Sucker Shapes, Skeletons, and Bioinspiration: How Hard and Soft Tissue Morphology Generates Adhesive Performance in Waterfall Climbing Goby Fishes.

Many teleost fish, such as gobies, have fused their paired pelvic fins into an adhesive disc. Gobies can use their pelvic suckers to generate passive adhesive forces (as in engineered suction cups), and different species exhibit a range of adhesive performance, with some even able to climb waterfalls. Previous studies have documented that, in the Hawaiian Islands, species capable of climbing higher waterfalls produce the highest passive pull-off forces, and species found at higher elevation sites are likely to have more rounded suction discs than those found in the lowest stream segments. Morphology of the pelvic girdle also varies between species, with more robust skeletons in taxa with superior passive adhesion. To investigate what factors impact the passive adhesive performance of waterfall climbing gobies, we tested biomimetic suction cups designed with a range of shapes and embedded bioinspired "skeletons" based on micro-CT scans of goby pelvic girdles. We found that while the presence of an internal skeleton may provide some support against failure, the performance of suction cups may be more strongly affected by their external shape. Nonetheless, factors besides external shape and skeletal morphology may still have a stronger influence on sucker tenacity. Our results suggest that the relationship between suction disc morphology and adhesive performance may be influenced by a variety of physical factors, and live animal performance likely is further complicated by muscle activation and climbing behavior. These results have implications for the evolution of suction disc shape in adhesive fish and for improving the design of biomimetic suction cups.

Temperature-Dependent Biomarkers of Estrogenic Exposure in a Piscivore Freshwater Fish.

The biological effects of endocrine-active compounds and increasing water temperatures as a result of climate change have been studied extensively and independently, but there is a dearth of research to examine the combined effect of these factors on exposed organisms. Recent data suggest that estrogenic exposure and rising ambient temperatures independently impact predator-prey relationships. However, establishing these connections in natural settings is complex. These obstacles can be circumvented if biomarkers of estrogenic exposure in resident fish can predict changes in predator-prey relationships. To test the effects of estrone and temperature, the piscivore bluegill sunfish (Lepomis macrochirus) was exposed for 30 days to estrone at concentrations (90 ± 17.6 ng/L [mean ± standard deviation] and 414 ± 146 ng/L) previously shown to reduce prey-capture success. Exposures were conducted at four temperatures (15 °C, 18 °C, 21 °C, 24 °C) to simulate breeding season ambient temperatures across the natural range of this species. A suite of morphological and physiological biomarkers previously linked to estrogenic exposures were examined. Biomarkers of estrone exposure were more commonly and severely impacted in male fish than in female fish. Notably, the gonadosomatic index was lower and gonads were less mature in exposed males. Additionally, temperature modulated the effects of estrone similarly in males and females with fish exposed at higher temperatures typically exhibiting a decreased morphological index. This study provides evidence that alterations in hepatic function and gonadal function may cause shifts in metabolism and energy allocation that may lead to declining prey capture performance.

Temperature and Estrogen Alter Predator-Prey Interactions between Fish Species.

A variety of environmental estrogens are commonly detected in human-impacted waterways. Although much is known about the effects of these environmental estrogens on the reproductive physiology and behavior of individuals within species, comparatively less is known about how these compounds alter the outcomes of interactions between species. Furthermore, few studies have considered how the effects of contaminants are modulated by natural variation in abiotic factors, such as temperature. To help fill this knowledge gap, we conducted a factorial experiment to examine the independent and combined effects of estrone (E1) and temperature on the outcome of predator-prey interactions between two common North American freshwater fishes, fathead minnows (Pimephales promelas) and bluegill sunfish (Lepomis macrochirus). Larval fathead minnows and adult sunfish were exposed to either a low (mean±standard deviation, 90.1 ± 18 ng/L; n = 16) or high (414 ± 147 ng/L; n = 15) concentration of E1 or to a solvent control for 30 days at one of four natural seasonal temperatures (15°C, 18°C, 21°C, and 24°C) before predation trials were performed. Exposure to E1 was associated with a significant increase in larval predation mortality that was independent of temperature. Across all temperature treatments, approximately 74% of control minnows survived; this survivorship significantly exceeded that of minnows exposed to either concentration of E1 (49% and 53% for minnows exposed to the low and high concentrations, respectively). However, exposure to E1 also impaired the prey-capture success of sunfish, partially mitigating predation pressure on exposed minnows. Overall prey-capture success by sunfish showed an inverted U-shaped distribution with temperature, with maximal prey consumption occurring at 21°C. This study illustrates the vulnerability of organismal interactions to estrogenic pollutants and highlights the need to include food web interactions in assessments of risk.

