Vertebrates 221

More than twenty years ago, Howell et al. [16] reported the presence of masculinised female mosquito fish (Gambusia affinis) in a small creek downstream of a paper mill discharging bleached kraft mill effluent. Subsequently, masculinised females of other (but not all) species were found, and laboratory studies showed that exposure to bleached kraft mill effluent induced male secondary sexual characteristics [17]; thus, it appeared that the effluent contained an androgenic chemical, or mixture of chemicals. A comprehensive review of all the work on endocrine disruption in mosquito fish can be found in [1]. Very recently,spawning female channel catfish (Tetaluruspunctatus) exhibiting male secondary sexual characteristics have been reported to be present in the Red River of the North in North Dakota, USA. [18]. There are no paper mills discharging effluent into this river, although it does receive waste water from a sewage treatment plant and a sugar beet processing plant; however, further investigation is required to identify the cause of masculinisation.

More recently, research has focused on the mechanisms whereby bleached kraft mill effluent has been shown to affect reproduction adversely by reducing plasma sex steroid hormone concentrations, delaying sexual maturity, reducing vitellogenin concentrations, and reducing gonad and egg size (e.g. [19, 20]). These multiple effects, which are undoubtedly effects of bleached kraft mill effluent on the endocrine system, are difficult, if not impossible, to classify as primarily androgenic or oestrogenic. This is unsurprising when one considers that the effluent is a highly complex mixture of natural and anthropogenic chemicals, many of which have been shown to alter the endocrine control of reproduction in various species.

It is also 20 years ago that grossly hermaphrodite (intersex) fish were found in the settlement lagoons of two sewage treatment works (STWs) in England. In the UK, and many other densely-populated, developed countries, effluents from STWs often contribute 50 % of the flow of rivers, a figure that can rise as high as 90% (or more!) in periods of low rainfall (when demand for water is highest). Thus, the fish in such rivers live in diluted, treated effluent, not clean water.

Research on the fisheries implications of effluents affecting sex determination began in the late 1980s. It was soon discovered that STW effluent was oestrogenic to fish [21]. Specifically, when caged trout were maintained in effluent channels, they responded by synthesising vitellogenin, which serves as a very sensitive and specific biomarker for exposure to 'oestrogens' [22]. Follow-up research in rivers receiving varying amounts of STW effluents showed that significant stretches of river downstream of major STWs were oestrogenic to caged fish [23,24]. In the worse case, an entire 5-km stretch of river downstream of a large STW was extremely oestrogenic; maximum vitellogenesis occurred in the caged fish [24]. Further, not only were plasma vitellogenin concentrations extremely high, but the testes of the caged male trout were much smaller than those of control fish.

Extensive studies, such as those done in the UK and summarised above, have not yet been conducted in other countries. However, fairly small scale, preliminary studies have shown that feral male carp (Cyprinus carpio) captured near a major sewage treatment plant in the US have elevated plasma vitellogenin concentrations [25], and that vitellogenin was present in some male and immature brown trout (Salmo trutta) captured downstream of sewage treatment plants in Switzerland [26].Very recently, a caged fish study in Sweden showed massive induction of vitellogenin in fish exposed to STW effluent [27]. All these studies suggest that the oestrogenic effects observed in the UK in fish exposed to effluent from STW [21] and in rivers receiving effluent [23,24] will prove not to be unique to the UK, but rather to be a general phenomenon. However, it is likely that the oestrogenic potency of effluent will vary from effluent to effluent (and hence from river to river), depending on the 'strength' of the effluent, the size and efficiency of the sewage treatment plant, and the degree of dilution of the

Fig. 1A-D. The histological apperance of the gonads of normal and intersex roach (Rutilis ru-tilis) caught in British rivers. A A normal male. The testis is full of lobules in which sper-matogenis takes place. B A normal female. The overy is full of oocytes at different stages of growth. The large oocytes are undergoing vitellogenesis, whereas the smaller (primary) oocytes have not yet entered vitellogenesis. C A mildly intersex fish. Most of the gonad has the appearance of a testis: it is full of lobules in which spermatogenesis is taking place. Six primary oocytes are scattered amongst the testicular tissue. D A severely intersex fish. This picture shows one large, vitellogenic oocyte and a number of smaller, primary oocytes, apparently physically separated from an area of reasonably normal-looking testicular tissue. All pictures were taken at the same magnification. (x 100).

