Sex-Related Embryotoxicity of Pulp Mill Effluent Extracts in Medaka (Oryzias latipes) Female Leucophore-free FLFII Strain.

The aim of the present study was to evaluate the effects of exposure to Chilean pulp mill effluent extracts on developing postfertilized medaka embryos before and after sex definition relative to sex steroids (testosterone and 17beta-estradiol) and a wood phytoestrogen (beta-sitosterol). Our study included 2 waterborne semichronic exposure experiments, using a 24-h post fertilization (hpf) unknown-sex FLFII (female leucophore free) group and a second 72-hpf FLFII phenotypic sex-identified group (male autofluorescence leucophore) strain of medaka embryos. Chronic exposure of both FLFII strain embryo groups showed similar delay in time to hatch and decreased hatchability. Teratogenic responses such as vertebral malformation (fusion, incomplete formation, and lack of vertebral formation process) and pericardial edema were observed in both experiments, with a high percentage related to FLFII fluorescent leucophore-identified males. In addition, high mortality associated with severe malformations was observed in male and female embryos exposed to testosterone. Our research has demonstrated that exposure to Chilean mill effluent extracts caused severe male medaka embryotoxicity (in postfertilized embryos) before and after sex definition and, irrespective of the experimental group and effluent treatment, suggests partial removal following secondary treatment. Furthermore, differences in the severity and type of teratogenic effects with previous experiments (d-rR medaka strain), are associated with the unique phenotypes of this medaka mutant strain. Environ Toxicol Chem 2021;40:2297-2305. © 2021 SETAC.

An 'omics approach to investigate the growth effects of environmentally relevant concentrations of guanylurea exposure on Japanese medaka (Oryzias latipes).

Metformin is a widely prescribed pharmaceutical used in the treatment of numerous human health disorders, including Type 2 Diabetes, and as a results of its widespread use, metformin is thought to be the most prevalent pharmaceutical in the aquatic environment by weight. The removal of metformin during the water treatment process is directly related to the formation of its primary degradation product, guanylurea, generally present at higher concentrations in surface waters relative to metformin. Growth effects observed in 28-day early life stage (ELS) Japanese medaka exposed to guanylurea were found to be similar to growth effects in 28-day ELS medaka exposed to metformin; however, effect concentrations were orders of magnitude below those of metformin. The present study uses a multi-omics approach to investigate potential mechanisms by which low-level, 1 ng · L-1 nominal, guanylurea exposure may lead to altered growth in 28-day post hatch medaka via shotgun metabolomics and proteomics and qPCR. Specifically, analyses show 6 altered metabolites, 66 altered proteins and 2 altered genes. Collectively, metabolomics, proteomics, and gene expression data (using qPCR) indicate that developmental exposure to guanylurea exposure alters a number of important pathways related to the overall health of ELS fish, including biomolecule metabolism, cellular energetics, nervous system function/development, cellular communication and structure, and detoxification of reactive oxygen species, among others. To our knowledge, this is the first study to both report the molecular level effects of guanylurea on non-target aquatic organisms, and to relate molecular-level changes to whole organism effects.

The Effects of 17α-Ethinylestradiol on the Heart Rate of Embryonic Japanese Medaka (Oryzias latipes).

Estrogen toxicity has been an area of priority in aquatic toxicology over the last 20 yr. Currently, estrogen toxicity is primarily linked to classical estrogen signaling, the interaction of estrogen receptors alpha and beta (ERα and ERß). Recent evidence has indicated that a rapid, nongenomic, nonclassical estrogen signaling pathway exists via the G protein-coupled estrogen receptor (GPER), which is expressed in many biological systems, with roles in the cardiovascular system. The objective of the present study was to investigate the effect of 17α-ethinylestradiol (EE2) on the heart rate of embryonic Japanese medaka (Oryzias latipes). A significant decrease (bradycardia) in embryonic heart rate was observed at all treatment concentrations (0.1, 1, 10, 100, and 1000 ng/L EE2) at 144, 168, and 192 h postfertilization (hpf; p ≤ 0.05), whereas 120 and 216 hpf embryos experienced a significant decrease from the control at 10, 100, and 1000 ng/L EE2 and 0.1, 100, and 1000 ng/L EE2, respectively (p ≤ 0.05). In addition, using select estrogen receptor modulators, it was demonstrated that estrogen-induced bradycardia appears to be linked to GPER and not ERα and ERß. The present study highlights GPER as a novel and alternative mode of action for EE2 toxicity at environmentally relevant concentrations. Environ Toxicol Chem 2020;39:904-912. © 2020 SETAC.

