Alexander P. Scott

The extent of oestrogenic contamination in the UK estuarine and marine environments — further surveys of flounder

In 1996, The Centre for Environment, Fisheries and Aquaculture Science (CEFAS) initiated a project to establish whether oestrogenic materials are present in UK estuarine and marine waters at biologically significant concentrations, and to investigate some of the possible effects which these may have in flounder (Platichthys flesus). Early results are published elsewhere; this paper describes the results of a second wider survey of vitellogenin and reproductive abnormalities in UK flounder. Vitellogenin levels in male blood plasma in the period from spring to winter 1997 were found to be significantly elevated (in comparison with a clean reference site on the Alde estuary) in at least one sample from most of the 11 estuaries investigated. The exceptions were the Tamar and the Dee where all fish appeared entirely normal. In broad terms, the degree of oestrogenic contamination as measured by male vitellogenesis in the various estuaries was ranked in the following descending order: Tees > Mersey > Tyne > Wear = Humber = Clyde = Southampton Water = Thames > Dee = Tamar. VTG concentrations in Tees, Mersey and Tyne male fish were extremely high (> 100,000 ng/ml), and often exceeded those normally found in sexually mature females. At most locations, ovotestis conditions in male flounder were entirely absent but 9% of male Mersey fish and 7% of male Tyne fish contained ovotestis. In a few cases, eggs were fully developed with yolk granules. Most testes did not show gross morphological abnormalities related to oestrogenic exposure, although one testis from a Mersey fish appeared to be almost entirely composed of eggs. Abnormal sex ratios were not seen in any estuary. The paper concludes by outlining a new research programme which will be addressing the biological significance of these observations.

Vitellogenesis as a biomarker of reproductive disruption by xenobiotics

There is increasing evidence that many xenobiotic chemicals can reduce the reproductive capacity of fish through disruption of the endocrine system. Hepatic stimulation of vitellogenesis in male and juvenile fish provides a sensitive indication of exposure to environmental oestrogens and oestrogenic chemicals. Moreover, perturbations of female vitellogenin levels can provide a useful marker of dysfunction caused by endocrine disruption at many sites in the reproductive axis. Vitellogenesis can therefore provide a useful indicator of endocrine disruption by xenobiotics. Both inhibition and stimulation of vitellogenesis can have direct repercussions on the reproductive capacity of fish. An important caveat in interpreting the results of such studies is that they must be related to other components in the reproductive system which may be more sensitive to disruption and have greater consequences for reproductive success.

SURVEYS OF PLASMA VITELLOGENIN AND INTERSEX IN MALE FLOUNDER (PLATICHTHYS FLESUS ) AS MEASURES OF ENDOCRINE DISRUPTION BY ESTROGENIC CONTAMINATION IN UNITED KINGDOM ESTUARIES: TEMPORAL TRENDS, 1996 TO 2001

Plasma vitellogenin (VTG) concentrations and the presence of the ovo-testis (intersex) condition have been recorded in male flounder (Platichthys flesus) captured from several United Kingdom (UK) estuaries since 1996 as part of the endocrine disruption in the Marine Environment (EDMAR) project and earlier programs. It has been confirmed that plasma VTG concentrations in male flounder have remained elevated in several UK estuaries (e.g., Tees, Mersey, and Tyne) throughout the period covered by this study. However, the time-series data indicate that plasma VTG, a measure of environmental estrogen contamination, has decreased in fish captured from several estuaries, especially those of the Tyne and Mersey. Shorter time-series data sets from the Forth and Clyde estuaries also suggest a decrease in estrogen contamination at these sites. Trends associated with specific point sources of estrogenic contamination show site-specific patterns. For instance, plasma VTG levels in male flounder captured near the Howdon sewage treatment outfall (Tyne) have shown a steady decline to near baseline levels in 2001, while the plasma of male fish captured at a site adjacent to the Dabholm Gut discharge in the Tees estuary have shown little evidence of a sustained decline. The occurrence of the intersex condition was detected at a low but consistent prevalence through the study period, with the majority of cases recorded in fish captured from the Tyne and Mersey estuaries. The data set does not allow conclusions to be drawn about any temporal trends associated with this condition. The significance of the findings and possible mitigating influences are discussed in terms of the impacts on wild fish and the role of effluent treatment in reducing these.

