John P. Sumpter

Estrogenic effects of effluents from sewage treatment works

Abstract The occurrence of hermaphrodite fish in the lagoons of sewage treatment works led us to hypothesize that sewage effluent might contain a substance, or substances, estrogenic to fish. to test this hypothesis, we placed cages containing rainbow trout in the effluent from sewage-treatment works, and one to three weeks later measured the vitellogenin concentration in the plasma of the fish. Vitellogenin is a protein synthesized by the liver of oviparous fish in response to estradiol stimulation; it is then conveyed by the blood to the ovary, where it is sequestered by oocytes to form the yolk. Thus, the presence of vitellogenin in the plasma is indicative of estrogenic stimulation of the liver. an initial study, at a sewage-treatment works, showed that plasma vitellogenin concentrations rose rapidly and very markedly (over 1000-fold in three weeks) when trout were maintained in the effluent. an extensive nationwide survey was then conducted. Results were obtained from fifteen sewage-treatment works d...

Detergent components in sewage effluent are weakly oestrogenic to fish: An in vitro study using rainbow trout (Oncorhynchus mykiss) hepatocytes

Abstract Alkylphenol-polyethoxylates (APnEO, n = 1–40) are a major group of surfactants and are normally present in raw sewage. Many of the products of the biodegradation of these compounds are both persistent and present in substantial quantities in effluent and in river water. We report here on the use of an in vitro bioassay to determine the oestrogenic potencies of these compounds to fish. The bioassay is based on the fact that the synthesis of vitellogenin by hepatocytes is oestrogen dependent. Of the compounds tested, 4-nonylphenol, 4-tert-octylphenol, 4-tert-butylphenol, 4-nonylphenol-diethoxylate, Tergitol-NP9, and 4-nonylphenoxycarboxylic acid were all weakly oestrogenic, with potencies between about 1 × 10−4 to 1 × 10−6 the activity of 17β-oestradiol The oestrogenic activity observed appeared to be confined to para or 4 substituted compounds, because 2-tert-butylphenol and 3-tert-butylphenol were inactive. The polyethoxylate compounds became less oestrogenic with increasing lengh of the ethoxy chain. Thus cells exposed to Tergitol NP40EO (with a chain length of 40) did not secrete vitellogenin. Simultaneous exposure of the hepatocytes to Tamoxifen (an oestrogen antagonist) and effective doses of representative compounds caused an inhibition of the oestrogenic effect in all cases, suggesting that the action of these compounds is mediated by the oestradiol receptor.

Oocyte growth and development in teleosts

Oocyte growth and development is an important issue in fish and fisheries biology. This paper reviews the information available on oocyte growth patterns and the rates and dynamics of oocyte growth in teleosts. In synchronous spawners, the weight of the gonad may represent as much as 40% of the overall body weight of the fish. In asynchronous spawners, the weight of the mature ovary is considerably less than in synchronous ovulators, but the ovary shows a more regular periodicity and may grow repeatedly many times during the breeding season. There is a huge variability in egg size in teleosts, with the largest known measuring up to 8 cm in diameter. Within the limits of variance set by genetic constraints, egg size may vary between populations of the same species. Oocytes in all teleosts undergo the same basic pattern of growth: oogenesis, primary oocyte growth, cortical alveolus stage, vitellogenesis, maturation and ovulation. The mechanisms that control oocyte growth are addressed in this review, albeit that the available information, as in all other vertebrates, is very limited. The main hormones that have been shown to affect ovarian growth are gonadotrophin, thyroid hormones, growth hormone, insulin and insulin-like growth factors. An overview of the determinants of fecundity, with particular reference to oocyte recruitment and atresia, is the focus of the second part of the paper. Genetics and nutrition have major effects on fecundity, and studies so far suggest that the determinants of fecundity usually operate during the early part of gametogenesis. The role of atresia in determining fecundity is less clear. The final part of this review highlights some areas of study that are priorities for research on ovarian development in fish.

Egg quality in fish: what makes a good egg?