Developmental and Full-Life Cycle Exposures to Guanylurea and Guanylurea-Metformin Mixtures Results in Adverse Effects on Japanese Medaka (Oryzias latipes).

Metformin is currently thought to be the highest drug by weight released into the aquatic environment, as a direct result of its widespread use in the treatment of a number of human health disorders. The removal of metformin from wastewaters is directly related to the formation of guanylurea (metformin's only known persistent degradation product), which is generally present at higher concentrations in surface waters than the parent compound. With metformin use rising steadily, it is important to characterize the effects of guanylurea on nontarget aquatic organisms. We recently demonstrated the effects of developmental exposure to environmentally relevant concentrations of metformin on the growth of early life stage (ELS) medaka as well as effects on the body weight of adult male fish following full-life cycle exposures. In the present study, we describe similar effects of guanylurea exposure on these endpoints and life stages. Guanylurea led to effects on growth in a 28-d ELS assessment that were similar to those of metformin; however, these effects occurred at concentrations in the ng/L range compared with the µg/L range for metformin. A possible sex-dependent association with body weight changes was also observed in adults following a 165-d full-life cycle exposure to guanylurea alone or in a mixture with metformin. To our knowledge, the present is the first study to report the toxicity of guanylurea to nontarget aquatic organisms. Environ Toxicol Chem 2019;00:1-6. © 2019 SETAC.

Effects of environmentally relevant metformin exposure on Japanese medaka (Oryzias latipes).

Metformin is one of the most prevalent pharmaceuticals in both surface and waste waters, yet little is known about the bioavailability and/or effects of developmental exposure on early life stage (ELS) fish. Here, we demonstrate that embryo-larval stages of medaka are capable of taking up metformin from the aquatic environment, provided exposure occurs prior to chorion hardening (∼6-hpf). Once transferred to clean water, ELS medaka are able to completely depurate metformin in <24-hours. Furthermore, ELS medaka exposed to a range of relevant concentrations of waterborne metformin (from 6 hpf through 28-days post hatch) had significantly reduced growth metrics, altered metabolomes, and changes in the expression of genes associated with cell growth. The range of concentrations investigated were 1.0, 3.2, 10, 32, and 100 µg·L-1. To examine effects of chronic, low level metformin exposure across the full medaka life-cycle, we exposed newly fertilized embryos to 3.2 µg L-1 waterborne metformin for 165-days. The weight and length of adult fish were examined, as were effects on the production of some steroid hormones, specifically a significant increase (control females: 0.161 ± 0.023 pg/mg; metformin treated females: 3.42 ± 0.543) in the production of 11-ketotestosterone was observed in adult female medaka. Collectively, these results suggest that current environmental exposure scenarios may be sufficient to cause effects on developing fish.

Evaluating the potential of effluent extracts from pulp and paper mills in Canada, Brazil, and New Zealand to affect fish reproduction: Estrogenic effects in fish.

The authors examined the potential of pulp mill effluent from pulp-producing countries (Canada, Brazil, New Zealand) to affect fish reproduction. Specifically, the estrogenic effects in juvenile rainbow trout (Oncorhynchus mykiss) pulse-exposed to 11 different mill effluent extracts (intraperitoneal injections of solid-phase extraction-dichloromethane nonpolar fraction). The results indicated that effluent extracts were estrogenic in juvenile trout irrespective of the gender, as reflected by increasing level of plasma vitellogenin (VTG; Brazil > New Zealand > Canada). Despite the high variability observed among mills, differences in VTG levels were related to the type of mill process (kraft > elementary chlorine-free kraft > thermomechanical pulping). Moreover, effluent treatments did not appear to significantly decrease VTG induction. A consistent estrogenic effect was observed in those mills that process a combination of feedstocks (softwood and hardwood), with the highest increase in VTG related to eucalyptus feedstock. The results demonstrate significant estrogenic effects of pulp mill effluents on chronically exposed juvenile trout, suggesting that in vivo metabolic activation of precursors is necessary to cause the observed increases in VTG levels. This molecular estrogenic response provides a useful starting point for predicting population-level impacts through the adverse outcome pathway methodology. Environ Toxicol Chem 2017;36:1547-1555. © 2016 SETAC.

