Biological response of high-back crucian carp (Carassius auratus) during different life stages to wastewater treatment plant effluent

This study presents the adverse effects of endocrine disrupting chemicals (EDCs) in effluent of wastewater treatment plants (WwTPs) on fish health. A study of chronic exposure to WwTPs effluent for 10 months was undertaken in high-back crucian carp (Carassius auratus) during different life stages, covering early-life-stage (ELS), prespawning period, and postspawning period. Condition factor (CF), gonadosomatic index (GSI), hepatosomatic index (HSI), and plasma vitellogenin (VTG) levels were employed as indicators to assess biological effects of effluent on this gynogenesis species. Meanwhile, some high-back crucian carp were caged in Demonstration Base of Biological Purification for Filter-feeding Fish (hereinafter, Demonstration Base), as WwTPs effluent exposure controls. In the meantime, a depuration study was carried out to determine whether or not the estrogenic effects caused by effluent exposure could be reduced after moving fish into EDCs-free water. CF, HSI, GSI, and plasma Vtg levels of high-back crucian carp caged in Demonstration Base were generally in accordance with seasonal change. Effluent exposure inhibited gonadal growth, reducing GSI in ELS while increasing it around spawning, sharpened liver burdens, increasing HSI, and induced abnormal Vtg expression in juvenile high-back crucian carp, augmenting Vtg concentrations in plasma. Around spawning period, Vtg in high-back crucian carp were mainly induced by endogenous estrogens, and EDCs in effluent had less influence on them. Staying in EDCs-free water for 30 days made high-back crucian carp recover from effects of previous effluent exposure, relieving inhibition of gonadal development and hypertrophy of liver as well as reducing Vtg induced by EDCs in effluent. The results revealed that high-back crucian carp in ELS are more sensitive to WwTPs effluent exposure. Additionally, the depuration study showed a clearance of the estrogenic effects caused by effluent.     

Plasma vitellogenin in male teleost fish from 43 rivers worldwide is correlated with upstream human population size

It has been previously demonstrated that vitellogenin (VTG) - a precursor egg yolk protein - is produced in male fish exposed to estrogenic compounds in wastewater treatment plant (WWTP) effluent. However, little attention has been given to examine whether any patterns of male VTG production exists across fish species on a global scale. We hypothesized that a composite measure of human population size over river discharge would best explain variations of protein levels in male fish. We compiled VTG data in 13 fish species from 43 rivers receiving municipal WWTP effluent on 3 continents. We found that human population size explained 28% of the variation in male VTG concentrations, whereas population/flow rate failed to significantly correlate with VTG. We suggest this result may be explained by the low solubility of estrogenic compounds, resulting in localized contamination near WWTP outfalls, rather than dilution by river water.     

Histological alternation and vitellogenin induction in adult rare minnow (Gobiocypris rarus) after exposure to ethynylestradiol and nonylphenol

