service (FRS) Marine Laboratory Aberdeen, the AstraZeneca Environmental Laboratory, Brixham and Glasgow Caledonian University. To obtain an invertebrate biomarker of exposure to environmental oestrogens analogous to vitellogenin in vertebrates, ELISAs for VTG were developed and tested for two species of marine crustaceans, brown shrimp (Crangon crangon) and shore crab (Carcinus maenas). However, this protein was not induced on exposure to model oestrogens and was absent in males from a number of oestrogen-contaminated field sites. Male crabs which had been feminised through parasitism by Sacculina carcini also did not express VTG. These results indicate that VTG induction does not provide evidence of endocrine disruption in response to oestrogens and androgens. This does not necessarily mean that endocrine disruption is not occurring in crustaceans . Protocols were developed for the immunocytochemical localisation of the oestrogen receptor and VTG in flounder liver and gonad tissue slices, and spiggin in stickleback kidney. Localisation of the oestrogen receptor, using mammalian antibodies, failed to produce reliable and reproducible data, but one antibody which gave a positive result indicated that it may have a diffuse localisation in liver hepatocytes, and be localised in the thecal/granulosa cell layer of female gonads and in the Sertoli cells and interstitium of male gonads. Immunocytochemical techniques for localising VTG in flounder liver and gonads and spiggin in stickleback kidney were partially successful. Localisation of VTG was indistinct in hepatocyte cytoplasm; in the gonad, staining was strongly positive in early VTG stage oocytes around the periphery. In the stickleback, spiggin was localised in the renal epithelium of kidney tubules. These techniques require further development and validation before being used as biomarkers for evaluating endocrine disruption. a Surveys of intersex and VTG induction in male flounder indicate that they are still being exposed to high concentrations of oestrogenic substances in some estuaries, particularly in the Clyde, Tyne, Tees and Mersey, although some sites may be showing a downward trend. To assess the extent and effects of endocrine disruption in both fish and invertebrates in the field, an extensive field survey programme was undertaken collecting a number of sentinel species from 14 estuaries and coastal areas around the UK flounder (Platichthys flesus), sand goby (Pomatoschistus minutus), viviparous blenny (Zoarces viviparus), salmon ( Salmo salar), sea trout ( Salmo trutta), brown shrimp ( Crangon crangon) and shore crab (Carcinus maenas). a VTG concentrations in male flounder continue to be elevated in some estuaries, particularly in the Clyde, Tyne, Tees and Mersey, although some sites may be showing a downward trend. Morphological abnormalities of secondary sexual characteristics, which have not been observed previously, were found in male sand goby. Although retarded growth in some characteristics of male shore crab was evident, it was difficult to identify any clear differences between the reference site and contaminated sites. a The possible effects of delayed migratory passage through contaminated estuaries on smolt physiology and seawater adaptation of Atlantic salmon and sea trout were investigated. Salmon smolts caged for a realistic migration time period of 5 days within the Tees estuary below the Barrage and in an adjacent control estuary (River Esk) showed no induction of VTG, and no adverse effects on gill physiology, which indicates no effect on seawater adaptation. Adult male migratory Atlantic salmon and sea trout obtained from several estuaries showed little or no induction of VTG. Those with detectable levels were from the Tees and Tyne estuaries and coastal waters outside the River Tyne. There were no effects on gill physiology and no evidence of intersex in any of the males. These results suggest that endocrine disruption in salmonids within estuaries is not widespread and it is unlikely that exposure to oestrogenic contamination is a major factor regulating salmonid populations in England and Wales. a The Toxicity Identification and Evaluation (TIE) technique with yeast oestrogen and androgen screens (YES and YAS) was used to identify and quantify oestrogenic and androgenic substances in samples from a range of estuaries. Oestrogenic activity was highest in the Tyne and Tees. In water and effluents substances identified included the natural steroids 17I-oestradiol and androsterone, and the synthetic compounds nonylphenol (a surfactant metabolite) and bis(2-ethylhexyl)phthalate. Much higher oestrogenic activity was found in sediments and nonylphenol, cinnarizine (an anti-histamine drug) and cholesa-4,6-dien-3-one (a natural cholesterol degradation product) were positively identified as minor contributors. Androgenic activity was highest in the Clyde estuary. In the Irvine Valley Sewer effluent the natural steroids/steroid metabolites dehydrotestosterone, androstenedione, androstanedione, 5 I-androstane-3I ,11I-diol-17one, androsterone and epi-androsterone were responsible for nearly all of the androgenic activity. a In keeping with previous studies, intersex in male flounder (i.e. ovotestis) was found to have a low but consistent prevalence. The highest occurrences were found in the Mersey and Tyne. Lower incidences were found in the Clyde and Thames. a No VTG or ZRP gene induction or male intersex were observed in wild sand goby caught from 10 estuaries, but this may be due to the short half-life of mRNA which could therefore be missed if exposures to oestrogens are transient. Some wild male sand goby exhibited morphological abnormalities in the uro-genital papilla (UGP), an organ which shows secondary sexual characteristics and is known to be involved in gamete deposition. These males had UGPs with a shape intermediate between male and female. The highest incidences of this condition, termed Morphologically Intermediate