Chronic effects of hydroxypropyl-ß-cyclodextrin on reproduction in the American flagfish (Jordanella floridae) over one complete life cycle.

Understanding the environmental risks of pharmaceuticals and personal care products (PPCPs) has become very important in the field of aquatic toxicology. Hydroxylpropyl-ß-cyclodextrin (HPßCD) is an amphiphilic, toroidal shaped molecule with the ability to form noncovalent inclusion complexes with a variety of guest molecules. The molecule can reduce volatility as well as improve the aqueous solubility of apolar guest compounds and is an emerging PPCP. As such, HPßCD is the active ingredient in Febreze (Procter & Gamble) and is extensively used as an excipient in the pharmaceutical industry. With the potential for entering the environment through waste-water treatment plant (WWTP) effluent, HPßCD poses an unknown risk to nontarget aquatic biota. A 145-d chronic full life-cycle exposure using American flagfish (Jordanella floridae) was completed using flow-through nominal concentrations of 0 µg/L (control), 5 µg/L, 16 µg/L, 50 µg/L, 160 µg/L, 500 µg/L, and 1600 µg/L of HPßCD maintained via a peristaltic pump. Fecundity, growth, and liver somatic index were all monitored, and no significant difference was found between treatments and controls (p > 0.05). However, a significant increase in the gonadosomatic index was observed in females exposed to HPßCD (p ≤ 0.05). Reduced offspring growth was observed after exposure in the same manner as the parental generation (p ≤ 0.05). Furthermore, an acute copper toxicity challenge assay was conducted on second-generation flagfish larvae, and a decrease in copper tolerance was observed in larval progeny from parents exposed to HPßCD. Environ Toxicol Chem 2016;35:1358-1363. © 2015 SETAC.

Pharmaceuticals and personal care products: A critical review of the impacts on fish reproduction.

Research in environmental toxicology involving pharmaceuticals and personal care products (PPCPs) has increased greatly over the last 10-15 years. Much research has been focused on the endocrine-disrupting potential of PPCPs, as they relate to negative population impacts of aquatic organisms. This review assesses the current data on the reported effects of PPCPs on fish reproduction with an emphasis on fecundity, a predictor of population effects. Studies of both individual PPCPs and PPCP mixtures are presented. As the majority of individual PPCP studies reviewed demonstrate negative effects on fish fecundity, we relate these findings to detected surface water concentrations of these compounds. Very few studies involving PPCP mixtures have been conducted; however, the need for these types of studies is warranted as fish are most likely exposed to mixtures of PPCPs in the wild. In addition, laboratory and field assessments of wastewater treatment plant (WWTP) effluents, a major source of PPCPs, are reviewed. Much of the data provided from these assessments are variable and do not generally demonstrate negative impacts on reproduction, or the studies are unable to directly associate observed effects with WWTP effluents. Finally, future research considerations are outlined to provide an avenue into understanding how wild populations of fish are affected by PPCPs. These considerations are aimed at determining the adaptation potential of fish exposed to mixtures of PPCPs over multiple generations. As global use of PPCPs continually rises, the need to discern the effects of chronic exposure to PPCPs is greatly increased.

Evaluating the potential of effluents and wood feedstocks from pulp and paper mills in Brazil, Canada, and New Zealand to affect fish reproduction: chemical profiling and in vitro assessments.

