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Not to be quoted without prior reference to the authors

Fisheries Research Services Report No 03/01

EFFECTS OF ETHYNYLOESTRADIOL AND SEWAGE EFFLUENT UPON MATURATION, MOLECULAR MARKERS OF OESTROGENIC EXPOSURE AND REPRODUCTIVE SUCCESS IN A MARINE TELEOST (POMATOSCHISTUS MINUTUS, PALLAS) Craig D Robinson, John A Craft, Ian M Davies, Colin F Moffat, David Pirie, Fiona E Robertson, Elaine Brown, Ronald M Stagg and Susan Struthers August 2001

Fisheries Research Services Marine Laboratory Victoria Road Aberdeen AB11 9DB

EFFECTS OF ETHYNYL OESTRADIOL AND SEWAGE EFFLUENT UPON MATURATION, MOLECULAR MARKERS OF OESTROGENIC EXPOSURE AND REPRODUCTIVE SUCCESS IN A MARINE TELEOST (POMATOSCHISTUS MINUTUS, PALLAS) Craig D Robinson†, John A Craft‡, Ian M Davies†, Colin F Moffat†, David Pirie§, Fiona E Robertson‡, Elaine Brown‡, Ronald M Stagg† and Susan Struthers§ †

Fisheries Research Services, Marine Laboratory, PO Box 101, Victoria Road, Aberdeen, AB11 9DB, UK ‡ School of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow, G4 0BA, UK § Scottish Environment Protection Agency (West Region), 5 Redwood Crescent, Peel Park, East Kilbride, G74 5PP, UK

ABSTRACT Sand goby (Pomatoschistus minutus, Pallas) were exposed for seven months to ethynyl oestradiol (EE2) or a sewage effluent containing known xeno-oestrogens (alkylphenol polyethoxylates). Nominal exposure concentrations were 6 ng l-1 EE2, 0.3% v/v or 0.03% v/v sewage effluent. The fish were bred using within treatment crosses during the final weeks of the exposures. At the end of the breeding trials expression of hepatic zona radiata protein (Zrp) and vitellogenin (Vtg) mRNA were determined using two newly developed cDNA probes, and were used to indicate oestrogenic exposure. Exposure to 6 ng l-1 EE2 induced Zrp and Vtg mRNA expression in male and female sand goby, impaired male maturation and reproductive behaviour, reduced female fecundity, egg fertility and reduced fertile egg production of the exposed population by approximately 95%. Exposure to sewage effluent (0.3% v/v) caused increased mortality, increased female Zrp and Vtg mRNA expression, and reduced population egg production by approximately 45% compared to 0.03% v/v effluent exposure. Exposure to 0.03% effluent significantly increased population fertile egg production relative to controls. EE2 and 0.3% v/v sewage effluent exposure also impaired development of the male urogenital papillae. It is concluded that male vitellogenesis can be used to indicate reduced reproductive success in an oestrogenically exposed population, but if used alone it may not be predictive of reduced reproductive success in a sewage exposed population. Keywords:

Endocrine disruption; fish; reproductive success; vitellogenin and zona radiata protein mRNA expression; ethynyl oestradiol; sewage effluent.

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Oestrogenic Effects on Sand Goby

