Article

Reproductive Health of Bass in the Potomac, USA, Drainage: Part 1. Exploring the Effects of Proximity to Wastewater Treatment Plant Discharge

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Abstract

Intersex (specifically, testicular oocytes) has been observed in male smallmouth bass (SMB; Micropterus dolomieu) and other centrarchids in the South Branch of the Potomac River, U.S.A., and forks of the Shenandoah River, U.S.A., during the past five years. This condition often is associated with exposure to estrogenic endocrine-disrupting chemicals in some fish species, but such chemicals and their sources have yet to be identified in the Potomac. In an attempt to better understand the plausible causes of this condition, we investigated the reproductive health of bass sampled up- and downstream of wastewater treatment plant (WWTP) effluent point sources on the Potomac River in Maryland, U.S.A. Smallmouth bass were sampled from the Conococheague Creek and the Monocacy River, and largemouth bass (LMB; Micropterus salmoides) were collected near the Blue Plains WWTP on the mainstem of the Potomac River. Chemical analyses of compounds captured in passive samplers at these locations also were conducted. A high prevalence of intersex (82-100%) was identified in male SMB at all sites regardless of collection area. A lower prevalence of intersex (23%) was identified in male LMB collected at the Blue Plains site. When up- and downstream fish were compared, significant differences were noted only in fish from the Conococheague. Differences included condition factor, gonadosomatic index, plasma vitellogenin concentration, and estrogen to testosterone ratio. In general, chemicals associated with wastewater effluent, storm-water runoff, and agriculture were more prevalent at the downstream sampling sites. An exception was atrazine and its associated metabolites, which were present in greater concentrations at the upstream sites. It appears that proximity to effluent from WWTPs may influence the reproductive health of bass in the Potomac watershed, but inputs from other sources likely contribute to the widespread, high incidence of testicular oocytes.

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... A field study on Micropterus dolomieu showed increased weight (males) and increased weight and length (females) at sites with greater average concentrations of atrazine (Iwanowicz et al. 2009). This was not deemed an adverse effect and a reduced SEJ was assigned. ...
... A physiological seawater challenge study with S. salar parr reported significantly reduced GSI in males but not in females (Nieves-Puigdoller et al. 2007). Finally, a field study was conducted over a 2-year period to investigate the potential impacts of environmental pollutants (including atrazine) on sexually mature M. dolomieu (Iwanowicz et al. 2009). Fish were collected above and below wastewater treatment plants (WWTPs), and it was found that the GSI was increased in both males and females at the upstream site relative to the downstream site. ...
... Likewise, no effect was observed in P. promelas exposed to atrazine at concentrations of 0.5, 5.0, and 50 mg/L for 14 or 30 days (Tillitt et al. 2010) or in repeated studies (2007 and 2008) in G. aculeatus exposed to atrazine (concentrations ranging from 0.1 to 100 mg/L) from hatching to 42 days (Le Mer et al. 2013). A field study conducted to assess the influence of WWTPs on a number of reproductive endpoints in M. dolomieu reported the presence of atrazine, as well as multiple other compounds, and found a high frequency of TOFs (90%-100%), but no significant differences between upstream and downstream sites (Iwanowicz et al. 2009). Because of exposures to multiple substances, causality could not be assigned. ...
Article
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Abstract A quantitative weight of evidence (WoE) approach was developed to evaluate studies used for regulatory purposes, as well as those in the open literature, that report the effects of the herbicide atrazine on fish, amphibians, and reptiles. The methodology for WoE analysis incorporated a detailed assessment of the relevance of the responses observed to apical endpoints directly related to survival, growth, development, and reproduction, as well as the strength and appropriateness of the experimental methods employed. Numerical scores were assigned for strength and relevance. The means of the scores for relevance and strength were then used to summarize and weigh the evidence for atrazine contributing to ecologically significant responses in the organisms of interest. The summary was presented graphically in a two-dimensional graph which showed the distributions of all the reports for a response. Over 1290 individual responses from studies in 31 species of fish, 32 amphibians, and 8 reptiles were evaluated. Overall, the WoE showed that atrazine might affect biomarker-type responses, such as expression of genes and/or associated proteins, concentrations of hormones, and biochemical processes (e.g. induction of detoxification responses), at concentrations sometimes found in the environment. However, these effects were not translated to adverse outcomes in terms of apical endpoints. The WoE approach provided a quantitative, transparent, reproducible, and robust framework that can be used to assist the decision-making process when assessing environmental chemicals. In addition, the process allowed easy identification of uncertainty and inconsistency in observations, and thus clearly identified areas where future investigations can be best directed.
... The phospholipoprotein vitellogenin (Vtg) is an egg yolk precursor protein necessary for normal oocyte maturation and development in most fish species with males expression generally at low to undetectable levels in the blood of normal males (Denslow et al., 1999;Devlin and Nagahama, 2002;Iwanowicz et al., 2009;Sumpter and Jobling, 1995). Generally, detection of TO in males is coupled with other ephemeral biomarkers of endocrine disruption, such as elevated plasma Vtg levels (Denslow et al., 1999;Devlin and Nagahama, 2002;Iwanowicz et al., 2009;Sumpter and Jobling, 1995). ...
... The phospholipoprotein vitellogenin (Vtg) is an egg yolk precursor protein necessary for normal oocyte maturation and development in most fish species with males expression generally at low to undetectable levels in the blood of normal males (Denslow et al., 1999;Devlin and Nagahama, 2002;Iwanowicz et al., 2009;Sumpter and Jobling, 1995). Generally, detection of TO in males is coupled with other ephemeral biomarkers of endocrine disruption, such as elevated plasma Vtg levels (Denslow et al., 1999;Devlin and Nagahama, 2002;Iwanowicz et al., 2009;Sumpter and Jobling, 1995). Vtg is produced in the liver and transported via blood and in the presence of exogenous EEDCs males will produce Vtg in a dose-dependent manner, this can cause damage to the liver and kidneys during development (Hutchinson et al., 2006). ...
... Shortly after, several reports of intersex SMB and LMB throughout much of the United States were published (Anderson et al., 2003;Baldigo et al., 2006;Blazer et al., 2007Blazer et al., , 2012Hinck et al., 2009;Iwanowicz et al., 2009Iwanowicz et al., , 2016Yonkos et al., 2014). ...
Thesis
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The appearance of testicular oocytes (TO) in wild fish populations has received considerable attention in the scientific literature and public media. Current methods to quantify TO are lethal; instead, a non-lethal alternative was examined. Laparoscopic insertion into the genital pore allowed internal visualization of the gonad and detection of TO by collecting five testis biopsies in smallmouth bass Micropterus dolomieu and largemouth bass Micropterus salmoides. Overall, biopsies quantified similar levels of TO detection and severity to conventional transverse sectioning with less than 10% mortality. Suitability of surgical anesthetics, tricaine methanesulfonate and electronarcosis were examined in laboratory and field applications. Electronarcosis had the added benefit of rapid sex identification and immediate release of female fish with minimal trauma, representing significant benefits when sampling small or compromised populations. Laparoscopy may be useful for monitoring the prevalence and severity of TO in these fish species when lethal sampling is not a desired outcome.
... 250 mm) were included. Smallmouth bass were selected for model development because the species has been found to have the highest testicular oocyte prevalence and severity in many US freshwater systems (as determined by well-established methods of testicular oocyte detection and enumeration) [8,9,[19][20][21]. Largemouth bass were employed to test the resulting biopsy model. ...
... In contrast, prevalence was only 31% in largemouth bass, with all site severity ranks 0.6. These findings mirror other reports in the scientific literature, with low testicular oocyte prevalence and severity in largemouth bass [19,26,27] contrasted with much higher values in smallmouth bass [8,9,[19][20][21]27]. In a retrospective survey of 8 US river basins (1995)(1996)(1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004), Hinck et al. [18] reported testicular oocyte prevalence of 33% in smallmouth bass (n ¼ 70) compared with 18% in largemouth bass (n ¼ 390; severity not reported). ...
... An ongoing challenge to the interpretation of intersex data in the scientific literature involves the lack of uniformity in practices employed to detect and characterize testicular oocyte abundance. Approaches used for black bass include subsampling transversely at 5 equidistant locations along 1 lobe [8,9,[19][20][21]26] and sampling longitudinally from 1 or both lobes [25,28]. Tissues collected by these various methods are then processed and observed singly or step-sectioned to produce multiple observable sections from proximate regions. ...
Article
Intersex in wild fish populations has received considerable attention in the scientific literature and public media. Conventional detection of testicular oocytes (TO), the presence of immature oocytes within testis of male fish, employs transverse sectioning of excised testis and is lethal. This present study used a non-lethal laparoscopic technique to collect biopsies of testis from black bass, entering the body cavity via the genital pore. Detection of TO was compared between biopsy and conventional methods using 79 smallmouth bass (SMB) Micropterus dolomieu from 8 sites and 68 largemouth bass (LMB) M. salmoides from 4 sites. Both methods performed similarly at sites where TO severity was moderate or high (6 of 8 SMB sites) while transverse sectioning resulted in superior TO detection at sites where severity was low (2 of 8 SMB sites and all 4 LMB sites). In SMB, TO prevalence by transverse and biopsy methods was strongly correlated across sites (r(2) = 0.81) and severity reported by enumeration of TO was moderately correlated across sites (r(2 ) = 0.59). Survival of a subset of LMB (n = 20) to 28-d after laparoscopic surgery was 90%. This research indicates that laparoscopy may be useful for monitoring the prevalence and severity of TO in Micropterus species, particularly when lethal sampling is precluded. This article is protected by copyright. All rights reserved.
... Co-infections of bacterial, viral, as well as parasitic infections have been associated with these events [4,6,7] suggesting immunosuppression. Estrogenic endocrine disruption as evidenced by testicular oocytes (intersex), vitellogenin in male fish [8][9][10] and the occurrence of contaminants linked to intersex [4,7,11,12] has been confirmed within the areas of the Chesapeake watershed where disease events occur. The observation of a complex disease etiology that includes multiple obligate and opportunistic pathogens and parasites as well as endocrine disruption necessitates laboratory investigations into the potential changes in physiologic functions including the immune response. ...
... While developing the criteria for maintaining fish for this purpose, a major issue that needs to be https://doi.org/10.1016/j.fsi.2019. 10 addressed is nutrition. The relationship between teleost nutrition, general health, and immune function is well established [13][14][15]. ...
Article
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Smallmouth bass (Micropterus dolomieu) are used as an indicator species in environmental monitoring and assessment studies. However, laboratory-based studies for methods development and effector assessment are limited for this species. Nutrition, a known modulator of teleost physiological responses including immune function, is a critical knowledge-gap sometimes overlooked in the design of laboratory studies. We report the results from a study evaluating a commercially available artificial pelleted diet for bass and live feed (fathead minnows). Following a six-month diet-acclimation period, age-0 smallmouth bass were assessed using morphometric measures, histologic and immune-function end points using conventional methods, miniaturized cell isolation and assay methods as well as imaging flow cytometry. Fish on the two diets did not significantly differ in length, weight, or condition factor, indicating that growth was similar in the two groups. Histologic examination revealed relatively higher levels of macrophage aggregates and accumulation of ceroid/lipofuscin in the spleen as well as hepatocyte changes in the pellet-fed group. Leukocytes from the pellet-fed group exhibited significantly elevated bactericidal activity and significantly depressed mitogen response compared to fish fed live feed. Following exposure to a known inducer of inflammatory responses, bacterial lipopolysaccharide, responses including the transition of leukocytes to an apoptotic/necrotic condition differed significantly based on diet. Histologic findings were consistent with the occurrence of diet-related oxidative stress in the pellet-fed fish. Oxidative stress can be induced by multiple factors including environmental pollutants. For a diet to be useful in laboratory-based studies, it cannot elicit response that could also be induced by experimental treatments. To do so greatly complicates the detection of experimental effects. Until an artificial diet is developed for smallmouth bass that does not produce potentially confounding conditions for laboratory-based studies, use of a live feed appears to be the best option.
... Numerous studies have attempted to establish relationships between TO and exposure to EDCs; however, the majority of these studies have failed to find strong cause-effect relationships (Reeder et al., 2005;Bjerregaard et al., 2006;Woodling et al., 2006;Douxfils et al., 2007). Only a handful of field studies have reported strong relationships between the TO prevalence and specific contaminants, such as steroids and atrazine (Iwanowicz et al., 2009;Kolpin et al., 2013). These findings were based only on field observations, so they do not necessarily imply causation. ...
