Fenholloway River Research Articles

Exposure to paper mill effluent at a site in North Central Florida elicits molecular-level changes in gene expression indicative of progesterone and androgen exposure.

Endocrine disrupting compounds (EDCs) are chemicals that negatively impact endocrine system function, with effluent from paper mills one example of this class of chemicals. In Florida, female Eastern mosquitofish (Gambusia holbrooki) have been observed with male secondary sexual characteristics at three paper mill-impacted sites, indicative of EDC exposure, and are still found at one site on the Fenholloway River. The potential impacts that paper mill effluent exposure has on the G. holbrooki endocrine system and the stream ecosystem are unknown. The objective of this study was to use gene expression analysis to determine if exposure to an androgen receptor agonist was occurring and to couple this analysis with in vitro assays to evaluate the presence of androgen and progesterone receptor active chemicals in the Fenholloway River. Focused gene expression analyses of masculinized G. holbrooki from downstream of the Fenholloway River paper mill were indicative of androgen exposure, while genes related to reproduction indicated potential progesterone exposure. Hepatic microarray analysis revealed an increase in the expression of metabolic genes in Fenholloway River fish, with similarities in genes and biological processes compared to G. holbrooki exposed to androgens. Water samples collected downstream of the paper mill and at a reference site indicated that progesterone and androgen receptor active chemicals were present at both sites, which corroborates previous chemical analyses. Results indicate that G. holbrooki downstream of the Fenholloway River paper mill are impacted by a mixture of both androgens and progesterones. This research provides data on the mechanisms of how paper mill effluents in Florida are acting as endocrine disruptors.

Reproductive Success of Eastern Mosquitofish (Gambusia affinis) Exposed to Pulp and Paper Dominated Receiving Streams and Effects of Masculinization Responses

Abstract Mosquitofish (Gambusia) are sexually dimorphic; adult males have an elongation of the anal fin to form a copulatory structure known as a gonopodium. Several studies since the early 1980s have reported elongated anal fins in female mosquitofish exposed to pulp and paper mill discharge, a phenomenon known as masculinization. Although adverse impacts have been suggested in these previous reports, the influence of masculinization on reproductive success has not been assessed for pulp mill effluent-exposed eastern mosquitofish (Gambusia holbrooki or affinis). The current study compared fecundity (number of fry per female at parturition) to an index of masculinization (ratio of anal fin rays 4 to 6). Pregnant females were collected from two effluent-receiving streams in Florida: Rice Creek and the Fenholloway River, over two reproductive seasons (2003 and 2004). Masculinization was consistent between years, with clear effects at the Fenholloway River site, while the response was minimal or nondetectable at the Rice Creek site. Masculinization was not correlated with the production of fry/fecundity at either site. Data suggest differing reproductive seasonal strategies between basins and populations but do not demonstrate any effects of pulp and paper exposure on reproductive success in mosquitofish.

Naturally occurring progesterone in loblolly pine (Pinus taeda L.): A major steroid precursor of environmental androgens

AbstractProgesterone, androstenedione, and androstadienedione were previously identified in the water and sediment of the Fenholloway River (Taylor County, FL, USA), a river that contains populations of masculinized female mosquitofish downstream of a paper mill, at levels higher than those in the nearby Spring Creek. Plant sterols, such as β‐sitosterol in mill effluent derived from pine tree pulp, were suggested to be metabolized by bacteria to progesterone and androgens to account for the masculinization phenomenon. The current study made use of standard solid‐phase methanol extraction procedures, high‐performance liquid chromatography, liquid chromatography‐mass spectrometry, and a cell‐based, androgen‐receptor transcription assay to determine naturally occurring progesterone levels in mature pine trees. Progesterone concentrations in the loblolly pine (Pinus taeda L.) were 49.34 ± 4.1 nmol/g dry mature wood (15.5 ± 1.29 μg/g), 12.26 ± 1.78 nmol/g pine needles (3.85 ± 0.56 μg/g), and 3.81 ± 0.36 nmol/g pine bark (1.19 ± 0.11 μg/g). The results suggest that naturally occurring progesterone from pine wood pulp contributes to increased progesterone levels downstream of paper mill effluent discharges and may serve as the natural steroid precursor for environmental androgen production that causes masculinization of female mosquitofish.

