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Feminisation of fish 7. Relation between environmental levels of estrogens/xenoestrogens and known effect concentrationsWhen comparing the concentrations of natural and synthetic estrogens in effluent and surface water with the dose-response relationships for the various reproductive effects described in the previous chapter, it is apparent that some concentrations have been found in various effluents and also in some aquatic environments which are above the LOECs described for i.e. induction of vitellogenin synthesis in male fish, induction of intersex and for causing other testicular effects. Concentrations of < 0.1 – 88 ng/l and 0.05 – 15.5 ng/l have been reported for estradiol in sewage effluent and surface water respectively. LOECs for vitellogenin induction and intersex have in comparison been reported to 5 and 10 ng/l and a range of other testicular effects have been seen at 10 – 50 ng/l. When the estrogen concentration has only been reported in sewage effluent it is, however, difficult to estimate whether this causes a resultant surface water concentration above the reported LOEC, since dilution and degradation in relation to the flow of the river will affect the fate of the steroid and therefore the final water concentration. Still, some reported surface water concentrations of estradiol have been high enough to predict feminisation of male fish. For estrone, concentrations in the range of <0.1 – 220 ng/l in sewage effluent and <0.1 – 17 ng/l in surface water have been detected and LOEC for induction of vitellogenin in male fish has been reported to approximately 30 ng/l (ten times lower for juvenile females). LOEC for induction of intersex in males has been reported to 10 ng/l which was the same as found for estradiol. Judged from these observations estrone levels in surface waters can be a candidate for causing feminisation of fish – also given the higher frequency of detection of this steroid compared to the other estrogens. Whether or not the other natural estrogen, estriol, participates in some of the observed reproductive disturbances reported from numerous countries is as yet not possible to assess since analyses of effluent and surface water concentrations plus in vivo water exposure experiments on estriol are sparse. One study has found a LOEC of intersex induction in medaka of 1 µg/l which is far above the few existing reports on estriol concentrations in surface water (< 0.1 – 3.4 ng/l) and sewage effluent (< 0.1 – 42 ng/l). Data on this steroid must, however, be considered inadequate for a reliable judgement of its contribution to the feminisation of male fish. As mentioned earlier in vitro studies have found estriol to be 30 times less potent than estradiol (42). Ethinylestradiol is generally detected less often than both estrone and estradiol and although it has been found in the range of < 0.1- 62 ng/l in effluent and 0.053 – 30.8 ng/l in surface water it is mostly detected below 5 ng/l (and often below 1 ng/l). Very low concentrations of the synthetic hormone are, however, capable of causing feminisation of male fish. Both vitellogenin induction and intersex have been reported at 0.1 ng/l, and although some have questioned the LOEC of intersex of that particular study (37) another experiment has demonstrated changed sex ratios at concentrations down to 0.6 ngEE2/l. A number of other testicular effects such as reduced testicular growth and inhibition of spermatogenesis have also been found at concentrations below 10 ng/l. Since the detection limit for many chemical analysis for EE2 is between 0.1 and 1 ng/l this impedes the judgement as to whether the concentrations in the environment is a risk factor in relation to feminisation of male fish. In some European waters ethinylestradiol has, however, been detected in concentrations which could explain all or part of the observed feminisation (3;46). Alkylphenols have in some hot-spot areas of England also been suspected as primary or contributory compounds to feminisation of male fish (34). Internationally, sewage effluent concentrations between 25 ng/l and 330 µg/l and surface water concentration between 5 ng/l to 180 µg/l have been reported for nonylphenol. A single river water concentration above 600 µg/l has been reported from Spain. Typically, concentrations in sewage effluent and surface water does, however, not exceed 10 and 1 µg/l, respectively. In the rivers with high concentrations of nonylphenol, these exceed well the LOEC for vitellogenin synthesis which is approximately 5 µg/l, and a long-term exposure study has moved the LOEC down to 1 µg/l (51). Exposure to between 30 and 100 µg/l has further resulted in inhibited testicular growth, changed sex ratio and degenerating testes. Degenerated testes have also been observed at low NP concentrations below 5 µg/l. In some rivers, nonylphenol will therefore be a likely endocrine disrupting compound. Octylphenol, as mentioned earlier, is generally assumed to have a higher estrogenic potency than nonylphenol but it is not as widely used in industry as nonylphenol. This is also reflected in the concentrations of octylphenol found in both sewage effluent and surface water in which it is found in concentrations of between 22 ng/l and 73 µg/l and 0.4 ng/l – 13 µg/l. The high end concentrations are above the LOEC of vitellogenin synthesis of 5 µg/l (50) and above the concentration which have caused intersex and a shift in sex ratio in the medaka (2µg/l) (49), and octylphenols might have participated in feminising effects in rivers with high concentrations. Few reports exist as mentioned on bisphenol A which together with alkylphenols are among the most potent of the presently known estrogenic chemicals. Highest concentrations reported in effluent and surface water is 6 and 1 µg/l, respectively with a single observation of 8 µg/l in a river site downstream of a manufacturer. Most effects of bisphenol A have been reported at concentrations above 40 µg/l, though intersex and a reduced number of spermatozoa have been found at exposure concentrations of 10 – 20 µg/l. Since only a single report exist of bisphenol A in concentrations near the lowest LOEC for any effects, this xenoestrogen is considered to play little or no part in the feminising effects observed at fish in different parts of the world. When assessing the possible implications for the reproductive health of male fish of
the estrogenic compounds in the aquatic environment it is, however, important not only to
look at the individual levels of single estrogens or xenoestrogens. The different
estrogens and xenoestrogens will act in a concerted manner with an additive activity (40). This means that concentrations below the LOECs
for the individual compounds can exert an estrogen activity when present in a mixture of
other estrogens and xenoestrogens. This has been demonstrated for both the combination of
estradiol and ethinylestradiol (57) and estradiol
and nonylphenol (40) in regard to vitellogenin
synthesis in rainbow trout. Another aspect which has to be taken into consideration when assessing the risk of sewage effluent outlet to the environment, is the consequences of intermittent release of high concentrations of endocrine disrupting compounds in pulses. Exposure of fathead minnow to shorter, repetitive high concentrations of estradiol resulted in concentrations of plasma vitellogenin which were higher than continuous exposure to the equivalent time-weighed average concentration. In general, therefore both the capacity of single estrogens and estrogenic chemicals, the additivity of the compounds with the same estrogenic mechanism of action and the unproportionate effects of intermittent exposure to high concentrations of the compounds have to be taken into account when evaluating the compound(s) responsible for the total estrogenicity of sewage effluent and the compounds responsible for the already observed endocrine disruption of the male fish reproductive system. It is also important to bear in mind that many of the controlled laboratory studies performed to assess LOECs and NOECs for the various estrogenic compounds often are short-term exposure experiments. As mentioned earlier long-term exposure experiments have been demonstrated to lower the concentration needed to result in testicular or other effects such as vitellogenin synthesis. Chronic exposure to low levels of estrogens might result in reduced LOECs for the different endocrine disrupting effects. Further, the effects of an exposure may depend on the timing of the exposure in relation to the sex differentiation and reproductive cycle of the fish. The most sensitive stage in regard to disruption of the reproductive system is generally thought to be the very early life stages in which the sex is determined and differentiated (58). The effects created at this stage is also often irreversible since it involved the formation of for instance female somatic structures. There might, however, also be periods in the reproductive cycle of adult, sexually mature fish in which they are more susceptible to estrogen exposure than others.
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