Adult rare minnow (Gobiocypris rarus) were exposed to 0, 1, 5, and 25 ng/l (nominal concentrations) of 17alpha-ethynylestradiol (EE2) and 3, 10, and 30 microg/l (nominal concentrations) of 4-nonylphenol (NP) under flow-through conditions for a period of 28 d. Low mortality was observed at 5 and 25 ng/l EE2 and the growth of fish reduced significantly at 25 ng/l EE2 compared to controls. However, the gonadosomatic indices (GSI) of male fish were significantly higher in 1 ng/l EE(2) treatments and in 10 and 30 microg/l NP treatments (p<0.05). Renal somatic indices (RSI) of male fish in EE2 treatments were significantly higher than those in controls (p<0.05). In contrast, significantly decreased GSI and RSI of female fish could only be observed in 5 and 25 ng/l EE(2) treatments (p<0.05). Hepatosomatic indices (HSI) of male fish were significantly higher in 25 ng/l EE(2) treatments. However, significantly increased of HSI of female fish could only be observed in 1 ng/l EE(2) treatments. Plasma vitellogenin (VTG) induction could be observed in males after exposed to different concentrations of EE2 and NP, and plasma VTG concentrations in females exposed to 5 and 25 ng/l EE(2) were also significantly higher than in controls (p<0.05). At level higher than 5 ng/l EE2 or 30 microg/l NP, hepatic tissue and renal tissue impairment of males could be observed. The pathological male liver was associated with a hypertrophy of hepatocytes and damages to cellar structure and accumulated eosinophilic material. Renal tissue showed different pathological effects which was reflected by accumulated eosinophilic material, hemorrhages within the kidney tubules and hypertrophy of the tubular epithelia. Also at these levels of exposure, feminization of male fish could be noticed and parts of males manifested the testis-ova phenomenon. Ovaries of female rare minnow in 25 ng/l EE2 treatment group were degenerated. Therefore when exposed to EE2 and NP even at environmental observed concentrations, adverse effects could occur in the reproductive system of adult fishes. The observed hepatic tissue and renal tissue impairment should be due to the induction and accumulation of VTG in organs, especially in males.     

Short-term responses of Oryzias latipes (Pisces: Adrianichthyidae) and Macrobrachium nipponense (Crustacea: Palaemonidae) to municipal and pharmaceutical waste water in Beijing, China: survival, behaviour, biochemical biomarkers

Whole effluent toxicity was assessed for the fish Oryzias latipes and the prawn Macrobrachium nipponense for 18 h in a dilution series (0-66%) of the inflow and effluent of a municipal waste water treatment plant as well as waste water from a teramycin producing pharmaceutical industry, before, during and after a pilot laboratory purification process. The waste waters caused acute toxicity as measured by inhibition of light emission in the luminiscent bacterium Vibrio qingaiensis sp. nov. (Q67). EROD and aryl hydrocarbon hydroxylase activity in in vitro carp liver-cells showed a dose-dependent toxic response to the municipal waste water. Behavioural responses and time-to-death of fish and prawn, recorded online with the "Multispecies Freshwater Biomonitor" proved to be concentration- and time-dependent sensitive toxicity indicators in both types of waste water. Behaviour changed stepwise from normal activity to (increased or decreased) activity to more time spent on ventilation and finally to increased morbidity at higher concentration and time of exposure. The municipal waste water treatment plant managed to reduce toxicity to bacteria (Q67), prawn and fish. The pharmaceutical waste water treatment process still has to be improved, in order to reduce toxicity for fish and prawn.     

Effects of long-term chlorpyrifos exposure on mortality and reproductive tissues of Banded Gourami (Trichogaster fasciata)

Abstract

This study assessed the long-term toxicity of chlorpyrifos on survival and reproduction of Banded Gourami by using mortality, gonado-somatic index (GSI) and histopathological observations as endpoints. Adult fish were exposed to five different concentrations of chlorpyrifos (0, 15, 50, 150, 500?µg/L) in 15 PVC tanks for 15, 30, 45, 60 and 75?days. Results showed that all male and female fish died after 15?days of 500?µg/L chlorpyrifos exposure. No consistent significant effect was observed for both male and female GSI. Furthermore, results showed dose- and time-dependent histopathological alterations for both ovary and testes. The 60-d No Observed Effect Concentration (NOEC) for most histopathological alterations of Banded Gourami ovary and testes was 50?μg/L, while 60-d NOEC for mortality of both male and female fish was < 15?μg/L. The results show that the long-term exposure to chlorpyrifos not only affect the reproductive tissues of Banded Gourami at exposure concentrations but also cause their mortality. Future studies should evaluate effects at lower concentrations.     

Vitellogenin gene expression in males of the Antarctic fish Trematomus bernacchii from Terra Nova Bay (Ross Sea): a role for environmental cadmium?

