Measuring the toxicity of alkyl-phenanthrenes to early life stages of medaka (Oryzias latipes) using partition-controlled delivery.

Alkyl-phenanthrenes are a class of compounds present in crude oil and toxic to developing fish. Most research on alkyl-phenanthrenes has focused on retene (7-isopropyl-1-methyl-phenanthrene), but little is known about the chronic toxicity of related congeners to the early life stages of fish. This project is the first to describe the chronic toxicity of a series of alkyl-phenanthrenes to the embryos of Japanese medaka (Oryzias latipes) using the partition-controlled delivery (PCD) method of exposure and is the first to establish a relationship between toxicity of alkyl-phenanthrenes and log P. With PCD, test concentrations were maintained by equilibrium partitioning of test chemicals from polydimethylsiloxane (PDMS) films containing various concentrations of C1 to C4 phenanthrenes. Log film:solution partition constants (log K(fs)) and aqueous solubility limits were determined for each alkyl-phenanthrene. The prevalence of abnormalities in fish embryos increased in an exposure-dependent manner, with median effective concentration (EC50) values lower than experimental solubility limits of the compounds, and typical of environmental concentrations. Alkyl-phenanthrenes were more toxic to medaka embryos than unsubstituted phenanthrene, with effects resembling those of dioxin and indicating a specific receptor-based mechanism of toxicity. These results extend conclusions for the Exxon Valdez oil spill, suggest a specific mechanism of toxicity for alkyl-phenanthrenes, and provide a model for assessing the risks of mixture toxicity.

Anthropometry and body composition do not predict bioavailable androgen or progesterone concentration in adolescent girls.

Our objective was to test the hypothesis that anthropometry and body composition were independently associated with sex-steroid concentration in adolescent girls. Premenarcheal (age, mean +/- SD = 10.9 +/- 0.6, N = 51), perimenarcheal (age = 13.7 +/- 0.6, N = 74), and postmenarcheal (age = 16.7 +/- 0.6, N = 44) cohorts provided saliva at morning recess during the schoolday over 6 weeks. Estradiol, testosterone, progesterone, and dehydroepiandrosterone (DHEA) were quantified. Age, developmental cohort, self-reported Tanner stage of breast development and pubic-hair distribution, gynecological age, height, weight, waist/hip ratio, and percent body fat (by impedance) were assessed. Repeated measures were reduced to a mean. Data were hierarchically standardized for sexual maturation status, and then used to predict anthropometry/percent body fat. After intensive repeated sampling, individuals still varied widely in testosterone, progesterone, and DHEA concentrations within a developmental cohort (35.0 < or = coefficient of variation < or = 66.5%). Individual hormone concentration was repeatable across 2 years (0.51 < or = r(2) < or = 0.73). In spite of the variability within a cohort, there was no evidence for an association between the standardized hormone and body mass index, percent body fat, or waist/hip ratio, whether or not measures were age-adjusted (21 univariate tests; 0.19 < or = P < or = 0.96). Stratification by developmental cohort also did not reveal associations. Low estradiol assay precision precluded analyses. In conclusion, despite considerable population variation in hormone concentration after repeated sampling, no evidence supported an association between anthropometry or pecent body fat and testosterone, progesterone, or DHEA in adolescent girls.

Laying-sequence-specific variation in yolk oestrogen levels, and relationship to plasma oestrogen in female zebra finches (Taeniopygia guttata).

We investigated the relationship between plasma and yolk oestrogens in laying female zebra finches (Taeniopygia guttata) by manipulating plasma oestradiol (E2) levels, via injection of oestradiol-17beta, in a sequence-specific manner to maintain chronically high plasma levels for later-developing eggs (contrasting with the endogenous pattern of decreasing plasma E2 concentrations during laying). We report systematic variation in yolk oestrogen concentrations, in relation to laying sequence, similar to that widely reported for androgenic steroids. In sham-manipulated females, yolk E2 concentrations decreased with laying sequence. However, in E2-treated females plasma E2 levels were higher during the period of rapid yolk development of later-laid eggs, compared with control females. As a consequence, we reversed the laying-sequence-specific pattern of yolk E2: in E2-treated females, yolk E2 concentrations increased with laying-sequence. In general therefore, yolk E2 levels were a direct reflection of plasma E2 levels. However, in control females there was some inter-individual variability in the endogenous pattern of plasma E2 levels through the laying cycle which could generate variation in sequence-specific patterns of yolk hormone levels even if these primarily reflect circulating steroid levels.

