Multiple biomarkers responses in Prochilodus lineatus allowed assessing changes in the water quality of Salado River basin (Santa Fe, Argentina)

This field study assessed water quality of Salado River basin by using a set of biomarkers in the fish Prochilodus lineatus. Multiple biomarkers were measured, including morphological indexes (condition factor, liver somatic index), hematological (red and white blood cells) and biochemical (glucose, total protein and cholinesterase activity) parameters. Besides, detoxication and oxidative stress markers (antioxidant enzymes, lipid peroxidation) were measured in liver, gills and kidney. Despite water quality assessment did not show marked differences among sites, biomarkers responses indicate that fish are living under stressful environmental conditions. According to multivariate analysis glucose, glutathione S-transferase activity, lipid peroxidation levels and the count of white blood cells are key biomarkers to contribute to discrimination of sites. So, we suggest use those biomarkers in future monitoring of freshwater aquatic systems.     

Temporal induction pattern of hepatic cytochrome P450 1A in thermally acclimated dab (Limanda limanda) treated with 3,3′,4,4′-Tetrachlorobiphenyl (CB77)

Mature male dab (Limanda limanda) acclimated at 10° and 16°C were orally administered a single dose of 0.5 mg/kg 3,3′,4,4′-tetrachlorobiphenyl (CB77). At both temperatures, levels of cytochrome P450 1A (CYP1A) protein and 7-ethoxyresorufin O-deethylase (EROD) activity showed a two to six fold induction 40 days after CB77 treatment compared to control groups. Maximum responses of both EROD activity and CYP1A protein for the warm-acclimated fish were observed at 5 days after treatment. For the cold-acclimated fish a slow, progressive elevation for both EROD activity and CYP1A protein was observed and maximum responses were measured 40 days after treatment. Absolute EROD activity and CYPIA protein levels of fish from both temperatures were equally high at 40 days after treatment. Since in the control groups EROD activity and CYP1A protein levels were higher in the cold-acclimated fish, the magnitude of induction was higher in the warm acclimated ones. The highest concentrations of CB77 in muscle of fish from both temperatures were found at 5 and 10 days after treatment. The liver somatic index (LSI) showed 1.5 fold significantly higher values for the fish acclimated at 10°C.     

The changes in common carp blood after short-term zinc exposure

Blood zinc level, hematological parameters and blood cell morphology were evaluated in common carp immediately after 3 h exposure to 20 mg dm(-3) of zinc (Zn0), and in 24, 48 and 96 hours after the end of it (Zn24, Zn48, Zn96). Blood zinc level in the non-exposed fish was 8 mg dm(-3), reached a maximum of 20 mg dm(-3) in Zn48, while it dropped to 9 mg dm(-3) in Zn96. Zinc caused a stress reaction in fish indicated by an increase in hematocrit value in Zn0, and elevated plasma glucose level and trombocytosis which persisted until the end of the experiment. Zinc-exposed fish showed an increased frequency of abnormal erythrocytes, and a compensatory release of immature erythrocytes to the blood stream. In zinc-treated fish, leukocyte count initially increased and subsequently decreased significantly below the control level due to a drop in lymphocyte number. Lymphocyte viability was reduced, and abnormal lymphocytes appeared. A decreased count of juvenile neutrophiles, and reduced phagocyte activity also occurred. The results indicate possible zinc-induced disturbances in both specific and non-specific immune mechanisms.     

Effect of organophosphorus (dimethoate) and pyrethroid (deltamethrin) pesticides on semen characteristics in rabbits

The present study was undertaken to determine the effect of chronic treatment with two sublethal doses of Dimethoate (organo-phosphorus) or Deltamethrin (pyrethroid) on body weight and semen characteristics in adult male rabbits. Pesticide treatment resulted in a decline in body weight, libido, ejaculate volume, sperm concentration and semen initial fructose; and an increase in abnormal and dead sperm and methylene blue reduction time. In this regard Dimethoate showed greater effects than Deltamethrin. The hazardous effect of these pesticides on semen quality continued during the post-treatment period, and was dose-dependent. This deleterious effect on sperm formation together with the decline in libido suggest a decrease in testosterone secretion by pesticide treatment.     

