| Front page | | Contents | | Previous | | Next |

Feminisation of fish

6. Established dose-response relations between the estrogens/xenoestrogens and their feminising potential.

Following the observations of feminised male fish in sewage effluent receiving waters and the increasing evidence that estrogens and in some cases xenoestrogens are the compounds responsible for the observed endocrine disruption, a number of experiments have been performed to assess the lowest observable effect concentrations (LOECs) and no effect concentration (NOECs) for various feminising effects of estrogens and xenoestrogens on male fish. These studies have demonstrated that different species show different sensitivities towards the feminising potential of the various compounds. The LOECs and NOECs mentioned for vitellogenin induction in male and immature fish and for development of intersex or other reproductive effects in the following sections will in general be the ones found for the most sensitive species by water exposure studies. It is also important to note that LOECs and the NOECs often are dependent on the duration of exposure. Longer exposure periods have been seen to lower the limit for feminisation (24;41).

6.1 17b -estradiol

Experiments with juvenile rainbow trout has demonstrated that vitellogenin can be induced in this species after 14 days exposure to 4.7 – 7.9 ng/l estradiol (40). NOEC was in another study of the same author assessed to <5 ng/l (41).

Very low LOECs and NOECs have also been reported for induction of intersex or testis-ova in Japanese medaka (Oryzias latipes). In a group exposed to a nominal concentration of 10 ng/l estradiol for approximately 100 days from hatch and onwards 10 % intersex was found (42). No difference in sex ratio was found at this concentration. Exposure to 100 ng/l estradiol altered the sex ratio towards females and all males had developed the intersex condition. The NOEC for intersex induction concentration was assessed to 1 ng/l, but no concentrations between 1 and 10 ng/l were tested (37).

In another study, exposure of the same species for 28 days from hatch to 10 ng/l E2 produced all females (43). Complete sex reversal of genotypic male medaka to females have also been obtained when exposing to 1 µg E2/l from day 0-8 from fertilisation until hatch at 10 days post fertilisation (176). These results demonstrate that the timing of exposure in relation to the sex differentiation is important for the resultant effect on the expression of the sex.

Exposure of male fish to E2 has also resulted in a number of other reproductive effects. Miles-Richardson have found alterations in the morphology of seminiferous tubules of the testis in fathead minnow from concentrations of 136 ng/l E2 after exposure for 14 days (44). Sertoli cells were hyperplastic and hypertrophied and degenerative changes such as loss of germ cells and presence of degenerated spermatozoa were observed. Electron microscopy revealed that the distended sertoli cells contained large phagolysosomes which contained various stages of degenerated spermatozoa and other cellular debris. This has also been seen after exposure of eelpout to E2 (177). An inhibition of spermatogenesis or inhibition of germ cell maturation was suggested in the study of medaka based on these observations plus the great number of earlier stages (spermatocytes) of spermatogenesis compared to controls (44). NOEC for these testicular effects was reported to 68 ng/l. Higher concentration of E2 (272 ng/l) also reduced the male secondary sex characteristics, fatpads and nuptial breeding tubercles.

In females changes in the follicular development such as a larger number of immature follicles has been observed after exposure to ³ 27 ng/l. NOEC for this effect was 17 ng/l.

Some of the disruptions in the sexual development of fish caused by estrogens might be due to changes in the activity of aromatase, the enzyme responsible for the synthesis of estrogens from aromatisable androgens. Exposure of maturing fathead minnows to E2 caused on up-regulation of P450aromataseB mRNA expression in the testis and ovary in a dose-dependent manner after 14 days exposure (45). LOEC was 320 ng/l for males and 100 ng/l for females. In the male brain, P450aromataseB mRNA levels were further significantly elevated after 14 days exposure to 32 ng E2/l. GSI was reduced in males exposed to 100 ng E2/l.

In line with the indicated up-regulation of estradiol synthesis, a four times higher plasma estradiol level was found in male goldfish after exposure to 1000 ng/l E2 (178).

Overall, for 17ß-estradiol it can be concluded that although high exposure concentrations have been used to induce some alterations of the male reproductive system, a number of adverse effects have been seen at concentrations between 10 and 50 ng/l and even lower in regard to vitellogenin synthesis.

6.2 Estrone

Less studies have in general been performed with both estrone and estriol compared to the other natural estrogen, 17ß-estradiol.