Endocrine disruption and altered gonadal development in white perch (Morone americana) from the lower Great Lakes region.

High prevalences of gonadal intersex have been observed in wild fish populations in areas affected by domestic and industrial effluents. For this study, fish were collected in 1998 from the Cootes Paradise region of Hamilton Harbour in western Lake Ontario, Canada, to determine whether gonadal abnormalities, including intersex, were present in young of the year (YOY) fish. No gonadal abnormalities were observed in goldfish (Carassius auratus), common carp (Cyprinus carpio), gizzard shad (Dorosoma cepedianum), brown bullhead (Ictalurus ameiurus), pumpkinseed (Lepomis gibbosus), and bluegill (Lepomis macrochirus). However, intersex gonads were observed in 8 of 16 male white perch (Morone americana) examined in this survey. Subsequently, in 1999 and 2000 white perch estimated to be YOY to approximately 2 years of age were collected from Cootes Paradise and from two other sites in the lower Great Lakes region. Gonadal intersex was observed in male white perch collected from the Bay of Quinte (22-44%) and Lake St. Clair (45%), although the prevalence and the extent of the intersex condition were lower relative to the 83% prevalence in white perch collected in Cootes Paradise. Intersex was not observed in hatchery-reared white perch or in white perch collected from an uncontaminated reference site (i.e., Deal Lake) in the United States. An analysis of plasma collected in the spring of 2002 from male adult white perch in Cootes Paradise revealed high concentrations of vitellogenin, ranging from 49 to 1,711 microg/mL. These observations indicate that male white perch are exposed to estrogenic endocrine-disrupting substances that may be responsible for the induction of gonadal intersex.

Effects of the isoflavones genistein and equol on the gonadal development of Japanese medaka Oryzias latipes.

The estrogenic isoflavone compound genistein recently has been found in the effluents of sewage treatment plants and pulp mills, and the related compound equol has been detected in the runoff from agricultural fields treated with hog manure. Waterborne exposures of Japanese medaka (Oryzias latipes) to equol from soon after hatch to approximately 100 days posthatch induced gonadal intersex (i.e., testis-ova) in males at incidences of 10 and 87% in equol treatments of 0.4 and 0.8 micro g/L, respectively. Exposure to the highest test concentration of genistein, 1,000 micro g/L, also caused a low incidence (i.e., 12%) of gonadal intersex in male medaka. The ovaries of female medaka from both equol and genistein treatments showed delayed oocyte maturation, atretic oocytes, an enlarged ovarian lumen, proliferation of somatic stromal tissue, and primordial germ cells; responses were concentration dependent. Alterations to externally visible secondary sex characteristics occurred in medaka exposed to both equol and genistein. In treatments with 1,000 micro g/L genistein, 72% of male medaka (as identified by the gonadal phenotype) showed feminized secondary sex characteristics. Gonadal intersex and alterations to secondary sex characteristics have been noted in several fish populations around the world. This laboratory study indicates that isoflavone compounds should be considered candidate estrogenic compounds that may be involved in the alteration of sexual development in feral fish populations.

Partiton-controlled delivery of toxicants: a novel in vivo approach for embryo toxicity testing.