This study investigates factors affecting reproduction in fish exposed to pulp and paper mill effluents by comparing effluents from countries with varying levels of documented effects. To explore the hypothesis of wood as a common source of endocrine disrupting compounds, feedstocks from each country were analyzed. Analyses included in vitro assays for androgenic activity (binding to goldfish testis androgen receptors), estrogenic activity (yeast estrogen screen), and neurotransmitter enzyme inhibition (monoamine oxidase and glutamic acid decarboxylase). Chemical analyses included conventional extractives, known androgens, and gas chromatograph index (GCI) profiles. All effluents and wood contained androgenic activity, particularly in nonpolar fractions, although known androgens were undetected. Effluents with low suspended solids, having undergone conventional biotreatment had lower androgenic activities. Estrogenic activity was only associated with Brazilian effluents and undetected in wood. All effluents and wood inhibited neurotransmitter enzymes, predominantly in polar fractions. Kraft elemental chlorine free mills were associated with the greatest neurotransmitter inhibition. Effluent and wood GCI profiles were correlated with androgenic activity and neurotransmitter enzyme inhibition. Differences in feedstock bioactivities were not reflected in effluents, implying mill factors mitigate bioactive wood components. No differences in bioactivities could be discerned on the basis of country of origin, thus we predict effluents in regions lacking monitoring would affect fish reproduction and therefore recommend implementing such programs.

The acute and chronic effects of endosulfan pulse-exposure on Jordanella floridae (Florida flagfish) over one complete life-cycle.

Endosulfan is an organochlorine pesticide, which is used worldwide and has known toxic effects on non-target organisms including fish. This research investigated the acute and chronic effects of pulse-exposed endosulfan on Florida flagfish (Jordanella floridae). A 4-h pulse-exposure of endosulfan to larval flagfish caused a significant increase in mortality after 96 h at nominal concentrations equal to or greater than 100 µg/L. Some of the acute sub-lethal observations included hyperactivity, convulsions, and axis malformation. Seven-eight day old post-hatch flagfish were pulse-exposed for 4h to endosulfan and then monitored over one full life-cycle for chronic effects on growth, reproduction, and survivability. There were no growth or reproductive effects of endosulfan pulse-exposure up to the highest exposure concentration of 10.8 µg/L. Thus, the life-cycle 4-h pulse-exposure no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) were 3.3 and 10.8 µg/L endosulfan, respectively, based on significantly higher larval and juvenile mortality.

Comparative embryotoxicity of pulp mill extracts in rainbow trout (Oncorhynchus mykiss), American flagfish (Jordanella floridae) and Japanese medaka (Oryzias latipes).

This study evaluated the effects of Chilean pulp mill effluent extracts (untreated, primary and secondary treated pulp mill effluents), along with steroid standards (testosterone and 17ß-estradiol) and a wood extractive standard (beta-sitosterol) on developing post-fertilized fish embryos. Our study included a cold freshwater species, rainbow trout (Oncorhynchus mykiss), and two warm freshwater species American flagfish (Jordanella floridae) and Japanese medaka (Oryzias latipes). Embryotoxicity results included delay in time to hatch and decreased hatchability but no significant egg and larvae mortality was observed in the pulp mill extract exposed embryos. By contrast, significant early hatching and increased hatchability were observed in beta-sitosterol exposed embryos, along with high mortality of testosterone exposed embryos across species. Teratogenic responses were observed in medaka embryos in all treatments. Abnormalities were detected starting at development stages 19-20 (2-4 somite stages) and included optical deformities (micro-opthalmia, 1 or 2 eyes) and lack of development of brains and hearts. Additionally, phenotypic sex identification of surviving offspring found female-biased sex-ratios in all treatments except testosterone across species. Overall, our study indicated that Chilean pulp and paper mill extractives caused embryotoxicity (post-fertilized embryos) across species and irrespective of the effluent treatment. The effects were mainly associated with delayed time to hatch, decreased hatchability, and species-specific teratogenesis.

Effects of pulp and paper mill effluent extracts on liver anaerobic and aerobic metabolic enzymes in rainbow trout.

This study investigates whether pulse exposure to Chilean pulp and paper mill effluent solid phase extracted (SPE) extracts via intraperitoneal injection (IP), would result in changes in the activities of the respiratory metabolic enzymes citrate synthase (CS) and lactate dehydrogenase (LDH) in rainbow trout livers. It also investigated if an alteration in liver metabolic capacity influenced the liver detoxification processes and estrogenic effects previously reported. Besides, a comparison of those enzymatic activities with fish IP injected with SPE extracts of two model effluents coming from industries that process 100% different type of feedstock (softwood, SW and hardwood, HW) was also evaluated. An initial induction of the anaerobic metabolism (increase in LDH enzymatic activity) was detected in all Chilean pulp mill effluent extracts evaluated, contrary to the initial unaltered aerobic metabolism (CS enzymatic activity) observed. A compensatory relationship in energy metabolism (Pasteur effect) was observed when comparing both enzymatic activities of fish exposed to those effluent extracts. LDH and CS activities observed in fish injected with Chilean extracts seem to be related to the effects observed in fish injected with SW extracts. This study showed that intraperitoneal injection of pulp and paper mill effluent extracts affected the anaerobic and aerobic metabolic capacities in rainbow trout livers, but this metabolic alteration did not affect detoxification capability or estrogenic effect previously reported.