The LOEC for estrone in regard to vitellogenin synthesis has been reported to be 3.2 ng/l for juvenile female rainbow trout (in (37)) after an exposure period of 14 days. Ten times as high a LOEC (31.8) was found for vitellogenin synthesis in male fathead minnow after 21 days of exposure (47) which compares well with a LOEC of between 25 and 50 ng/l for adult male rainbow trout exposed for the same period of time (46).

Induction of intersex in medaka has been obtained with a LOEC of estrone of 10 ng/l af 100 days exposure from hatch and onwards (42). No NOEC was assessed since 10 ng/l was the lowest tested concentration.

Testicular growth has been inhibited in male fathead minnow by 21 days of exposure to 318 ng/l (47).

Estrone is widely considered to be of similar or slightly lower in vivo estrogenic potency than estradiol as also seen from the above mentioned experiments. A 3-5 times lower potency of estrone has been suggested (37).

6.3 Estriol

The potency of estriol in regard to vitellogenin induction in male fish has not been determined by water exposure experiments. Estriol is in general considered to be the least estrogenic of the three natural estrogens. An in vitro study has demonstrated estriol to be 30 times less potent than 17ß-estradiol (42).

In medaka exposed to estriol from hatch to 100 days after hatch a LOEC of 1 µg/l for induction of intersex was reported (42). One of 40 males had testes-ova at this concentration. At an exposure concentration of 10 µ/l all males had testis-ova.

6.4 Ethinylestradiol

A number of studies have examined the feminising potential of the synthetic estrogen, ethinylestradiol (EE2) on various endpoints. The NOEC for vitellogenin in fish has been tested in numerous species (61;119;179-182). Lowest concentration which have been found to induce vitellogenin in male fish is 0.1 ng/l (nominal concentration) after exposure of adult male rainbow trout for 10 days (119). Other studies with zebrafish and rainbow trout have reported vitellogenin induction at 1 – 5 ng/l EE2 in short-term exposure experiments (50;61;66;179-181). This agrees with the general finding that rainbow trout is the more sensitive of a number of test species with regard to vitellogenin induction (181).

Intersex in medaka has been observed after exposure to 0.1 ng EE2/l for 100 days from hatch (42). The assessment of this concentration as LOEC has been criticised (37) since intersex was only detected as a single oogonium within the testes of a single individual and no intersex individuals were observed at 10 ng/l. The concentration of 100 ng/l, however, clearly changed the sex ratio producing 91 % females and all males has testis-ova. Therefore a LOEC of intersex in medaka in this study has instead been proposed to be between 10-100 ng/L EE2 which is similar to the potency of estradiol reported in the same study. Another study with medaka obtained complete sex reversal with 100 ng EE2/l (183). In zebrafish, however, a change in sex ratio has been obtained at a much lower concentration (48). Exposing the fish to concentrations of 0.6 ng EE2/l from 20 to 60 day after hatch caused the sex ratio to change from an approximately 50:50 ratio to approximately 20:80 % (M:F).

In the sheepshead minnow (Cyprinodon variegatus) exposed for 59 days from a subadult stage to sexual maturity, LOEC and NOEC for induction of testis-ova or intersex were 20 and 2 ng EE2/l, respectively (31).

In a full life-cycle test on fathead minnow where newly fertilised eggs were exposed to EE2 for 305 days, a male:female ratio of 5:84 with intersex in 11 % of fish was seen at day 56 post hatch with an exposure concentration of 4 ng/l. A sex ratio of approximately 50:50 was seen in the control group (184). No testicular tissue was observed in any fish after 172 days post hatch and male fish exposed to the same concentration failed to develop secondary sexual characteristics. NOEC for these effects was 1 ng/l.

Finally, though not a water exposure study, it should be mentioned that a single injection of 0.5 –2.5 ng EE2/egg has been shown to cause sex reversal in genetic male medaka (185). The sex reversed females had more atretic (degenerating) oocytes and fewer mature oocytes than unexposed females.

Other testicular effects such as fibrosis have been reported with a LOEC of 2 and NOEC of 0.2 ng EE2/l in the study with sheepshead minnow (31). An inhibition in the testicular growth seen as lowered GSI has been observed with 2 ng EE2/l in adult male rainbow trout (50)and with 10 ng EE2/l in zebrafish (61;182) after exposure for 21-24 days. Analysis of the distribution of the different developmental stages of germ cells in the rainbow trout and zebrafish testes revealed a higher proportion of the early stages compared to the stages in control fish (50;182). A similar altered pattern of germ cell development has been seen after exposure of newly fertilised eggs of fathead minnows to 10 ng EE2/l (in (37)). The fish were exposed at different time windows throughout the embryo development. Fewer spermatozoa in exposed male fish compared to control fish were also observed as was induction of the female duct, the ovarian cavity. The treatment, however, did not induce testis-ova.