Functional Diversity of Evolutionary Novelties: Insights from Waterfall-Climbing Kinematics and Performance of Juvenile Gobiid Fishes.

The evolution of novel functional traits can contribute substantially to the diversification of lineages. Older functional traits might show greater variation than more recently evolved novelties, due to the accrual of evolutionary changes through time. However, functional complexity and many-to-one mapping of structure to function could complicate such expectations. In this context, we compared kinematics and performance across juveniles from multiple species for two styles of waterfall-climbing that are novel to gobiid fishes: ancestral "powerburst" climbing, and more recently evolved "inching", which has been confirmed only among species of a single genus that is nested within the clade of powerburst climbers. Similar net climbing speeds across inching species seem, at first, to indicate that this more recently evolved mode of climbing exhibits less functional diversity. However, these similar net speeds arise through different pathways: Sicyopterus stimpsoni from Hawai'i move more slowly than S. lagocephalus from La Réunion, but may also spend more time moving. The production of similar performance between multiple functional pathways reflects a situation that resembles the phenomenon of many-to-one mapping of structure to function. Such similarity has the potential to mask appropriate interpretations of relative functional diversity between lineages, unless the mechanisms underlying performance are explored. More specifically, similarity in net performance between "powerburst" and "inching" styles indicates that selection on climbing performance was likely a limited factor in promoting the evolution of inching as a new mode of climbing. In this context, other processes (e.g., exaptation) might be implicated in the origin of this functional novelty.

FRENCH: Diversité fonctionnelle des innovations évolutives: l'exemple de la cinématique et des performances de grimpe des chutes d'eau des juvéniles de gobies Résumé L'évolution de nouveaux traits fonctionnels peut contribuer significativement à la diversification des lignées. Les traits fonctionnels les plus anciens peuvent montrer plus de variabilité que les plus récents du fait de l'accumulation de changements évolutif au cours du temps. Cependant, ces prédictions peuvent être complexifiées par la diversité des fonctions et par l'implication de plusieurs structures dans une même fonction. Dans ce contexte, nous avons étudié la cinématique et les performances de grimpe des chutes d'eau de plusieurs espèces de gobies utilisant deux styles de grimpe originaux au sein de cette famille: le mode « powerburst ¼ plus ancestral et le mode « inching ¼ qui a évolué plus récemment. Le mode inching n'a été confirmé que pour deux espèces du même genre incluses au sein du clade des powerburst. Des vitesses de grimpe similaires entre les espèces utilisant le mode inching paraissent indiquer que ce mode de grimpe, qui a évolué plus récemment, présente une diversité fonctionnelle moins élevée. Toutefois, la similarité des vitesses de grimpe entre les deux espèces s'explique par des processus différents: le Sicyopterus stimpsoni d'Haiwaï se déplace plus lentement que le S. lagocephalus de La Réunion mais passe plus de temps en mouvement. La production de performances similaires, résultant de processus différents, reflète un phénomène semblable à celui de l'implication de plusieurs structures dans une même fonction. Si les mécanismes sous-jacents ne sont pas explorés, ces similarités peuvent perturber l'interprétation des différences relatives de diversité fonctionnelle entre les lignées. Par ailleurs, les performances de grimpe similaires entre certaines espèces utilisant le mode inching et d'autres le mode powerburst paraissent indiquer que la force de sélection sur les performances de grimpe est sans doute un facteur réduisant l'avantage évolutif du mode de grimpe inching. Dans ce contexte, d'autres mécanismes (e.g., exaptation) pourraient être à l'origine de cette innovation fonctionnelle. Translated to French by Raphael Lagarde (raph.lagarde@gmail.com).

