A protocol for the evaluation of genotoxicity in bile of carp (Cyprinus carpio) exposed to lake water treated with different disinfectants

A sensitive and rapid method to evaluate toxic and genotoxic properties of drinking water supplied from Lake Trasimeno (Umbria, Central Italy) was worked out analysing bile in Cyprinus carpio exposed for 20 d to lake water treated with 3 different disinfectants, sodium hypochlorite (NaClO), chlorine dioxide (ClO₂) and peracetic acid (PAA). Fish were sacrificed at 0, 10 and 20 d in order to investigate the time course of these endpoints. An aliquot of bile samples was fractionated by adsorption on C₁₈ silica cartridges and the genotoxic potential of whole bile and of bile fractions was evaluated by the single-cell microgel-electrophoresis (comet) assay on human colonic adenocarcinoma cells (Caco-2). Bile (both whole and fractionated) from specimens exposed to the three disinfectants always showed a genotoxic activity as compared to the control group. The results of this study provide evidence that all three disinfectants cause an increase in bile genotoxicity of chronically exposed fish.     

Effects of concentrated drinking water injection on glutathione and glutathione-dependent enzymes in liver of Cyprinus carpio L

Two drinking water production plants located in North Italy, collecting water from the River Po (Plants 1 and 2) were chosen for this study. Water samples were collected before and after the disinfection process and at two points along the piping system. Water samples were concentrated by the solid-phase extraction system and injected intraperitoneally into specimens of Cyprinus carpio. The concentration of water samples was 3 l/equiv. In order to assess the effects of the water samples on carp liver, total glutathione and glutathione-dependent enzymes, such as glutathione S-transferase, glutathione peroxidase, glutathione reductase and glyoxalase I, were measured following this treatment for 6 days at two experimental times (3 and 6 days). Both water plant-treated carp showed a general increase of the enzymatic activities of glutathione S-transferase, and glutathione reductase which might be employed as potential biomarkers of oxidative stress induced by disinfected river water. Plant 1-treated carp showed higher glyoxalase I and glutathione levels and lower glutathione peroxidase activity. A depleted level of total glutathione and of glyoxalase I for specimens of water plant 2 (for both experimental times), without correlation with the distances in the pipeline, suggests that river plant water can also lead to potentially adverse effects on selected biochemical parameters in C. carpio.     

Effects of gallium on common carp (Cyprinus carpio): acute test, serum biochemistry, and erythrocyte morphology

Gallium (Ga) is one of the intermetallic elements that are increasingly being used in making high-speed semiconductors such as gallium arsenide. The purposes of this study were to investigate the effects of gallium on acute toxicity, on serum biochemical variables as well as on erythrocyte morphological changes in the blood stream of common carp (Cyprinus carpio). Median lethal concentrations were determined in acute tests. The 96-h LC50 value was 19.78 (18.49-21.16) mgl(-1). Common carp were exposed to different gallium concentrations (2.0, 4.0, and 8.0 mgl(-1)) for 28 days in laboratory toxicity tests. Means of the measured serum biochemistry parameters (including glucose, blood urea nitrogen, creatinine, cholesterol, and triglyceride) of these exposed groups significantly differed from those of the untreated group. Deformation of erythrocytes suggest disturbance of respiration as an additional indicator of Ga exposure. Our results suggest that 2.0 mgl(-1) is proposed as a biologically safe concentration which can be used for establishing tentative water quality criteria concerning of same size common carp. In addition, serum biochemical parameters as well as erythrocyte morphological changes are promising clinical diagnostic tools for assessing the effects of gallium compounds on common carp.     

Effect of ibuprofen exposure on blood,gill, liver,and brain on common carp (Cyprinus carpio) using oxidative stress biomarkers

Although trace concentrations of ibuprofen (IBP) have been detected in diverse water bodies, there is currently insufficient information on the potentially deleterious effects of this xenobiotic. The present study aimed to determine whether IBP induces oxidative stress in brain, liver, gill, and blood of the common carp Cyprinus carpio. To this end, the median lethal concentration at 96 h (96-h LC₅₀) was determined and the lowest observed adverse effect level was established. Carp were exposed to the latter concentration (17.6 mg L^?1) for 12, 24, 48, 72, and 96 h, and the following biomarkers were evaluated: lipid peroxidation (LPX) and activity of the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase. Results indicated that LPX and antioxidant enzymes’ activity increased significantly (p?<?0.05) with respect to the control group in liver, gill, and blood, while no significant differences occurred in brain. In conclusion, IBP induced oxidative stress on C. carpio, the liver being the organ most affected by this damage.     