Altered development and reproduction in mosquitofish exposed to pulp and paper mill effluent in the Fenholloway River, Florida USA

Female mosquitofish exposed to pulp and paper mill effluent (PME) in the Fenholloway River, Florida, USA have masculinized secondary sex characteristics and altered aromatase enzyme activity. We and others have shown that the Fenholloway River PME contains androgenic and progestogenic substance(s). The present study was designed to test the hypothesis that the development and reproductive health of PME-exposed Fenholloway River mosquitofish are altered compared to mosquitofish living in Econfina River, which is the reference site. Fish were collected on a single day from both sites in June and August 1999 and January and June 2000. We compared standard length, anal fin length and segment number; body, liver, and gonad mass; and number of eggs and embryos from Fenholloway and Econfina River mosquitofish. The data were analyzed collectively for generalized site effect, for site effects during reproductive and nonreproductive seasons, and for repeatability of site effects between years. Mosquitofish exposed to PME in the Fenholloway River were generally smaller in length and mass, anal fin segment number was greater, and the number of embryos, but not oocytes, was significantly decreased compared to the reference site fish. Anal fin length and segment number and liver and testis masses were generally greater in Fenholloway compared to the Econfina River males. The importance of this study is that we have documented masculinized development and decreased embryo production in PME-exposed mosquitofish and that these site effects are generally consistent across seasons and between years.

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.

Altered social behavior and sexual characteristics in mosquitofish ( Gambusia holbrooki) living downstream of a paper mill

Several environmental contaminants have been found in paper mill effluent including substances with androgenic properties. The presence of androgenic substances in paper mill effluent was also indicated in the present study which demonstrated masculinized anal fins of female mosquitofish from the paper mill effluent contaminated Fenholloway River. In addition, when compared to the nearby Econfina River, which does not receive paper mill effluent, fewer females from the contaminated river were pregnant, they were smaller and their estradiol concentration exhibited greater variation. Males from the Fenholloway River and the Econfina River had similar sperm counts, but the testes were larger and greater variation in testosterone concentration was observed in male fish from the Fenholloway River. In males and females from the Fenholloway River, liver weights were increased and computer-aided behavior analysis demonstrated a reduction in their social behavior when compared to reference fish. We conclude that a number of sexual characteristics were affected in mosquitofish living in the paper mill contaminated Fenholloway River, with possible adverse effects on the reproduction of this population.

Production of Androgens by Microbial Transformation of Progesterone in Vitro : A Model for Androgen Production in Rivers Receiving Paper Mill Effluent

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., β-sitosterol) derived from the pulping of pine trees are transformed by bacteria into progesterone and subsequently into 17α-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 Δ1 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.

Evaluation of androstenedione as an androgenic component of river water downstream of a pulp and paper mill effluent

This study evaluates a recent report indicating that androstenedione (4-androsten-3, 17-dione) contributes to the androgenicity of water downstream of a pulp and paper mill discharge on the Fenholloway River (FL, USA). Extraction and concentration of Fenholloway water with C18 solid-phase extraction columns followed by reverse-phase high-pressure liquid chromatography resulted in clearly defined fractions with in vitro androgenic activity in CV-1 cells that had been transiently cotransfected with human androgen receptor and reporter gene constructs. However, we were unable to detect androstenedione in the active fractions by gas chromatography/mass spectrometry. Mass spectrometry analyses of deionized and Fenholloway River water samples that had been spiked with androstenedione, then extracted and fractionated, revealed that the androgen was found only in inactive fractions. We conclude that, although androstenedione was present at easily detectable concentrations in the river water (> 100 ng/L), this compound is not associated with androgenic activity of water from the site.

Aromatase activity in the ovary and brain of the eastern mosquitofish (Gambusia holbrooki) exposed to paper mill effluent.

Studies have shown that female mosquitofish living downstream of a paper mill located on the Fenholloway River, Florida, have masculinized secondary sex characteristics, including altered anal fin development and reproductive behavior. Masculinization can be caused by exposure to androgens in the water or from an alteration in aromatase activity in the fish. We hypothesized that aromatase activity would be inhibited by a component(s) of the paper mill effluent. Aromatase inhibition could masculinize the hormonal profile and, subsequently, secondary sex characteristics of the exposed females. Therefore, we predicted that ovarian and brain aromatase activity would be lower in the female mosquitofish from the Fenholloway River compared with the reference site, the Econfina River. Adult females were collected and standard length, body mass, anal fin length, and segment number were measured. Ovarian and brain aromatase activity were determined using a tritiated water assay. Fenholloway females had masculinized anal fin development as indicated by an increase in the number of segments in the longest anal fin ray (p < 0.0001), yet the length of the ray did not differ between sites (p = 0.95). Fenholloway females exhibited higher ovarian (p = 0.0039) and brain (p = 0.0003) aromatase activity compared with reference site fish. These data do not support aromatase inhibition as the mechanism for masculinization, suggesting that the masculinization of the Fenholloway female mosquitofish is due to androgenic contaminants. Future studies should examine the relationship between aromatase enzyme activity and exposure to environmental androgens.