Estrogenic and anti-estrogenic effects of wood extractives present in pulp and paper mill effluents on rainbow trout.

Wood extractives present in pulp and paper mill effluents may cause reproductive disturbances in fish. A chronic-exposure toxicity experiment using immature rainbow trout (Oncorhynchus mykiss) was conducted in order to assess the endocrine disrupting effects of two Chilean pulp and paper mill specific extracts (solid phase extraction, SPE) obtained from primary and secondary treated effluents. The (anti)estrogenic potencies and toxicity of the wood extractives regularly present in pulp mill effluent such as dehydroabietic acid (DHAA), beta-sitosterol (BS), and model estrogen 17beta-estradiol (E2) were evaluated by analysis of plasma vitellogenin (VTG) levels, gonadal somatic index (GSI) and liver ethoxyresorufin-O-deethylase (EROD) activity, respectively. The protocol involved the use of multiple intra-peritoneal injections (1 injection every 7 days for a total exposure period of 28 days). Analysis of variance/covariance, demonstrated no differences associated with fish gender other than GSI. The phytosterol BS, E2 and both pulp mill effluent extracts showed significant inductions of EROD and increased VTG levels after 4, 7, 14, 21 and 28 of exposure. While fish injected with secondary treated effluent extract showed a delayed induction in VTG levels compared to primary effluent injected fish, no effects on VTG and EROD levels were observed in DHAA injected fish. Moreover simultaneous injection of DHAA+E2 reduced the VTG levels found in E2 injected fish, indicating a potential indirect anti-estrogenic effect of this resin acid. The results of this study indicate that Chilean pulp and paper mill effluent extracts are estrogenic in rainbow trout males and females.

Effects of pulp and paper mill effluent extractives on aromatase CYP19a gene expression and sex steroid levels in juvenile triploid rainbow trout.

We evaluated plasma testosterone (T) and 17beta-estradiol (E2) levels and ovarian aromatase CYP19a gene expression following a single intraperitoneal injection of Chilean pulp and paper mill effluent extracts into juvenile triploid rainbow trout. Fish injected with untreated effluent extracts had increased plasma T after 4 days, while plasma E2 concentration was increased in fish injected with both primary and secondary treated effluent extracts at the same sampling period. Ovarian CYP19a gene expression as measured by qRT-PCR was significantly induced in fish injected with the untreated, primary and secondary treated pulp and paper mill effluent extracts. Similar induction of CYP19a expression was found in fish injected with the androgens androstenedione (ADD) and T. A Principal Component Analysis (PCA) was conducted in order to identify structure in relationships between all measured variables and identifying which factors were most responsible for the variance observed within the plasma steroid levels, upregulation of ovarian CYP19a gene expression and the final estrogenic effect of increased plasma VTG levels. This analysis indicated a cluster correlation between plasma T levels and CYP19a gene expression (Factor 1, explaining 27.2% of total variance), a cluster including condition factor and liver somatic index (Factor 2, explaining 17.3%) and an additional cluster including plasma E2 and vitellogenin levels (Factor 3, explaining an additional 15.8%). The present results indicate that Chilean pulp and paper mill effluent extracts cause estrogenic effects in triploid rainbow trout. These effects could be related to the compounds present in the effluent that act as estrogen receptor agonists, or that induce changes leading to increased amounts of endogenous estrogens, reflected by increased E2 levels and induced aromatase expression/activity.

Physiological changes in reproductively active rainbowfish (Melanotaenia fluviatilis) following exposure to naphthalene.