Factors affecting egg quality are determined by the intrinsic properties of the egg itself and the environment in which the egg is fertilized and subsequently incubated. Egg quality in fish is very variable. Some of the factors affecting egg quality in fish are known, but many (probably most) are unknown. Components that do affect egg quality include the endocrine status of the female during the growth of the oocyte in the ovary, the diet of the broodfish, the complement of nutrients deposited into the oocyte, and the physiochemical conditions of the water in which the eggs are subsequently incubated. In captive broodfish, the husbandry practices to which fish are subjected are probably a major contributory factor affecting egg quality. Our knowledge of the genetic influences on egg quality is very limited indeed. We know that parental genes strongly influence both fecundity and egg quality, but almost nothing is known about gene expression and/or mRNA translation in fish oocytes/embryos. This is surprising because the products synthesized in ovoand the mechanisms controlling their expression are likely to play a central role in determining egg quality. The genetic mechanisms underpinning oocyte and embryo growth and development are a priority for research

Fish stress and health in aquaculture

Preface 1. Stress in finfish: past, present and future - a historical perspective B. A. Barton 2. Effects of rearing conditions on the health and physiological quality of fish in intensive culture G. A. Wedermeyer 3. Effects of stress on reproduction and growth of fish N. W. Pankhurst, and G. van der Kraak 4. The endocrinology of stress J. P. Sumpter 5. Ionic, osmotic and acid-base regulation in stress G. McDonald, and L. Milligan 6. Behavioural response to stress C. B. Schreck 7. Genetic basis to the stress response: selective breeding for stress-tolerant fish T. G. Pottinger and A. D. Pickering 8. Immune-endocrine interactions P. Balm 9. Dietary effects on stress and health T. C. Fletcher 10. Measurements of stressed states in the field J. D. Morgan, and G. K. Iwama Index.

Structural Features of Alkylphenolic Chemicals Associated with Estrogenic Activity

The ability of certain man-made chemicals to mimic the effects of natural steroid hormones and their potential to disrupt the delicate balance of the endocrine system in animals are of increasing concern. The growing list of reported hormone-mimics includes the alkylphenolic (AP) compounds, a small number of which have been reported to be weakly estrogenic. In their most basic form, APs are composed of an alkyl group, which can vary in size, branching, and position, joined to a phenolic ring. The aim of this project was to identify the important structural features responsible for the estrogenic activity of AP chemicals. This was achieved by incubating APs with different structural features in a medium containing a previously described estrogen-inducible strain of yeast (Saccharomyces cerevisiae) expressing the human estrogen receptor and comparing their activity spectrophotometrically by the resulting color change of the medium. The results were compared to the effects of the main natural estrogen 17β-estradiol. The data indicate that both the position (para > meta > ortho) and branching (tertiary > secondary = normal) of the alkyl group affect estrogenicity. Optimal estrogenic activity requires a single tertiary branched alkyl group composed of between 6 and 8 carbons located at the para position on an otherwise unhindered phenol ring. The results are discussed in relation to the purity and composition of the chemicals tested.

Altered Sexual Maturation and Gamete Production in Wild Roach (Rutilus rutilus) Living in Rivers That Receive Treated Sewage Effluents

Abstract Disruption in gonadal development of wild roach living in U.K. rivers receiving large volumes of treated sewage effluent is manifest in a variety of ways, ranging from malformation of the germ cells and/or reproductive ducts to altered gamete production. Intersex fish were also found to have an altered endocrine status and an elevated concentration of plasma vitellogenin. Gonadal growth was inhibited only in severely intersex fish, whereas progression of spermatogenesis was delayed in a large proportion of all intersex and exposed male fish. In contrast to the effects observed in the intersex and exposed male fish, the maturation of ovaries in female fish inhabiting effluent-contaminated rivers appeared to be less obviously affected, although a higher incidence of oocyte atresia was found in the effluent-exposed fish compared with the reference fish. A positive correlation was found between the proportion of female tissue in the gonads of intersex fish and their plasma vitellogenin concentration, suggesting that vitellogenin can be an indicator for the level of gonadal disruption in intersex roach. The estradiol-17β concentration in intersex fish was intermediate between the concentration found in males and females, and the plasma testosterone was between 2- and 3-fold higher in intersex fish compared with male fish. These data suggest a link between altered endocrine status in intersex and female fish and gonadal disruption. Spermiation was also affected in roach living in effluent-impacted rivers: a lower proportion of fish were found releasing sperm, and in those intersex fish that were spermiating, a reduced milt volume and a reduced sperm density were found. All intersex fish had malformations of the reproductive duct(s), and in severely affected fish, the ducts were occluded, thus preventing release of gametes. In view of the widespread occurrence of intersexuality in wild fish populations in rivers throughout the United Kingdom, assessment of the reproductive capabilities of these intersex roach is clearly needed to understand the impact of this phenomenon on roach fertility.