In conventional static or semi-static embryo toxicity assays with fish, the nominal concentrations of hydrophobic chemicals are often used to establish the toxic thresholds, which often far exceed the solubility limits of test compounds. Saturators and continuous-flow diluters have been used to provide stable concentrations below solubility but are complex, use large amounts of test substance, and produce large volumes of waste. We present a partition-controlled delivery (PCD) method that maintains the concentrations of chemicals in test solutions at or below solubility limits for extended exposure times. Concentrations are maintained by equilibrium partitioning of test chemicals from a series of poly(dimethylsiloxane) films loaded with a range of concentrations of each chemical. The efficacy of the PCD assay was tested by comparisons with static (no renewal) and semi-static (24-h renewal) embryo-larval toxicity tests. The test species was Japanese medaka (Oryzias latipes) exposed to retene (7-isopropyl-1-methylphenanthrene), a compound causing blue sac disease (BSD) in fish embryos. In the PCD assay, the median effective concentration (EC50) for BSD was 10 microg/L, below retene's solubility of 17 microg/L. In contrast, the nominal EC50 values for the semi-static 24-h and static assays were about 10 (150 microg/L) and 150 times (2500 microg/L) greater than solubility, respectively. The PCD method is a more sensitive and realistic method for assessing toxicity of nonpolar compounds than (semi)-static assays.

Effects of the antiandrogens, vinclozolin and cyproterone acetate on gonadal development in the Japanese medaka (Oryzias latipes).

This study was focused on determining the effects of exposure to antiandrogens on the gonadal development of Japanese medaka (Oryzias latipes). Test compounds included the fungicide, vinclozolin and the clinical antiandrogen, cyproterone acetate. Newly hatched medaka were exposed to aqueous solutions of vinclozolin (2500 microg/l) and the vinclozolin fungicide formulation, Ronilan (1000 and 5000 microg/l) and cyproterone acetate (1 and 10 microg/l), for 3 months. Histological evaluation of the gonadal tissues of exposed fish indicated that the 5000 microg/l concentration of the vinclozolin formulation (Ronilan) induced a low incidence of intersex (i.e. testis-ova) and the 2500 microg/l concentration of vinclozolin-affected spermatogenesis in males. Also, the vinclozolin treatments induced moderate ovarian atresia. Cyproterone acetate also induced a low incidence of testis-ova, but in contrast to the vinclozolin treatment the amount of ovarian tissue in the testis-ova was equal to or greater than the amount of testicular tissue. In the cyproterone acetate treatments, both oogenesis and spermatogenesis were moderately inhibited at all test concentrations. The results of this study indicate that antiandrogens have the potential to alter testicular development and gametogenesis in fish. However, research is needed to determine the mechanisms by which antiandrogens affect fish.

Increased cellular apoptosis after chronic aqueous exposure to nonylphenol and quercetin in adult medaka (Oryzias latipes).

Increasing evidence suggests that sublethal effects of natural or xenobiotic chemicals in the environment may be mediated via the stimulation of apoptosis. To investigate whether apoptosis can be induced in fish by weakly estrogenic and androgenic chemicals, adult male Japanese medaka (Oryzias latipes) were exposed to 100 ppb of the estrogenic alkylphenol, 4-nonylphenol, and adult female medaka were exposed to 100 ppb of the aromatase-inhibiting bioflavonoid, quercetin, for 6 weeks. Exposure to nonylphenol and quercetin had no significant effect on the length, weight or condition factors compared to solvent (acetone) controls in male or female medaka. Apoptosis was evaluated in blinded histological sections of whole medaka using terminal dideoxynucleotidyl transferase dUTP nick end labeling (TUNEL) that labels nuclei of cells containing apoptotic (fragmented) DNA. There was a six-fold greater extent of apoptosis in spermatocytes, Sertoli cells and Leydig-homologue cells, but not in spermatids of testes from nonylphenol-exposed male medaka compared to testes of solvent controls. No significant differences in the extent of apoptosis were detected in intestine, liver or kidney from the same male fish. Quercetin-treated female medaka had a significantly increased number of atretic ovarian follicles, but no significant differences in the extent of apoptosis in intestine, liver or kidney. These results suggest that nonylphenol caused testicular degeneration via increased testicular cell apoptosis, while quercetin may be ovotoxic via increased follicular atresia.