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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.

We report here on studies designed to asses the effects of paper mill effluents on non-reproductive functions of free-ranging and captive Florida largemouth bass (Micropterus salmoides floridanus). This was accomplished by conducting an outdoor tank study, in which fish were exposed to well water or to 10%, 20%, 40%, and 80% full strength effluent for 28 or 56 days, and by sampling largemouth bass from sites within the St. Johns River, Florida, upstream and downstream from a paper mill plant. Blood and plasma samples from fish from the tank study and from fish sampled from the ambient sites were analyzed for over 20 variables. We also determined liver and spleen weights and examined them histologically. The most significant finding from the tank study was an increase in the concentration of albumin and hepatosomatic index for bass exposed to > or = 20% effluents for 56 days. Spleenosomatic index and number of melanomacrophage centers were decreased in bass from effluent-dominated sites (Palatka and Rice Creek), whereas concentrations of calcium, phosphorous, glucose, and creatinine were elevated in fish from these sites, compared to fish from reference streams. Fish from Rice Creek also had fewer red blood cells, and male bass from Palatka had lower concentrations of cholesterol. Plasma concentrations of albumin and hepatic concentrations of glutathione were elevated in males from Palatka, and both females and males from Rice Creek had higher concentrations of globulin. These results indicate a complex pattern of effects of paper mill effluents on several physiological functions. However, despite the myriad of treatment and site-related effects, most physiological parameters fell within normal ranges when compared to reports on largemouth bass and other freshwater species.

The objective of this study was to evaluate the effects of bleached/unbleached kraft mill effluents (B/UKME) on the reproductive parameters of free-ranging Florida largemouth bass (Micropterus salmoides floridanus). The reproductive parameters measured included gonadosomatic index (GSI), histological evaluation of gonads, and plasma concentrations of vitellogenin (VTG), 17beta-estradiol, and 11-ketotestosterone (11-KT). Hepatic ethoxyresorufin-O-deethylase (EROD) activity was measured as a marker of exposure to cytochrome P450-inducing agents in these effluents. Endpoints were compared among adult bass sampled from tributary and mainstream effluent-contaminated and reference sites. Females sampled from the site closest to the mill outfall had a significant five-fold increase in EROD activity compared to bass sampled from reference streams. Although sex hormones were significantly reduced in bass from exposed sites, there were no differences in VTG and GSI across sites. The absence of organism-level responses was probably not related to a lack of sensitivity, as previous studies in our laboratory have shown that bass exposed to these effluents exhibit changes in GSI and in other measures associated with reproductive success. In females, inverse relationships were observed between VTG and GSI and EROD activity. These relationship, however, were not consistent within all of the sites studied. Collectively, our findings indicate that hepatic EROD induction is an effective marker of B/UKME exposure in largemouth bass and that it might be associated with antiestrogenic effects in this species.

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.

Water samples were collected on three dates at 24 sites influenced by effluent from Georgia-Pacific's Palatka Pulp and Paper Mill Operation, a bleached and unbleached kraft mill near Palatka, Florida, USA. The sampling sites were located within the mill retention ponds, Rice Creek, and the St. John's River. Samples were analyzed by gas chromatography-mass spectrometry for abietic, dehydroabietic, and isopimaric acids, all of which are potentially toxic by-products of pulp production. Isopimaric acid concentrations greater than 12 mg/L were measured at the mill's effluent outfall but were less than 20 microg/L at the end of Rice Creek. This result indicates that the waters of Rice Creek provide dilution or conditions conducive for degradation or sorption of these compounds. Large differences in resin acid concentrations were observed between sampling events. In two sampling events, the maximum observed concentrations were less than 2 mg/L for each analyte. In a third sampling event, all of the compounds were detected at concentrations greater than 10 mg/L. Data from the three sample dates showed that resin acid concentrations were below 20 microg/L before the confluence of Rice Creek and the St. John's River in all cases.

The objective of this study was to describe and compare several reproductive parameters for Florida largemouth bass (Micropterus salmoides floridanus) inhabiting the St. Johns River and exposed to different types and/or degrees of contamination. Welaka was selected as the reference site in this study because of its low urban and agricultural development, Palatka is in close proximity to a paper mill plant, the Green Cove site is influenced by marine shipping activities and Julington Creek site receives discharges of domestic wastewater and storm water runoff from recreational boating marinas. For this study, bass were sampled both prior to (September 1996) and during the spawning season (February 1997). In order to characterize chemical exposure, bass livers were analyzed for up to 90 trace organics and 11 trace metal contaminants. Reproductive parameters measured included gonadosomatic index (GSI), histological evaluation of gonads and plasma concentrations of vitellogenin (VTG), 17beta-estradiol (E2) and 11-ketotestosterone (11-KT). In general, the sum of organic chemicals was highest in livers from Palatka bass and bass from Green Cove and Julington Creek had higher hepatic concentrations of low molecular polycyclic aromatic hydrocarbons and polychlorinated biphenyls when compared to fish from Welaka. Metals were more variable across sites, with highest mean concentrations found in bass from either Julington Creek (Ag, As, Cr, Cu, Zn) or Welaka (Cd, Hg, Pb, Se, Tn). Female bass from Palatka and Green Cove had lower concentrations of E2, VTG and lower GSI in relation to Welaka. Males from Palatka and Green Cove showed comparable declines in 11-KT in relation to males from Julington Creek and GSI were decreased only in Palatka males. These results indicate a geographical trend in reproductive effects, with changes being most pronounced at the site closest to the paper mill (Palatka) and decreasing as the St. Johns River flows downstream. Since reproductive alterations were most evident in bass sampled from the site closest to the paper mill discharge, it is possible that exposure to these effluents might explain at least some of the results reported here. However, the presence of reproductive alterations in fish sampled at a considerable distance from the mill discharge (Green Cove, 40 km) would suggest exposure to chemicals released from sources other than the paper mill plant. It is clear that additional studies are needed to evaluate the potential impact of these reproductive changes in populations of Florida largemouth bass inhabiting the St. Johns River.

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.