Effects of different inorganic arsenic species in Cyprinus carpio (Cyprinidae) tissues after short-time exposure: Bioaccumulation, biotransformation and biological responses

Differences in the toxicological and metabolic pathway of inorganic arsenic compounds are largely unknown for aquatic species. In the present study the effects of short-time and acute exposure to As^III and As^V were investigated in gills and liver of the common carp, Cyprinus carpio (Cyprinidae), measuring accumulation and chemical speciation of arsenic, and the activity of glutathione-S-transferase omega (GST Ω), the rate limiting enzyme in biotransformation of inorganic arsenic. Oxidative biomarkers included antioxidant defenses (total glutathione-S-transferases, glutathione reductase, glutathione, and glucose-6-phosphate dehydrogenase), total scavenging capacity toward peroxyl radicals, reactive oxygen species (ROS) measurement and lipid peroxidation products. A marked accumulation of arsenic was observed only in gills of carps exposed to 1000 ppb As^V. Also in gills, antioxidant responses were mostly modulated through a significant induction of glucose-6-phosphate dehydrogenase activity which probably contributed to reduce ROS formation; however this increase was not sufficient to prevent lipid peroxidation. No changes in metal content were measured in liver of exposed carps, characterized by lower activity of GST Ω compared to gills. On the other hand, glutathione metabolism was more sensitive in liver tissue, where a significant inhibition of glutathione reductase was concomitant with increased levels of glutathione and higher total antioxidant capacity toward peroxyl radicals, thus preventing lipid peroxidation and ROS production. The overall results of this study indicated that exposure of C. carpio to As^III and As^V can induce different responses in gills and liver of this aquatic organism.     

The combined use of chemical and biochemical markers to assess water quality along the Ebro River

Carp (Cyprinus carpio) and barbel (Barbus graellsii) were collected from five sites along the Ebro River. The study was designed to assess levels of persistent organic pollutants and metals bioaccumulated by fish, and some biochemical responses (cytochrome P450 system, phase II activities, and metallothioneins) against those pollutants. The highest levels of PCBs and DDTs were detected in carp from industrialised areas, which also showed high levels of mercury and cadmium in the liver, and high levels of nonylphenol in bile. Significant alterations in some biochemical markers were observed and associated to combined exposure to pollutants. The activity 7-ethoxyresorufin O-deethylase ranged from 69 pmol/min/mg protein in carp from the reference site to 415 pmol/min/mg protein in those from polluted sites. Carp from the Ebro Delta, an agricultural area, had depressed acetylcholinesterase in muscle tissue. Metallothionein concentrations were positively correlated with copper residues, but no significant differences among sampling points were observed.     

Transformation of phenol, catechol, guaiacol and syringol exposed to sodium hypochlorite

Germs, xenobiotics and organic matter that influence the colour, turbidity and organoloeptic properties of water are removed by chlorination. Unfortunately, chlorine oxidants including sodium hypochlorite, used in water treatment induce processes that partly convert the treated compounds to unwanted chlorinated derivatives. The purpose of this work was to analyse the efficiency of transformation of phenol, catechol, guaiacol and syringol exposed to sodium hypochlorite and determine the intermediates formed during oxidative conversion of these compounds. The analysis was performed in aerobic conditions, both in acidic (pH 4.0) and alkaline (pH 8.0) medium. The effectiveness of transformation was slightly higher in acidic in comparison to alkaline conditions. Some chlorophenols, such as 2-chlorophenol, 2,4-dichlorophenol, 2,4,5-trichlorophenol and pentachlorophenol were determined as the products of phenol conversion. Chlorophenols were also formed during catechol, guaiacol and syringol transformation by replacement of hydroxy and methoxy residues by chlorine atoms. Moreover, some chlorocatechols and chlorinated methoxyphenols were determined during catechol and methoxyphenols transformations. Higher concentrations of chlorinated compounds were observed in the alkaline environment during phenol transformation. Conversion of catechol and methoxyphenols generated higher amounts of chlorinated intermediates in the acidic medium. In samples carboxylic acids like acetic and formic acids were determined. The formation of these compounds was the result of the cleavage of aromatic structure of phenols.     