PORTUGESE: Diversidade funcional de novidades evolucionárias: percepções da cinemática da escalada em cascatas e desempenho de peixes juvenis gobiídeos ResumoA evolução de novos traços funcionais pode contribuir substancialmente para a diversificação de linhagens. Os traços funcionais mais antigos podem mostrar maior variação do que as novidades desenvolvidas mais recentemente, devido ao acúmulo de mudanças evolutivas ao longo do tempo. No entanto, a complexidade funcional e os inúmeros mapeamentos de uma estrutura para uma única função podem complicar essas expectativas. Nesse contexto, comparamos a cinemática e a performance em juvenis de várias espécies para dois estilos de escalada em cascata que são novidades em peixes gobiídeos: a ancestral escalada por "explosão" e o evolutivamente mais recente chamado de "avançamento", que foi confirmado apenas entre espécies de um único gênero que dentro do clado de escaladores por explosão. Velocidades finais de escalada semelhantes entre espécies usando "avançamento" parecem, inicialmente, indicar que esse modo de escalada evolutivamente mais recente exibe menor diversidade funcional. No entanto, essas velocidades finais similares ocorrem por diferentes formas: Sicyopterus stimpsoni do Havaí se move mais devagar que S. lagocephalus das Ilhas Reunião, mas pode gastar mais tempo se movendo. Desempenhos semelhantes entre várias vias funcionais refletem uma situação que se assemelha ao fenômeno de mapeamento de uma estrutura para um única função. Essa semelhança tem o potencial de ocultar interpretações apropriadas sobre relativa diversidade funcional entre linhagens, a menos que os mecanismos que afetam o desempenho sejam explorados. Mais especificamente, a semelhança no desempenho final entre os estilos "explosão" e "avançamento"indica que a seleção na performance em escalada provavelmente foi um fator limitante na promoção da evolução por "avançamento" como um novo modo de escalada. Nesse contexto, outros processos (e.g., exaptação) podem estar relacionados com a origem dessa novidade funcional. Translated to Portuguese by Diego Vaz (dbistonvaz@vims.edu).

Do environmental factors affect male fathead minnow (Pimephales promelas) response to estrone? Part 1. Dissolved oxygen and sodium chloride.

Laboratory exposures indicate that estrogens and their mimics can cause endocrine disruption in male fishes, yet while studies of resident fish populations in estrogen-polluted waters support these findings, biomarker expression associated with field versus laboratory exposure to estrogenic endocrine disruptors (EDs) often differ dramatically. Two of the environmental parameters often found to vary in dynamic aquatic ecosystems were chosen (dissolved oxygen [DO] and sodium chloride concentrations) to assess their potential impact on ED exposure. In separate experiments, male fathead minnows (Pimephales promelas) were exposed to estrone (E1) a natural ED, under either two concentrations of DO, or two concentrations of sodium chloride, in a laboratory flow-through system. Morphological and hematological parameters were assessed. While vitellogenin concentrations were elevated with exposure to estrone (29 to 390ng/L), the effect on other indices were variable. Estrone exposure altered SSC, blood glucose, hematocrit, and hepatic and gonado-somatic index in 1 of 4 experiments, while it decreased body condition factor in 3 of 4 experiments. At the concentrations tested, no main effect differences (P<0.05) were found associated with DO or sodium chloride treatments, except in one experiment low DO resulted in a decrease in secondary sex characteristic score (SSC). The combination of DO or sodium chloride and E1 altered blood glucose in one experiment each. These results indicate the variability of fathead minnow response to estrone, even within the confines of controlled laboratory conditions.

Do environmental factors affect male fathead minnow (Pimephales promelas) response to estrone? Part 2. Temperature and food availability.