Synthesis of vitellogenin (VTG) in male fish is a widely recognized effect for estrogenic pollutants in temperate environments, while similar investigations are still lacking for Antarctic organisms. In this study, a preliminary characterization of vitellogenin gene expression was performed by RT-PCR in the key species Trematomus bernacchii sampled in different phases of reproductive cycle and food availability. Females exhibited the highest gene expression during the spawning period, but VTG mRNA was always detected also in males; a significant increase of gene expression was observed both in males and females at the end of the feeding season. These results were not fully supported by a differential exposure to phyto- or anthropogenic estrogens during the planctonic cycle; on the other side, the endocrine properties of cadmium, naturally elevated in Terra Nova Bay and increasing during algal bloom, would explain both the presence of VTG mRNA in males and the seasonal changes of gene induction. Laboratory exposures did not reveal short-term estrogenic effects of cadmium while an elevated responsiveness of T. bernacchii was observed toward a classical estrogenic receptor agonist (17beta-estradiol). Different hypotheses were considered to suggest delayed endocrine effects of cadmium, including the early interaction with other cellular detoxification systems or alterations at multiple levels of the hypothalamus-pituitary-gonad-liver axis. Although molecular mechanisms of VTG gene expression in males of T. bernacchii remain unclear, obtained results provide interesting insights on this species which should stimulate future research activities.     

Effects of environmental methylmercury on the health of wild birds, mammals, and fish

Wild piscivorous fish, mammals, and birds may be at risk for elevated dietary methylmercury intake and toxicity. In controlled feeding studies, the consumption of diets that contained Hg (as methylmercury) at environmentally realistic concentrations resulted in a range of toxic effects in fish, birds, and mammals, including behavioral, neurochemical, hormonal, and reproductive changes. Limited field-based studies, especially with certain wild piscivorous bird species, e.g., the common loon, corroborated laboratory-based results, demonstrating significant relations between methylmercury exposure and various indicators of methylmercury toxicity, including reproductive impairment. Potential population effects in fish and wildlife resulting from dietary methylmercury exposure are expected to vary as a function of species life history, as well as regional differences in fish-Hg concentrations, which, in turn, are influenced by differences in Hg deposition and environmental methylation rates. However, population modeling suggests that reductions in Hg emissions could have substantial benefits for some common loon populations that are currently experiencing elevated methylmercury exposure. Predicted benefits would be mediated primarily through improved hatching success and development of hatchlings to maturity as Hg concentrations in prey fish decline. Other piscivorous species may also benefit from decreased Hg exposure but have not been as extensively studied as the common loon.     

Assessing the biological status of fish in a river receiving pulp and paper mill effluents

This study compared the use of sentinel species- and community-based field approaches for assessing the biological status of fish living in a river receiving pulp and paper mill effluents. Three approaches were compared. Two approaches used sentinel species. One of these involved an internal/external examination of the fish that leads to the calculation of a fish health assessment index (HAI) and the other involved biochemical measurements of hepatic mixed function oxidase (MFO) activity and plasma steroid levels. The third approach characterized the fish community structure according to an index of biotic integrity (IBI). The comparison focused on how the methods respond to the hypothesis that recent process modifications/effluent treatment changes, resulting in demonstrable improvements in effluent quality, have beneficial effects on fish. Neither of the approaches using sentinel fish indicated clear mill-related influences either before or after the process modifications/effluent treatment changes. There was no evidence of depressed plasma steroids and increased MFO activity in fish frequently associated with mill effluent exposure in previous studies. While the HAI was higher at stations downstream from two mills, this could not be linked to effluent exposure alone. In contrast, the study of community structure showed a substantial improvement in fish assemblages at all the mill sites.     