Investigation of acute toxicity and the effect of 2,4-D (2,4-dichlorophenoxyacetic acid) herbicide on the behavior of the common carp (Cyprinus carpio L., 1758; Pisces,Cyprinidae)

A 96-h LC(50) values of 2,4-D [(2,4-dichlorophenoxy)acetic acid], a common contaminating agricultural herbicide, were determined on the adult common carp (Cyprinus carpio L., 1758; Pisces, Cyprinidae). The study was conducted in two stages using 130 carp. The data obtained were statistically evaluated by the use of the EPA computer program based on Finney's Probit Analysis Method and a 96-h LC(50) value for C. carpio L., 1758 was found to be 63.24 mg/l in a static bioassay test system. 95% lower and upper confidence limits for the LC(50) were 55.03 and 71.92 mg/l, respectively. Water temperature was 17+/-1 degrees C. Behavioral changes of the above mentioned species were examined for various herbicide concentrations.     

Reaction of silver nanoparticles in the disinfection process

This study investigated the dissolution, aggregation, and reaction kinetics of silver nanoparticles (AgNPs) with the three types of water disinfectants (ultraviolet, sodium hypochlorite, and ozone) under the different conditions of pH, ionic strength, or humic acid (HA). The physicochemical changes of AgNPs were measured by using UV–Vis spectroscopy, transmission electron microscopy, and inductively coupled plasma optical emission spectrometer. The results showed that when AgNPs contacted the disinfectants, oxidative dissolution was the primary reaction. In addition, the reaction kinetics studies revealed that the reaction rate of AgNPs with disinfectants was significantly influenced by different disinfectants along with different pH and the presence of sodium nitrate and HA. Our research demonstrated the potential effect of disinfectants on AgNPs, which will improve our understanding of the fate of AgNPs in the disinfection processes in the water and wastewater treatment plant.     

Biomarkers assessment in juvenile Cyprinus carpio exposed to waterborne cadmium

The impact of long-term exposure to waterborne cadmium (Cd) on Cyprinus carpio was evaluated through changes of selected parameters considered as biomarkers of toxicity. Fish were exposed to 1.6 mg l(-1) Cd for 14 days and then transferred to Cd-free water for 19 days. The measured parameters were gill ATPases, brain acetylcholinesterase (AchE), liver glutamate oxaloacetate (GOT) and glutamate pyruvate (GPT) transaminases, muscle water content, and protein content of liver, gills and brain. Condition factor and liver somatic index were also calculated. Branchial ATPase activities were impaired in a dissimilar way: the (Na(+),K(+))-ATPases were inhibited by approximately 30%, while the Mg(2+)-ATPase was significantly activated by 70%. Brain AchE showed no changes after Cd exposure. Both liver GOT and GPT activities were increased by the metal by 63 and 98%. Water content of the skeletal muscle showed no significant alterations. After the 19-day recovery phase, changes in the Mg(2+)-ATPase and GPT were reversed to values similar to controls, but the Cd exposure resulted in an irreversible alteration in GOT activity. Results indicate that the sublethal Cd concentrations are stressful to carp, particularly with reference to branchial enzymes which may disrupt the osmotic and ionic balance of the animals.     