Fish are subject to constantly changing environmental conditions and food availability, factors that may impact their response to endocrine disruptors (EDs). This may, in part, explain outcome discrepancies between field studies and laboratory exposures to EDs. This study assessed whether standard laboratory conditions for fish exposures adequately represent effects of ED exposure at two environmentally realistic temperatures. The impact of temperature and food availability on male fathead minnow response to estrone (E1) exposure was studied in two experiments (3×2×2 factorial design) with three E1 concentrations (range 0-135ng/L); two temperatures (18°C and 26°C, the latter the prescribed laboratory temperature), and two feeding treatments (full fed vs. 25% of full fed) in a 21-day flow-through system. Morphometric endpoints [including body condition factor, somatic index of gonad (GSI) and liver (HSI), and secondary sex characteristics (SSC)], blood parameters [hematocrit (HCT), blood glucose, cortisol, and vitellogenin (VTG) concentrations], and histology of liver and testis were determined on day 22. High E1 consistently increased VTG, though interactions among E1, temperature and/or food on liver weight, HSI, and HCT were inconsistent between experiments. High temperature impacted the greatest number of parameters, independent of E1 treatment. Three sex-linked parameters were lower at high temperature (testis weight, GSI and VTG), and in Exp. 2SSC and gonad maturity rating were lower. At 26°C, in Exp. 1 HSI and HCT decreased, and in Exp. 2 length, body and liver weight, and body condition factor were lower. Food restriction decreased GSI in Exp. 1, and blood glucose and liver weight in Exp. 2. At 26°C several parameters were altered independent of E1 exposure, including three out of four measurements of sperm differentiation. Concordance between laboratory and field investigations of the biological effects of EDs may improve if environmentally-relevant exposure conditions, especially temperature, are employed.

Temperature modulates estrone degradation and biological effects of exposure in fathead minnows.

Environmental pollutants, including estrogens, are widespread in aquatic environments frequently as a result of treated wastewater effluent discharged. Exposure to estrogens has been correlated with disruption of the normal physiological and reproductive function in aquatic organisms, which could impair the sustainability of exposed populations. However, assessing the effects of estrogen exposure on individuals is complicated by the fact that rates of chemical uptake and environmental degradation are temperature dependent. Because annual temperature regimes often coincide with critical periods of biological activity, temperature-dependent changes in estrogen degradation efficacy during wastewater treatment could modulate biological effects. We examined the interactions between ambient water temperature and degradation of estrone (E1) during wastewater treatment. In addition, we exposed mature fathead minnows (Pimephales promelas) to three environmentally relevant concentrations of E1 at four different water temperatures (15°C, 18°C, 21°C, and 24°C) to reflect natural seasonal variation. E1 degradation occurred with and without the support of robust nitrification at all temperatures; however, the onset of E1 degradation was delayed at cooler water temperatures. In addition, we observed significant interactive effects between temperature and E1 exposure. Female morphometric endpoints were more susceptible to temperature-modulating effects while physiological endpoints were more strongly affected in males. Collectively, the data demonstrate that natural seasonal fluctuations in temperature are sufficient to affect E1 degradation during wastewater treatment and induce sex-dependent physiological and anatomical changes in exposed fish.

Local adaptation despite high gene flow in the waterfall-climbing Hawaiian goby, Sicyopterus stimpsoni.

Environmental heterogeneity can promote the emergence of locally adapted phenotypes among subpopulations of a species, whereas gene flow can result in phenotypic and genotypic homogenization. For organisms like amphidromous fishes that change habitats during their life history, the balance between selection and migration can shift through ontogeny, making the likelihood of local adaptation difficult to predict. In Hawaiian waterfall-climbing gobies, it has been hypothesized that larval mixing during oceanic dispersal counters local adaptation to contrasting topographic features of streams, like slope gradient, that can select for predator avoidance or climbing ability in juvenile recruits. To test this hypothesis, we used morphological traits and neutral genetic markers to compare phenotypic and genotypic distributions in recruiting juveniles and adult subpopulations of the waterfall-climbing amphidromous goby, Sicyopterus stimpsoni, from the islands of Hawai'i and Kaua'i. We found that body shape is significantly different between adult subpopulations from streams with contrasting slopes and that trait divergence in recruiting juveniles tracked stream topography more so than morphological measures of adult subpopulation differentiation. Although no evidence of population genetic differentiation was observed among adult subpopulations, we observed low but significant levels of spatially and temporally variable genetic differentiation among juvenile cohorts, which correlated with morphological divergence. Such a pattern of genetic differentiation is consistent with chaotic genetic patchiness arising from variable sources of recruits to different streams. Thus, at least in S. stimpsoni, the combination of variation in settlement cohorts in space and time coupled with strong postsettlement selection on juveniles as they migrate upstream to adult habitats provides the opportunity for morphological adaptation to local stream environments despite high gene flow.