Distribution of antidepressants and their metabolites in brook trout exposed to municipal wastewaters before and after ozone treatment--evidence of biological effects

This study examined the tissues distribution of selected serotonin reuptake inhibitors (SSRIs) in brook trout exposed for 3 months to continuous flow-through primary-treated effluent before and after ozone treatment. A reliable analytical method was developed for the quantification of trace amounts of antidepressants in small tissue homogenate extracts. Levels of six antidepressants and four of their N-desmethyl metabolites were determined using liquid chromatography-tandem mass spectrometry. Significant amounts of the SSRIs were found in fish tissue-in decreasing order: liver>brain>muscle. Sertraline and its metabolite desmethylsertraline were the predominant substances observed in most tissues (0.04-10.3 ng g(-1)). However, less SSRIs (0.08-1.17 ng g(-1)) were bioaccumulated in the ozonated effluent. The early molecular effects of these SSRIs on the Na/K-dependent ATPase pump activity in brain synaptosomes where also investigated in vitro and in fish exposed to the municipal effluents. With respect to their potential biological effects, in vitro exposure to selected SSRIs induced a reduction of the brain Na/K-ATPase activity in synaptosomes in a dose-dependent manner. Results showed that Na/K-ATPase activity was readily inhibited by exposure to municipal effluent before and, to a lesser extent, after ozone treatment. Moreover, the Na/K-ATPase activity was significantly and negatively correlated with brain tissue concentrations of fluoxetine (r=-0.57; p<0.03), desmethylsertraline (r=-0.84; p<0.001), and sertraline (r=-0.82; p<0.001). The present study reveals that SSRIs are readily available in fish, biologically active and corroborates previous findings on the serotonergic properties of municipal effluents to aquatic organisms.     

Toxicity of herbal pharmaceutical wastewater on fish--Lebistes reticulatus (Peter)

Toxic effects of herbal pharmaceutical wastewater on common fish (Lebistes reticulatus) under laboratory conditions were studied. LC50 values for raw, neutralized, and physico-chemically treated effluent for 24, 48, 72, and 96 h ranged between 3.0-9.5%, 5.0-10.5%, and 35-41%, respectively. It was evident from the studies that physico-chemical treatment reduces the toxicity by more than 30%. This article discusses in detail the bioassay evaluation of raw, neutralized, and primary treated effluent. Physico-chemically treated effluent was further subjected to biological treatment by activated sludge system. Effluent from biological treatment revealed no toxic effect even after one month of exposure.     

Effects of waterborne exposure to 4-nonylphenol on plasma sex steroid and vitellogenin concentrations in sexually mature male carp (Cyprinus carpio)

4-Nonylphenol (NP) has been shown to elicit estrogenic responses both in vivo and in vitro. The mechanism by which NP exerts estrogenic and other endocrine-modulating effects in vivo remains unclear, however. The goal of this study was to evaluate the ability of NP to elicit estrogenic responses through indirect mechanisms of action involving the modulation of endogenous steroid hormone concentrations. Sexually mature male common carp (Cyprinus carpio) were exposed to aqueous NP concentrations ranging from <0.05 to 5.4 microg NP/l for 28-31 d. Approximately 0.5-3.5 ppm of NP was detected in pooled plasma samples or tissue samples from the carp studied. NP exposure did not significantly increase plasma concentrations of 17beta-estradiol (E2), testosterone (T) or vitellogenin (VTG). Excluding outliers, plasma E2 concentrations ranged from <175 to 700 pg E2/ml. T concentrations ranged from 940 to 24,700 pg T/ml plasma. The greatest VTG concentration detected was 52 microg/ml. One-third of the plasma samples tested contained <1 microg VTG/ml. Overall, the results of this study did not support the hypothesis that exposure to waterborne NP can modulate concentrations of steroid hormones in the plasma of sexually mature male carp. The results did, however, raise a number of questions regarding the utility of estradiol equivalent (EEQ) estimates as a means of predicting in vivo effects of estrogenic substances. Furthermore, they provide information regarding the concentrations and variability of E2, T, and VTG in the plasma of sexually mature male carp, which may aid in design and interpretation of future studies.     