The same concentration of 10 ng EE2 /l did in a 2 month exposure study with freshly hatched medaka result in aromatase activity in the testes of exposed male fish (183). The activity was normally only detectable in female medaka. As mentioned earlier aromatase is the enzyme which converts androgens to estradiol.

In conclusion, LOECs for vitellogenin and intersex induction by EE2 are very low observed at concentrations of 0.1 ng/l and a wide range of testicular effects have been seen at concentrations from 1-10 ng/l. This illustrates that ethinylestradiol is even more potent in regard to feminisation of male fish than 17ß-estradiol.

6.5 Alkylphenols

6.5.1 Nonylphenol

Xenoestrogens in general have a lower estrogenic potential compared to the natural and synthetic estrogens. Hemmer et al. found elevated vitellogenin levels in male sheepshead minnows (Cyprinodon variegatus) after 5 days exposure to 5.4 µg/l nonylphenol (186). No effect was found at a concentration of 0.64 µg/l. A LOEC in the same range (6.1 – 6.4 µg/l) has been found for juvenile rainbow trout following a 14 day exposure period (40). Long-term and intermittent exposure to nonylphenol can decrease the LOEC for vitellogenin synthesis, since exposure of adult male rainbow trout for ten days in every month for a total of 4 months caused vitellogenin induction by 1 µg NP/l (51). Exposure of juvenile rainbow trout for one year during embryonic, larval and juvenile life stages has also been demonstrated to increase vitellogenin expression in liver at 1.05 ng/l (52).

However, an example of an inverted dose-response curve for vitellogenin concentrations has also been reported in male fathead minnow exposed for 0.05 – 3.4 m g/l. Significantly higher vitellogenin levels were found among males exposed to the lowest nonylphenol concentration but not at higher concentrations (187). The effects of nonylphenol in this study were expected to be caused by changes in the endogenous levels of estradiol since increased plasma levels of E2 were found in plasma at the lower nonylphenol concentrations. Only 4 % of the activity was estimated to be caused by the estrogen agonist activity of nonylphenol itself. This study therefore challenges the normal concept of dose-response studies.

Intersex has been induced in Japanese Medaka at nominal concentrations of 50 µg NP/l after exposure from hatch to 3 month of age. 50 % of the males had testes-ova at this concentration whereas 86 % showed the condition after exposure to 100 µg/l (53). The sex ratio was also significantly changed at this concentration to 1M:2F compared to 2M:1F in the control group.

A concentration of 30 µg NP/l has been capable of inhibiting testicular growth (lowered GSI) in mature male rainbow trout after an exposure period of 3 weeks (50). The inhibition was confirmed histologically by the predominance of less mature stages of sperm cells in exposed fish when compared to unexposed fish.

Degenerative effects on testes have also been seen after both high and low dose exposure to nonylphenol. Exposure of sexually mature male fathead minnow to 1.1 and 3.4 µg NP/l for 42 days caused necrotic aggregates of various stages of germ cells and the presence of phagocytic cells (54). Higher dose exposure (100 µg/l) of adult male medaka has been demonstrated to result in a six-fold greater extent of apoptosis (programmed cell death) in spermatocytes, sertoli cells and leydig cells (188) and similar observations of apoptosis and degenerated cells in interstitium and in cell types in the seminiferous tubules along with suppressed spermatogenesis have been observed after just 3 days exposure of swordfish (Xiphophorus helleri) to the same concentration (189). Longer exposure for 60 days resulted in reduced sword length, a secondary sexual characteristic in males used to attract females. Abnormal, female-like anal fins have also been induced in male medaka after exposure to 100 µg NP/l 200-230 days from the embryonic stage. Abnormal testes were also seen in this study by Tabata et al. 2001.

Importantly, transgenerational effects of nonylphenol have also been observed since exposure of both sexes of adult fathead minnows intermittently for four months prior to spawning for 1 and 10 µg/l resulted in a two-fold increase in the plasma level of estradiol in plasma of male offspring and a 13-fold increase in the testosterone level in plasma of female offspring (51).

