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.

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-17beta 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.

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SUMMARYThe major protocol features of the immature rat uterotrophic assay have been evaluated using a range of reference chemicals. The protocol variables considered include the selection of the test species and route of chemical administration, the age of the test animals, the maintenance diet used and the specificity of the assay for estrogens. It is concluded that three daily oral old rats, followed by determination of absolute uterus weights on the fourth day, provides a sensitive and toxicologically relevant in vivo estrogenicity assay. Rats are favoured over mice for reasons of toxicological practice, but the choice of test species is probably not a critical protocol variable, as evidenced by the similar sensitivity of rats and mice to the uterotrophic activity of methoxychlor. Vaginal opening is chemicals to reduce or abolish the uterotrophic response of estradiol is suggested to provide a useful extension of the uterotrophic assay foThe results of a series of studies on the environmental estrogen nonylphenol (NP), and its ng of the aliphatic sidechain is important for activity. 17 similar activity to estradiol in the uterotrophic assay, and is suggested to represent the 'parent' estrogen of NP. Benzoylation of r uterotrophic activity, in contrast to the enhancing effect of benzoylation on estradiol.Selected chemicals shown to be active in the immature rat uterotrophic assay were also evaluated in an in vitro yeast human estrogen receptor transactivation assay. Most of the chemicals gave similar qualitative responses to those seen in the uterotrophic assay, and the detection of the estrogen methoxychlor by the yeast assay evidenced a degree of intrinsic metabolic competence. However, the assay had a reduced ability (compared to rodents) to hydrolyse the benzoate ester of estradiol, and the estrogenic benzoate derivative of NP was not active in the yeast assay. These last results indicate that current metabolic deficiencies of in vitro estrogenicity assays will limit the value of negative data for the immediate future.The results described illustrate the intrinsic complexity of evaluating chemicals for estrogenic activities and confirm the need for rigorous attention to experimental design and criteria for assessing estrogenic activity.3

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.