Naphthalene makes up a substantial fraction of polycyclic aromatic hydrocarbons (PAHs) in crude oil and is an important by-product of industry; however, few studies have investigated the toxicity of naphthalene to aquatic organisms. We examined the toxicity of increasing concentrations (0, carrier control, 130, 200 and 400microg/l) of naphthalene to adult rainbowfish (Melanotaenia fluviatilis) for 3 and 14 days to determine its potential to act as an endocrine disruptor. After exposure for 3 days, no changes in sex steroids were measured. After 14 days, a decrease of serum estradiol in females and an increase in serum testosterone in males was observed. These results suggest that naphthalene has the potential to act as an endocrine disruptor, although since no changes in plasma vitellogenin concentrations were observed in either sex, it is unlikely that naphthalene is acting as a xenoestrogen. There was a positive correlation between the incidences of deformities in larval offspring with increasing naphthalene concentrations, suggesting parental transfer of the toxicant. Egg production, hatchability and larval lengths remained unaltered, whilst few changes were measured in gamma-glutamyltranspeptidase (GTP), an enzymatic indicator of spermatogenesis. Contrary to other PAHs, hepatic ethoxyresorufin-O-deethylase (EROD) activities declined with increasing exposure concentration, suggesting that naphthalene was either having a cytotoxic effect or disrupting enzyme synthesis.

Pulp and paper mill effluent treatments have differential endocrine-disrupting effects on rainbow trout.

Endocrine disruption (ED) effects due to pulp and paper mill effluents extracts involving different industrial procedures and effluent treatments (nontreated, primary, and secondary treated) were evaluated using immature triploid rainbow trout in a pulse-exposure toxicity experiment. The protocol involved the use of intraperitoneal injection of mill extracts (solid-phase extraction [SPE]) corrected for individual fish weight and included several laboratory standards (steroidal hormones and phytosterols). Biological endpoints at two different levels of biological organization were analyzed (molecular and individual organism). Results indicated that nonsignificant changes were observed in the individual physiological indices represented by condition factor, liver somatic index, and gonad somatic index during the experiment. Significant induction of liver ethoxyresorufin-O-deethylase activity was observed between different effluent treatments and experimental controls. Significant endocrine-disrupting effects at the reproductive level were observed in all effluent treatments involving significant increments in plasma vitellogenin (VTG) levels. Fish exposed to untreated effluent extracts had significantly higher VTG levels compared to fish exposed to primary and secondary treatment effluent extracts, indicating a decrease of the estrogenic effect due to the effluent treatment. The present study has shown that for the Chilean pulp and paper mill SPE extracts evaluated, an endocrine disruption effect was induced in immature triploid rainbow, reaffirming the significant estrogenic effects demonstrated previously in laboratory and field experiments.

Multigeneration assessment of nonylphenol and endosulfan using a model Australian freshwater fish, Melanotaenia fluviatilis.

Changes over two generations in offspring and reproductive ability were recorded in crimson-spotted rainbowfish (Melanotaenia fluviatilis), a model Australasian freshwater fish, following a 24 h exposure to nominal nonylphenol concentrations of 50, 100, 500, 1000, 2250, and 5000 microg/L and following a 4 h exposure to nominal endosulfan concentrations of 1.0, 5.0, 10, 22, 33, and 50 microg/L. There were also four replicated control treatments: control 1 and 2 and solvent control 1 and 2, as well as "positive" female and male controls: 1 microg/L estradiol 1 and 2 and 1 microg/L testosterone 1 and 2. Results suggested that there may be some parental transfer of toxicants to embryos even over this short exposure period. Fertility of M. fluviatilis was reduced by a 24 h pulse exposure of adults to 100 microg/L nonylphenol and a 4 h exposure to 1.0 microg/L endosulfan. Hatch rates were significantly reduced after exposure to nonylphenol, endosulfan, and estradiol control but not in solvent controls and testosterone control. Significant correlations were found between reproductive and physiological parameters for nonylphenol and endosulfan exposed F0 adult rainbowfish. The major reproductive effects were on hatchability of the F1 generation and the gonadosomatic indices of male F1. The respective nominal NOEC and LOEC's for nonylphenol were 50 and 100 microg/L, and for endosulfan were <1.0 and 1 microg/L. These observed effects have the potential to significantly impact exposed rainbowfish populations through the observed approximately 45% reduction of hatchability and thus larval production.