The mutagenic effect of tobacco smoke on male fertility

Despite the association between tobacco use and the harmful effects on general health as well as male fertility parameters, smoking remains globally prevalent. The main content of tobacco smoke is nicotine and its metabolite cotinine. These compounds can pass the blood-testis barrier, which subsequently causes harm of diverse degree to the germ cells. Although controversial, smoking has been shown to cause not only a decrease in sperm motility, sperm concentration, and an increase in abnormal sperm morphology, but also genetic and epigenetic aberrations in spermatozoa. Both animal and human studies have highlighted the occurrence of sperm DNA-strand breaks (fragmentation), genome instability, genetic mutations, and the presence of aneuploids in the germline of animals and men exposed to tobacco smoke. The question to be asked at this point is, if smoking has the potential to cause all these genetic aberrations, what is the extent of damage? Hence, this review aimed to provide evidence that smoking has a mutagenic effect on sperm and how this subsequently affects male fertility. Additionally, the role of tobacco smoke as an aneugen will be explored. We furthermore aim to incorporate the epidemiological aspects of the aforementioned and provide a holistic approach to the topic.

     

Cytotoxicity,redox and immune status in African catfish,Clarias gariepinus (Burchell, 1822) exposed to bisphenol A (BPA) and its analogues

The objective of the study was to determine the comparative toxicities and immune dysfunction in the African catfish, Clarias gariepinus, exposed to bisphenol A (BPA) and its two analogues: bisphenol AP (BPAP) and bisphenol P (BPP). Juveniles of C. gariepinus were exposed to sublethal concentrations (70 and 140 μg/L) of BPA, BPAP and BPP for 7, 14 or 21 days after which various endpoints which are indicative of cytotoxicity, oxidative stress and haematological and innate immune parameters were determined in the liver homogenates or blood plasma. The exposure of C. gariepinus to BPA and its analogues caused significant increased activities of lactate dehydrogenase, catalase and superoxide dismutase. The exposed fish had increased levels of DNA fragmentation, lipid peroxidation, white blood cells, nitric oxide and respiratory burst, while the red blood cell counts and the percentage packed cell volume decreased significantly in the exposed fish compared to control. The toxic effects elicited by the bisphenols were both concentration- and duration-dependent. Generally, BPA exerted the most toxic effects on the fish, followed by BPAP, while BPP exerted the least toxic effects to C. gariepinus. Summarily, the findings indicated that BPA and its two analogues studied in the research are capable of causing cytotoxicity, oxidative stress and immune dysfunction in C. gariepinus.

     

PAH Metabolites in Bile Fluids of Dab (Limanda limanda) and Flounder (Platichthys flesus): Spatial Distribution and Seasonal Changes (7 pp)

Background, Aim and Scope Polycyclic aromatic hydrocarbons (PAH) are important environmental contaminants which may lead to increased levels of neoplastic aberrations or tumours in fish liver. Therefore, monitoring of PAH and their effects are part of several international environmental programmes. The aim of the present field study was to investigate the concentrations of the PAH metabolites in fish bile, to elucidate spatial, seasonal and species differences as well as to discuss different strategies of normalisation with regard to environmental monitoring. Materials and Methods: PAH metabolites were determined in the bile fluid of dab (Limanda limanda) and flounder (Platichthys flesus) caught in the North Sea and Baltic Sea between 1997 and 2004. After enzymatic deconjugation, two metabolites were determined by means of HPLC. The limit of detection and the limit of quantification were calculated. The accuracy of the method was tested with a standard reference material. Results were referred to bile volume as well as to biliverdin. Results: The main metabolite, 1-hydroxypyrene, was determined in concentrations from <0.7 to 838 ng/ml in bile of dab (Limanda limanda) and flounder (Platichthys flesus) caught between 1997 and 2004. The values for 1-hydroxyphenanthrene in fish bile were considerably lower (<0.4 – 87 ng/ml). Significant differences in the 1-hydroxypyrene levels were found between summer and winter surveys as well as between the sampling sites in the data set from 2004 (383 dabs and 62 flounders): Highest levels of PAH contamination were found in dab from the German Bight and in flounder from the Baltic Sea. Discussion: Spatial differences in 1-hydroxypyrene concentrations between North Sea and Baltic Sea were discussed, as well as differences in relation to season, sex and species. Three parameters of normalisation (biliary protein, biliverdin and bile pigments) were discussed. Biliverdin was identified as a suitable parameter for the normalisation of PAH metabolites in field samples. Conclusions: Spatial differences in 1-hydroxypyrene concentrations of dab demonstrate the usefulness of PAH metabolites in fish bile as a monitoring parameter in marine regions. Significant differences in 1-hydroxypyrene concentrations were found between summer and winter sampling campaigns. This may be linked to an annual cycle of 1-hydroxyprene in dab. It is also possible that bile synthesis/release in dab differs between the seasons. There is no indication for a time trend from 1997 to 2004. Recommendations and Perspectives: It is recommended to relate PAH metabolites in fish bile to biliverdin concentrations. Although the concentrations are low in offshore regions and bile volumes are small, the method presented here allows one to measure PAH metabolites on an individual level which is a crucial prerequisite for meaningful monitoring studies.     