... The contribution of compounds to estrogenicity other than known estrogens is supported by studies that have established significant positive correlations between the prevalence and severity of TO occurrence and land use metrics (Blazer et al., 2007(Blazer et al., , 2012. Interestingly, TO prevalence and severity has been shown to be more correlated to the intensity of agricultural practices than to wastewater treatments plant discharge contributions (Blazer et al., 2007Iwanowicz et al., 2009;Ciparis et al., 2012). ...
Article
Over the past decade, studies have shown that exposure to endocrine disrupting chemicals (EDCs) can cause gonadal intersex in fish. Smallmouth bass (Micropterus dolomieu) males appear to be highly susceptible to developing testicular oocytes (TO), the most prevalent form of gonadal intersex, as observed in various areas across the U.S. In this study, prevalence and severity of TO was quantified for smallmouth bass sampled from the St. Joseph River in northern Indiana, intersex biomarkers were developed, and association between TO prevalence and organic contaminants were explored. At some sites, TO prevalence reached maximum levels before decreasing significantly after the spawning season. We examined the relationship between TO presence and expression of gonadal and liver genes involved in sex differentiation and reproductive functions (esr1, esr2, foxl2, fshr, star, lhr and vtg). We found that vitellogenin (vtg) transcript levels were significantly higher in the liver of males with TO, but only when sampled during the spawning season. Further, we identified a positive correlation between plasma VTG levels and vtg transcript levels, suggesting its use as a non-destructive biomarker of TO in this species. Finally, we evaluated 43 contaminants in surface water at representative sites using passive sampling to look for contaminants with possible links to the observed TO prevalence. No quantifiable levels of estrogens or other commonly agreed upon EDCs such as the bisphenols were observed in our contaminant assessment; however, we did find high levels of herbicides as well as consistent quantifiable levels of PFOS, PFOA, and triclosan in the watershed where high TO prevalence was exhibited. Our findings suggest that the observed TO prevalence may be the result of exposures to mixtures of nonsteroidal EDCs.
... Micropterus species have been shown to be sensitive species for endocrine disruption studies, specifically responding to estrogenic compounds by induction of vitellogenin (Vtg) and TO in male bass (Blazer et al. 2014d(Blazer et al. , 2007Hinck et al. 2009;Iwanowicz et al. 2009;Kellock et al. 2014;Yonkos et al. 2014;Iwanowicz et al. 2016;Abdel-moneim et al. 2017;Lee Pow et al. 2017). White sucker (WS) have also been used for monitoring effects of CECs on wild fish populations (Munkittrick et al. 1998;Doherty et al. 2003;Dorval et al. 2005;Woodling et al. 2006;Vajda et al. 2008) and are an indicator species for the fish tumor BUI at Great Lakes AOCs (Blazer et al. 2014a(Blazer et al. , 2017. ...
... The AOCs sampled here are in close proximity to major urban areas. In general, the prevalence and severity of TO were lower than what has been reported in bass from agriculturally impacted sites, where it is not uncommon to have 80-100% prevalence (Iwanowicz et al. 2009;Blazer et al. 2012Blazer et al. , 2014d. These areas have also experienced bass mortality and population declines (Blazer et al. 2010;Smith et al. 2015). ...
Article
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Adverse effects resulting from potential exposure of wild fishes to estrogenic endocrine disruptors were assessed at seven United States Great Lakes Areas of Concern using biomarkers ranging from organismal (gonadosomatic indices) to tissue/plasma (histology, plasma vitellogenin) and molecular (hepatic gene transcripts) levels. Biomonitoring was conducted on pelagic, top predator species, largemouth Micropterus salmoides and smallmouth M. dolomieu bass and benthic, omnivorous white sucker Catostomus commersonii. Seasonal (spring and fall) comparisons were conducted at select sites. Intersex (testicular oocytes), plasma vitellogenin, and hepatic vitellogenin transcripts were commonly observed in bass species. Testicular oocyte severity was positively, although weakly, correlated with plasma vitellogenin, hepatic transcripts of vitellogenin, estrogen receptor α, and estrogen receptor β2, while negatively correlated with androgen receptor β and phosphoenolpyruvate carboxykinase. No testicular oocytes were observed in white sucker; however, plasma vitellogenin and hepatic vitellogenin transcripts were commonly detected in the males. The results demonstrate the importance of utilizing multiple endpoints to assess exposure to estrogenic compounds as well as the importance of choosing sensitive species.
... Tetreault [25] reported longer white stickleback collected downstream of the WWTP, but fatheads from the same site were shorter. Generally increased condition and growth is referred to fish exposed to WWE [26,43,44]. The increase in fish condition is explained by the enhanced productivity of waters affected by urban effluents [37]. ...
... The increase in fish condition is explained by the enhanced productivity of waters affected by urban effluents [37]. However, the enhance growth refers mainly to older fish classes [26,43,44]. In juveniles, the toxic effect may have a greater impact than the benefits of nutrient enlargement [45]. ...
Article
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Our study focuses on the European perch (Perca fluviatilis) regarding differences in concentrations of the biogenic (P, S, Cl, K, Ca, Mn, Zn) and metallic (Sr, Sb, Rb, Pb, Mo, Cr, Fe) elements relative to perch morphology and environmental factors at the largest dam in Slovakia. The juvenile perch were sampled at four capture sites from June to October. The highest concentration of biogenic elements and metals was found in perch caught in the site disposed to wastewater effluent (WWE). The concentrations of biogenic elements in perch decrease with the size of the fish; at most the perch affected by WWE had the smallest size. The concentration of biogenic elements in perch increased with the date from June to October and with water temperature for all sites. Lead accumulated by the perch in all sites exceeded more than five times the maximum permissible limits provided by international institutions.
... Testicular oocytes are commonly observed in smallmouth bass and there are significant positive correlations between the incidence and severity of this condition with land-use metrics (Blazer et al., , 2007. Wastewater treatment plants seem to be a minor contributor to TOs in locations investigated within the Chesapeake Bay drainage (Blazer et al., 2011(Blazer et al., , 2007Iwanowicz et al., 2009) as the density of AFOs and other agricultural practices better correlate with the severity of and estrogenic potential of chemical mixtures in the water (Ciparis et al., 2012). Research in other geographic areas, with other species, have noted associations between the occurrence and degree of intersex and the extent of urban land-use (Tanna et al., 2013). ...
... In addition to possible movement of fish between sites, factors such as agricultural run-off originating above the upper most collection site may also explain a lack of differences between the paired sites. This was suggested as an explanation for the lack of an observed WWTP effect on smallmouth bass in a previous study of the Potomac River drainage (Iwanowicz et al., 2009). Observation of differences in the TO and Vtg endpoints at the upriver and downriver locations at the Great Meadows, Blackwater (Garrett Island Division), Mason Neck, Ohio River Islands, Moosehorn, and Sunkhaze NWRs suggests that point source contributions may be important. ...
... Some CECs disrupt the endocrine systems of animals by interfering with the action of hormones involved with reproduction or growth. Research in regions outside southern California suggests that environmental concentrations of some CECs may be sufficient to produce endocrine disruption in fish living in coastal waters (Rule et al. 2006, Deng et al. 2007, Alvarez et al. 2009, Björkblom et al. 2009, Iwanowicz et al. 2009). ...
... Some CECs disrupt the endocrine systems of animals by interfering with the action of hormones involved with reproduction or growth. Research in regions outside southern California suggests that environmental concentrations of some CECs may be sufficient to produce endocrine disruption in fish living in coastal waters (Rule et al. 2006, Deng et al. 2007, Alvarez et al. 2009, Björkblom et al. 2009, Iwanowicz et al. 2009). ...
Technical Report
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Thousands of chemicals are in daily use for which little is known about their fate and effects on aquatic life. These compounds include pharmaceuticals and personal care products (PPCPs), current use pesticides (CUPs), natural and synthetic hormones, and industrial and commercial compounds (ICCs). Collectively known as contaminants of emerging concern (CECs), many of these compounds are discharged into coastal waters from point and nonpoint sources and have the potential to cause adverse biological effects. There is little information to assess the ecological impacts of CECs, partly because environmental monitoring programs usually focus on priority pollutants such as trace metals, chlorinated pesticides, PCBs, and petroleum hydrocarbons that historically contaminated coastal waters. Concern over the environmental impacts of CECs has increased in recent years as a result of studies showing their common occurrence in waste discharges and receiving waters, and instances of biological effects such as endocrine disruption on fish and wildlife associated with some CECs. Over a billion gallons of treated municipal wastewater are discharged into southern California coastal waters every day. These discharges represent a potentially significant source of CEC exposure for marine life. However, only limited information is available regarding the types, concentrations, and fate of CECs discharged to the Southern California Bight (SCB) from treated wastewater discharges and their potential for ecological impacts. This 2006 Coastal Effects Study was a collaborative effort among SCCWRP, major southern California municipal wastewater agencies, and universities. This study was designed to investigate the impacts of CECs from ocean wastewater discharges on fish in the SCB. Samples of effluent, ocean water, sediment, and fish were collected from multiple locations and analyzed to address six key questions:  What types of CECs are discharged into the SCB from municipal wastewater outfalls?  Are SCB marine life exposed to CECs from municipal wastewater discharges?  Is there evidence of endocrine disruption or other physiological effects in SCB fish?  Are effects on fish related to historical contaminants or current municipal wastewater discharges?  Are specific chemicals responsible for the effects?  Are the physiological effects adversely impacting fish populations or communities?
... While not a major food fish, they may be eaten by anglers. Within the Chesapeake Bay watershed, adult smallmouth bass have experienced disease and mortality events [8] and signs of reproductive endocrine disruption such as testicular oocytes and vitellogenin in males [9][10][11]. Lesions and mortalities of young-ofyear smallmouth bass [12,13] are also observed. ...
... Total plasma perfluoroalkyl compounds followed the same pattern (Table 3). 4.9 ± 0.5 c (2)(3)(4)(5)(6)(7)(8)(9)(10)(11) 68.6 ± 7.0 c (28-250) 1 Includes PFNA and PFOSA when detected; a,b,c Values within a column followed by the same letter are not significantly different. ...
Article
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Smallmouth bass Micropterus dolomieu is an economically important sportfish and within the Chesapeake Bay watershed has experienced a high prevalence of external lesions, infectious disease, mortality events, reproductive endocrine disruption and population declines. To date, no clear or consistent associations with contaminants measured in fish tissue or surface water have been found. Therefore, plasma samples from two sites in the Potomac River and two in the Susquehanna River drainage basins, differing in land-use characteristics, were utilized to determine if perfluoroalkyl substances were present. Four compounds, perfluorooctane sulphonic acid (PFOS), perfluoroundecanoic acid (PFUnA), perfluorodecanoic acid (PFDA) and perfluorododecanoic acid (PFDoA), were detected in every fish. Two additional compounds, perfluorooctane sulphonamide (PFOSA) and perfluorononanoic acid (PFNA), were less commonly detected at lower concentrations, depending on the site. Concentrations of PFOS (up to 574 ng/mL) were the highest detected and varied significantly among sites. No seasonal differences (spring versus fall) in plasma concentrations were observed. Concentrations of PFOS were not significantly different between the sexes. However, PFUnA and PFDoA concentrations were higher in males than females. Both agricultural and developed land-use appeared to be associated with exposure. Further research is needed to determine if these compounds could be affecting the health of smallmouth bass and identify sources.
... Evidence of endocrine disruption in wild fish has been observed in many areas. A high prevalence of testicular oocytes, an intersex condition, has been observed in smallmouth bass within the Potomac (Blazer et al., 2007, 2010Iwanowicz et al., 2009) and Susquehanna (Blazer et al., 2014) river basins of the Chesapeake Bay watershed (CBW) in the eastern United States. Throughout North America, intersex gonads have been found in black bass species including smallmouth bass (Micropterus dolomieu) and largemouth bass (Micropterus salmoides) (Abdel-Moneim et al., 2015;Blazer et al., 2018;Grieshaber et al., 2018;Hinck et al., 2009;Iwanowicz et al., 2016;Kellock et al., 2014;Yonkos, Friedel & Fisher, 2014). ...
