Acute and chronic effects of carrier solvents in aquatic organisms: a critical review.

Recognising the scientific and regulatory need for testing relatively hydrophobic or 'difficult substances', the OECD currently recommends that selected organic solvents may be used in aquatic toxicity testing in order to help achieve more effective dispersion of the toxicant. The OECD recommends a maximum solvent concentration of 100 microl l(-1) (with specific gravity equivalents to 100 microl l(-1) in parentheses) for acetone (79 mg l(-1)), dimethylformamide (95 mg l(-1)), dimethylsulfoxide (1.10 mg l(-1)), ethanol (78.9 mg l(-1)), methanol (79.2 mg l(-1)) and triethylene glycol (1.12 mg l(-1)). While this recommendation is supported by historical data, we have recently observed evidence that some solvents may affect the reproduction of certain fish species, and also impact biomarkers of endocrine disruption. This review presents available data on the effects of solvents in aquatic organisms, supplemented by relevant information from mammalian studies (e.g. effects on liver enzyme induction potentially altering the metabolism of sex hormones). In conclusion, it is recommended that maximum effort should be given to avoiding the use of carrier solvents wherever possible, for example through the use of saturation columns or other physical methods (e.g. stirring or ultrasonification). Where solvent use is necessary, however, it is recommended that in reproduction studies with aquatic organisms, the maximum solvent concentration should not exceed 20 microl l(-1) of dilution water.

Ecological risk assessment and testing for endocrine disruption in the aquatic environment.

Field observations of endocrine disruption (ED) in fish and other aquatic species have contributed to a global effort to establish test methods for detecting ED effects in wildlife species. For ecological risk assessment, validated tests are needed for Amphibia, fish and aquatic invertebrates, supported by a tiered approach incorporating mechanistic data and exposure characterisation. The potential for extrapolation of ED data from mammalian to aquatic species may be limited, however, due to significant physiological differences in function and regulation of hormone systems in (aquatic) lower vertebrates and invertebrates. Presently, the OECD is considering a tiered approach for ED risk assessment, incorporating a fish 14-day screening assay (Tier 1); fish development and reproduction tests (both Tier 2); and a fish full life-cycle test (Tier 3). For detection of (anti-)oestrogens, the yolk-precursor protein vitellogenin is an ideal biomarker of exposure and functionally equivalent biomarkers are being sought for (anti-)androgens in fish. At the two higher tiers, impacts are assessed in terms of apical endpoints (e.g. development, breeding behaviour and fecundity) and also gonadal histopathology. Validation of these higher tier tests should include comparison of sensitivity of biochemical and apical endpoints to optimise the value of biomarkers for predicting adverse health effects (e.g. impaired reproduction). The specificity of future OECD fish and amphibian test guidelines for endocrine disrupters needs further consideration through inclusion of mechanistic endpoints based on state-of-the-art molecular endocrinology.

Plasma steroid concentrations and male phallus size in juvenile alligators from seven Florida lakes.

Neonatal and juvenile alligators from contaminated Lake Apopka in central Florida exhibit abnormal plasma sex steroid concentrations as well as morphological abnormalities of the gonad and phallus. This study addresses whether similar abnormalities occur in juvenile alligators inhabiting six other lakes in Florida. For analysis, animals were partitioned into two subsets, animals 40-79 cm total length (1-3 years old) and juveniles 80-130 cm total length (3-7 years old). Plasma testosterone (T) concentrations were lower in small males from lakes Apopka, Griffin, and Jessup than from Lake Woodruff National Wildlife Refuge (NWR). Similar differences were observed in the larger juveniles, with males from lakes Jessup, Apopka, and Okeechobee having lower plasma T concentrations than Lake Woodruff males. Plasma estradiol-17beta (E(2)) concentrations were significantly elevated in larger juvenile males from Lake Apopka compared to Lake Woodruff NWR. When compared to small juvenile females from Lake Woodruff NWR, females from lakes Griffin, Apopka, Orange, and Okeechobee had elevated plasma E(2) concentrations. Phallus size was significantly smaller in males from lakes Griffin and Apopka when compared to males from Lake Woodruff NWR. An association existed between body size and phallus size on all lakes except Lake Apopka and between phallus size and plasma T concentration on all lakes except lakes Apopka and Orange. Multiple regression analysis, with body size and plasma T concentration as independent covariables, explained the majority of the variation in phallus size on all lakes. These data suggest that the differences in sex steroids and phallus size observed in alligators from Lake Apopka are not limited to that lake, nor to one with a history of a major pesticide spill. Further work examining the relationship of sex steroids and phallus size with specific biotic and abiotic factors, such as antiandrogenic or estrogenic contaminants, is needed.

Effects of endocrine-disrupting contaminants on amphibian oogenesis: methoxychlor inhibits progesterone-induced maturation of Xenopus laevis oocytes in vitro.