Do laboratory species protect endangered species? Interspecies variation in responses to 17ß-estradiol, a model endocrine active compound.

Although the effects of estrogens on model laboratory species are well documented, their utility as surrogates for other species, including those listed as endangered, are less clear. Traditionally, conservation policies are evaluated based on model organism responses but are intended to protect all species in an environment. We tested the hypothesis that the endangered Rio Grande silvery minnow (Hybognathus amarus) is more vulnerable to endocrine disruption-as assessed through its larval predator-escape performance, survival, juvenile sex ratios, and whole-body vitellogenin concentration-than the commonly used toxicological model species fathead minnow (Pimephales promelas) and the bluegill sunfish (Lepomis macrochirus). Fish were exposed concurrently for 21 days to the model endocrine active compound (EAC) 17ß-estradiol (E2) at 10 ng E2/L and 30 ng E2/L in a flow-through system using reconstituted water that simulated the physicochemical conditions of the Middle Rio Grande in New Mexico, USA. No significant differences were observed between the fathead and silvery minnow in larval predator-escape response or juvenile sex ratio. Rio Grande silvery minnow survival decreased significantly at day 14 compared with the other two species; by day 21, both cyprinid species (silvery minnow and fathead minnow) exhibited a significant decrease in survival compared with bluegill sunfish, a member of the family Centrarchidae. Male Rio Grande silvery minnow showed a significant increase in whole-body vitellogenin concentration in the 10 ng/L treatment, whereas fathead minnow and bluegill sunfish showed no significant increases in vitellogenin concentrations across treatments. Our study showed response differences to estrogen exposures between the two cyprinid species and further divergence in responses between the families Cyprinidae and Centrarchidae. These results suggest that commonly used laboratory model organisms may be less sensitive to EACs than the endangered Rio Grande silvery minnow. However, this study supports the continued use of surrogate species for the beneficial implementation of water-quality regulations for the protection of threatened and endangered species if phylogenetic relationships are taken into consideration.

Technical note: The equivalency of sodium results in cheese digested by either dry ashing or microwave-accelerated digestion.

Analysis of dairy products for minerals such as sodium requires mineralization of the sample, which is typically done by either dry ashing or atmospheric wet ashing; both methods are time consuming and wet ashing requires the repeated handling of hot acid. A rapid method using microwave-accelerated acid digestion before atomic absorption spectrometry to measure sodium was compared with dry ashing in 138 samples of blue cheese (in duplicate) that varied in sodium content and age. Linear regression of the results obtained within different cheese salting treatments and sampling locations over time showed that the methods were equivalent in terms of linearity and the slope of the line. A consistent bias was observed, with lower sodium concentrations being quantified during atomic absorption spectrometry for the microwave-digested samples. Evaluation of this difference by the 2 one-sided test (TOST) procedure showed that the confidence intervals of the percentage difference between the methods fell within the predetermined acceptable percentage difference. We conclude that this rapid microwave digestion procedure of blue cheese yielded equivalent results to dry ashing.

Sex-specific gonadal and gene expression changes throughout development in fathead minnow.