Article
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Endocrine disrupting contaminants are of continuing concern for potentially contributing to reproductive dysfunction in largemouth and smallmouth bass in the Chesapeake Bay watershed (CBW) and elsewhere. Exposures to atrazine (ATR) have been hypothesized to have estrogenic effects on vertebrate endocrine systems. The incidence of intersex in male smallmouth bass from some regions of CBW has been correlated with ATR concentrations in water. Fish early life stages may be particularly vulnerable to ATR exposure in agricultural areas, as a spring influx of pesticides coincides with spawning and early development. Our objectives were to investigate the effects of early life stage exposure to ATR or the model estrogen 17α-ethinylestradiol (EE2) on sexual differentiation and gene expression in gonad tissue. We exposed newly hatched largemouth bass (LMB, Micropterus salmoides ) from 7 to 80 days post-spawn to nominal concentrations of 1, 10, or 100 µg ATR/L or 1 or 10 ng EE2/L and monitored histological development and transcriptomic changes in gonad tissue. We observed a nearly 100% female sex ratio in LMB exposed to EE2 at 10 ng/L, presumably due to sex reversal of males. Many gonad genes were differentially expressed between sexes. Multidimensional scaling revealed clustering by gene expression of the 1 ng EE2/L and 100 µg ATR/L-treated male fish. Some pathways responsive to EE2 exposure were not sex-specific. We observed differential expression in male gonad in LMB exposed to EE2 at 1 ng/L of several genes involved in reproductive development and function, including star , cyp11a2 , ddx4 (previously vasa ), wnt5b , cyp1a and samhd1 . Expression of star , cyp11a2 and cyp1a in males was also responsive to ATR exposure. Overall, our results confirm that early development is a sensitive window for estrogenic endocrine disruption in LMB and are consistent with the hypothesis that ATR exposure induces some estrogenic responses in the developing gonad. However, ATR-specific and EE2-specific responses were also observed.
... The toxicological implication of PCSXs in surface waters is largely unknown since very few of these chemicals are regulated, but fish reproductive abnormalities in rivers have been correlated with chemicals in WTP effluent, [12][13][14] including the Potomac River. [15] The upstream tidal freshwater region of the Potomac River lies near the urban center of metropolitan Washington, DC. In this stretch of the Potomac River, the mainstem river channel expands into embayments, regions where the tidal tributaries widen into coves or small bays upon convergence with the mainstem river. ...
Article
Selected pharmaceutical chemicals, steroids and xenoestrogens (PCSXs) consisting of 29 endocrine modulators, therapeutic drugs, pesticides, detergents, plastics, and active ingredients in household products were measured in water, riverbed sediments and fish collected in a tributary embayment of the Potomac River (Hunting Creek, Alexandria, VA, USA) in the vicinity of wastewater discharge. A total of 17 PCSXs were found in the Hunting Creek samples, with steroid hormones (e.g., progesterone and 17α-ethinylestradiol), triclosan, dextromethorphan and bisphenol A being the most prominent micropollutants detected.The geospatial distribution of the PCSXs in Hunting Creek indicated that the steroids correlated with wastewater treatment plant discharge in all matrices, but such an association is tentative in Hunting Creek given the complex nature of urban sources of PCSXs and hydrodynamics in an urban tidal river. The sediment PCSX concentrations correlated with sediment total organic carbon content at all sampling sites. For the most part, the PCSXs showed an enrichment in fish tissue relative to sediments when concentrations were normalized to lipids and sediment organic carbon contents, but the influence of endogenous steroids is also an important consideration for these chemicals.
... Plasma concentrations of E2 and T were measured by specific radioimmunoassay according to previously published procedures [21]. The T antiserum (R156/7 provided by Coralie Monroe, University of California, Davis School of Veterinary Medicine, Department of Reproduction) has a reported cross-reactivity of 57% with DHT. ...
Article
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The contribution of sex steroids to nutrient partitioning and energy balance during gonad development was studied in rainbow trout. Specifically, 19-mo old triploid (3N) female rainbow trout were fed treatment diets supplemented with estradiol-17β (E2), testosterone (T), or dihydrotestosterone (DHT) at 30 mg steroid/kg diet for a 1-mo period. Growth performance, nutrient partitioning, and expression of genes central to growth and nutrient metabolism were compared to 3N and age-matched diploid (2N) female fish consuming a control diet not supplemented with steroids. Only 2N fish exhibited active gonad development, with gonad weights (GSI) increasing from 3.7% to 5.5% of body weight throughout the study while GSI in 3N fish remained at 0.03%. Triploid fish consuming DHT exhibited faster specific growth rates than 3N controls (P < 0.05). Consumption of E2 in 3N fish reduced fillet growth and caused lower fillet yield compared to all other treatment groups (P < 0.05). In contrast, viscera fat gain was not affected by steroid consumption (P > 0.05). Gene transcripts associated with physiological pathways were identified in maturing 2N and E2-treated 3N fish that differed in abundance from 3N control fish (P < 0.05). In liver these mechanisms included the growth hormone/insulin-like growth factor (IGF) axis (igf1, igf2), IGF binding proteins (igfbp1b1, igfbp2b1, igfbp5b1, igfbp6b1), and genes associated with lipid binding and transport (fabp3, fabp4, lpl, cd36), fatty acid oxidation (cpt1a), and the pparg transcription factor. In muscle these mechanisms included reductions in myogenic gene expression (fst, myog) and the proteolysis-related gene, ctsl, suggesting an E2-induced reduction in the capacity for muscle growth. These findings suggest that increased E2 signaling in the sexually maturing female rainbow trout alters physiological pathways in liver, particularly those related to IGF signaling and lipid metabolism, to partition nutrients away from muscle growth towards support of maturation-related processes. In contrast, the mobilization of viscera lipid stores appear to be mediated less by E2 and more by energy demands associated with gonad development. These findings improve understanding of how steroids regulate nutrient metabolism to meet the high energy demands associated with gonad development during sexual maturation.
... Papoulias et al. (2006) reported on the incidence of intersex in other Asian carps (bighead carp Hypophthalmichthys nobilis and silver carp H. molitrix) in the Missouri River, and they were not able to link the incidence of intersex to a specific cause. Endocrine-disrupting compounds such as mercury, PCB, DDT and its derivatives, sex hormones, and other chemicals from municipal wastewater treatment have been linked to incidence of intersex in fish (Mills and Chichester 2005;Hinck et al. 2009;Iwanowicz et al. 2009;Schwindt et al. 2009). However, it is not clear whether the procedure used to induce triploidy creates intersex in our grass carp. ...
Article
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Grass carp Ctenopharyngodon idella are sometimes used as a biological tool for managing aquatic vegetation in reservoirs. Sterile, triploid fish were stocked in Devils Lake, Oregon, during 1986, 1987, and 1993 to control aquatic vegetation. We present a case study for using multiple measures on the same fish to determine whether illegal stocking of fertile, diploid grass carp occurred. An investigation into the estimated age of a dead grass carp found in Devils Lake suggested that it was significantly younger than would otherwise be expected, given the only stocking events occurred during 1986, 1987, and 1993. To determine whether illegal stocking or reproduction by presumed sterile grass carp had occurred in Devils Lake, we conducted a study that balanced the needs of lethally sampling grass carp for biological measures with the socially and politically sensitive sentiment of the pro–grass carp citizenry of Devils Lake. These considerations, in combination with a low catch per-unit effort, resulted in a modest sample size for grass carp. We sampled grass carp and recorded multiple measures for each fish. Ploidy testing of blood samples indicated the grass carp were all triploid. Based on gonadal histopathology, six fish were male, two were female, and two were sex-indeterminate with severe gonadal dysgenesis. Age estimates from lapillus otoliths were consistent with fish originating from the legal stocking events in Devils Lake. The grass carp were 21–30 y old, and we were unable to find published reports of grass carp anywhere else in the world that are older. The grass carp were significantly smaller than much younger fish from other regions. The small size of these grass carp relative to their age in Devils Lake suggests food limitations that stunted growth. The dead grass carp that was the impetus for this study was aged by anatomical structures that we have since found to be unreliable. This suggests that the dead grass carp was probably in fact older and originated from the legal stockings. The use of multiple biological measurements on a modest sample size of grass carp, combined with the knowledge that no juvenile grass carp have been observed since legal stocking occurred, lead us to conclude that the grass carp in Devils Lake are sterile fish that originated from legal stocking events.
... Resident fish species are ideal bioindicators and integrators of chemical mixture effects as they are continually exposed to CECs and legacy contaminants and serve as sentinels for the evaluation of the impacts in aquatic ecosystems. Contaminant monitoring studies have identified indications of reproductive endocrine disruption, such as intersex (testicular oocytes), in the pelagic smallmouth bass Micropterus dolomieu and largemouth bass M. salmoides (Blazer et al., 2007;Blazer et al., 2012;Hinck et al., 2009;Iwanowicz et al., 2009;Sepulveda et al., 2002;Tetreault et al., 2011). Benthic species, including the brown bullhead Ameiurus nebulosus and white sucker Catostomus commersonii, have been widely used in ecotoxicological studies evaluating the presence of liver and skin tumors and other adverse effects (Baumann et al., 1996;Blazer et al., 2014;Blazer et al., 2009aBlazer et al., , 2009bHayes et al., 1990;Pinkney et al., 2011;Premdas et al., 1995;Pyron et al., 2001;Rafferty et al., 2009;Smith et al., 1989), immune dysfunction and reproductive endocrine disruption (McMaster, 2001;Woodling et al., 2006). ...
... The presence and effects of EDCs in aquaculture environments have been poorly studied, and this area currently represents a frontier of much needed research. Recent investigation has focused on environmental EDCs and their impact on fish populations in natural settings (Blazer et al. 2007;Iwanowicz et al. 2009). Blazer et al. (2012) examined intersex male smallmouth bass (i.e., male fish with testicular oocytes) in the Potomac River basin and found a spatial-temporal relationship between intersex severity and potential sources of EDCs, such as wastewater treatment plants and areas of intensive agriculture, particularly poultry operations. ...
Article
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Sexual maturation of Atlantic salmon, Salmo salar, is a complex process, with many variables having the capacity to influence the timing and prevalence of maturation and acting as promoters and/or inhibitors of sexual development. Precocious maturation has the capacity to seriously impact production in commercial aquaculture settings, and in response there has been a significant amount of research devoted to understanding this issue in order to develop remedial strategies. Very little research has been conducted specifically examining salmon maturation in land-based, closed containment water recirculation aquaculture systems, which have recently received attention as an alternative technology for the sustainable production of market-size Atlantic salmon. Unfortunately, the nascent closed containment salmon industry has thus far experienced high levels of precocious maturation, for reasons that are presently unclear. Given the economic challenges facing the closed containment industry's expansion, it is imperative that best management practices be developed to reduce economic losses from early maturation, in order to assist the sustainable growth of farmed Atlantic salmon production. This review provides a brief summary of published research on factors associated with early salmonid maturation, as well as information from research examining maturation and growout performance of Atlantic salmon in closed containment aquaculture systems.
... Chemical methods for detection of EDCs have revealed the presence of five classes of steroid hormones comprising estrogens, androgens, progestogens, glucocorticoids, and a mineralocorticoid in water sources in China 12 . Studies in the US have also revealed contamination with some of these classes of EDCs [5][6][7][8][9]13 . However, the presence of steroids such as glucocorticoids or mineralocorticoids was never studied. ...
... Our results suggest that prevalence of intersex is similar among species of the same genera. This is unique to current literature that typically documents higher prevalence of intersex in smallmouth bass relative to largemouth bass found within the same system [16,21,35,36]. However, smallmouth bass and largemouth bass were never sampled at the same location within our present study. ...
Article
Male fish are susceptible to developing intersex, a condition characterized by the presence of testicular oocytes. In the present study, the relationship between intersex and exposure to estrogenic endocrine active contaminants (EACs) was assessed for 2 genera of sport fish, Micropterus and Lepomis, at 20 riverine sites. Seasonal trends and relationships between EACs and intersex (prevalence and severity) were examined at varying putative sources of EACs throughout North Carolina, identified as 'point', 'non-point' and 'reference' sites. Intersex was identified in both genera, where we documented it for the first time in wild-caught Lepomis. Intersex was more prevalent (59.8%) and more severe (1.6 mean rank) in Micropterus, which was highly correlated to EACs in sediment. In contrast, intersex was less common (9.9%) and less severe (0.2 mean rank) in Lepomis and was highly correlated to EACs in the water column. We found that concentrations of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, industrial EACs and estrogens were highest at point source sites, but identified no source type variation in the prevalence or severity of intersex; nor were there seasonal trends in intersex or EAC concentrations. Our results associate genus-specific prevalence of intersex with specific EAC classes in common sport fishes with biological, ecological and conservation implications. This article is protected by copyright. All rights reserved.