Comparison of effects of paraquat and methidation on enzyme activity and tissue necrosis of carp, following exposure to the pesticides singly or in combination

Under aquarium conditions, treatment with the herbicide paraquat (PQ) and with the insecticide methidation (MD) caused cell damage and stress in carp (Cyprinus carpio L.), as shown by increases in glutamate-dehydrogenase (GIDH, EC 1.4.1.2), glutamate-oxalacetate transaminase (GOT, EC 2.6.1.1) and lactate dehydrogenase (LDH, EC 1.1.1.27) activities and in blood sugar levels. PQ proved synergistic with MD in certain cases as regards the harmful effect exerted. On combined treatment, dilated extracellular spaces were visible by light microscope in the liver, while electronmicroscopic studies revealed signs indicative of cell autolysis in the same organ.     

Perfluorooctane sulfonic acid and organohalogen pollutants in liver of three freshwater fish species in Flanders (Belgium): relationships with biochemical and organismal effects

A perfluorooctane sulfonic acid (PFOS) assessment was conducted on gibel carp (Carassius auratus gibelio), carp (Cyprinus carpio), and eel (Anguilla anguilla) in Flanders (Belgium). The liver PFOS concentrations in fish from the Ieperlee canal (Boezinge, 250-9031 ng/g wet weight, respectively) and the Blokkersdijk pond (Antwerp, 633-1822 ng/g wet weight) were higher than at the Zuun basin (Sint-Pieters-Leeuw, 11.2-162 ng/g wet weight) and among the highest in feral fish worldwide. Eel from the Oude Maas pond (Dilsen-Stokkem) and Watersportbaan basin (Ghent) had PFOS concentrations ranging between 212 and 857 ng/g wet weight. The hepatic PFOS concentration was significantly and positively related with the serum alanine aminotransferase activity, and negatively with the serum protein content in eel and carp. The hepatic PFOS concentration in carp correlated significantly and negatively with the serum electrolyte concentrations whereas a significant positive relation was found with the hematocrit in eel. Although 13 organochlorine pesticides, 22 polychlorinated biphenyl (PCB) congeners and 7 polybrominated diphenyl ethers (PBDEs) were also measured in the liver tissue, only PCB 28, PCB 74, gamma-hexachlorocyclohexane (gamma-HCH) and hexachlorobenzene (HCB) were suggested to contribute to the observed serological alterations in eel.     

Antioxidant response versus selenium accumulation in the liver and kidney of the Siberian sturgeon (Acipenser baeri)

The aim of this study was to examine the effects of selenium on concentrations of metabolites and enzyme activities acting as antioxidant markers in liver and kidney of Siberian sturgeon Acipenser baeri. Sturgeons were fed selenium cysteine for 30 and 60 d at 1.25, 5, 20 mg Se kg⁻¹. Selenium level in the control feed was 0.32 mg kg⁻¹. Se concentration was measured in liver, kidney and muscle of every specimen. Sturgeon accumulated Se in tissues with a clear dose-response relationship and the highest Se concentration was recorded in liver. This outcome is lined up with the findings obtained on the antioxidant markers evaluated in both tissues, and in which a dose-response for several biomarkers was recorded in liver. The superoxide dismutase activity in Se-treated fish was generally induced, while catalase activity was lower in liver or unaltered in kidney. The concentrations of glutathione S-transferase, glutathione reductase and total glutathione responded differently for both tissues and were induced in a different way at both endpoints. No changes of glyoxalase I activity were noted for both Se-treated tissues, while for glyoxalase II enzyme in liver a dose-related pattern was found showing a reversible effect (decreased and increased counteractive response) only in the 5 mg kg⁻¹ group. Moreover, the highest Se concentrations did not cause marked changes in malondialdehyde levels of liver and kidney. The enhancement of glutathione peroxidase activity in Se-treated sturgeon might have prevented the lipid peroxidation in both tissues, providing to the Siberian sturgeon a great defense ability versus the prooxidant effect of selenium.     