Masculinization of female mosquitofish in Kraft mill effluent-contaminated Fenholloway River water is associated with androgen receptor agonist activity.

Female mosquitofish (Gambusia affinis holbrooki) downstream from Kraft paper mills in Florida display masculinization of the anal fin, an androgen-dependent trait. The current investigation was designed to determine if water contaminated with pulp-mill effluent (PME) from the Fenholloway River in Florida displayed androgenic activity in vitro and to relate this activity to the reproductive status of female mosquitofish taken from this river. We tested water samples for androgenic activity from a reference site upstream of a Kraft pulp and paper mill on the Fenholloway River, from 3 sites downstream from the mill, and from another reference site on the Econfina River, also in Florida, where there is no paper mill. We also examined anal fin ray morphology in mosquitofish from these rivers for evidence of masculinization. Eighty percent of the female mosquitofish from the Fenholloway River were partially masculinized while another 10% were completely masculinized, based upon the numbers of segments in the longest anal fin ray (18.0 +/- 0.4 vs. 28.1 +/- 0.9 [p < 0.001]) in the Econfina River vs. the Fenholloway River, respectively). In a COS whole cell-binding assay, all 3 PME samples displayed affinity for human androgen receptor (hAR) (p < 0.001). In addition, PME induced androgen-dependent gene expression in CV-1 cells (cotransfected with pCMV hAR and MMTV luciferase reporter), which was inhibited by about 50% by coadministration of hydroxyflutamide (1 microM), an AR antagonist. Water samples collected upstream of the Kraft mill or from the Econfina River did not bind hAR or induce luciferase expression. When CV-1 cells were transfected with human glucocorticoid receptor (hGR) rather than hAR, PME failed to significantly induce MMTV-luciferase expression. Further evidence of the androgenicity was observed using a COS cell AR nuclear-translocalization assay. PME bound hAR and induced translocalization of AR into the nucleus. In contrast, AR remained perinuclear when treated with water from the control sites (indicating the absence of an AR ligand). Interestingly, PME also displayed "testosterone-like" immunoreactivity in a testosterone radioimmunoassay, whereas water from the reference sites did not. In summary, water collected downstream of the Kraft mill on the Fenholloway River contains unidentified androgenic substances whose presence is associated with masculinization of female mosquitofish.

Identification of androstenedione in a river containing paper mill effluent

AbstractEffluent from a paper mill discharging into the Fenholloway River, Taylor County, Florida, USA, contains chemicals that masculinize females of the resident population of eastern mosquitofish (Gambusia holbrooki), as evidenced in females by elongated anal fins, which is normally a male‐specific trait. To identify androgenic components in the effluent, water collected from the Fenholloway River and a control tributary was fractionated using solid‐phase extraction and reverse‐phase high‐performanceliquid chromatography. Two Fenholloway River fractions induced androgen receptor‐dependent transcriptional activity in transient transfection cell culture assays. Of these, androstenedione was confirmed by liquid chromatography‐mass spectrometry with multiple reaction monitoring.

Identification of androstenedione in a river containing paper mill effluent.

Effluent from a paper mill discharging into the Fenholloway River, Taylor County, Florida, USA, contains chemicals that masculinize females of the resident population of eastern mosquitofish (Gambusia holbrooki), as evidenced in females by elongated anal fins, which is normally a male-specific trait. To identify androgenic components in the effluent, water collected from the Fenholloway River and a control tributary was fractionated using solid-phase extraction and reverse-phase high-performance-liquid chromatography. Two Fenholloway River fractions induced androgen receptor-dependent transcriptional activity in transient transfection cell culture assays. Of these, androstenedione was confirmed by liquid chromatography-mass spectrometry with multiple reaction monitoring.

Tidal Characteristics of Two Estuaries in Florida

Although the Waccasassa and Fenholloway River estuaries are superficially similar, there are differences in their mixing characteristics resulting from steepness of gradients and exposure to winds and the tidal front. Wind effects are especially important in the shallow waters of the Florida West Coast. Determination of mean velocity variation in such streams can be readily effected by a process of cubature, provided that good tidal records are available at a few key points between the mouth and the head of the estuary.