Although fathead minnows (Pimephales promelas) are commonly used as a model fish in endocrine disruption studies, past studies have not characterized sex-specific baseline expression of genes involved in sex differentiation during development in this species. Using a sex-linked DNA marker to verify gender, we evaluated the expression over time of genes involved in sex differentiation (dmrt1, cyp19a, cyp17, star, esr1, ar) in developing fathead minnows (10-45 days post hatch). Evaluation of these molecular markers in combination with gender identification help us to better understand the mechanisms regulating sex differentiation in fathead minnows and how endocrine-disrupting chemicals may alter these processes.

Serum estrogenicity and biological responses in African catfish raised in wastewater ponds in Ghana.

Reuse of wastewater for aquaculture improves the efficient use of water and promotes sustainability but the potential effects of endocrine disrupting compounds including estrogens in wastewater are an emerging challenge that needs to be addressed. We examined the biological effects of wastewater-borne estrogens on African catfish (Clarias gariepinus) raised in a wastewater stabilization pond (WSP) of a functioning municipal wastewater treatment plant, a wastewater polishing pond (WWP) of a dysfunctional treatment plant, and a reference pond (RP) unimpacted by wastewater, located in Ghana. Measurements of estrogen concentrations in pond water by liquid chromatography/tandem mass spectrometry showed that mean 17 ß-estradiol concentrations were higher in the wastewater ponds (WWP, 6.6 ng/L±2.7 ng/L; WSP, 4.9 ng/L±1.0) than the reference (RP, 3.4±1.1 ng/L). Estrone concentrations were found to be highest in the WSP (7.8 ng/L±1.7) and lowest in the WWP (2.2 ng/L±2.4) with the RP intermediate (4.7±5.0). Fish serum estrogenicity assayed by E-SCREEN was significantly higher in female vs. male catfish in the RP and WSP but not in the WWP (p≤0.05). Histological examination of liver and gonad tissue showed no apparent signs of intersex or pathology in any ponds. The similarities in various measures of body indices between fish of this study and African catfish from freshwater systems suggest that aquaculture may be a suitable reuse option for treated municipal wastewater.

Finding paradise: cues directing the migration of the waterfall climbing Hawaiian gobioid Sicyopterus stimpsoni.

A series of waterfall-climbing trials were conducted to identify cues that direct the climbing of juvenile Sicyopterus stimpsoni. In the first experiment, whether climbing juveniles preferentially ascend water sources with conspecifics or whether the presence of just stream water is sufficient to attract fish to ascend a climbing path were assessed. In the second experiment, whether climbing juveniles create a trail of mucus that facilitates the ability of conspecifics to follow their lead was determined. The results indicate that juvenile S. stimpsoni are less likely to climb in waters devoid of organic cues but are strongly attracted to stream water with or without the odour of conspecifics. Once climbing, performance did not differ for juveniles climbing in differing water choices, suggesting an all-or-nothing commitment once climbing commences. Climbing S. stimpsoni did produce a mucous trail while climbing that was associated with a mucous gland that dramatically increases in size just prior to juveniles gaining the ability to climb. The trail was not followed closely by subsequent juveniles traversing the same channel, however, suggesting only weak trail-following in waterfall climbing S. stimpsoni. Previous genetic studies suggest that juvenile S. stimpsoni do not home to natal streams in the face of strong near-shore oceanic currents. Instead, these fish appear primarily to rely on cues that suggest the presence of organic growth in streams, a factor that may indicate suitable habitat in an ever-changing stream environment but which may also be vulnerable to interference through human activity.

Comparing biological effects and potencies of estrone and 17ß-estradiol in mature fathead minnows, Pimephales promelas.

