Endocrine-disrupting effects of spironolactone in female western mosquitofish, Gambusia affinis.

The discovery of pharmaceuticals in effluent from wastewater treatment plants and drug manufacturing facilities and in receiving waters has raised environmental concern. Because these compounds are ending up in the environment, it is important to investigate the effects of these compounds on wildlife as well as humans. The present study used a fish model to investigate the endocrine-disrupting effects of spironolactone (SPL), an aldosterone antagonist used as a diuretic, but which also exhibits antiandrogenic effects in humans. A dose-response study measured the effects of SPL on anal fin ray elongation, an androgen-dependent secondary sex trait, and expression of the vitellogenin gene, an estrogen-dependent trait, in female western mosquitofish, Gambusia affinis. Fish were exposed to SPL in the water for 35 d at four nominal concentrations: 10, 100, 250, and 500 nM (4.2, 41.7, 104.1, and 208.3 µg/L, respectively) via the static renewal method. Masculinization of females, as evidenced by development of an elongated and modified anal fin, was observed in the fish exposed to the three highest concentrations. Anal fin elongation was observed in the group exposed to the lowest SPL concentration, but without the development of a tip apparatus. These results confirm the results of a preliminary study that, in contrast to antiandrogenic effects seen in humans, SPL has androgenic and/or antiestrogenic activity in a fish.

Triclosan has endocrine-disrupting effects in male western mosquitofish, Gambusia affinis.

Triclosan (TCS) is an antibacterial agent used in a variety of personal care and industrial products. Triclosan and its environmentally transformed derivative, methyl-TCS, have been detected in waters receiving effluent from public wastewater treatment plants. Previous studies have demonstrated that TCS has the potential to act as an endocrine disruptor. The present study tested the hypothesis that TCS acts as an endocrine-disrupting agent in fish. Mature male western mosquitofish, Gambusia affinis, were exposed to TCS concentrations of 100, 200, and 350 nM (29.0, 57.9, and 101.3 microg/L) for 35 d by the static renewal method. Induction of the normally female-limited vitellogenin gene expression and reduction in sperm count were quantified as biomarkers of endocrine disruption. Vitellogenin mRNA expression was significantly elevated in the 350 nM TCS treatment. Sperm counts in the same treatment group were significantly decreased. The mean hepatosomatic index in the 350 nM treatment group was significantly increased. This study demonstrates that TCS has the potential to act as an endocrine disruptor in male mosquitofish.

Effects of sonication and advanced chemical oxidants on the unicellular green alga Dunaliella tertiolecta and cysts, larvae and adults of the brine shrimp Artemia salina: a prospective treatment to eradicate invasive organisms from ballast water.

Uptake and release of ship-borne ballast water is a major factor contributing to introductions of aquatic phytoplankton and invasive macroinvertebrates. Some invasive unicellular algae can cause harmful algal blooms and produce toxins that build up in food chains. Moreover, to date, few studies have compared the efficacy of ballast water treatments against different life history phases of aquatic macroinvertebrates. In the present study, the unicellular green alga Dunaliella tertiolecta, and three discrete life history phases of the brine shrimp Artemia salina, were independently used as model organisms to study the efficacy of sonication as well as the advanced oxidants, hydrogen peroxide and ozone, as potential ballast water treatments. Algal cells and brine shrimp cysts, nauplii, and adults were subjected to individual and combined treatments of sonication and advanced oxidants. Combined rather than individual treatments consistently yielded the highest levels of mortality in algal cells (100% over a 2 min exposure) and in brine shrimp (100% and 95% for larvae and adults, respectively, over a 2 min exposure). In contrast, mortality levels in brine shrimp cysts (66% over 2 min; increased to 92% over a 20 min exposure) were moderately high but consistently lower than that detected for larval or adult shrimp. Our results indicate that a combination of sonication and advanced chemical oxidants may be a promising method to eradicate aquatic unicellular algae and macroinvertebrates in ballast water.

In vivo assessment of the capacity of androstenedione to masculinize female mosquitofish (Gambusia affinis) exposed through dietary and static renewal methods.

In this study, we investigated the capacity of androstenedione to masculinize female mosquitofish. Previous studies have identified androstenedione in the water and sediment of the Fenholloway River, a Florida, USA, coastal river that receives paper mill effluent and contains masculinized eastern mosquitofish (Gambusia holbrooki). Females of the closely related western mosquitofish, Gambusia affinis, were exposed to androstenedione through both dietary and static renewal treatments. Morphological masculinization of female mosquitofish is characterized by the development of a male secondary sexual trait: an elongated and modified anal fin (gonopodium). Dietary exposure to 0.7, 7, 70, and 700 microg of androstenedione per gram of food failed to induce gonopodial development at any concentration within the six-week exposure period. Static renewal treatments used androstenedione concentrations of 0.14, 1.4, 14, 140, and 350 nM. Significant anal fin ray elongation was observed in all but the lowest exposure group. Fish growth during the static renewal exposure experiment was negatively correlated with androstenedione concentration. No significant effects were observed for gonadosomatic index, vitellogenin expression, or ovarian area in fish exposed to androstenedione via either the dietary or static renewal methods. These results indicate that exposure to androstenedione via water can cause masculinization of adult female mosquitofish in a relatively short period of time and that acute dietary exposure to androstenedione at the concentrations used is not sufficient to induce masculinization.

Elevated fluctuating asymmetry in eastern mosquitofish (Gambusia holbrooki) from a river receiving paper mill effluent.