Suppression of sexual behavior in male Japanese medaka (Oryzias latipes) exposed to 17beta-estradiol

Mating pairs of Japanese medaka (Oryzias latipes) received 17beta-estradiol (E2; dose, 3 and 30 microg/g body weight daily) in their diet and E2-free diet (control) for two weeks. The effect on reproductive success was assessed in the mating pairs, and sexual and nonsexual behaviors in the male were quantified. The fecundities of the E2-treated groups were reduced significantly (p < 0.05) compared with those of control, and the sexual behaviors (following, dancing, floating, and crossing) of male fish in response to prostaglandin F2alpha (PG)-injected E2-unexposed females were suppressed dramatically. While the fertility of spawned eggs and the frequencies of the nonsexual behaviors of resting and swimming in a straight line performed by male were unchanged in E2-exposed groups. Therefore, sexual behavior in male may be linked with reproductive success in mating pair of madaka exposed to E2. This assay for sexual behavior using PG may provide a useful tool for assessing the effect of endocrine-disrupting chemicals.     

Effects of distillery waste on the performance of Cicer arietinum L

The impact of distillery effluent in various concentrations (1, 2.5, 5, 10, 25, 50, 75 and 100%) on the seed germination, Speed of Germination Index, growth behaviour, leaf area, biomass, net primary productivity, pigment content, reproductive capacity, seed output, seed weight, seed density and the seed protein content of Cicer arietinum L. plants was investigated. The percentage and speed of germination of seeds were increasingly retarded with increase in effluent concentration and at 100% concentration there was no germination. The seedlings exhibited maximum shoot length at 5% concentration and maximum root length at 2.5% concentration. The values of root and shoot lengths, leaf area, biomass, net primary productivity, pigment content, reproductive capacity, seed output, seed weight, seed density and seed protein content in pot plants exhibited a gradual increase from the control up to 5% concentration and decreases at higher concentrations. The very high BOD load and the presence of excessive concentrations of soluble salts could be responsible for the toxicity of the effluent. The effluent at up to 5% concentration was, however, beneficial for the overall growth parameters studied and can thus be used as a liquid fertilizer.     

Trophic transfer of pyrene metabolites and nonextractable fraction from Oligochaete (Lumbriculus variegatus) to juvenile brown trout (Salmo trutta)

Cell lines of Etroplus suratensis established in our laboratory were evaluated for their potential use as screening tools for the ecotoxicological assessment of tannery effluent. The cytotoxic effect of tannery effluent in three cell lines derived from eye, kidney and gill tissue of E. suratensis was assessed using multiple endpoints such as Neutral Red (NR) assay, Coomassie Blue (CB) protein assay and Alamar Blue (AB) assay. Acute toxicity tests on fish were conducted by exposing E. suratensis for 96 h to tannery effluent under static conditions. The toxic effect of tannery effluent on the survival of fish was found to be concentration and time dependent. The tannery effluent at the concentration of 15% caused 100% mortality at 96 h whereas the lower concentration (0.5%) caused 13.33% mortality. The cytotoxicity of tannery effluent was found to be similar in the three cell lines tested, independent of the toxic endpoints employed. EC₅₀ values, the effective concentration of tannery effluent resulting in 50% inhibition of cytotoxicity parameters after 48 h exposure to tannery effluent were calculated for eye, kidney and gill cell lines using NR uptake, AB and cell protein assays. Statistical analysis revealed good correlation with r² = 0.95-0.99 for all combinations between endpoints employed. Linear correlations between each in vitro EC₅₀ and the in vivo LC₅₀ data, were highly significant p < 0.001 with r² = 0.977, 0.968 and 0.906 for AB₅₀, NR₅₀, and CB₅₀, respectively.     