Uranium bioaccumulation and biological disorders induced in zebrafish (Danio rerio) after a depleted uranium waterborne exposure

Because of its toxicity and its ubiquity within aquatic compartments, uranium (U) represents a significant hazard to aquatic species such as fish. In a previous study, we investigated some biological responses in zebrafish either exposed to depleted or to enriched U (i.e., to different radiological activities). However, results required further experiments to better understand biological responses. Moreover, we failed to clearly demonstrate a significant relationship between biological effects and U radiological activity. We therefore chose to herein examine U bioaccumulation and induced effects in zebrafish according to a chemical dose-response approach. Results showed that U is highly bioconcentrated in fish, according to a time- and concentration-dependent model. Additionally, hepatic antioxidant defenses, red blood cells DNA integrity and brain acetylcholinesterase activity were found to be significantly altered. Generally, the higher the U concentration, the sooner and/or the greater the effect, suggesting a close relationship between accumulation and effect.     

Genotoxic effect of copper on salivary gland polytene chromosomes of Chironomus riparius Meigen 1804 (Diptera, Chironomidae)

The genotoxic action of copper (Cu) on the polytene chromosomes of Chironomus riparius was investigated by analysing structural and functional chromosome aberrations of fourth instars larvae hatched from eggs subject to acute (48 h) exposure with three environmentally relevant concentrations of aqueous Cu (0.005, 0.01, 0.05 mg/l). A dose dependent relationship was observed between Cu concentration and frequency of chromosomal aberrations. A significantly higher frequency of functional alterations, specifically decondensed centromeres and telomeres, and reduction in activity of Balbiani rings, was observed in treated material compared to control. A comparison of breakpoints resulting from treatment with chromium and lead from earlier studies with those Cu-induced identified a series of chromosomal weak points particularly vulnerable to trace metals. We also show that the appearance of structural and functional chromosome aberrations are more sensitive indicators of acute Cu toxicity in chironomid larvae than changes in external morphology.     

Vitellogenin content in European eel (Anguilla anguilla) in Flanders, Belgium

As part of a large-scale monitoring program of bioaccumulating contaminants in the European eel (Anguilla anguilla) in Flanders (Belgium), we investigated potential effects of xenoestrogens in these fish. The present paper describes the results of the plasma vitellogenin (VTG) content, measured in 142 eels sampled at 20 different locations, in relation to the internal pollution levels. To validate the blood VTG assays, a small number of eels (n=8) was exposed to 10 microg ethinylestradiol/l (EE2) for 9 days. In this experiment, VTG was detected as a protein with a molecular weight of 214 kDa and confirmed by Western blotting. Compared with the solvent controls, significantly higher concentrations of VTG were measured in EE2 exposed eel. However, the VTG content was relatively low compared with other fish species exposed to high concentrations of estrogens. The plasma VTG content of eels from the field study was very low, despite a very high internal load of endocrine disrupters. These results, together with previously published studies, suggest that immature yellow European eel might not be the best sentinel species to study the effects of estrogenic compounds on VTG levels of wild fish populations.     

A comparative study on characterization of textile wastewaters (untreated and treated) toxicity by chemical and biological tests

Toxicity of textile wastewaters (untreated and treated) and their ingredient chemicals was quantified in terms of their chemical characteristics, fish (Gambusia affinis) mortality and end point growth responses of duckweed (Lemna aequinoctialis) in short-term bioassays. Other parameters of fish bioassay were erythrocyte morphology and its counts. Despite of a definite correlation between data of biological tests (LC/EC(50) values) with that of chemical tests, biological tests were found to be relatively more sensitive to both wastewaters and ingredient chemicals. Amongst all the examined parameters of test organisms, fish RBCs (morphology and counts) sensitivity to pollutants in the wastewaters was usually maximum and therefore, their study should be included in the routine fish bioassay. Other advantage of biological test such as on Lemna is even detection of eutrophic potential of wastewaters, as noted at their higher dilutions. The ingredient chemicals (major) contributing maximum toxicity to textile dye wastewater were, acids (HCl and H(2)SO(4)), alkali (Na(2)O SiO(2)), salt (NaNO(2)) and heavy metal (Cu), whereas dyes (4) were relatively less toxic.     