... These mobile laboratory and field observations were consistent with previous studies in SRW that reported wild fish kills, immunosuppression, intersex, and other health effects related to multiple environmental stressors, including chemical mixtures. [28][29][30]83,140 SRW has robust fish and invertebrate sampling efforts across multiple agencies to evaluate aquatic community biotic integrity ( Figure S7A). 141−145 Using the ecological integrity assessment, fewer than 10% of streams in SRW were considered to be ecologically healthy, and the fish community modified index of biotic integrity scores indicated an absence of intolerant species ( Figure S7B). ...
Article
River waters contain complex chemical mixtures derived from natural and anthropogenic sources. Aquatic organisms are exposed to the entire chemical composition of the water, resulting in potential effects at the organismal through ecosystem level. This study applied a holistic approach to assess landscape, hydrological, chemical, and biological variables. On-site mobile laboratory experiments were conducted to evaluate biological effects of exposure to chemical mixtures in the Shenandoah River Watershed. A suite of 534 inorganic and organic constituents were analyzed, of which 273 were detected. A watershed-scale accumulated wastewater model was developed to predict environmental concentrations of chemicals derived from wastewater treatment plants (WWTPs) to assess potential aquatic organism exposure for all stream reaches in the watershed. Measured and modeled concentrations generally were within a factor of 2. Ecotoxicological effects from exposure to individual components of the chemical mixture were evaluated using risk quotients (RQs) based on measured or predicted environmental concentrations and no effect concentrations or chronic toxicity threshold values. Seventy-two percent of the compounds had RQ values <0.1, indicating limited risk from individual chemicals. However, when individual RQs were aggregated into a risk index, most stream reaches receiving WWTP effluent posed potential risk to aquatic organisms from exposure to complex chemical mixtures.
... Recent reports of high (to 100%) intersex rates in male black bass (genus, Micropterus) from various sites across the south-eastern (Hinck et al. 2009;Ingram et al. 2011) and eastern (Blazer et al. 2007;Iwanowicz et al. 2009) USA have all involved fish from riverine environments. Recently, Blazer et al. (2012) described significant correlations between land-use characteristics (percent agriculture and animal density) and intersex prevalence in Smallmouth Bass Micropterus dolomieu in Potomac River catchments. ...
Conference Paper
Recently, high incidences of intersex fish (males with testicular oocytes) were reported in some rivers in the southeastern U.S. To date, a systematic evaluation of the extent, severity, and causes of intersex fish has not been completed in Georgia. Our objectives were to (1) assess intersex condition in black bass collected from rivers and impoundments across Georgia, and (2) determine estrogenic potency (a measure of the estrogens and estrogen-like substances) of surface waters where fish were collected. Fish (n=15-35) and water samples were collected from sites near municipal wastewater effluent discharges in each of four rivers and from a river that does not receive major wastewater effluent discharges. Fish and water were also sampled from 12 impoundments without major wastewater inputs to determine if a natural ‘background’ rate of intersex could be established for fish from relatively unpolluted water bodies. Gonads from all male fish were examined and the incidence and severity of the intersex condition were compared among sites. Potency of estrogens in surface waters was determined by use of an in vitro yeast-based reporter gene (YES) assay. The overall percentage of intersex for male bass collected in rivers was 30%, whereas 40% of male fish collected from impoundments were intersex. Incidence of intersex varied substantially (0-88%) among impoundments, and surface area of the impoundment was a strongly negatively correlated with intersex incidence. Severity of intersex was not predicted by impoundment surface area. Causes of intersex remain unknown, but the high incidence of intersex males in some impoundments suggests that factors other than municipal wastewater are involved.
... In addition to these environmental factors that potentially limit recruitment, the abundance or health of males may limit recruitment. For our study, we assumed that recruitment resulted from equal and redundant contributions by males and females; however, negative effects of organic pollutants (e.g., estrogen) on males (Iwanowicz et al. 2009) or fitness differences among more-often-caught males (Sutter et al. 2012) could lead to some males playing a more important role than others in contributing to recruitment. Unfortunately, these complicated issues are not controlled by sole actions of anglers or fishery managers. ...
Article
Conservation of Largemouth Bass (LMB) Micropterus salmoides populations requires an understanding of population dynamics that are influenced by environmental challenges, such as the spread of invasive species. We used an age-structured population model to compare population growth rates (λ) between a simulated population that included invasive Northern Snakehead (NSH) Channa argus as a competitor and predator and a simulated population that did not. We then assessed the sensitivity of our results to natural variation in LMB recruitment. When recruitment of LMB was already poor, there was a high risk of population decline that did not depend on whether NSH was included in the model scenario. When recruitment of LMB was high, the risk of population decline was only 40% when NSH was not included in the model scenario; however, predation by and competition with NSH caused a higher risk of LMB population decline. Regardless of the level of LMB recruitment, the size of the LMB population at equilibrium was 20% lower (on average) when including NSH in the model. We conclude that when habitat conditions do not already significantly limit recruitment, populations of LMB may be adversely affected by cohabitation with NSH. Preventing the spread of NSH will lessen ecosystem pressures that negatively affect the LMB population. We encourage continued vigilance in conserving LMB populations by encouraging actions that promote recruitment and prevent spread of invasive species.
... Histopathological effects associated with MWWTP effluents have also been widely reported, particularly intersex (simultaneous presence of male and female gonad tissue) condition in male fish Bjerregaard et al., 2006;Woodling et al., 2006;Blazer et al., 2007;Tetreault et al., 2011;Abdel-moneim et al., 2015). Changes in organ weight (e.g., gonad and liver size) and body size (condition factor) have also been observed and are indicators of changes in energy allocation and storage (Vajda et al., 2008;Iwanowicz et al., 2009). Impacts at the population level include changes in sex ratios (Jeffries et al., 2008;Vajda et al., 2008) and reduced fertilization success (Jobling et al., 2002;Fuzzen et al., 2015). ...
Thesis
Impacts on aquatic biota residing near municipal wastewater treatment plant (MWWTP) outfalls have been documented globally. These impacts may be directly or indirectly associated with elevated contaminants such as nutrients, metals, suspended solids (SS), biochemical oxygen demanding matter (BOD), pharmaceuticals, and personal care products. A variety of effects have been well documented in the Grand River watershed of southern Ontario below the outfalls of the MWWTPs of the cities of Kitchener and Waterloo. Responses in wild fish have been reported at multiple levels of biological organization, ranging from altered gene expression to changes in fish communities, as well as changes in nutrient cycling within the aquatic food web. The most consistently observed effect has been high occurrences and severe cases of intersex (ova-testes) in the male rainbow darter (Etheostoma Caeruleum); this finding represents one of the worst examples of pollution-caused intersex reported anywhere in the world. Primarily in response to the introduction of new effluent quality standards, the Region of Waterloo has invested millions of dollars to upgrade several of its MWWTPs including the facility servicing Kitchener, creating a unique opportunity to conduct a before-and-after study. The main objective of this thesis was to assess if treatment upgrades, which were targeted at conventional contaminants (i.e., ammonia, BOD, SS, and chloride), effectively remediated the responses previously reported in wild fish downstream of the MWWTP. To test this, historical, archived, and new data collections were used to assess changes at multiple levels of biological organization, including changes in nutrient cycling in the aquatic food web, reproductive effects in the male rainbow darter (e.g., intersex), and changes in fish community composition. For comparative purposes, responses in rainbow darter were also examined at numerous reference sites and below the smaller Waterloo MWWTP, which did not undergo any major upgrades during the study period. The treatment upgrades at the Kitchener MWWTP (which included nitrifying activated sludge) improved the overall quality of the effluent; these improvements included reductions in nutrients (total ammonia), pharmaceuticals, and total estrogenicity (E2eq). In contrast, the Waterloo MWWTP had deteriorating effluent quality, with ammonia levels increasing over the course of the study. Changes in effluent quality at both the Kitchener and Waterloo MWWTPs were detected in the downstream aquatic food webs using stable isotope ratios (δ15N and δ13C). Patterns of δ15N in a primary consumer (benthic invertebrate) and a secondary consumer (rainbow darter) reflected the exposure to MWWTP effluents and changes in nutrient cycling in response to the changing effluent quality. A major reduction in intersex in the male rainbow darter below the Kitchener MWWTP outfall was also associated with the improvements in effluent quality. Rates of intersex were reduced by as much as 70% in the first year post-upgrade and dropped to near background levels within three years. Detecting change in fish communities below MWWTP outfalls (including before and after the upgrades) was more challenging. While subtle changes were detected (e.g., increases in pollution-tolerant species below the MWWTP outfalls), these could not be directly associated with MWWTP effluents because they were confounded by a watershed gradient (e.g., stream size). Fish communities were highly variable both spatially and temporally, limiting our ability to associate changes with local environmental conditions (i.e., effects of MWWTP outfalls). Although rainbow darter has been used as a sentinel species for detecting impacts of MWWTP effluents in many studies, little is known about its movement patterns. Elevated intersex was observed historically at the near-field upstream site of the Kitchener MWWTP outfall, leading to a hypothesis that wastewater-exposed fish may be moving upstream. To inform the interpretation of responses in rainbow darter as a sentinel species, a mark-and-recapture study was conducted at an upstream reference site to better understand their movement. Although the majority of fish (85%) had high site fidelity, a small proportion of fish moved considerable distances (up to 975 m). This study confirmed that there is potential for some fish to move and thereby confound the interpretation of near-field upstream sites that are not physically separated from the sites below the MWWTP outfall. The decline in intersex in rainbow darter after the upgrades at the site immediately upstream of the Kitchener outfall supports the view that at least some of the responses seen at this site were probably associated with fish movements. Overall, this thesis advances our understanding of the impacts of MWWTP effluents on wild fish and their response to improved effluent quality (i.e., treatment). The relatively simple (conventional) upgrades at the Kitchener MWWTP resulted in improvements in the aquatic receiving environment, indicating that more advanced treatment may not be required to address these effects of concern. However, other impacts may be occurring that were not measured in this study. The results drawn from this thesis may have implications for future wastewater management strategies for other MWWTPs across Canada and around the globe. In addition, these studies may provide insight into key biological endpoints that could be useful for future biomonitoring programs for MWWTP effluents.
... Studies of fish from these areas documented multiple bacterial and viral infections, high parasite loads, and periphytic cyanobacterial compounds (Blazer et al. 2010;Smith et al. 2015;Foss et al. 2017;Walsh et al. 2018), with no factor consistently identified as the cause. The finding of intersex (testicular oocytes) and plasma vitellogenin in male fish (Blazer et al. 2007(Blazer et al. , 2014Iwanowicz et al. 2009) from the same areas suggested that chemical contaminants and other environmental stressors may contribute to the reduced health of these populations. Smallmouth Bass and GDR are the most common species experiencing observable mortality during fish kill investigations. ...
Article
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Over recent decades, the South Branch Potomac River, West Virginia, has experienced fish kills and episodes of suppressed health in adult fish that have spanned small stretches to nearly 120 km of contiguous habitat. Although factors such as endocrine disruption, chemical contaminants, and infectious agents have been detected, no single causal mechanism has been identified. To gain information about the temporal nature of abnormalities, differences in life stage impacts, and potential risk factors, investigations of rank scores of macroscopic fish health indicators were conducted utilizing seasonal and annual boat electrofishing surveys for Smallmouth Bass Micropterus dolomieu (SMB) and Golden Redhorse Moxostoma erythrurum (GDR). Gill and body abnormalities were assigned rank scores for each fish based on visual severity and were tested for correlation with seasonal climatic (flow and stream temperature) and environmental (stream pH, dissolved oxygen, and turbidity) factors. Comparisons between juveniles and adults for these species indicated that body lesions were more common for adult GDR and gill lesions were more common for adult SMB. Significantly higher rank sums of adult gill abnormalities corresponded with heavy annual mortality of SMB between ages 2 and 3 (86%), the period during which this species transitions from juvenile to adult length. Higher ranks were frequently assigned to fish of both species for gill and body/fin lesions in summer and fall samples. Low stream discharge and lower pH correlated with elevations of body raised lesions (GDR) and body erosions (GDR and SMB) as well as erosions of the gill lamellae (SMB). This study connects the disciplines of fisheries management, fish health, and environmental monitoring, providing information gained through tracking the macroscopic conditions of these two indicator species to focus future studies and better understand risks to fish health.
... These results do not change the fact that long-term exposure to even low concentrations of active endocrine compounds may adversely affect human health [41]. Increased presence of the hermaphrodite fish populations in the reservoirs fed with purified water flowing from sewage treatment plants was observed in the US, Asia and Europe [42][43][44]. Still, however, there is a need to prove a direct relation between the presence of estrogens in surface waters, and changes in the sexuality of fish populations found in rivers, lakes and streams. ...