INTRODUCTION Xeno-oestrogenic exposure of freshwater fish was first reported in English rivers (Purdom et al., 1994) and the source traced to discharges from sewage treatment works (Harries et al., 1996, 1997). Subsequently, oestrogenically exposed fish have also been seen in North America and continental Europe (eg Folmar et al., 1996; Knudsen et al., 1997; Flammarion et al., 2000). Rather fewer studies have been made of marine fish. In the UK, flounder (Platychthys flesus) exhibiting effects of oestrogenic exposure have been reported from some industrialised estuaries (Lye et al., 1997; Matthiessen et al., 1998). Japanese flounder (Pleuronectes yokohamae) from Tokyo Bay exhibited elevated plasma Vtg, and intersex male fish (with ovarian tissue in their testes) also occurred (Hashimoto et al., 2000). In North America, affected fish have been reported from industrialised harbours (Pereira et al., 1992). Typically, the evidence of exposure to xeno-oestrogens is by the detection of the female specific egg yolk precursor protein vitellogenin (Vtg) in the blood plasma of male fish (Sumpter and Jobling, 1995). However, more recent studies have examined eggshell zona radiata protein (Zrp) production (Arukwe et al., 1998) or the expression of oestrogen receptor, Zrp or Vtg mRNA in liver samples (Yadetie et al., 1999). A range of different chemicals have been shown to induce an oestrogenic response in fish. Most common amongst these chemicals are natural oestrogens (17β-oestradiol and oestrone), synthetic oestrogens (ethynyl oestradiol), alkylphenolic compounds and certain phthalates (see review by Kime, 1998). These compounds can be discharged to the aquatic environment in industrial and domestic sewage effluents (Blackburn and Waldock, 1995; Pirie et al., 1996; Desbrow et al., 1998). They interact with the oestrogen receptor which is up-regulated, as it would be through the normal increase in circulating oestradiol in maturing female fish. This results in an increase of hepatic Zrp and Vtg mRNA expression and protein production. In a normally maturing female, these proteins are transported via the blood to the ovary where they are internalised via receptor-mediated processes (Davial et al., 1998; Perazzolo et al., 1999). Zrp forms the protective shell of the oocyte and vitellogenin is incorporated into the egg yolk of the developing oocytes (see Kime, 1998). In immature or male animals which have little oestrogen present naturally, xeno-oestrogen exposure can be detected as an increase in hepatic oestrogen receptor, Zrp and Vtg mRNA expression, or increased concentrations of Zrp or Vtg proteins circulating in the blood plasma. As male fish have no oocytes to remove Vtg from the bloodstream, exposure of wild fish to xenooestrogens may result in plasma Vtg concentrations increasing by several orders of magnitude (Purdom et al., 1994; Matthiessen et al., 1998). Additionally, other effects seen on fish exposed experimentally to xeno-oestrogens include altered serum hormone concentrations (Folmar et al., 1996; Giesy et al., 2000), inhibited gonad growth during sexual maturation (Jobling et al., 1996; Panter et al., 1998), regression of testes in mature fish (Gimeno et al., 1998a), induction of intersex (Gray and Metcalfe, 1997). Exposure of juvenile, genetically male fish may lead to the development of ovaries rather than testes (Gimeno et al., 1998b) and populations of all-female fish can be produced following exposure of early post-hatch larvae (Nimrod and Benson, 1998), or by microinjection of fish embryos (Papoulias et al., 1999). In the UK, the principal oestrogenic components of effluents are natural hormones, particularly oestradiol (E2; Desbrow et al., 1998), and alkylphenol polyethoxylates (APEOs; Harries et al., 1997). The oestrogenicity of domestic effluents is primarily due to hormones, while that of industrial effluents is primarily due to APEOs. APEOs consist of the parent alkylphenol with a chain of ethoxylate groups (APnEO), where n can be between one and 100. APEOs rapidly biodegrade aerobically in sewage treatment works through the loss of

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Oestrogenic Effects on Sand Goby

the ethoxylate groups, becoming more lipophilic and more oestrogenic as a consequence (Jobling and Sumpter, 1993). Typically, about 85% of APnEO is degraded within five days, although much of the remainder can persist for 30 days or more (Manzano et al., 1998). Once in the environment, short-chain APEOs and parent alkylphenols bind to particulate material and degrade slowly, if at all, in marine sediments (Shang et al., 1999). Consequently both short-chain APEOs and parent alkylphenols are environmentally persistent. Following the initial reports of the effects of xeno-oestrogens on wild fish, many experimental studies have been undertaken to examine effects on the physiology, histology and molecular biology of impacted fish (eg Harries et al., 1996, 1997; Gray and Metcalfe, 1997; Christiansen et al., 1998; Gimeno et al., 1998a, 1998b; Panter et al., 1998; Christensen et al., 1999; Yadetie et al., 1999; Folmar et al., 2000). There have been fewer studies of the potential effects of xeno-oestrogen exposure at population level, for example on fish reproductive success. Short-term exposure to high concentrations of oestradiol (E2) has been shown to reduce egg production (EC50 of 120 ng l-1) in fathead minnows (Kramer et al., 1998). Exposure of fresh hatched medaka (Oryzias latipes) to ethynyl oestradiol (EE2) as they were grown to maturity and bred showed effects on sex ratio, ovary size, percentage of females carrying eggs and population fecundity (Scholz and Gutzeit, 2000). However, there appear to have been no studies on the reproductive success of marine fish exposed to environmentally relevant concentrations of oestrogens. The aim of the current work was to investigate effects of chronic exposure to environmentally realistic concentrations of a model oestrogen (EE2) and a sewage effluent on the breeding success of a marine fish. Irvine Valley (Ayrshire, Scotland) Sewer (IVS) effluent was used because of its historically high levels of alkylphenolic compounds (about 1,500 µg l-1 in 1996; Pirie et al., 1996). The Irvine Valley sewerage system serves a population equivalent of 237,600 and the effluent receives limited treatment (screening and maceration) prior to discharge via a 2.2 km long sea outfall into Irvine Bay (the major nursery and feeding ground for flatfish from the Clyde Sea Area), south west Scotland. In addition to domestic waste, several industrial discharges enter the IVS system. These include waste from the wool processing, aerospace, textile and electronics industries. The exposure concentrations (0.3% or 0.03% v/v) represent concentrations that could occur immediately outwith the 'mixing zone' and after initial dilution and dispersion by natural hydrographic processes. As a discharge plume rises from the seabed it entrains water and is diluted until it reaches the surface as a 'boil'. In Scotland, the mixing zone around the surface boil is normally 100 m in diameter from the centre of the boil and the discharge is controlled so that no Environmental Quality Standard is breached outwith the mixing zone, and so that, the 3 hour acute No Observed adverse Effects Concentration (NOEC) for any individual contaminant, is not exceeded within the mixing zone (SEPA, 1998). Ethynyl oestradiol (EE2) is a synthetic analogue of 17β-oestradiol. It is manufactured for use as a human contraceptive and has been detected in rivers and sewage effluents at concentrations in the range 0.1-10 ng l-1 (Aherne and Briggs, 1989; Desbrow et al., 1998). It was used in this experiment as a positive control. This experiment used long-term exposures and the nominal concentration was chosen so as to be similar to the lowest observable effect concentration (LOEC) of other species, and also to be environmentally realistic. The sand goby (Pomatoschistus minutus) is an inshore and estuarine fish, common in northern European waters, which reaches maturity within a single year. It is small (up to 8 cm) and females undergo multiple spawning in late spring and early summer. During the