The role of the maturation-inducing steroid, 17,20β-dihydroxypregn-4-en-3-one, in male fishes: a review

The major progestin in teleosts is not progesterone, as in tetrapods, but 17,20beta-dihydroxypregn-4-en-3-one (17,20beta-P) or, in certain species, 17,20beta,21-trihydroxy-pregn-4-en-3-one (17,20beta,21-P). Several functions for 17,20beta-P and 17,20beta,21-P have been proposed (and in some cases proved). These include induction of oocyte final maturation and spermiation (milt production), enhancement of sperm motility (by alteration of the pH and fluidity of the seminal fluid) and acting as a pheromone in male cyprinids. Another important function, initiation of meiosis (the first step in both spermatogenesis and oogenesis), has only very recently been proposed. This is a process that takes place at puberty in all fishes and once a year in repeat spawners. The present review critically examines the evidence to support the proposed functions of 17,20beta-P in males, including listing of the evidence for the presence of 17,20beta-P in the blood plasma of male fishes and discussion of why, in many species, it appears to be absent (or present at low and, in some cases, unvarying concentrations); consideration of the evidence, obtained mainly from in vitro studies, for this steroid being predominantly produced by the testis, for its production being under the control of luteinizing hormone (gonadotrophin II) and, at least in salmonids, for two cell types (Leydig cells and sperm cells) being involved in its synthesis; discussion of the factors involved in the regulation of the switch from androgen to 17,20beta-P production that seems to occur in many species just at the time of spermiation; discussion of the effects of in vivo injection and application of 17,20beta-P (and closely related compounds) in males; a listing of previously published evidence that supports the proposed new function of 17,20beta-P as an initiator of meiosis; finally, discussion of the evidence for environmental endocrine disruption by progestins in fishes.

Non-invasive measurement of steroids in fish-holding water: important considerations when applying the procedure to behaviour studies

Fish behaviourists are increasingly turning to non-invasive measurement of steroid hormones in holding water, as opposed to blood plasma. When some of us met at a workshop in Faro, Portugal, in September, 2007, we realised that there were still many issues concerning the application of this procedure that needed resolution, including: Why do we measure release rates rather than just concentrations of steroids in the water? How does one interpret steroid release rates when dealing with fish of different sizes? What are the merits of measuring conjugated as well as free steroids in water? In the ‘static’ sampling procedure, where fish are placed in a separate container for a short period of time, does this affect steroid release—and, if so, how can it be minimised? After exposing a fish to a behavioural stimulus, when is the optimal time to sample? What is the minimum amount of validation when applying the procedure to a new species? The purpose of this review is to attempt to answer these questions and, in doing so, to emphasize that application of the non-invasive procedure requires more planning and validation than conventional plasma sampling. However, we consider that the rewards justify the extra effort.

The potential of the three-spined stickleback (Gasterosteus aculeatus L.) as a combined biomarker for oestrogens and androgens in European waters

The majority of endocrine disruption studies in Europe have been on non-indigenous species (some of them tropical!)--and none of which has traits that make them suitable for the detection of androgenic compounds. To overcome these problems, we have been developing the stickleback as a model biomarker for testing the effect of endocrine disrupters in European waters. Its advantages are: it is the only fish with a quantifiable in vivo androgen and anti-androgen endpoint (the production of the glue protein, spiggin, by the kidney); it is the only fish in which it will be possible to simultaneously test oestrogenic and androgenic properties of compound; it has a genetic sex marker; it is found in all EU countries; it survives and breeds in both seawater and freshwater; it is extremely robust and can be readily deployed in situ; it displays a variety of pronounced reproductive behaviours; it has a simple and short life cycle, low fecundity and high egg/fry survival rates.

Several Synthetic Progestins with Different Potencies Adversely Affect Reproduction of Fish

Synthetic progestins are widely used as a component in both contraceptives and in hormone replacement therapy (HRT), both on their own and in combination with EE2. Their presence in the environment is now established in wastewater effluent and river water and this has led to concerns regarding their potential effects on aquatic organisms living in these waters. We carried out in vivo experiments to determine the potencies of four different synthetic progestins on the reproductive capabilities of the fathead minnow (Pimephales promelas). We then performed a series of in vitro assays to try and determine the reason for the effects seen in the in vivo experiments. In the first experiment, fathead minnow exposed to a single concentration of 100 ng/L of either Levonorgestrel or Gestodene stopped spawning almost completely. The same nominal concentration of Desogestrel and Drospirenone did not affect reproduction (21 d NOECs of 100 ng/L). The second experiment investigated two progestins of different potency: Gestodene at 1, 10, and 100 ng/L and Desogestrel at 100 ng/L, 1 μg/L, and 10 μg/L. Gestodene concentrations as low as 1 ng/L had significant effects on reproduction over 21 d, whereas concentrations of Desogestrel at or above 1 μg/L were required to significantly reduce egg production. The synthetic progestins also masculinized the female fish in a concentration-dependent manner. Results from yeast-based in vitro assays demonstrated that the progestins are all strongly androgenic, thereby explaining the masculinization effects. The results strongly suggest that synthetic progestins merit serious consideration as environmental pollutants.