Article
Full-text available
Concern for the natural environment increasingly devotes more attention to growing potential hazards resulting from the release of various substances. Currently, one of the main problems associated with environmental pollution is the derivation of organic compounds from wastewater. Substances derived from sewage leaks into the environment in the form of a multicomponent mixtures often enhances the toxic effects caused by these compounds. While analyzing the reports in the literature of the last two decades it can be seen that substantial efforts are devoted to the determination of selected trace contaminants present in wastewater. Among the most marked there are endocrine disrupting compounds, residues of pharmaceuticals and personal care products, plastics and sunscreens. Recently, a new group of compounds joined the aforementioned contaminants, namely drugs, whose legality and availability is increasing every year. Exposure to these type of compounds, named in the literature as emerging contaminants (ECs), involves, among others, such effects as hormonal imbalance, reduction of the survivability of aquatic organisms and reproductive problems. This paper provides a review of the types of emerging organic groundwater contaminants (EGCs) which are beginning to be found in the natural environment in many countries all around the world.
... The nutrient enrichment can impact energy allocation manifested as changes in GSI, LSI and K (Allen et al. 1999;McMaster et al. 2005). However, other studies found fish exposed to MWWE experienced no significant changes in energy allocation van Aerle et al. 2001;Hinck et al. 2009;Iwanowicz et al. 2009). Effects of nutrient-laden MWWE additions to nutrient poor environments are more likely to be observed as changes in nutrient uptake, energy assimilation, and somatic distribution relative to those already saturated with nutrients ( mg/L (Grand River Conservation Authority, personal communication). ...
... Water pH can be a critical factor determining the solubility, treatment removal efficiency, and bioavailability of certain pharmaceuticals and other CECs [20]. Water temperature may increase effects of diseases, infections, and other stressors from CEC-caused immune system modifications in certain species [21]. The types of species that normally could inhabit the site may also influence the likelihood that CECs have or could cause population or community effects. ...
Article
Trace levels of a variety of currently unregulated organic chemicals have been detected in treated wastewater effluents and surface waters that receive treated effluents. Many of these chemicals of emerging concern (CECs) originate from pharmaceuticals and personal care products that are used widely and that frequently are transported "down the drain" to a wastewater treatment plant (WWTP). Actual effects of CECs on aquatic life have been difficult to document, although biological effects consistent with effects of some CECs have been noted. There is a critical need to find appropriate ways to screen wastewater sites that have the greatest potential of CEC risk to biota. Building on the work of several researchers, the authors present a screening framework, as well as examples based on the framework, designed to identify high-risk versus lower-risk sites that are influenced by WWTP effluent. It is hoped that this framework can help researchers, utilities, and the larger water resource community focus efforts toward improving CEC risk determinations and management of these risks. Environ Toxicol Chem 2015;34:2671-2681. © 2015 SETAC.
... Exposure of humans and aquatic species through water to substances that cause disruption of the endocrine system, called endocrine disrupting chemicals (EDCs), is becoming a serious environmental and health problem [1,2]. Scientific research studies have for instance linked the intersex in male fish condition to exposure of fish to estrogens in the aquatic environment [3][4][5][6]. ...
Article
The removal and recovery of highly potent endocrine disrupting chemicals (EDCs) estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) and the oxidation product 2-hydroxyestradiol (2OHE2) in water was achieved on Polyamide 6 (PA6) particles. Hydrogen bonding was the main mechanism driving the adsorption of these EDCs on PA6 at pHs lower than the EDCs pKas (∼10.5) and their adsorption was not affected by the water matrix nor by solute-solute interaction. The adsorption isotherms were linear and the values of the linearity constants for E2 and EE2 were almost double those for E1 and 2OHE2. This was correlated to the number of intermolecular hydrogen bonds via –OH groups of the EDCs (H-bond donors) available for interaction with PA6’s surface via the amide groups (H-bond acceptors). The effect of pH on the adsorption of the EDCs on PA6 was significant only at pHs > EDCs pKa (∼10.5). The breakthrough curves of the EDCs on PA6 particles in a fixed-bed column were successfully modelled using a linearised mass transfer model. This study shows that PA6 appears an effective sorbent for the removal as well as the enrichment and pre-concentration of EDCs in wastewater samples.
... Several studies have reported high TO prevalence in Micropterus from regions with human activities such as WWTPs, agriculture, and industry, which are known to release EDCs; nevertheless, evidence for a causal relationship between EDC exposure and TOs in these species is weak and inconsistent. Many of these studies did not assess TO occurrence in bass near suspected EDC sources in comparison with reference sites [3,10,23], or reported that TO prevalence was not consistently or significantly lower at reference sites compared with sites near known EDC sources [9,[24][25][26][27][28]. For example, Hinck et al. [10] reported that high TO prevalence did not always correspond with a high number of detected contaminants, and that at least one site with a high TO prevalence had no obvious sources of EDCs. ...
Article
Full-text available
Testicular oocytes (TOs) have been found in black basses (Micropterus spp.) from many locations in North America. The presence of TOs is often assumed to imply exposure to estrogenic endocrine disrupting compounds (EDCs), however, a definitive causal relationship has yet to be established, and TO prevalence is not consistently low in fish from areas lacking evident EDC sources. This might indicate any of a number of situations: (a) unknown or unidentified EDCs or EDC sources, (b) induction of TOs by other stressors, or (c) TOs occurring spontaneously during normal development. In the present study we analyzed TO occurrence in smallmouth bass (M. dolomieu) from eight populations in Northeastern Minnesota watersheds with differing degrees of human development and hence, presumed likelihood of exposure to anthropogenic chemicals. Three watersheds were categorized as moderately developed, based on the presence of municipal wastewater discharges and higher human population density (4-81 per km2), and five watersheds were minimally developed, with very low human population density (0-1 per km2) and minimal built environment. Testicular tissues from mature fish were evaluated using a semi-quantitative method that estimated TO density, normalized by cross-sectional area. TO prevalence and density among populations from moderately developed watersheds was higher than in populations from minimally developed watersheds. However, TO prevalence was unexpectedly high and variable (7-43%) in some populations from minimally developed watersheds, and only weak evidence was found for a relationship between TO density and watershed development, suggesting alternative or more complex explanations for TO presence in smallmouth bass from this region. This article is protected by copyright. All rights reserved
... Regardless of a compound's designation as a POP or CEC, the detection of anthropogenic contaminants remains a top priority in environmental monitoring studies, especially those where there are threats to organism health. In the Susquehanna and Potomac River basins of the Chesapeake Bay Watershed, USA, smallmouth bass (Micropterus dolomieu, SMB) have exhibited signs of adverse health effects such as dermal lesions, reproductive endocrine disruption, and population declines due to low chronic mortality and sporadic fish kills (Blazer et al., 2007(Blazer et al., , 2012(Blazer et al., , 2014Chaplin et al., 2009;Iwanowicz et al., 2009;Schall et al., 2018;Walsh et al., 2018). Adult male SMB have been documented expressing the egg precursor protein vitellogenin and having testicular oocytes, both markers of intersex due to endocrine disruption. ...
Article
Smallmouth bass in the Susquehanna River Basin, Chesapeake Bay Watershed, USA, have been exhibiting clinical signs of disease and reproductive endocrine disruption (e.g., intersex, male plasma vitellogenin) for over fifteen years. Previous histological and targeted chemical analyses have identified infectious agents and pollutants in fish tissues including organic contaminants, mercury, and perfluorinated compounds, but a common causative link for the observed signs of disease across this widespread area has not been determined. This study examines 146 young-of-year smallmouth bass collected from 14 sampling sites in the Susquehanna River Basin, Pennsylvania, USA with varying levels of disease prevalence. Whole fish were extracted by a recently developed modification to the quick, easy, cheap, effective, rugged, and safe extraction method and analyzed by comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry. A targeted analysis was conducted to identify the presence and quantity of 127 known contaminants, including polychlorinated biphenyls, brominated diphenyl ethers, organochlorinated pesticides, and pharmaceutical and personal care products. A non-targeted analysis was conducted on the same data set to identify analytes of interest not included on routine target compound lists. Chromatographic alignment through Statistical Compare (ChromaTOF GC) was followed by Fisher ratio and principal component analysis to reduce the data set from thousands of peaks per sample to a final data set of 65 analytes of interest. Comparisons of these 65 compounds between Normal (no observed health anomalies) and Lesioned (observed health anomaly at time of collection) fish revealed increased levels of three chemical families in Lesioned fish including esters, ketones, and nitrogen containing compounds.
... Resident fish species are ideal bioindicators and integrators of chemical mixture effects as they are continually exposed to CECs and legacy contaminants and serve as sentinels for the evaluation of the impacts in aquatic ecosystems. Contaminant monitoring studies have identified indications of reproductive endocrine disruption, such as intersex (testicular oocytes), in the pelagic smallmouth bass Micropterus dolomieu and largemouth bass M. salmoides (Blazer et al., 2007;Blazer et al., 2012;Hinck et al., 2009;Iwanowicz et al., 2009;Sepulveda et al., 2002;Tetreault et al., 2011). Benthic species, including the brown bullhead Ameiurus nebulosus and white sucker Catostomus commersonii, have been widely used in ecotoxicological studies evaluating the presence of liver and skin tumors and other adverse effects ( Baumann et al., 1996;Blazer et al., 2014;Blazer et al., 2009aBlazer et al., , 2009bHayes et al., 1990;Pinkney et al., 2011;Premdas et al., 1995;Pyron et al., 2001;Rafferty et al., 2009;Smith et al., 1989), immune dysfunction ( Iwanowicz et al., 2012) and reproductive endocrine disruption (McMaster, 2001;Woodling et al., 2006). ...
Conference Paper
A recent shift in environmental monitoring of the Great Lakes watershed includes the evaluation of a new group of compounds collectively referred to as contaminants of emerging concern (CECs). Perhaps one of the best studied classes of CECs is that of the endocrine disrupting compounds (EDCs). A multiagency project is in progress to evaluate the significance of such chemicals on aquatic biota inhabiting the Great Lakes basin. A component of this project involves the evaluation of biological perturbations in resident pelagic smallmouth bass and largemouth bass, and the benthic brown bullhead and white sucker throughout the Great Lakes Basin, but primarily in locations proximate to Great Lakes Areas of Concern (AOCs). Using next generation sequencing technologies biomarker genes were identified in these non-model species and gene expression analyses were conducted via qPCR or nCounter analysis. Gene expression analysis was designed to complement the histopathological, plasma and other observations on the same individuals. It will also be compared to similar data available from caged fathead minnow studies performed by the Environmental Protection Agency. In addition, genomic DNA has been collected for microsatellite analysis and population assignment. The results and significance of these analyses will be discussed.
... Similar observations were made in tributaries of the Great Lakes, where the occurrence of TO ranged from 33 to 78% in SMB and 0e25% in LMB, but were not observed in white sucker, although Vtg was induced in all species (Blazer et al., 2018b). In the above studies and within the Potomac drainage there is some indication SMB may be more likely than LMB to have TO (Blazer et al., 2007;Iwanowicz et al., 2009). However, a survey conducted at 20 sites throughout North Carolina did not document an overall difference in TO prevalence or severity between SMB and LMB, although Micropterus species were 12.9 times more likely to exhibit TO than sunfish species (Lee Pow et al., 2017). ...
Article
Ecological risk assessments play an important role in environmental management and decision-making. Although empirical measurements of the effects of habitat changes and chemical exposure are often made at molecular and individual levels, environmental decision-making often requires the quantification of management-relevant, population-level outcomes. In this study, we generalized a modeling framework to evaluate population-level ecological risk of environmental stress and bioactive chemicals. The modeling framework includes (1) a biological model module that incorporates complex and interacting biological and ecological processes, and environmental stochasticity, (2) an effect module that links the impacts of environmental changes and chemical exposure to individual characteristics, and (3) a population module that makes decisions on the choice of population-level properties to best capture the effects and thus to track in the model based on the target species and the research and management interest. This framework is a 3-module procedure that provides an alternative way for researchers to organize, present and communicate the risk assessment modeling studies. To demonstrate this framework, we used a socioeconomically important riverine fish species, smallmouth bass Micropterus dolomieu, as the model species. We developed an individual-based model as the biological model module. We evaluated the impacts of changing water temperature and flow regimes, and the impacts of exposure to estrogenic endocrine disrupting compounds (EEDC) on smallmouth bass populations in the Chesapeake Bay Watershed, USA. Warm summer water temperatures and year-round high flows had the most severe impacts on the smallmouth bass population. An increase in exposure level to EEDC, both year-round and in summer months, substantially reduced population size, spawner and recruit abundance, and the proportion of quality-length individuals. Acute exposure to EEDC was more detrimental to the population than chronic exposure. Acute exposure during spawning season had the most severe impacts. This modeling framework can be extended to other species, environmental factors and chemicals, and can be used to inform management and conservation decisions.