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Oestrogenic Effects on Sand Goby

breeding season, male sand goby build a nest under a suitable stone or shell and court females to lay eggs in it. The male fertilises the eggs and guards them until the larvae hatch (Wheeler, 1969). This strategy makes it useful as a test species for examining effects of contaminants on reproductive output and has previously been utilised by FRS Marine Laboratory to investigate reproductive success following exposure to sewage sludge (Waring et al., 1996). The sand goby is also being used as a sentinel organism for studies of environmental oestrogenic exposure within the UK Endocrine Disruption in the Marine Environment (EDMAR) programme.

MATERIALS AND METHODS The experiment took place at the Fisheries Research Services Aultbea field station on the NW coast of Scotland (NGR NG 844 911) and consisted of a seven month continuous flowthrough exposure of sand goby to sewage effluent or EE2. Immature fish were exposed throughout gonadal maturation, and then during an eight week breeding period.

1.

IVS Effluent

Composite 24 hour samples of IVS effluent were obtained fortnightly between October 1998 and February 1999 (batches 1-9) and weekly thereafter (batches 10-29). Effluent was collected frequently in order to reflect possible seasonal changes in the composition, and avoid decomposition during storage. The effluent was shipped overnight to Aultbea, and stored at ambient temperature during use. A two stage dilution was required to achieve nominal exposure concentrations of 0.3% and 0.03% v/v. The effluent was initially diluted to 55% and 5.5% v/v with freshwater piped from a nearby moorland lochan and then pumped into mixing chambers on the seawater intakes of the exposure tanks. Batches 1-9 of IVS effluent were sampled for chemical analysis immediately on receipt at the field station and at the end of their usage period. Batches 10-29 were also sampled at time of collection. Samples were preserved by the addition of either 50 ml hexane (for polychlorinated biphenyl (PCB) and pesticides analysis) or 100 ml 40% formaldehyde (for alkylphenol analysis) and stored below 5oC until analysis. Samples were analysed for 18 pesticides and breakdown products, seven PCBs, nine alkylphenols, total alkylphenol ethoxylates (n= 4-17; AP4-17EO), suspended solids, chemical oxygen demand, ammonia and pH. In addition, the samples taken at time of collection were analysed for six heavy metals. Nonylphenol (NP) concentrations were determined according to Wahlberg et al. (1990). NP4-17EO was determined, with slight modifications, according to Ahel and Giger (1985) and Marcomini et al. (1987). Organochlorine pesticides and PCBs were analysed using standard GC-ECD techniques (HMSO, 1985) and heavy metals by ICP-MS. Detection limits were: pesticides 7-100 ng l-1; individual PCBs 2-20 ng l-1; individual alkylphenols 1.5 µg l-1; total APEO 2.5 µg l-1; metals 0.1-5.8 µg l-1. All chemicals used were of analytical or higher grade and purchased from Merck Ltd (Leicestershire, UK), Rathburn Chemicals Ltd (Peebleshire, UK), or Sigma-Aldrich Chemical Company Ltd (Dorset, UK).

2.

Fish

Juvenile sand goby, identified as Pomatoschistus minutus by the patterns of sensory papillae on their cheeks (Webb, 1980), were collected in October 1998 by drawing a 1 m beam trawl through shallow water at the mouth of a tributary burn of the Ythan estuary, Aberdeenshire, Scotland (NGR NJ 999272). In mid-November 1998, approximately

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Oestrogenic Effects on Sand Goby

125 goby were allocated to each of four, weathered 1 m diameter fibreglass tanks. Each tank was supplied with gravity-fed seawater at 60 l hr-1 and goby were fed to satiation twice per day with frozen mysids, occasionally supplemented with finely chopped squid. One tank per treatment was nominally dosed with 0.03% v/v IVS, 0.3% v/v IVS, 6 ng l-1 EE2 or control (methanol, 17 µl l-1). Fish were subject to natural light and temperature fluctuations. Water temperature (4.5 to 10.5oC over the period of the experiment) was measured daily, whilst water pH (7.9 to 8.2), dissolved oxygen saturation (>85%) and ammonia concentrations (