Wild Intersex Roach (Rutilus rutilus) Have Reduced Fertility

Abstract Endocrine-disrupting chemicals, known to be present in the environment, have great potential for interfering with reproductive health in wildlife and humans. There is, however, little direct evidence that endocrine disruption has adversely affected fertility in any organism. In freshwater and estuarine fish species, for example, although a widespread incidence of intersex has been reported, it is not yet known if intersexuality influences reproductive success. The purpose of this study was, therefore, to determine gamete quality in wild intersex roach (Rutilus rutilus) by assessing sperm characteristics, fertilization success, and ability to produce viable offspring. The results clearly demonstrate that gamete production is reduced in intersex roach. A significantly lower proportion of moderately or severely feminized fish (17.4% and 33.3%, respectively) were able to release milt compared with normal male fish from contaminated rivers (in which 97.6% of the males were able to release milt), reference male fish (97.7%), or less severely feminized intersex fish (experiment 1: 85.8%, experiment 2: 97%). Intersex fish that did produce milt produced up to 50% less (in terms of volume per gram of testis weight) than did histologically normal male fish. Moreover, sperm motility (percentage of motile sperm and curvilinear velocity) and the ability of sperm to successfully fertilize eggs and produce viable offspring were all reduced in intersex fish compared with normal male fish. Male gamete quality (assessed using sperm motility, sperm density, and fertilization success) was negatively correlated with the degree of feminization in intersex fish (r = −0.603; P < 0.001) and was markedly reduced in severely feminized intersex fish by as much as 50% in terms of motility and 75% in terms of fertilization success when compared with either less severely feminized intersex fish or unaffected male fish. This is the first evidence documenting a relationship between the morphological effects (e.g., intersex) of endocrine disruption and the reproductive capabilities of any wild vertebrate. The results suggest that mixtures of endocrine-disrupting substances discharged into the aquatic environment could pose a threat to male reproductive health.

Accurate Prediction of the Response of Freshwater Fish to a Mixture of Estrogenic Chemicals

Existing environmental risk assessment procedures are limited in their ability to evaluate the combined effects of chemical mixtures. We investigated the implications of this by analyzing the combined effects of a multicomponent mixture of five estrogenic chemicals using vitellogenin induction in male fathead minnows as an end point. The mixture consisted of estradiol, ethynylestradiol, nonylphenol, octylphenol, and bisphenol A. We determined concentration–response curves for each of the chemicals individually. The chemicals were then combined at equipotent concentrations and the mixture tested using fixed-ratio design. The effects of the mixture were compared with those predicted by the model of concentration addition using biomathematical methods, which revealed that there was no deviation between the observed and predicted effects of the mixture. These findings demonstrate that estrogenic chemicals have the capacity to act together in an additive manner and that their combined effects can be accurately predicted by concentration addition. We also explored the potential for mixture effects at low concentrations by exposing the fish to each chemical at one-fifth of its median effective concentration (EC50). Individually, the chemicals did not induce a significant response, although their combined effects were consistent with the predictions of concentration addition. This demonstrates the potential for estrogenic chemicals to act additively at environmentally relevant concentrations. These findings highlight the potential for existing environmental risk assessment procedures to underestimate the hazard posed by mixtures of chemicals that act via a similar mode of action, thereby leading to erroneous conclusions of absence of risk.

Adverse reproductive effects in male fathead minnows (Pimephales promelas) exposed to environmentally relevant concentrations of the natural oestrogens, oestradiol and oestrone

Recent research has shown that low concentrations of natural and synthetic oestrogens are present in sewage treatment works effluents. However, the effects of these oestrogens on fish, at environmentally relevant concentrations, are unknown. Therefore, male fathead minnows (Pimephales promelas) were exposed to nominal concentrations of 17β-oestradiol (10, 32, 100, 320 and 1000 ng l−1) and molar equivalent concentrations of oestrone (9.9, 31.8, 99.3, 318 and 993 ng l−1) for 21 days, to determine the effects of these chemicals on plasma vitellogenin levels and gonad weight. Exposure to both steroidal compounds resulted in an elevation of plasma vitellogenin levels, in a concentration-related manner. Significant (P 100 ng oestradiol l−1 and 31.8 ng oestrone l−1. This increase in plasma vitellogenin levels was accompanied by an inhibition of testicular growth, which for the highest two concentrations of oestradiol was total. The results from this study indicated that although oestrone induced significant effects at a lower concentration, oestradiol was more potent than oestrone at higher concentrations, causing greater vitellogenin synthesis and testicular inhibition. The key result is that low concentrations of oestradiol and oestrone, similar to the reported concentrations in effluent, have profound effects on male fish.