... Similar observations were made in tributaries of the Great Lakes, where the occurrence of TO ranged from 33 to 78% in SMB and 0e25% in LMB, but were not observed in white sucker, although Vtg was induced in all species (Blazer et al., 2018b). In the above studies and within the Potomac drainage there is some indication SMB may be more likely than LMB to have TO (Blazer et al., 2007;Iwanowicz et al., 2009). However, a survey conducted at 20 sites throughout North Carolina did not document an overall difference in TO prevalence or severity between SMB and LMB, although Micropterus species were 12.9 times more likely to exhibit TO than sunfish species (Lee Pow et al., 2017). ...
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The Chesapeake Bay is the largest estuary in the United States and its watershed includes river drainages in six states and the District of Columbia. Sportfishing is of major economic interest, however, the rivers within the watershed provide numerous other ecological, recreational, cultural and economic benefits, as well as serving as a drinking water source for millions of people. Consequently, major fish kills and the subsequent finding of estrogenic endocrine disruption (intersex or testicular oocytes and plasma vitellogenin in male fishes) raised public and management concerns. Studies have occurred at various sites within the Bay watershed to document the extent and severity of endocrine disruption, identify risk factors and document temporal and spatial variability. Data from these focal studies, which began in 2004, were used in CART (classification and regression trees) analyses to better identify land use associations and potential management practices that influence estrogenic endocrine disruption. These analyses emphasized the importance of scale (immediate versus upstream catchment) and the complex mixtures of stressors which can contribute to surface water estrogenicity and the associated adverse effects of exposure. Both agricultural (percent cultivated, pesticide application, phytoestrogen cover crops) and developed (population density, road density, impervious surface) land cover showed positive relationships to estrogenic indicators, while percent forest and shrubs generally had a negative association. The findings can serve as a baseline for assessing ongoing restoration and management practices.
... Plasma levels of E2 and T were measured by specific RIA according to previously published procedures (Iwanowicz et al., 2009). The T antiserum (R156/7 provided by Coralie Monroe, University of California, Davis School of Veterinary Medicine, Department of Reproduction) has a reported cross reactivity of 57% with DHT, which is normally not detectable in fish plasma (Borg, 1994). ...
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Effects of a single injection of 17β-estradiol (E2), testosterone (T), or 5β-dihydrotestosterone (DHT) on expression of genes central to the growth hormone (GH)/insulin-like growth factor (IGF) axis, muscle-regulatory factors, transforming growth factor-beta (TGFβ) superfamily signaling cascade, and estrogen receptors were determined in rainbow trout (Oncorhynchus mykiss) liver and white muscle tissue. In liver in addition to regulating GH sensitivity and IGF production, sex steroids also affected expression of IGF binding proteins, as E2, T, and DHT increased expression of igfbp2b and E2 also increased expression of igfbp2 and igfbp4. Regulation of this system also occurred in white muscle in which E2 increased expression of igf1, igf2, and igfbp5, suggesting anabolic capacity may be maintained in white muscle in the presence of E2. In contrast, DHT decreased expression of igfbp5. DHT and T decreased expression of myogenin, while other muscle regulatory factors were either not affected or responded similarly for all steroid treatments. Genes within the TGFβ superfamily signaling cascade responded to steroid treatment in both liver and muscle, suggesting a role for sex steroids in the ability to transmit signals initiated by TGFβ superfamily ligands, with a greater number of genes responding in liver than in muscle. Estrogen receptors were also regulated by sex steroids, with era1 expression increasing for all treatments in muscle, but only E2- and T-treatment in liver. E2 reduced expression of erb2 in liver. Collectively, these data identify how physiological mechanisms are regulated by sex steroids in a manner that promotes the disparate effects of androgens and estrogens on growth in salmonids. Copyright © 2014. Published by Elsevier Inc.
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In recent years, wide-scale mortality of young-of-year (YOY) Smallmouth Bass Micropterus dolomieu has affected recruitment in the Susquehanna River and a number of its tributaries. Investigations have determined that these mortality events are associated with changes in various components of water quality in the presence of multiple pathogens. Outbreaks have been characterized by lesions colonized by several species of bacteria, including motile Aeromonas spp., Flavobacterium columnaris, and Pseudomonas aeruginosa. Further, the myxozoan parasite Myxobolus inornatus and trematode metacercariae have been documented in affected fish. Many of the specimens submitted for analysis have also been infected by Largemouth Bass virus. However, the relationship between any particular pathogen and the mortalities remains unclear. Histological analysis of adult bass has demonstrated frequent and severe cases of intersex (i.e., testicular oocytes) and measurable plasma concentrations of vitellogenin in male Smallmouth Bass, suggesting the presence of endocrine-disrupting compounds in the system. It is uncertain what compounds are present or how these compounds may contribute to immunosuppression of YOY Smallmouth Bass, allowing for bacterial and parasite colonization. A complex relationship of several, sublethal stressors are contributing to repeated occurrence of disease outbreaks. Although the Smallmouth Bass is not native to the Susquehanna system, it could be used as a case study as to how changes in water quality and multiple pathogens could pose a threat to conservation of populations of black bass species with low densities and limited distribution.
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Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P < 0.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin-fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa. Published by Elsevier Inc.
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There is a growing interest in the health risk posed by endocrine-disrupting chemicals (EDCs). Steroidal EDCs can interfere with the normal function of the endocrine system, and have been associated with developmental defects, metabolic disorders and cancer. Identification of EDCs relies on a laborious analysis of chemical structures. Considering that many natural steroids are rapidly metabolized, their derivatives are not present in the currently existing libraries, and thus cannot be identified by chemical methods. We developed an inexpensive high-throughput assay for biological testing of EDCs using mammalian cells that express GFP-tagged nuclear steroid receptor constructs [1]. This assay is based on translocation of a fluorescent fusion protein from the cytoplasm to the nucleus in the presence of a ligand. Using this assay we screened water samples collected from 14 states in the US and found androgen activity in 35% of samples, and a previously unrecognized glucocorticoid (GC) activity in 27% of the samples. Widespread contamination with the two classes of steroidal EDCs represents a potential health hazard not only for the aquatic ecosystems, but also for humans. Our automated, highly reproducible, and low cost assay detects biologically active steroidal EDCs and is suitable for wide application in testing water samples.
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Studies quantifying the influence of reference site selection on transcriptomic profiles in aquatic organisms exposed to complex mixtures are lacking in the literature, despite the significant implications of such research for the interpretation of omics datasets. We measured hepatic transcriptomic responses in fish across an urban environment in the central Grand River watershed (Ontario, Canada). Adult male rainbow darter (RBD) (Etheostoma caeruleum) were collected from 9 sites at varying distances from 2 major municipal wastewater treatment plants (MWWTPs) (Waterloo, Kitchener), including 3 upstream reference sites. The transcriptomic response in RBD was independently compared with that of fish from each of the 3 reference sites. Data collected in fish downstream of the Waterloo MWWTP (poorest effluent quality) suggested ~15.5 % of the transcriptome response was influenced by reference site selection. In contrast, at sites where the impact of MWWTPs was less pronounced and fish showed less of a transcriptome response, reference site selection had a greater influence (e.g., ~56.9% of transcripts were different depending on the site used). This study highlights the importance of conducting transcriptomics studies that leverage more than 1 reference site and it broadens our understanding of the molecular responses in fish in dynamic natural environments.
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A reconnaissance project completed in 2009 identified intersex and elevated plasma vitellogenin in male smallmouth bass inhabiting the Missisquoi River, VT. In an attempt to identify the presence and seasonality of putative endocrine disrupting chemicals or other factors associated with these observations, a comprehensive reevaluation was conducted between September 2012 and June 2014. Here, we collected smallmouth bass from three physically partitioned reaches along the river to measure biomarkers of estrogenic endocrine disruption in smallmouth bass. In addition, polar organic chemical integrative samples (POCIS) were deployed to identify specific chemicals associated with biological observations. We did not observe biological differences across reaches indicating the absence of clear point source contributions to the observation of intersex. Interestingly, intersex prevalence and severity decreased in a stepwise manner over the timespan of the project. Intersex decreased from 92.8% to 28.1%. The only significant predictor of intersex prevalence was year of capture, based on logistic regression analysis. The mixed model of fish length and year-of-capture best predicted intersex severity. Intersex severity was also significantly different across late summer and early spring collections indicating seasonal changes in this metric. Plasma vitellogenin and liver vitellogenin Aa transcript abundance in males did not indicate exposure to estrogenic endocrine disrupting chemicals at any of the four sample collections. Analysis of chemicals captured by the POCIS as well as results of screening discrete water samples or POCIS extracts did not indicate the contribution of appreciable estrogenic chemicals. It is possible that unreported changes in land-use activity have ameliorated the problem, and our observations indicate recovery. Regardless, this work clearly emphasizes that single, snap shot sampling for intersex may not yield representative data given that the manifestation of this condition within a population can change dramatically over time.
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In this study the activity of the biomarker acetylcholinesterase (AChE) in freshwater snails (Pachychilus sp.) was determined, with the aim of identifying by pesticides through the biomarker, besides using freshwater snails as potential bioindicators of freshwater bodies such as wetlands. In the "Ciénaga de Cabezas" wetland, Tamasopo, S.L.P. (Ramsar site), in 2017, in 20 different sampling stations, 20 snails were collected per site. A total of 60 organisms per sample, giving a total of 240 snails, were collected. The sampling months were taken based on the rainy season (June and September) and dry season (March and November). The morphometric values (weight and height) of the organisms were recorded and, subsequently, the organisms were sacrificed to obtain the tissue. Later, it will be transferred to a laboratory for analysis. On the other hand, physicochemical parameters in the water were evaluated to identify the quality of this, and the relationship as a cofactors in the activity of AChE in freshwater snails. From the biological samples, the activity of the AChE biomarker (nmol of thiocholine formed/min x mg of proteins) was determined by applying the spectrophotometric method, as well as the concentration of total proteins of the homogenized complete tissue. The general results are compared with the activity of AChE for months, with a 2.6-fold increase in AChE activity in November compared to the other three months, being statistically significant (p<0.001). Likewise, comparing the general results in the season, there is a decrease of 1.6 times in rainfall with respect to statistically significant statistics (p <0.001). On the other hand, the correlations between the AChE results with the physicochemical parameters, a statistically significant association with the temperature in the season (p<0.05), which may be a factor in the results of the ACHE activity. The results in our study show an impact on the AChE activity of Pachychilus sp. possibly in the use of organophosphorus pesticides in the study area.
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During recent decades, survey studies have documented the widespread presence of oocytes in the testes of male Smallmouth Bass Micropterus dolomieu collected from surface waters throughout the United States. There are few published reports of testicular oocytes (TO) in Smallmouth Bass before the 1990s, so it is difficult to know how long this has been occurring. Consequently, this study was conducted to evaluate the prevalence and severity of TO occurrence in whole fish specimens from two archival collections-the Smithsonian Institution's National Museum of Natural History in Suitland, Maryland, and Cornell University's Museum of Vertebrates in Ithaca, New York. Gonads were excised from 167 preserved male Smallmouth Bass that were originally collected between 1875 and 2004, and routine histologic sections were prepared and examined. The severity of TO was determined using a semiquantitative scoring system. Overall, 52.1% of male Smallmouth Bass were found to have TO. Affected fish had been collected in 11 of the 18 represented states, and TO were found in specimens harvested during decades as early as the 1880s and 1900s. Unfortunately, the small number of samples acquired at the earliest time periods precluded analyses of prevalence and severity trends over time. The results of this study demonstrated that the phenomenon of TO in male Smallmouth Bass is at least a century old and confirmed the widespread nature of this finding throughout the species' historic range. Further research efforts should focus on determining the baseline prevalence of TO in laboratory-reared male Smallmouth Bass that have not been exposed to endocrine active substances or the effects of experimental estrogen exposure on such fish.