The presence of endocrine active compounds such as estrogens in treated wastewater effluent and their effects on aquatic life are causing concern among aquatic resource managers. In contrast to 17ß-estradiol (E2), the steroid hormone produced by all vertebrates, the biological effects of estrone (E1), one of its breakdown products are less understood, even though the aquatic concentrations of E1 are often higher than those of E2. The central hypothesis of this study was that at environmental concentrations, E1 has estrogenic effects in fish, with increased vitellogenin concentrations and decreased reproductive success in both male and female fathead minnows, as found with E2. In two replicate experiments, we exposed mature fathead minnows to three concentrations of each estrogen for 21 days in a flow-through exposure system and measured a broad suite of anatomical (body indices, histopathology), physiological (plasma vitellogenin), behavioral (nest defense), and reproductive (fecundity, fertility, hatching) endpoints. These endpoints have previously been associated with adverse effects of estrogenic exposures. While body length and weight parameters were unaltered by exposure, secondary sex characteristics exhibited an exposure concentrated-related decline in male fathead minnows. Interestingly, low concentrations of estrone (≈ 15 ng/L) enhanced the aggressiveness of male fathead minnows in a behavioral assay. Vitellogenin concentrations in male fish increased with higher concentrations of both estrogens, but remained unchanged in all female treatments. A decrease in fecundity was observed at high concentrations of E2 as compared with control minnows. These results suggest that E1, at concentrations previously found in waters receiving wastewater effluent, can have reproductive effects on fish.

Comparative biological effects and potency of 17α- and 17ß-estradiol in fathead minnows.

17ß-Estradiol is the most potent natural estrogen commonly found in anthropogenically altered environments and has been the focus of many toxicological laboratory studies. However, fewer aquatic toxicological data on the effects of 17α-estradiol, a diastereoisomer of 17ß-estradiol, exists in the literature even though it has been found in the aquatic environment, sometimes at higher concentrations than 17ß-estradiol. The central objective of this study was to determine how the anatomical, physiological, and behavioral effects of exposure to 17α-estradiol compare to the well-documented effects of 17ß-estradiol exposures in aquatic vertebrates. A 21-day flow-through exposure of mature male and female fathead minnows to three concentrations each of 17α- and 17ß-estradiol (averaged measured concentrations 27, 72, and 150 ng/L for 17α-estradiol, and 9, 20, and 44 ng/L for ß-estradiol, respectively) yielded significant, concentration-dependent differences in plasma vitellogenin concentrations among estradiol-exposed males when compared to fish from an ethanol carrier control. Interstitial cell prominence in the testis of fish was elevated in all estradiol treatments. Aggressiveness of male fish to defend nest sites appeared depressed in many of the higher concentration estradiol treatments (albeit not significantly). No clear effects were observed in female fish. Based on plasma vitellogenin data, it appears that 17ß-estradiol is 8-9 times more potent than 17α-estradiol and that the lowest observable effect concentration (LOEC) for 17α-estradiol in fathead minnows is greater than 25 ng/L and may be less than 75 ng/L.

Assessing the effects of exposure timing on biomarker expression using 17beta-estradiol.

Temporal and spatial variability in estrogenicity has been documented for many treated wastewater effluents with the consequences of this variability on the expression of biomarkers of endocrine disruption being largely unknown. Laboratory exposure studies usually utilize constant exposure concentrations which may produce biological effects that differ from those observed in organisms exposed in natural environments. In this study, we investigated the effects of differential timing of exposures with 17beta-estradiol (E2) on a range of fathead minnow biomarkers to simulate diverse environmentally relevant exposure profiles. Two 21-day, replicate experiments were performed exposing mature male fathead minnows to E2 at time-weighted mean concentrations (similar average exposure to the contaminant during the 21-day exposure period; 17ng E2/L experiment 1; 12ng E2/L experiment 2) comparable to E2 equivalency values (EEQ) reported for several anthropogenically altered environments. A comparable time-weighted mean concentration of E2 was applied to five treatments which varied in the daily application schema: E2 was either applied at a steady rate (ST), in a gradual decreasing concentration (HI), a gradual increasing concentration (LO), applied intermittently (IN), or at a randomly varying concentration (VA). We assessed a range of widely used physiological (vitellogenin mRNA induction and plasma concentrations), anatomical (body and organ indices, secondary sex characteristics, and histopathology), and behavioral (nest holding) biomarkers reported to change following exposure to endocrine active compounds (EACs). All treatments responded with a rise in plasma vitellogenin concentration when compared with the ethanol carrier control. Predicatively, vitellogenin mRNA induction, which tracked closely with plasma vitellogenin concentrations in most treatments was not elevated in the HI treatment, presumably due to the lack of E2 exposure immediately prior to analysis. The ability of treatment male fish to hold nest sites in direct competition with control males was sensitive to E2 exposure and did yield statistically significant differences between treatments and carrier control. Other biological endpoints assessed in this study (organosomatic indices, secondary sex characteristics) varied little between treatments and controls. This study indicates that a broad suite of endpoints is necessary to fully assess the biological consequences of fish exposure to estrogens and that for at least field studies, a combination of vitellogenin mRNA and plasma vitellogenin analysis are most promising in deciphering exposure histories of wild-caught and caged fishes.