Use of the Australian crimson-spotted rainbowfish (Melanotaenia fluviatilis) as a model test species for investigating the effects of endocrine disruptors.

Few studies have investigated the potential reproductive effects of toxicants on Australian freshwater fish species. The present study uses the Australian rainbowfish (Melanotaenia fluviatilis) as a model for testing the potential effects of 17beta-estradiol. Groups of reproductively active rainbowfish were exposed to waterborne 17beta-estradiol (control, carrier control, and 30, 100, 300, and 1,000 ng/L) for 3- and 14-d periods. Biomarkers of both low ecological relevance (plasma estradiol and testosterone, phosphoprotein, gamma-glutamyltranspeptidase [GTP]) and high ecological relevance (egg counts, hatchability, larval lengths, histopathology) were measured and the relationships between these examined. Relative to the controls, exposed female rainbowfish had a decline in plasma estradiol. An increase in phosphoprotein (vitellogenin) also was observed after exposure to 1,000 ng/L after 3 d, and histological examination of the gonads showed an increase in oocyte atresia at 1,000 ng/L on days 3 and 14. Changes in egg production were observed at 300 and 1,000 ng/L. No changes were detected to egg hatchability and larval lengths of offspring. Although there were no changes in male rainbowfish plasma testosterone or the histological organization of testes, levels of phosphoprotein increased and GTP activities were increased after 3 d of exposure at all test concentrations, but these declined at day 14. The present study demonstrated that, though measurements at the biochemical level were responsive to 17beta-estradiol exposure, fewer changes were observed in markers of higher ecological relevance at the exposures concentrations and durations tested.

The use of respiratory enzymes as biomarkers of petroleum hydrocarbon exposure in Mytilus edulis planulatus.

The effect of exposure to petroleum hydrocarbons via the water column and through contaminated sediment upon changes in respiratory enzymes in the common mussel (Mytilus edulis planulatus) was investigated. Mussels were exposed to three concentrations of the water-accommodated fraction (WAF) of Bass Strait crude oil, for 24, 48, and 96 h. In a second study mussels were exposed to three concentrations of crude oil-contaminated sediment for 2 weeks and 1, 2, 4 and 6 months. Activities of citrate synthase (CS) and lactate dehydrogenase (LDH) were measured in the gills. In mussels exposed to WAF, a significant decrease in CS activity was observed over time (P<0.05), whereas treatment did not cause a significant change in CS activity (P>0.05); neither treatment nor time had an effect on LDH activity. Exposure to contaminated sediment did not have a significant effect on CS activity, however, time had a significant effect on CS activity (P<0.05). Both time and treatment had an effect on LDH activity (P<0.05). Results demonstrated that changes in gill CS and LDH are not sensitive biomarkers of petroleum hydrocarbon exposure in M. edulis planulatus.

Hydrocarbon-induced changes to metabolic and detoxification enzymes of the Australian crimson-spotted rainbowfish (Melanotaenia fluviatilis).

The toxicity of petroleum hydrocarbons to marine aquatic organisms has been widely investigated; however, the effects on freshwater environments have largely been ignored. Selected biomarkers were measured in a freshwater species, the crimson-spotted rainbowfish (Melanotaenia fluviatilis). Fish were exposed to either a water-accommodated fraction (WAF) of crude oil or a dispersed crude oil water-accommodated fraction (DCWAF) for 3 days and were depurated for 14 days. Generally, biomarkers were altered following the short-term exposures but recovered after 14 days of depuration. Metabolic enzymes measured in gill tissue were citrate synthase and lactate dehydrogenase (LDH). As a result of WAF and DCWAF exposures, citrate synthase and LDH activities increased. Enzyme activities returned to control levels following depuration. Subsequent to the WAF exposure, hepatic ethoxyresorufin-O-deethylase (EROD) activity levels were higher than controls and they returned to control levels during depuration. For the DCWAF exposure, EROD was induced by a TPH (total petroleum hydrocarbons) concentration of 14.5 mg/L; however, after depuration the 14.5 mg/L TPH group had lower EROD activity than did controls. There were no changes in liver- to body-weight ratios or the histopathological organization of gill or liver tissues. As the majority of biomarkers returned to control levels after 14 days of depuration, rainbowfish were able to recover from short-term exposures to crude oil and dispersed crude oil.