Evaluation of reproductive safety of beta-sitosterol on the American mink (Neovison vison)

beta-Sitosterol is a weakly estrogenic phytosterol used extensively in functional foods to lower elevated serum cholesterol concentrations due to its inhibitory action on intestinal cholesterol absorption. It caused previously decreased sex steroid concentrations in fish and lowered sperm counts in rats. In the American mink (Neovison vison), litter size increased slightly due to dietary beta-sitosterol supplement. The aim of the present experiment was to conduct a dose-response study on the effects of beta-sitosterol on the reproduction of the American mink. Juvenile male and female mink (n=480) were exposed to 0, 5, 10 or 50 mg of peroral beta-sitosterol kg(-1)d(-1) for 10 months. After 3 months of exposure in November, 15 males per group were sacrificed and general biochemical variables reflecting overall health were determined. The beta-sitosterol-treated male mink had increased absolute and relative masses of intraabdominal fat and higher blood hemoglobin and serum high-density lipoprotein cholesterol concentrations. In spring, the top-rated male mink were mated with multiple females within each study group and reproductive success was assessed. No differences in the reproductive performance of the males (10-11 per group) or females (47-50 per group) could be detected in the exposed groups and the kits of all groups developed in a similar manner. The results suggest that dietary beta-sitosterol presents no significant risk to mammalian fertility.     

Assessing the genotoxic potentials of arsenic in tilapia (Oreochromis mossambicus) using alkaline comet assay and micronucleus test

This experiment was conducted to study the genotoxic potentials of sodium arsenite (NaAsO₂) in freshwater fish Oreochromis mossambicus by using alkaline comet assay and micronucleus (MN) test. Fish were exposed to three different concentrations (3 ppm, 28 ppm and 56 ppm) of arsenic and gill, liver and blood tissue samples were collected after 48 h, 96 h and 192 h of exposure. Arsenic exposure induced DNA damage in all tissues examined in a concentration dependent manner. A significant (p < 0.05) increase in the comet tail DNA (%) of the exposed fish liver, gill, and blood was observed after 48 h and 96 h of exposure, but a decline in DNA damage was recorded in all the tissues at all the three concentrations studied after 192 h of exposure. Liver tissue exhibited significantly (p < 0.05) higher DNA damage at all the concentrations examined, followed by gill and blood. Higher liver tail DNA (51.38 ± 0.21%) refers that it is more prone to injury to arsenic toxicity than the gill and blood. In blood samples arsenic induced micronucleus formation in a concentration dependent manner and highest (5.8 ± 0.46%) value was recorded in 56 ppm after 96 h of exposure, whereas, it was decreased after 192 h of exposure at all the three concentrations of NaAsO₂ examined which refers to the DNA repairing ability of fish to arsenic toxicity. The results of this study depict the genotoxic potentials of arsenic to fish which in turns provide insight on advanced study in aquatic toxicology.     

Toxicity of leather tanning wastewater effluents in sea urchin early development and in marine microalgae

This study was designed to investigate the composition and the toxicity of leather tanning wastewater and conditioned sludge collected at the leather tanning wastewater treatment plant (CODISO) located in Solofra, Avellino (Southern Italy). Samples were analyzed for their conventional parameters (COD, TSS, chromium and ammonia) and for metal content. Effluent samples included raw wastewater, and samples collected following coagulation/flocculation process and biological treatment. A set of toxicity endpoints were tested using sea urchin and marine microalgal bioassays by evaluating acute embryotoxicity, developmental defects, changes in sperm fertilization success and transmissible damage from sperm to the offspring, and changes in algal growth rate. Dose-related toxicity to sea urchin embryogenesis and sperm fertilization success was exerted by effluent or sludge samples according to the following rank: conditioned sludge > coagulated effluent > or = raw influent > effluent from biological treatment. Offspring quality was not affected by sperm exposure to any wastewater or to sludge samples. Algal growth was inhibited by raw or coagulated effluent to a similar extent and, again, the effluent from the biological treatment resulted in a decreased toxicity. The results suggest that coagulated effluent and conditioned sludge result in higher toxicity than raw influent in sea urchin embryos and sperm, whereas the biological wastewater treatment of coagulated effluent, in both sea urchins and algae, cause a substantial improvement of wastewater quality. Hence a final biological wastewater treatment should be operated to minimize any environmental damage from tannery wastewater.