Treated wastewater effluent reduces sperm motility along an osmolality gradient.

Many toxic effects of treated wastewater effluent on organismal and reproductive health have been documented. However, the physicochemical environment of treated wastewater effluent frequently differs considerably from that of its receiving waters and may affect organismal function independently of toxic effects. Teleost sperm, for example, may be affected by the higher osmolality of treated wastewater, as this sperm is activated for a brief period of time following ejaculation due to the sudden decrease in osmolality of its surrounding environment. In this study, we examined the effects of treated wastewater effluent on sperm motility to test the hypothesis that the higher osmolality of effluent compared to river water will adversely affect sperm activation in a concentration-dependent relationship. Treated wastewater effluent was collected on 5 days from the outflow of the Metropolitan Wastewater Treatment Plant, St. Paul, Minnesota, and from an upstream site on the Mississippi River. Milt aliquots collected from goldfish were diluted in an isotonic extender solution and subsequently activated in either deionized water, 100%, 50%, or 10% effluent, a synthetic ion mixture, or river water. Sperm motility and velocity were assessed at 15-s intervals for 1 min using a computer assisted sperm analyzer. Significant differences in performance parameters were found only at 15 s, with sperm motility and velocity declining rapidly at later sampling times. Predictably, deionized water resulted in the greatest activation of sperm motility, while motility exhibited a concentration-dependent decline in 10%, 50%, and 100% treated wastewater effluent. Interestingly, Mississippi River water and a synthetic ion mixture with an osmolality comparable to 50% effluent both resulted in the least amount of sperm activation. However, sperm activation in river water varied between collection days during the study. River water and 100% effluent both had low sperm activation characteristics despite a 10-fold difference in osmolality between these two treatments (1 and 10 mOsmol kg(-1), respectively). Results of this study indicate a concentration-dependent decrease in sperm motility in treated wastewater effluent as well as significant fluctuations of sperm activation in Mississippi River water. This study illustrates the complexity of assessing the effects of treated wastewater effluents and the difficulty of determining appropriate reference sites for such studies.

Impairment of the reproductive potential of male fathead minnows by environmentally relevant exposures to 4-nonylphenolf.

The synthetic organic compound 4-nonylphenol (NP) has been detected in many human-impacted surface waters in North America. In this study, we examined the ability of NP to alter reproductive competence in male fathead minnows after a 28 day flow-through exposure in a range of environmentally relevant concentrations bracketing the U.S. Environmental Protection Agency toxicity-based NP chronic exposure criterion of 6.1 microg NP/L. Exposure to NP at and above the EPA chronic exposure criterion resulted in an induction of plasma vitellogenin (VTG) within 14 days. However, 7 days after the cessation of exposure, VTG concentrations had dropped more than 50% and few males expressed VTG above the detection threshold. All of the morphological endpoints, including gonadosomatic index, hepatosomatic index, secondary sexual characters, and histopathology, were unaltered by all NP treatments. However, when NP-exposed male fish were allowed to compete with control males for access to nest sites and females, most treatments altered the reproductive competence of exposed males. At lower NP concentrations, exposed males out-competed control males, possibly by being primed through the estrogenic NP exposure in a fashion similar to priming by pheromones released from female fathead minnows. At higher NP exposure concentrations, this priming effect was negated by the adverse effects of the exposure and control males out-competed treated males. Results of this study indicate the complexity of endocrine disrupting effects and the need for multiple analysis levels to assess the effects of these compounds on aquatic organisms.