Prenatal and postnatal exposure to diazinon and its effect on spermatogram and pituitary gonadal hormones in male offspring of rats at puberty and adulthood

The objective of this research is to study the possible reproductive adverse effects of diazinon on rat offspring exposed in utero and during lactation. Twenty-four Sprague–Dawley female rats (10–12 week old) were randomly assigned to four groups, each consisting of six rats. Group 1 served as the control and these rats were given normal saline orally. Rats in groups 2, 3, and 4 were administered diazinon, dissolved in saline at 10, 15, 30 mg/ kg^?1 body weight, per oral, once daily, during mating, pregnancy and lactation. The male offsprings were examined at puberty and adulthood for body weight, testis weight, epididymis weight, sperm count, motility and morphology, pituitary-gonadal hormone levels. At 30 mg kg^?1 dose, the male offsprings showed a decrease in testicular weight, sperm count, motility, with an increase in abnormal sperm percentage and a decline in pituitary-gonadal hormones, at puberty. Upon attaining adulthood, there was a decrease in testicular weight, sperm count and motility with an increase in abnormal sperm percentage and a decrease in pituitary hormone level. There was evidence of some adverse reproductive effects on the male offspring at the 15 mg/ kg^?1 dose. Most of the adverse effects were irreversible and were evident at both puberty and adulthood in the offsprings, although a few parameters reverted to the normal growth pattern. Diazinon is a reproductive toxicant for male offsprings if exposed during prenatal and postnatal phases.     

Upgrading food wastes by means of bromelain and papain to enhance growth and immunity of grass carp (<Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis>)

The fast growing of global aquaculture industry accompanied with increasing pressure on the supply and price of traditional feed materials (e.g., fish meal and soy bean meal). This circumstance has urged the need to search alternative sources of feed stuff. Food waste was used as feed stuff in rearing fish which possess substantial protein and lipid. Grass carp are major species reared in Hong Kong with lower nutritional requirements; it is also an ideal species for investigating the feasibility of using food waste as fish feeds for local aquaculture industry. The growth and immunity, reflected by total protein, total immunologlobulin (IgI), and nitroblue tetrazolium (NBT) activity of grass carp blood, were depressed when feeding with food waste feeds without enzymes. However, the supplementation of bromelain and papain in fish feed enhanced the efficient use of food waste by grass carp, which in turn improved the fish immunity. The present results indicated that the addition of those enzymes could enhance the feed utilization by fish and hematological parameters of grass carp, and the improvement on growth and immunity superior to the control (commercial feed) was observed with the addition of bromelain and papain supplement. Addition of 1 and 2 % mixture of bromelain and papain could significantly enhance the lipid utilization in grass carp.     

Contamination of the River Ganga and its toxic implication in the blood parameters of the major carp Labeo rohita (Ham)

A field study was conducted to examine different physicochemical properties of water and various haematological and biochemical parametres of the fish Labeo rohita collected from the Ganga River (National river of India) at Varanasi district, India. The water was found to be greatly contaminated with a number of dissolved metals (Fe, Cr, Zn, Cu, Mn, Ni and Pb) whose concentrations were above the safe limits suggested by Bureau of Indian Standard (BIS 1991) for drinking water (Fe, 1,353.33 %; Cr, 456 %; Mn, 553.33 %; Ni, 4,490 % and Pb, 1,410 %). The metal accumulation in the fish blood was very high (Fe, 2,408 %; Cr, 956.57 %; Zn, 464.90 %; Cu, 310.57 %; Mn, 1,115.48 %) in comparison to the control fish maintained under strict quality control. Lower values of the various haematological parameters (total erythrocytes count, haemoglobin, haematocrit, mean corpuscular volume and O₂-carrying capacity) in the river fish in comparison to the control indicate toxic manifestation exerted by the contaminated river water on the fish. The higher level of total leucocytes count further illustrates stressed condition of the river fish. The toxic impact of the Ganga water is also expressed in the fish by the presence of higher levels of cholesterol, glucose, elevated activities of the enzymes aspartate amino transferase and alanine amino transferase, and lowered protein concentration.     