Fig. 1A-D. The histological apperance of the gonads of normal and intersex roach (Rutilis ru-tilis) caught in British rivers. A A normal male. The testis is full of lobules in which sper-matogenis takes place. B A normal female. The overy is full of oocytes at different stages of growth. The large oocytes are undergoing vitellogenesis, whereas the smaller (primary) oocytes have not yet entered vitellogenesis. C A mildly intersex fish. Most of the gonad has the appearance of a testis: it is full of lobules in which spermatogenesis is taking place. Six primary oocytes are scattered amongst the testicular tissue. D A severely intersex fish. This picture shows one large, vitellogenic oocyte and a number of smaller, primary oocytes, apparently physically separated from an area of reasonably normal-looking testicular tissue. All pictures were taken at the same magnification. (x 100).

effluent in the receiving water, besides (probably) many other factors that will emerge as research proceeds.

The discovery that effluent from STWs is oestrogenic to fish raises the issue of whether wild fish, living in rivers receiving significant inputs of effluent, are being adversely affected. Until very recently, there was very little evidence to suggest that they are. If effluents were 'feminising' wild fish populations, then one might expect to find elevated vitellogenin concentrations, and intersexuality (specifically, ovarian tissue in the testes of males), possibly even all-female populations. In fact, reports of occasional, individual intersex fish, or of a small proportion of intersex fish amongst a large sample, have regularly appeared (e.g. [28, 29]). Sometimes these instances have even been linked to environmental conditions [28], although the specific cause has remained unknown. This situation has now changed, with the publication of an extensive field study of intersexuality in one species of native freshwater fish, the roach (Rutilis rutilis) [2]. This study set out to address the question of whether exposure to effluent from STWs caused oes-trogenic responses in wild fish. Populations of roach were sampled both upstream and downstream of STWs on eight rivers and from five reference (control) sites throughout the British Isles; the rivers selected represented a range with regard to general water quality (from very good to poor). Histological examination of the gonads revealed that a high proportion of the 'males' were, in fact, intersex, as defined by the simultaneous presence of both male and female gonadal characteristics (Fig. 1). Intersex fish were found at all sites, although the incidence was much higher in rivers that received STW effluents than at the control sites; the incidence of intersexuality in 'male' fish ranged from 4% (at two control sites) to 100% in two populations of roach living downstream of major STWs in heavily impacted rivers. There was a highly significant positive correlation between the degree of intersexuality in the 'male' fish and their plasma vitellogenin concentrations [2], suggesting (but not proving) that both parameters were caused by the same factor (STW effluent). These results provide compelling evidence that populations of wild fish inhabiting many rivers in the UK are being exposed to oestrogenic chemicals, and that these are, in most cases, present at higher concentrations in stretches of river directly downstream from large STWs. However, the ecological significance of these results remains unclear presently.

Endocrine disruption has also been observed in marine fish. Following up on the report by Lye et al. [30] of reproductive problems in flounders (Platichthys flexus) exposed to effluents from a STW, Matthiessen and colleagues have conducted a very extensive investigation of endocrine disruption in flounder from estuaries and marine waters around the UK [31]. Vitellogenin concentrations were found to be significantly elevated, often markedly so, in flounder living in many estuaries, especially those that receive large amounts of industrial effluent. At two locations, some of the fish were hermaphrodite, with their testes containing large numbers of oocytes amongst apparently normal looking tes-ticular tissue. The causative substances are unknown, but there was a clear relationship between vitellogenin induction and the volume of industrial (but not domestic) effluent discharged into the estuaries.

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