There is currently little evidence of pollution-induced endocrine dysfunction in amphibia, in spite of widespread concern over global declines in this ecologically diverse group. Data regarding the potential effects of endocrine-disrupting contaminants (EDCs) on reproductive function in amphibia are particularly lacking. We hypothesized that estrogenic EDCs may disrupt progesterone-induced oocyte maturation in the adult amphibian ovary, and tested this with an in vitro germinal vesicle breakdown assay using defolliculated oocytes from the African clawed frog, Xenopus laevis. While a variety of natural and synthetic estrogens and xenoestrogens were inactive in this system, the proestrogenic pesticide methoxychlor was a surprisingly potent inhibitor of progesterone-induced oocyte maturation (median inhibitive concentration, 72 nM). This inhibitory activity was specific to methoxychlor, rather than to its estrogenic contaminants or metabolites, and was not antagonized by the estrogen receptor antagonist ICI 182,780, suggesting that this activity is not estrogenic per se. The inhibitory activity of methoxychlor was dose dependent, reversible, and early acting. However, washout was unable to reverse the effect of short methoxychlor exposure, and methoxychlor did not competitively displace [3H]progesterone from a specific binding site in the oocyte plasma membrane. Therefore, methoxychlor may exert its action not directly at the site of progesterone action, but downstream on early events in maturational signaling, although the precise mechanism of action is unclear. The activity of methoxychlor in this system indicates that xenobiotics may exert endocrine-disrupting effects through interference with progestin-regulated processes and through mechanisms other than receptor antagonism.

Alterations in steroidogenesis in alligators (Alligator mississippiensis) exposed naturally and experimentally to environmental contaminants.

Many environmental contaminants alter the reproduction of animals by altering the development and function of the endocrine system. The ability of environmental contaminants to alter the endocrine system of alligators was studied both in a descriptive study in which juvenile alligators from a historically contaminated lake were compared to animals from a control lake and in an experimental study in which hatchling control alligators were exposed in ovo to several endocrine-disrupting standards and two modern-use herbicides. Endocrine status was assessed by examining plasma hormone concentrations, gonadal-adrenal mesonephros (GAM) aromatase activity, and gonadal histopathology. In the descriptive study, juvenile alligators from the contaminated lake had significantly lower plasma testosterone concentrations (29.2 pg/ml compared to 51.3 pg/ml), whereas plasma 17 beta-estradiol concentrations did not vary when compared to controls. GAM aromatase activity was significantly decreased n the alligators from the contaminated lake (7.6 pmol/g/hr compared to 11.4 pmol/g/hr). In the experimental study, the endocrine-disrupting standards had the expected effects. 17 beta-Estradiol and tamoxifen caused sex reversal from male to female, with a corresponding increase in aromatase activity. Vinclozolin had no apparent effect on male or female alligators. Among the herbicides tested, atrazine induced GAM aromatase activity in male hatchling alligators that was neither characteristic of males nor females, although testicular differentiation was not altered. Exposure to 2,4-dichlorophenoxyacetic acid had no effect on the endocrine parameters that were measured. Together, these studies show that exposure to some environmental chemicals (such as atrazine) can alter steroidogenesis in alligators, but the endocrine alterations previously noted for Lake Apopka, Florida, alligators can not be fully explained by this mechanism.

Sequences of the lizard cDNAs encoding lactate dehydrogenase (LDH) isozymes A (muscle) and B (heart).

The nucleotide and deduced amino acid sequences of cDNAs encoding L-lactate dehydrogenase (LDH) isozymes A (muscle) and B (heart) from the lizard, Sceloporus undulatus, were determined. The evolutionary relationships among LDH isozymes from animals, plants and bacteria are presented.

Reduction in penis size and plasma testosterone concentrations in juvenile alligators living in a contaminated environment.

The development of the male reproductive ducts and external genitalia in vertebrates is dependent on elevated androgen concentrations during embryonic development and the period of postnatal growth. We have observed that a population of juvenile alligators living on Lake Apopka exhibit significantly smaller penis size (24% average decrease) and lower plasma concentrations of testosterone (70% lower concentrations) when compared to animals of similar size on Lake Woodruff. In addition to smaller phalli, no relationship exists between plasma testosterone concentrations and penile size in males from Lake Apopka, whereas a positive relationship exists for males from Lake Woodruff. The alligators on Lake Apopka are known to have elevated concentrations of the antiandrogenic DDT breakdown product p.p'-DDE stored in their fat. We suggest a number of hypotheses that could explain the modification in the phenotype of the juvenile male living in Lake Apopka. These modifications in phenotype include a smaller penis size, lower plasma androgen concentrations, and lack of responsiveness of the penis to the plasma androgens present.

Organization versus activation: the role of endocrine-disrupting contaminants (EDCs) during embryonic development in wildlife.

Many environmental contaminants disrupt the vertebrate endocrine system. Although they may be no more sensitive to endocrine-disrupting contaminants (EDCs) than other vertebrates, reptiles are good sentinels of exposure to EDCs due to the lability in their sex determination. This is exemplified by a study of alligators at Lake Apopka, Florida, showing that EDCs have altered the balance of reproductive hormones resulting in reproductive dysfunction. Such alterations may be activationally or organizationally induced. Much research emphasizes the former, but a complete understanding of the influence of EDCs in nature can be generated only after consideration of both activational and organizational alterations. The organizational model suggests that a small quantity of an EDC, administered during a specific period of embryonic development, can permanently modify the organization of the reproductive, immune, and nervous systems. Additionally, this model helps explain evolutionary adaptations to naturally occurring estrogenic compounds, such as phytoestrogens.