Use of the three-spined stickleback (Gasterosteus aculeatus) as a sensitive in vivo test for detection of environmental antiandrogens.

We have previously shown that exposure to exogenous androgens causes female sticklebacks (Gasterosteus aculeatus) to produce the glue protein, spiggin, in their kidneys. This protein can be quantified by an enzyme-linked immunosorbent assay developed and validated at the Centre for Environment, Fisheries and Aquaculture Science. Here we report the development of an in vivo test for the detection of environmental antiandrogens. The system involves the simultaneous exposure of female sticklebacks to 17α-methyltestosterone (a model androgen) at 500 ng/L and suspected environmental antiandrogens over a period of 21 days. The spiggin content of the kidneys is then measured, and any antiandrogenic activity is evaluated by comparing the spiggin levels of female fish exposed to antiandrogens to those of female fish exposed solely to the model androgen. The assay detects the antiandrogenic activity of flutamide, vinclozolin (both used at 250 μg/L), linuron (at 150 μg/L), and fenitrothion (at 15 and 150 μg/L). These results provide the first evidence of in vivo antiandrogenic activity of both linuron and fenitrothion in teleosts. Although there are other suggested fish species that could be used for this purpose, the stickleback is the only widely available species in which it is now possible to study both estrogenic and antiandrogenic end points in the same individual. Furthermore, the species is endemic and ubiquitous in Europe, and it possesses many ecological traits that make it better suited than other potential species for field research into endocrine disruption.

Principles of sound ecotoxicology.

We have become progressively more concerned about the quality of some published ecotoxicology research. Others have also expressed concern. It is not uncommon for basic, but extremely important, factors to apparently be ignored. For example, exposure concentrations in laboratory experiments are sometimes not measured, and hence there is no evidence that the test organisms were actually exposed to the test substance, let alone at the stated concentrations. To try to improve the quality of ecotoxicology research, we suggest 12 basic principles that should be considered, not at the point of publication of the results, but during the experimental design. These principles range from carefully considering essential aspects of experimental design through to accurately defining the exposure, as well as unbiased analysis and reporting of the results. Although not all principles will apply to all studies, we offer these principles in the hope that they will improve the quality of the science that is available to regulators. Science is an evidence-based discipline and it is important that we and the regulators can trust the evidence presented to us. Significant resources often have to be devoted to refuting the results of poor research when those resources could be utilized more effectively.

Fertility and motility of sperm from Atlantic halibut (Hippoglossus hippoglossus) in relation to dose and timing of gonadotrophin-releasing hormone agonist implant

Abstract In broodstocks of Atlantic halibut, Hippoglossus hippoglossus , male and female gamete production often becomes unsynchronised towards the end of the spawning season—milt becomes very viscous and difficult to express while the females are still producing batches of good quality eggs. Gonadotrophin-releasing hormone agonist (GnRHa) has been shown to stimulate spermiation in a number of fish species. Therefore, we conducted two experiments where male halibut were implanted intramuscularly with pellets containing GnRHa. The effect of the pellets was tested at three periods: before, at the height of and at the end of spermiation. In the middle period, GnRHa was tested at two doses (5 and 25 µg/kg bodyweight). Measurements were made of milt hydration, sperm motility and fertilisation rate. Implanted males began spermiation at least 4 weeks before control males. Both doses of GnRHa increased the fluidity of the milt. This effect lasted for at least 20 days in the low dose group and for 40 days in the high dose group. When applied at the end ofthe season, GnRHa reversed the normal trend for the milt to become more viscous. GnRHa treatments did not affect fertilisation rates obtained with the sperm. However, towards the end of the spawning season, sperm motility was enhanced in males treated with the high dose of GnRHa (25 μg/kg) compared to controls. As described previously, plasma concentrations of the gonadal steroids, 5β-pregnane-3β,17,20β-triol 20-sulphate and 17,20α-dihydroxy-4-pregnen-3-one, were significantly enhanced by GnRHa treatment. Concentrations of testosterone on the other hand decreased when spermiating males were treated with GnRHa. Our data suggest that 17,20β-dihydroxy-4-pregnen-3-one or its metabolites are involved in milt hydration, possibly through affecting ion transport. Crown Copyright