The present study investigated whether fluctuating asymmetry can serve as a useful biomarker of environmental stress in the eastern mosquitofish (Gambusia holbrooki). Fluctuating asymmetry was measured in sexually mature females sampled from two Florida, USA, coastal streams: The Fenholloway River, which is dominated by effluent from a paper mill, and Spring Creek, a tributary to the Fenholloway River (Taylor County, FL, USA) that does not receive paper mill effluent. Nine morphometric (lengths of the A and B scales, fifth pectoral fin ray, supraorbital canal, and preorbital canal and the sixth gill raker on the first brachial arch; orbit diameter; distance from the dorsal-ventral midpoint of the eye socket to the base of the pectoral fin; and distance from the postorbital canal to the operculum) and five meristic traits (numbers of scales in the lateral line, radii on the A and B scales, pectoral fin rays, and gill rakers on the first brachial arch) were included. For each of the three indexes of fluctuating asymmetry that were used, the majority of the traits showed a higher level of fluctuating asymmetry in fish from the Fenholloway River than in fish from Spring Creek. For two of the indexes, the difference was significant. Comparisons of mean values for fluctuating asymmetry (over all traits) for each fish and the means of a composite index of asymmetry both indicated that fish from the Fenholloway River had significantly greater overall fluctuating asymmetry than those from Spring Creek. Results of the present study are consistent with the hypothesis that environmental stress increased the level of fluctuating asymmetry in fish from the Fenholloway River. Thus, fluctuating asymmetry appears to be a useful biomarker for stress-induced developmental instability in the eastern mosquitofish.

Effects of 17alpha-ethynylestradiol on sexual development of male western mosquitofish (Gambusia affinis).

Juvenile male western mosquitofish (Gambusia affinis) were exposed to different concentrations of 17alpha-ethynyl estradiol (EE2) in the diet during the period of sexual maturation. A clear inhibiting influence of EE2 on sexual development was apparent. The proportion of males in each treatment group that failed to complete gonopodial development during the 150-day observation period increased significantly with EE2 concentration. There were significant nonlinear trends toward shorter gonopodia in groups exposed to higher EE2 concentrations. Vitellogenin (VTG) was detectable in the blood of all fish exposed to 1.0 or more micro/g food and the concentration increased dramatically with increasing EE2 exposure. A significant negative association was seen between EE2 concentration and spermatophore counts. This study has demonstrated deleterious effects of EE2 exposure on sexual maturation and several indirect measures of reproductive fitness. It supports the biological relevance of vitellogenin in the blood and reduced gonopodium length as biomarkers for estrogen exposure and endocrine disruption in mosquitofish.

Production of androgens by microbial transformation of progesterone in vitro: a model for androgen production in rivers.

We have previously documented the presence of progesterone and androstenedione in the water column and bottom sediments of the Fenholloway River, Taylor County, Florida. This river receives paper mill effluent and contains masculinized female mosquitofish. We hypothesized that plant sterols (e.g., ss-sitosterol) derived from the pulping of pine trees are transformed by bacteria into progesterone and subsequently into 17alpha-hydroxyprogesterone, androstenedione, and other androgens. In this study, we demonstrate that these same androgens can be produced in vitro from the bacterium Mycobacterium smegmatis. In a second part to this study, we reextracted and reanalyzed the sediment from the Fenholloway River and verified the presence of androstadienedione, a delta1 steroid with androgen activity.

Androstenedione and progesterone in the sediment of a river receiving paper mill effluent.

The Fenholloway River near Perry, Florida, receives effluent from a paper mill and contains populations of masculinized female eastern mosquitofish, Gambusia holbrooki. A previous study identified the androgen precursor androstenedione at a low concentration (0.14 nM) in water samples from the river. The present study makes use of a toxicity identification and evaluation approach that includes solid phase extraction and high pressure liquid chromatography purification, androgen receptor transcription assays, and liquid chromatography mass spectroscopy to identify and characterize steroids in the Fenholloway River sediment. Androstenedione (2.4 nM) and progesterone (155 nM) were identified in the river sediment at concentrations greater than in the river water column (0.14 nM androstenedione, and 6.5 nM progesterone). Spring Creek, a comparison stream that does not receive mill effluent, contained low levels of progesterone (0.3 nM) but no androstenedione in the sediment. The data are consistent with the hypothesis that pine pulp-derived phytosteroids in the paper mill effluent accumulate in river sediment where they are converted by microbes into progesterone and this into androstenedione and other bioactive steroids. Equally important is that normal streams with much less organic matter still contain progesterone, but at dramatically lower levels. The presence of androgens and androgen precursors in the river water and sediment likely contributes to the masculinized phenotype of the female Gambusia holbrooki in the Fenholloway River.

Reproductive characteristics of male mosquitofish (Gambusia affinis) inhabiting a small southeastern U.S. river receiving treated domestic sewage effluent.

A population of western mosquitofish (Gambusia affinis) living below a wastewater treatment plant in the vicinity of Birmingham (AL, USA) was studied for evidence of exposure to estrogens. Mosquitofish are sexually dimorphic live-bearing fish. Males have an elongated and modified anal fin, called a gonopodium, used in mating. It has been hypothesized that exposure to estrogens and/or anti-androgens in treated wastewater might inhibit the androgen-dependent development of the gonopodium. The population in this study showed no evidence of having been exposed to endocrine-disrupting chemicals. The difference in adjusted (for overall fish size via analysis of covariance) mean gonopodium length between effluent-exposed and control populations was not significant. No detectable levels of vitellogenin were observed in the blood of any of the male mosquitofish, either from the effluent-exposed or the control population. Testes and livers were weighed and examined histologically. The fish exposed to treated wastewater effluent had significantly larger adjusted mean weights for both testis and liver, but no histological changes indicating exposure to estrogens were observed.