Papilla Syndrome (MIPS) were found in males from the Tees, Mersey and Clyde; it was rare or absent in the Alde (the clean reference estuary), Crouch and Thames. The biological significance of this is as yet unclear. a More limited surveys of the viviparous blenny showed evidence for oestrogenic endocrine disruption. Up to 100% of males produced VTG in some estuaries (Tyne, Tees and Clyde) and intersex males were found in the Clyde and Tyne. a Potential effects of oestrogenic endocrine disruption on exoskeletal morphology of wild shore crabs were investigated using males from four sites where strong oestrogenic contamination is present - the Tyne, Tees, Clyde and Nene - and the Alde reference site. 6 Both natural steroids and industrial chemicals are responsible for the oestrogenicity in the estuaries sampled. Androgenicity has so far been attributed to natural steroids. a ecdysteroid (moulting hormone) 20hydroxyecdysone and the reference oestrogen diethylstilbestrol (DES) did cause significant inhibition of development and reproduction of these copepods. A newly developed in vitro ecdysteroid receptor (EcR) binding assay showed that natural and synthetic oestrogens/androgens and their antagonists did not bind to the EcR, but Bisphenol-A, diethylphthalate and lindane were weak EcR antagonists. Natural vertebrate steroids did not affect the lifecycle of a model crustacean. Several synthetic compounds, including a pesticide, were antagonists for the invertebrate moulting hormone receptor. Male flounder caged on sediment for 2 weeks in the Tyne and Tees showed no VTG induction, so feeding experiments with shrimp and mussels from the Tees were initiated to investigate other exposure pathways. No VTG induction was observed in male flounder from the Alde reference site fed with wild-caught shrimp from the Tees, but those fed on mussels that had been caged for several months on the Tees showed some induction. This increase was much lower than the levels observed in wild-caught flounder. Although food chain exposure of wild fish to oestrogenic substances appears to be a possibility, the results indicate that several exposure factors may be interacting in the natural environment. a a As part of EDMAR associated research, a laboratory based programme was initiated using a marine copepod (Tisbe battagliai) life-cycle test to investigate the potential for environmental endocrine disrupters to affect population relevant parameters (survival, development, reproduction and sex ratio) in indigenous Crustacea. It was demonstrated that a range of steroidal oestrogens and androgens showed no reproductive or developmental toxicity to T. battagliai. The In summary, the EDMAR project has shown that oestrogenic effects at the biochemical, cellular or gross morphological levels are present in five species of estuarine fish from a range of industrialised (past and/or present) estuaries. Insufficient field data are yet available to indicate whether these changes have consequences for reproductive success, although experimental data from sand gobies suggests that this is a possibility. A range of natural and synthetic oestrogens is present in the estuaries where the most marked effects have been observed. Most of the activity is strongly adsorbed on sediment particles and much remains to be identified. However, it seems likely that flounder (and possibly other fish) obtain some oestrogenic exposure through feeding on benthic invertebrates. Whether crustaceans themselves are susceptible to oestrogenic exposure remains an open question; apparent changes of shore crab exoskeletons from oestrogen-contaminated locations are not conclusive and require further investigation. In comparison with oestrogenic effects, those caused by exposure to androgens appear to be weak or non-existent in fish (stickleback), although few data are yet available. However, weak androgenic activity is present in the vicinity of poorly treated sewage discharges and is composed exclusively of natural substances in the single sewage effluent subjected to TIE. Various research recommendations flowing from EDMAR and other UK research programmes on endocrine disruption in freshwaters were discussed at a DEFRA sponsored workshop at CEFAS Weymouth in September 2001. The report of this workshop a Future directions for government funded research on endocrine disruption in the aquatic environment a, can be obtained from the EDMAR secretariat at DEFRA, 3/E6 Ashdown House, 123 Victoria Street, London SW1E 6DE. The effects of long-term exposure to sewage effluent (from the River Irvine in Scotland), and known oestrogenic substances on biomarker responses (VTG mRNA), maturation indices and reproductive success were investigated in the sand goby. Exposure to known oestrogens induced VTG mRNA expression, reduced testis and seminal vesicle size and delayed/inhibited nuptial colouration. Long-term exposure to environmentally realistic concentrations (0.3 and 0.03%) of Irvine Valley Sewage (IVS) effluent produced only weak (not statistically significant) effects on markers of oestrogenic exposure (VTG mRNA, seminal vesicle size, nuptial colouration) at the higher concentration. Although exposure to known oestrogens significantly reduced population reproductive output (fertile egg production), it was unaffected by exposure to IVS, but reduced adult survival resulted in reduced overall output. Long term exposure to sewage effluent weakly affected primary and secondary sexual characteristics but did not affect reproductive parameters. Survival of adults was affected resulting in impacts on laboratory population size. a a 7 2. gonad development (e.g. Jobling et al, 1996, 1998; Gimeno et al, 1996) and alterations in sex steroid titres (Folmar et al, 1996). BACKGROUND AND
Publisher: Environment Agency
Subject Keywords: Pollution; Chemicals; Environmental protection; Endocrine disruptors; Public health; Hazardous substances
Extent: 70
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