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Estrogens and estrogen mimics are commonly found in surface waters and are associated with deleterious effects in fish populations. Impaired fertility and fecundity in fish following chronic exposures to estrogens and estrogen mimics during critical windows in development are well documented. However, information regarding differential reproductive effects of exposure within defined developmental stages remains sparse. In this study, reproductive capacity was assessed in Japanese medaka (Oryzias latipes) after exposure to two concentrations of 17β-estradiol (E2β; 2 ng/L and 50 ng/L) during four distinct stages of development: gonad development, gonad differentiation, development of secondary sex characteristics (SSC) and gonad maturation. Exposure to E2β did not adversely impact survival, hatch success, growth, or genotypic ratios. In contrast, exposure to 50 ng/L E2β during SSC development altered phenotypic ratios and SSC. Exposure to both E2β treatments reduced reproductive capacity (fertility, fecundity) by 7.3-57.4% in adult medaka breeding pairs, with hindrance of SSC development resulting in the largest disruption in breeding capacity (51.6-57.4% decrease) in the high concentration. This study documents differential effects among four critical stages of development and provides insight into factors (window of exposure, exposure concentration and duration of exposure period) contributing to reproductive disruption in fish.
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We compared the precision and bias of age estimates derived from scales, whole otoliths, and sectioned otoliths and by readers of varying experience level for largemouth bass Micropterus salmoides, smallmouth bass M. dolomieu, and spotted bass M. punctulatus from Skiatook Lake, Oklahoma. Precision was assessed with the coefficient of variation of age estimates for each fish. Bias was determined among readers and structures from age bias graphs derived from least squares regression. Precision was similar among species for all three structures and among structures for any species, except for smallmouth bass; its age estimates were more precise with whole otoliths. Bias between pairs of readers was found for scales and whole otoliths among all three species but never for sectioned otoliths. The experience level of the reader influenced the bias between readers for scales but not for otoliths. Bias between structures was found between scales and whole otoliths for all three species and between scales and sectioned otoliths for smallmouth bass and spotted bass. Age estimates were unbiased between whole and sectioned otoliths for all three species. Although sectioned otoliths required more preparation time, they provided the best age estimates for the three populations in Skiatook Lake.
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Histology offers a powerful tool in the study of reproductive health of fishes. It is routinely used for sex verification, identifying stage of development, documenting presence of intersex, tumors, parasites and other abnormalities and quantifying atresia. It can also be used for more subtle changes such as thickness of the vitelline envelope at various stages, yolk appearance, necrosis of sperm, and Sertoli cell proliferation. Gonadal histology, in conjunction with hormone and vitellogenin measurements, morphological and fecundity studies, can provide insights into the effects of various environmental stressors on reproductive health. However, much research, both field and laboratory, is needed to understand cause and effect for observed changes and to understand the meaning of many of the histological observations made in field studies, in terms of reproductive success of fish populations.
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Vitellogenin, the egg yolk precursor protein, has become a popular biomarker for measuring exposure of oviparous animals to estrogen or estrogen mimics. Vitellogenin is normally produced by females in response to normal cycles of estradiol during oogenesis. The gene for vitellogenin is also present in the livers of males but it is normally silent. Upon exposure to estrogen or to an estrogen mimic, the gene is turned on and vitellogenin is synthesized. After synthesis, it is exported into the blood where, in males, it remains until it is degraded or cleared out by the kidneys. In females, vitellogenin is taken up by the developing oocyte through receptor mediated endocytosis. There are several assays in the literature for measuring vitellogenin levels in plasma. The easiest method is through antibody based assays including ELISA (enzyme-linked immunosorbent assay) or by western blot. Competition or sandwich ELISAs are the most sensitive assays and they can detect vitellogenin in plasma in the nanogram to milligram per ml range. This chapter discusses methods for purifying vitellogenin from plasma, generating antibodies, and performing assays to measure vitellogenin.
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Numerous studies are available reporting the effects of pesticides on reproductive activity in Indian fishes. The majority of these reports deals with histopathological changes in gonads and endocrine glands involved in the regulation of reproduction following treatment with different pesticides. Pesticides are reported to cause degenerative changes in gonads and arrest gametogenic processes either by acting directly on the gonads or by interfering with the secretory activity of the hypothalamo-hypophyseal-gonadal/thyroid axis that regulates various reproductive events. Secretion of hormones such as gonadotropin-releasing hormone (GnRH), gonadotropin, growth hormone, adrenocorticotropic hormone (ACTH), testosterone, estrogens, 17,20β-dihydroxyprogesterone and thyroid hormones are in general lowered, leading to cessation of gametogenesis, vitellogenesis, oocyte maturation, ovulation, spermiation, etc. Adverse effects of pesticides have also been demonstrated on fecundity, fertilization, hatching, and postembryonic development. The effects are highly variable and depend on the nature, dose, and mode of application of the pesticides.
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A Basin-wide Index of Benthic Integrity (B-IBI) was developed to uniformly assess aquatic ecosystem health of non-tidal, wadeable streams in the multi-jurisdictional Potomac River basin (US). Multiple datasets were merged and used to identify stream classes and discriminating biological metrics. The index (aggregated metric scores) accurately identified 95% of impaired sites. A jackknife cross-validation procedure confirmed the accuracy of the B-IBI. B-IBI assessments generally compare favorably to basin states’ assessments derived from the same data. A habitat quality matrix which includes an indicator of anthropogenic alterations and disturbances is recommended. The Potomac B-IBI is more useful than existing state-specific indexes for stream health comparisons across jurisdictional boundaries and basin-wide. The Potomac B-IBI can improve understanding of water quality issues in the basin and enhance the abilities of water quality managers to make well-informed decisions concerning the basin's non-tidal waters.
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In respect to the differences found for vitellogenin (VTG) expression in male and immature flounder, general biological aspects and a set of chemical residues in the liver were compared between flounder from the Mersey and Dee estuaries (UK). Except for alpha-HCH, all pollutant chemicals analysed in flounder liver differed highly significantly between Mersey and Dee fish. Overall, the higher liver contaminant concentrations were found in VTG-induced Mersey flounder. The biomarkers studied were not found to indicate significant differences between Mersey and Dee fish. However, when all of the minor differences are taken into consideration, it appears that the slightly lower growth rates after age 2 in Mersey flounder, lower gonadosomatic index in both mature male and female animals, less precise seasonal patterns of condition factor (CF), hepatosomatic index and gonadosomatic index, and lower CF in immature Mersey fish may well be signs of a contaminant-affected Mersey population.
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The annual reproductive cycle of hatchery-raised largemouth bass (Florida subspecies Micropterus salmoides floridanus) was characterized over a one-year period. Largemouth bass have a distinct annual reproductive cycle with a spring spawning season (approximately between mid-January and mid-June). Cycle characterization focused on an evaluation of gonadal development and plasma concentrations of several sex steroids and vitellogenin (VTG). Adult largemouth bass (n = 20: 10 females and 10 males) were collected monthly from hatchery ponds for one full calendar year. Plasma samples were analyzed for estradiol-17β (E2), 11-ketotestosterone (11-KT), testosterone (T), progesterone (P), and VTG. Gonadal tissues were weighed to calculate gonadosomatic index (GSI) and evaluated histologically to characterize reproductive stage. In both sexes, GSI began to increase in November, and peaked in February-March. Increases in gonad weights were correlated with maturation of gonads as evidenced by histological evaluations. Bass exhibited seasonal changes in plasma sex steroids and VTG. In males, 11-KT was the only sex steroid that showed strong seasonality, with highest values in February. In females, although E2 and T concentrations followed a similar annual cycle, with highest and lowest values in February and August, respectively, the strongest pattern was observed with E2. 11-KT concentrations were less variable across months, and values were about half of those observed in males. In females, P peaked two months after E2, with high values still in May and June and decreased thereafter, and VTG began to increase in October, but peaked a month prior to the observed peaked in E2. VTG was also detected in males but at concentrations that were about 1/12 that of females, and no seasonal pattern was evident. This study is the first to fully characterize the seasonal endocrine cycle for largemouth bass. These data will be useful when conducting reproductive evaluations of free-ranging populations of largemouth bass and for assessing potential reproductive effects due to environmental contaminants in this species.
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Much recent interest has focused on 'xenoestrogens' in the environment. The potential for these chemicals to disrupt hormonal function in wildlife has led to the development of techniques to monitor the effects of discharges of these compounds particularly in aquatic environments. Increases in the concentration of the egg yolk precursor protein, vitellogenin (Vtg), in the plasma of fish and other vertebrates have been identified as a potentially useful biomarker of exposure to and the effects of chemicals with estrogen like properties. Here, we review the mechanisms underlying Vtg production, methods for the analysis of Vtg, and studies that have measured Vtg production in response to chemical or effluent exposure. These studies indicate that the production of Vtg may be a useful biomarker of exposure to estrogenic chemicals but that a greater understanding of the mechanism of action of these chemicals is required. In cyprinid fishes reproductive endpoints are more sensitive to exposure to xenoestrogens than Vtg induction. These observations indicate that Vtg induction is of more use as a biomarker of exposure to xenoestrogens rather than a predictor of adverse effects.
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Pharmaceuticals are a class of emerging contaminants whose fate in the wastewater treatment process has received increasing attention in past years. Acidic pharmaceuticals (ibuprofen, naproxen, mefenamic acid, ketoprofen, and diclofenac), caffeine, and the antibacterial triclosan were quantified at four different steps of wastewater treatment from three urban wastewater treatment plants. The compounds were extracted from wastewater samples on Waters Oasis hydrophilic-lipophilic balance solid-phase extraction columns, silylated, and analyzed by gas chromatography-mass spectrometry. For the chemicals studied, it was found that the majority of the influent load was removed during secondary treatment (51-99%), yielding expected surface water concentrations of 13 to 56 ng/L.
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We monitored the movement and habitat use of 34 stream-dwelling smallmouth bass Micropterus dolomieui in Jacks Fork River, Missouri, by radiotelemetry. Observations were made 24 h/d in all seasons. Definite patterns of diel activity and habitat use were evident and were modified by seasonal changes in water temperature. Fish remained in restricted home ranges for most of the year but tended to disperse in spring when all of the radio-tagged fish left their home pool; 75% returned during the same season. Equal numbers of fish moved upstream and downstream. However, the median distance moved upstream was greater. Intrapool movement of smallmouth bass peaked soon after sunrise and again after sunset in all seasons. Average intrapool movement was 120 m/d when water temperatures were lowest (4°C) and 980 m/d when temperatures were highest (27.5°C). In the warmer seasons, fish preferred logjams and root wads by day and increased their use of boulders at night. In cooler seasons, fish used boulders almost exclusively. Regardless of season, open water without cover was used most during the period just after sunrise but was never used in proportion to its availability. Boulders were the most preferred substrate, and gravel was the least preferred. Small mouth bass used intermediate depths the most and showed no daily or seasonal changes in depth preferences. Fish preferred velocities less than 0.2 m/s at all times of day and in all seasons. Movements in floods did not differ from those observed during normal discharges; however, single logs were selected in significantly greater proportion than logjams.
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Movement patterns of 305–406-mm smallmouth bass Micropterus dolomieu were observed by use of radiotelemetry in main-stem Elkhorn Creek and the Kentucky River, Kentucky, in 2000 and 2001 to determine impacts of movement and homing ability on a special regulation area within the open-river system. Smallmouth bass are protected from harvest in main-stem Elkhorn Creek by a 305–406-mm protective slot limit, but can be harvested in the Kentucky River under a 305-mm minimum length limit. In 2000, 39 smallmouth bass radio-tagged in May and June after spawning were composed of migratory (69%) and sedentary (31%) groups, with 13% utilizing the Kentucky River. In 2001, 15 smallmouth bass radio-tagged in late March prior to spawning were year-round sedentary residents; 20% made limited movements out of their original capture pool. In addition, we displaced 15 smallmouth bass from Elkhorn Creek into the Kentucky River in March 2001 to observe and quantify homing; 60% of the fish returned to the creek from their displacement locations. Managers should consider movement patterns when developing special regulation areas, particularly as seasonal movements may affect the size and scope of the area necessary for adequate protection.
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Oocyte maturation and yolk incorporation (vitellogenesis) is a hormonally controlled and regulated process. In fish, the primarily exogenous synthesis of vitellogenin is initiated by gonadotropins and regulated by estrogens. Vitellogenin is a species-specific protein synthesized by hepatocytes, released into the bloodstream and actively sequestered by maturing oocytes. A large amount of research has been directed at assessing the effects of polycyclic aromatic hydrocarbons (PAHs) on different phases of vitellogenesis in fish and identifying their mechanisms of action. PAHs have been found to have a deleterious effect on the vitellogenesis of fish from feral populations as well as in laboratory experiments. This is of particular concern as any of the stages of the vitellogenic cycle could be affected which would result in harmful sub-lethal effects with trans-generational consequences. The reported effects include reduction in circulating hormones and plasma vitellogenin, estrogenic and antiestrogenic effects, retardation of oocyte maturation and reduction of reproductive success. However, the lack of agreement between some of the results indicates that some of the mechanisms at play are not yet completely understood and require further investigation. This review attempts to summarize the present understanding of the effects of PAHs on vitellogenesis in fish.