Environmental risk appraisement of disinfection by-products (DBPs) in plant model system: Allium cepa

The organic toxicants formed in chlorinated water cause potential harm to human beings, and it is extensively concentrated all over the world. Various disinfection by-products (DBPs) occur in chlorinated water are genotoxic and carcinogenic. The toxicity is major concern for chlorinated DBPs which has been present more in potable water. The purpose of the work was to evaluate genotoxic properties of DBPs in Allium cepa as a plant model system. The chromosomal aberration and DNA laddering assays were performed to examine the genotoxic effect of trichloroacetic acid (TCAA), trichloromethane (TCM), and tribromomethane (TBM) in a plant system with distinct concentrations, using ethyl methanesulfonate (EMS) as positive control and tap water as negative control. In Allium cepa root growth inhibition test, the inhibition was concentration dependent, and EC₅₀ values for trichloroacetic acid (TCAA), trichloromethane (TCM), and tribromomethane (TBM) were 100 mg/L, 160 mg/L, and 120 mg/L respectively. In the chromosome aberration assay, root tip cells were investigated after 120 h exposure. The bridge formation, sticky chromosomes, vagrant chromosomes, fragmented chromosome, c-anaphase, and multipolarity chromosomal aberrations were seen in anaphase–telophase cells. It was noticed that with enhanced concentrations of DBPs, the total chromosomal aberrations were more frequent. The DNA damage was analyzed in roots of Allium cepa exposed with DBPs (TCAA, TCM, TBM) by DNA laddering. The biochemical assays such as lipid peroxidation, H₂O₂ content, ascorbate peroxidase, guaiacol peroxidase, and catalase were concentration dependent. The DNA interaction studies were performed to examine binding mode of TCAA, TCM, and TBM with DNAs. The DNA interaction was evaluated by spectrophotometric and spectrofluorometric studies which revealed that TCAA, TCM, and TBM might interact with Calf thymus DNA (CT- DNA) by non-traditional intercalation manner.

     

EROD and CYP1A protein in channel catfish (Ictalurus punctatus) from an urban estuary relative to that in benzo[a]pyrene-exposed hatchery specimens

Bottom-feeding fish such as flounder and killifish have been widely used in monitoring hepatic monooxygenase induction in polluted water bodies. While channel catfish are often utilized in tissue monitoring of fresh and estuarine water bodies, few data are available on their use in environmental monitoring of hepatic monooxygenase activity. In this project, the presence of CYP1A protein was verified in channel catfish through recognition by Mab 1-12-3, an antibody which recognizes the CYP1A homologue in a variety of teleost species. CYP1A protein levels and 7-ethoxyresorufin-o-deethylase (EROD) activity in laboratory control and benzo-a-pyrene (BaP)-challenged channel catfish were compared to those in feral channel catfish from Back River, an urban estuarine tributary to Chesapeake Bay. Though more variable, mean CYP1A protein levels in the field-collected fish were similar to those of the BaP-induced laboratory fish. However, EROD activity of the Back River fish was less than one half that observed in the BaP-induced laboratory fish. When normalized to CYP1A protein levels, EROD activity was slightly lower in the Back River fish than either the laboratory control or BaP-treated fish. This finding may indicate possible inhibition or inactivation of the CYP1A protein in the feral fish.     

Di(2-ethylhexyl)phthalate and mono(2-ethylhexyl)phthalate in media for in vitro fertilization

In vitro fertilization (IVF) is one of the most important treatments of infertility to provide a chance of conceiving. In IVF treatment, sperm are washed and motile sperm are isolated with sperm washing media (SWM) for the purpose of fertilization; fertilized ova are then incubated for a maximum of 5 or 6 d in media for IVF (IVFM). The exposure of fertilized ova to chemicals via such media has not been studied. We determined the concentrations of two contaminants; di(2-ethylhexyl)phthalate (DEHP) and its hydrolyzed product mono(2-ethylhexyl)phthalate (MEHP) in IVFM, SWM, and protein sources (PS: human serum albumin or serum substitute) for IVFM and SWM. The DEHP and MEHP in these media were extracted by a liquid-liquid extraction method and their concentrations determined by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Fifteen IVFM, nine SWM, and six PS obtained in Japan were examined. The concentrations of DEHP and MEHP in IVFM and SWM were <10-114 and <2.0-263 ng mL⁻¹, respectively. The concentrations of both DEHP and MEHP were higher in the media containing PS than in those without PS. Either MEHP alone or both DEHP and MEHP were detected in PS. The concentrations of DEHP and MEHP in PS were <10-982 and 47.0-1840 ng mL⁻¹, respectively. The DEHP and MEHP detected in these media were derived from PS. This is the first study on the chemical contamination of IVFM, SWM, and PS.     

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.