In vitro biotransformation of surfactants in fish. Part I: linear alkylbenzene sulfonate (C12-LAS) and alcohol ethoxylate (C13EO8)

Developing regulatory activities (e.g., REACh, [DGEE. 2003. Directorates General Enterprise and Environment. The new EU chemicals legislation REACH. DG Enterprise, Brussels, Belgium. (http://www.europa.eu.int/comm/enterprise/reach/index_en.htm)]) will require bioaccumulation to be assessed for thousands of chemicals. Further, there is increasing pressure to reduce, refine or replace animal tests. Given this scenario, there is an urgent need to evaluate the feasibility of in vitro systems to supply data useful for bioaccumulation estimation. Subcellular and cellular hepatic systems were tested to determine the biotransformation of two surfactants: C12-2-LAS (2-phenyl dodecane p-sulfonate) and an alcohol ethoxylate C13EO8 (Octaethylene glycol monotridecyl ether). The subcellular systems tested were liver homogenates and microsomes from the common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss). Cellular systems consisted of primary hepatocytes from the common carp (Cyprinus carpio) and PLHC-1 cells, hepatocarcinoma cells from the desert topminnow (Poeciliopsis lucida). All in vitro systems were exposed to radiolabeled test compounds and assayed for biotransformation using liquid scintillation and thin layer chromatographic methods. First-order kinetics were used to estimate rates of biotransformation. Bioconcentration of test materials in fish were predicted using an in vitro to in vivo metabolic rate extrapolation model linked to a mass-balance model commonly used to predict bioaccumulation in fish. Subcellular biotransformation rates for each of the surfactants were greatest with microsomes. Cellular loss rates exceeded subcellular rates, leading to lower predicted BCF values. Predicted BCFs corresponded closely to measured values in several fish species, verifying the utility of in vitro systems in refining Kow-only-based BCFs via the inclusion of biotransformation rates.     

Effects of hexachlorobenzene on antioxidant status of liver and brain of common carp (Cyprinus carpio)

Hexachlorobenzene (HCB)-induced oxidative damages have been published in rats while the effects have not yet been reported in fishes. Juvenile common carps (Cyprinus carpio) were exposed to waterborne HCB from 2 to 200 microg l-1 for 5, 10 or 20 days. Liver and brain were analyzed for various parameters of oxidative stress. There were no significant changes of glutathione (GSH) content and superoxide dismutase (SOD) activity in liver after 5 or 10 days exposure, whereas obvious drops were observed at higher concentrations after 20 days exposure. Significant decreases of GSH content and SOD activity in brain were found during all the exposure days. In brain, HCB also significantly elevated the contents of reactive oxygen species (ROS), thiobarbituric acid- reactive substances (TBARS, as an indicator of lipid peroxidation products), glutathione disulfide (GSSG), and activities of nitric oxide synthase (NOS), glutathione peroxidase (GPx), and glutathione reductase (GR), and inhibited activities of acetylcholinesterase (AchE) and glutathione S-transferase (GST). The results clearly demonstrated that environmentally possible level of HCB could result in oxidative stress in fish and brain was a sensitive target organ of HCB toxicity.     

Accumulation and partition of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F) in the muscle and liver of fish

The present study monitored fish from sites contaminated for many years, and the data reflected the results of actual, long-term environmental exposure. The sum concentrations and TEQs of PCDD/F in the muscle of common carp (Cyprinus carpio (linnaeus)) and big head (Aristichthys nobilis) from the different site of a heavily polluted lake in China are decreased with decrease of PCDD/F concentration in sediment and water. Concentration in fish can be highly variable, but the concentration is comparable in muscle between different species of fish if the concentrations are normalized by lipid content. PCDD/F accumulate in certain tissues with the highest proportions found in the liver of fish. The concentrations and TEQs in livers were also decreased with concentrations of sediments and water. The concentrations of PCDD/F in muscle are significantly correlated with those in liver. BCFs of 2,3,7,8-substituted PCDD/F calculated directly from water were consistent with that calculated indirectly from sediment. PCDD/F concentrations in sediment are much higher than that in water, therefore the measurement is much easier and the results are more accurate. Estimated BCFs for superhydrophobic chemicals derived from sediment may also serve as acceptable surrogates for "true" bioaccumulation potential.     