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The reproductive cycle of smallmouth bass Micropterus dolomieu naturalized in Lake Akimoto at Fukushima Prefecture was investigated to obtain biological information for developed techniques to exterminate this species in Japan. Samples were captured monthly from April to December 2002. In male testes, spermatozoa or spermatids were observed from April to July and from September to December. In August, males showed low gonadosomatic index (GSI) and the testes were occupied by spermatocytes. In feature, oocytes with early or late perinucleolus and yolk stages were observed from April to June and from October to December. From July to September, females showed low GSI and a decrease in the number of oocytes with yolk stages. In both males and females, plasma testosterone and 17,20beta-dihydroxy-4-pregen-3-one levels reached peaks in May. In males, 11-ketotestosterone reached a peak in May. In females, estradiol-17beta showed high levels in April. From these results, the reproductive period of smallmouth bass can be considered to be from spring to early summer. However, they have gonads keeping a certain degree of maturity from autumn to early winter.
Article
This document describes procedures used by biologists to collect information, tissues, and fluids useful for determining the exposure of fish to environmental contaminants. Fish are to be captured and held alive (generally <1 h), then weighed, measured, and examined for grossly visible external lesions. A blood sample is collected by caudal veinipuncture. The fish is subdued, and its abdominal cavity is opened with a mid-ventral incision. The internal organs are dissected from the fish for examination. Selected organs are weighed, and tissues are collected for laboratory analyses. All remaining tissues and fluids are then returned to the carcass, which is prepared for chemical analysis. Individual fish are composited by station, species, and gender; frozen; and shipped to the analytical laboratory. Procedures are also described for record keeping; processing blood to obtain serum and plasma; flash-freezing samples; cleaning equipment; and preventing the transport of living organisms among waterways. A list of necessary equipment and supplies is also provided.
Article
We collected examined and analyzed 1378 fish of 99 species from 47 sites in the Mississippi River basin (MRB) during 1995 and from a reference site in 1996. The sampling sites in the MRB represented National Contaminant Biomonitoring Program (NCBP) stations situated at key points on major rivers and National Water Quality Assessment Program (NAWQA) stations located on lower-order rivers and streams in the Eastern Iowa Basins (FIB) and Mississippi Embayment (MSE) Study Units. The reference site was the water supply system of the USGS-Leetown Science Center in rural Jefferson County WV Common carp (Cyprinus carpio; carp) and black basses (Micropterus spp., bass) the targeted species together represented 82% of the fish collected. Each fish was examined in the field for externally and internally visible gross lesions selected organs were weighed to compute various ponderal and organo-somatic indices and selected tissues and fluids were obtained and preserved for analysis of biomarkers. Fish health indicators included splenic macrophage aggregates lysozyme activity and hispathological anlysis of liver kidney and of liver tissues. Reproductive biomarkers included analysis of plasma concentrations of vitellogenin (vtg) and the sex steroid hormones 17beta-estradiol (E2) and 11-ketotestosterone (11- kt), and the histological determination of percent oocyte atresia (in female fish) and gonadal stage. Hepatic ethoxyresorufin O-deethylase (EROD) activity was also measured. Composite samples of whole fish from each station were grouped by species and gender and analyzed for persistent organochlorine and elemental contaminants and for dioxin-like activity (TCDD-EQ) using the H411E rat hepatoma cell bioassay.
Article
In the present study, the estrogenic response in the reproductive cycle of female carp, Cyprinus carpio, in relation to a sewage treatment works (STWs) was measured as: alterations in plasmatic and hepatic vitellogenin (VTG) content, plasma levels of testosterone (T) and estradiol (E2), and effects on key enzymes of phases I and II biotransformation, ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST). Low plasma VTG content in females collected 4 km downstream of an effluent discharge suggested possible estrogenic disturbances; however, hepatic VTG content and sex hormone levels did not mirror this. In fact, E2 and T largely fluctuated among fish, with individual variations even greater than among sampling stations or periods. This suggests that at moderately polluted sites, biological variations of female carp and genetic particularities are prevalent. Hepatic biotransformation enzymes, such as EROD, increased with increasing water temperature while, in contrast, GST was maximal at the lowest water temperature.
Article
In a partial life-cycle test, the impact of 17alpha-ethinylestradiol (EE2) and 17alpha-methyltestosterone (MT) on juvenile zebrafish was evaluated by use of vitellogenin measurements and gonadal development. Exposure to EE2 (1-25 ng/l) resulted in a dose-dependent increase in vitellogenin production starting at 2 ng/l. Significant changes in sex ratios in female direction were detected at 1 ng/l, with complete sex reversal taking place after exposure to 2 ng/l. No intersex fish were observed after exposure to EE2. Exposure to MT resulted in decreased vitellogenin concentrations. Complete sex reversal was detected in all MT concentrations used (26-1000 ng/l). A large proportion of intersex fish was observed after exposure to 1000 ng MT/l. The period of gonadal sex reversal in non-exposed zebrafish was also studied. The main morphological features of the transformation of ovaries into testis were observed 4-5 weeks after hatching.
Article
The occurrence of hermaphrodite fish in the lagoons of sewage treatment works led us to hypothesize that sewage effluent might contain a substance, or substances, estrogenic to fish. to test this hypothesis, we placed cages containing rainbow trout in the effluent from sewage-treatment works, and one to three weeks later measured the vitellogenin concentration in the plasma of the fish. Vitellogenin is a protein synthesized by the liver of oviparous fish in response to estradiol stimulation; it is then conveyed by the blood to the ovary, where it is sequestered by oocytes to form the yolk. Thus, the presence of vitellogenin in the plasma is indicative of estrogenic stimulation of the liver. an initial study, at a sewage-treatment works, showed that plasma vitellogenin concentrations rose rapidly and very markedly (over 1000-fold in three weeks) when trout were maintained in the effluent. an extensive nationwide survey was then conducted. Results were obtained from fifteen sewage-treatment works distributed throughout England. in all cases, exposure of trout to effluent resulted in a very pronounced increase (500 to 100,000-fold, depending on site) in the plasma vitellogenin concentration. Induction of vitellogenesis was also observed in carp, but to a much lesser extent than in trout.The identity of the estrogenic substance is unknown. It is suggested that the two most likely possibilities are ethynylestradiol, originating from pharmaceutical use, or alkylphenol-ethoxylates (APE), originating from the biodegradation of surfactants and detergents during sewage treatment.Laboratory studies on the potency of ethynylestradiol demonstrated that levels as low as 1 to 10 ng 1 could generate the response shown by the caged fish and that positive responses may arise at 0.1 to 0.5 ng 1. Further work is in progress on the potency of APE.
Article
A number of chemicals present in the environment have been shown to mimic or antagonize the actions of steroid hormones, an issue often described as "endocrine disruption/modulation". There is very little evidence, however, to support the hypothesis that exposure to endocrine-disrupting chemicals is a global environmental health problem. In this paper, we demonstrate a high incidence of intersexuality in wild populations of riverine fish (roach; Rutilus rutilus) throughout the United Kingdom. These reproductive disturbances are consistent with exposure to hormonally active substances and are associated with discharges from sewage treatment works that are known to contain estrogenic chemicals. This is the first documented example of a widespread sexual disruption in wild populations of any vertebrate and indicates that reproductive and developmental effects do result from exposure to ambient levels of chemicals present in typical British rivers.
Article
Annual cycles of growth and reproduction of hatchery–reared Florida largemouth bass, Micropterus salmoides floridanus, were investigated. Animals were raised on either forage (goldfish, Carassius auratus) or a pelleted salmon feed. Male and female year–class 1 largemouth bass were sampled throughout one complete yearly cycle (January–December). A biphasic growth cycle was observed in both forage–fed and pellet–fed fish. No increase in body length or weight was observed until approximately midway through the spawning period (May), after which fish grew at a consistent rate for the remainder of the study. The reproductive cycle of forage–fed fish was characterized by a rapid increase in gonadosomatic index (GS1) between January and April, followed by a prolonged spawning period (April–July) during which GSI progressively declined. Fully regressed gonads were observed in September and October, and a resumption of gonadal recrudescence was observed between October and December. Visceral adipose deposits (expressed as mesenteric fat index; MFI) were resorbed during gonadal growth and the initial stages of the spawning period, and restored during the post–spawning phase. Fish raised on pelleted feed had growth and reproductive cycles that parallelled those of forage–fed fish, but several significant differences were observed between the two diet groups. During the growth phase of the cycle, pellet–fed largemouth bass grew significantly faster than forage–fed largemouth bass, and had significantly larger MFIs than forage–fed largemouth bass at all times of the year. Pellet–fed fish also had significantly larger GSIs than forage–fed fish. These data indicate that diet composition may be an important determinant of growth and reproductive function in this species.
Article
Oocyte growth and development is an important issue in fish and fisheries biology. This paper reviews the information available on oocyte growth patterns and the rates and dynamics of oocyte growth in teleosts. In synchronous spawners, the weight of the gonad may represent as much as 40% of the overall body weight of the fish. In asynchronous spawners, the weight of the mature ovary is considerably less than in synchronous ovulators, but the ovary shows a more regular periodicity and may grow repeatedly many times during the breeding season. There is a huge variability in egg size in teleosts, with the largest known measuring up to 8 cm in diameter. Within the limits of variance set by genetic constraints, egg size may vary between populations of the same species. Oocytes in all teleosts undergo the same basic pattern of growth: oogenesis, primary oocyte growth, cortical alveolus stage, vitellogenesis, maturation and ovulation. The mechanisms that control oocyte growth are addressed in this review, albeit that the available information, as in all other vertebrates, is very limited. The main hormones that have been shown to affect ovarian growth are gonadotrophin, thyroid hormones, growth hormone, insulin and insulin-like growth factors. An overview of the determinants of fecundity, with particular reference to oocyte recruitment and atresia, is the focus of the second part of the paper. Genetics and nutrition have major effects on fecundity, and studies so far suggest that the determinants of fecundity usually operate during the early part of gametogenesis. The role of atresia in determining fecundity is less clear. The final part of this review highlights some areas of study that are priorities for research on ovarian development in fish.
Article
The goals of this study were to: (1) measure atrazine and metolachlor concentrations during both high and low use periods in the Chesapeake Bay's mainstem/major tributaries, smaller tributaries and representative small agricultural streams during 1995 and 1996; (2) compare these exposure data with toxicity benchmarks for each herbicide to predict ecological risk and (3) use in-stream fish community data collected in the streams to provide supportive data for ecological risk characterization. Spatially, atrazine (
Article
Environmental contaminants with estrogenic activity have recently received attention because of their potential effects on the reproductive efficiency of humans and wildlife. This study was conducted with the endogenous estrogen, 17 β-estradiol (E2), to establish the histologic response of the fathead minnow (Pimephales promelas) as a model organism. Sexually mature fathead minnows were exposed for 14 days to waterborne concentrations of 1000, 100, 10, 2, 1, 0.5, 0.25, 0.125, 0.1 or 0.0625 nM E2. Exposure to E2 caused a reduction in size of the prominent male secondary sex characteristics, the fatpads and nuptial breeding tubercles. Histological lesions observed in the testes included proliferation of Sertoli cells and degenerative changes. Electron microscopy of seminiferous tubules and their Sertoli cells revealed large phagolysosomes filled with degenerating spermatozoa and other cellular debris. Females had ovaries in which most of the follicles were in the primary stage of development. There were also more atretic follicles and fewer secondary and Graafian follicles than in unexposed females. These findings demonstrate components of sexually mature fish which may be altered by compounds that mimic E2. To determine if lesions observed in males were permanent, 50 sexually mature males and females were exposed to a single concentration of 10 nM E2 for 10 days. Samples were collected from males on the final day of E2 exposure and over a period of 16 weeks after the exposure was stopped. No E2-induced lesions were observed beyond 16 weeks post E2 exposure. Results of these studies suggest that histological lesions could occur at ecologically-relevant exposures to ‘estrogenic’ compounds. However, certain lesions caused by exposure of adult fathead minnows are not permanent.