6.5.2 Octylphenol

Octylphenol has generally been reported to have a higher estrogenic potential than nonylphenol when compared in the same test system (50;190).

Lowest LOEC which has been reported for octylphenol is, however, not different from the one reported for nonylphenol. This discrepancy might be due to fewer studies performed with octylphenol than nonylphenol. In a three week exposure study with rainbow trout vitellogenin was induced by 4.8 µg/l with a reported NOEC of 1.6 µ/l (50). The concentration of 4.8 µg OP/l reduced GSI.

Development of intersex in medaka and a shift in sex ratio towards females have been found with 2 - 50 µg/l OP when exposing from hatch to maturation (49). Concentrations greater than 41 µg/l have resulted in an inhibition of testicular growth in the same species, seen as an increase in the early spermatogenetic stages (191). In rainbow trout a similar skewed germ cell distribution has been induced by 30 µg OP/l. Testicular fibrosis in adult medaka has been caused by 100 µg OP/l (192).

As described in section 10.3 alkylphenols are degradation products of alkylphenolpolyethoxylates – a degradation which takes place in the sewage treatment process. Other degradation products such as alkylphenolmono- and –diethoxylates and alkylphenolmono- and diethoxycarboxylates are also formed (193). Compared to alkylphenols, nonylphenolmonoethoxylate and nonylphenoldiethoxylate have in some studies been shown to have approximately equal potency to nonylphenol but lower potency than octylphenol (50;194) while other studies have found weaker estrogenic activity of these short chained ethoxylate- and carboxylate-derivatives compared to the alkylphenols (42;54;195;196). In vitro the ethoxylates and carboxylates have been demonstrated to be 10⁴-10⁵ less potent than 17ß-estradiol (42;195). In vivo 100 µg/l of either nonylphenolmono- or diethoxylate or nonylphenolmono- or diethoxycarboxylate could not induce intersex in medaka (42). In general, however, little knowledge exist on the estrogenic activity in fish of these compounds compared to nonylphenol and the role of these degradation products of alkylphenolpolyethoxylates in the total emission of estrogenic chemicals is not clear. They might not be disconsidered in the total picture of estrogenic chemicals in sewage effluent.

6.5.3 Bisphenol A

Bisphenol A is less potent as an estrogenic agonist compared to the alkylphenols. In rainbow trout the LOEC for vitellogenin synthesis in males has been found to be between 40 and 70 µg/l (55). Occurrence of intersex has, however, been reported in male medaka exposed to 10 µg/l for approximately 100 days from hatch (42). In fathead minnow 16 µg/l bisphenol A reduced the number of mature spermatozoa produced by sexually mature males after 164 days exposure (56).

In general higher concentrations seem to be needed to produce reproductive effects by bisphenol A. Inhibition of gonadal growth was observed in male medaka with a LOEC of 640 µg/l (56) and degenerative and necrotic effects on germ cells which have been seen with 100 µg/l NP exposure of swordfish were not seen with a concentration of 10 mg/l bisphenol A (189).

6.5.4 Phthalates

The health risk associated with the use of phthalates has recently received much attention in the Danish media. It has long been known that phthalates are testicular toxicants (197;198) but they do not appear exclusively to exert their action though a direct estrogenic mechanism in which they bind to the estrogen receptor. In vitro only 5 out of 35 of the commercially most used phthalates have been demonstrated to have a very weak estrogenic activity (199). The phthalate, butylbenzylphthlate (BBP), which has been demonstrated to have the highest estrogenic activity, was 1-milllion-fold less potent than 17ß-estradiol in vitro (199). This is also the only phthalate which has been demonstrated to have a very weak in vivo estrogenic activity in fish and this only at environmentally unrealistic doses (41). Some phthalates have also been demonstrated to have antiandrogenic activity (200). The most widely used phthalate, diethylhexylphthalate (DEHP) was in the in vitro study above not found to have any estrogenic activity (199). A single study has with caution identified DEHP as a possible contributor to in vitro estrogenic activity in a water sample from an English estuary but also points the possibly that this might be due to contamination. DEHP is a common laboratory contaminant (129).

Since the present report concentrates on estrogens and estrogenic chemicals which exert their effects by mimicking estradiol and also considering the very weak estrogenic activity of phthlates, they are not included in this report.

| Front page | | Contents | | Previous | | Next | | Top |