Effects of long-term alachlor exposure on hepatic antioxidant defense and detoxifying enzyme activities in crucian carp (Carassius auratus)

Alachlor has been widely used in agriculture all over the world. It is suggested that it may be a carcinogen and also an environmental estrogen. In this paper, the physiological and biochemical perturbations of crucian carp (Carassius auratus) exposed to alachlor at different concentrations over 60 days were investigated. The gonadosomatic index (GSI) and hepatosomatic index (HSI) were measured. The activity of hepatic antioxidant defense and detoxifying enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) and the content of glutathione (GSH) were determined and compared with the control group. The result showed that GSI and HSI decreased significantly (P<0.05) in almost all treatments. The activities of SOD, CAT and GST were induced continuously (P<0.05), while the content of reduced glutathione (GSH) was inhibited on the whole. These changes reflect that the antioxidant systems of the tested fishes were affected. The possible defense mechanistic implications about the changes were thus discussed. Furthermore, hepatic SOD and GST were sensitive to alachlor at low concentration, indicating that they might be potential biomarkers in early detection of alachlor contamination in aquatic ecosystems.     

Impacts of quicklime (CaO) on the toxicity of copper (CuSO4, 5H2O) to fish and fish food organisms

Static bioassays of 96 h duration were conducted in the laboratory using fry of common carp (Cyprinus carpio), adult tubificid worm (Branchiura sowerbyi) and adult copepod plankton (Cyclops viridis) to determine LC50 values of Cu and CaO to these organisms and effects of interaction between Cu and CaO. Ninety-six hour LC(50) values of Cu to fry of common carp, worm and copepod were found to be 1.40 mgl(-1), 0.08 mgl(-1) and 0.03 mgl(-1) respectively. CaO up to 500 mgl(-1) did not produce any mortality of the fry of common carp up to 96 h. But 96 h LC50 values of CaO to worm and copepod were 83.00 mgl(-1) and 27.80 mgl(-1) respectively. When common carp fry, worm and the copepod were exposed to respective LC50 dose of Cu in presence of varying concentration of CaO, mortality of the organisms significantly reduced and was found inversely correlated with the doses of CaO [y = 48.36-0.807x, r = -0.99 (n = 7) for fish; y = 44.46-0.146x, r = -0.97 (n = 7) for worm; y = 49.46-0.66x, r = -0.99 (n = 7) for the copepod]. The present results indicate that CaO is non-toxic to fish and is capable of reducing the toxicity of Cu to fish while CaO and Cu are antagonistic to each other for the worm and the copepod. Potential of using CaO as antitoxic agent for Cu in water is discussed.     

Evaluation of disinfection techniques in the treatment of advanced primary treated wastewater for Ciudad Juárez, México.

The purpose of this study was to develop and evaluate the effectiveness of alternative disinfection techniques at the bench-scale level using wastewater from Ciudad Juarez, Mexico, as model feed. This paper presents findings on the effectiveness of UV radiation, peracetic acid (PAA), chlorine dioxide (ClO2), and hypochlorous acid (HOCl) as disinfectants for advanced primary treatment (APT) plant effluent. Wastewater samples for bench-scale testing were collected from an agua negra ("black water") ditch that is part of the combined sewer system in Ciudad Juarez. Bench-scale simulations of the APT process used in Ciudad Juarez were run using a jar test apparatus and aluminum sulfate [Al2(SO4)3] as the coagulant. Jar test effluent from the bench system was used for disinfection testing. The Mexican discharge quality standard for total coliforms is 10 000/100 mL. Ultraviolet radiation met this standard at a dose of 47.5 mW-s/cm2. Ultraviolet disinfection proved reliable and effective despite the presence of suspended solids, and UV dose effectiveness expressed as a total coliforms survival ratio was best explained by a linear regression model. The ClO2 dose ranged from 10 to 20 mg/L and was only effective under ambient temperature conditions found during the winter months; PAA disinfection never met Mexican standards. Chlorine disinfection was effective at a dose range of 8 to 10 mg/L on samples collected at low temperature conditions. Since the completion of this research, Ciudad Juarez has discontinued the use of chlorine disinfection because of its high cost and ineffectiveness.     

Assimilation efficiencies of Cd and Zn in the common carp (Cyprinus carpio): effects of metal concentration, temperature and prey type

The impact of several factors on the assimilation efficiency (AE) of Cd and Zn from food in the common carp (Cyprinus carpio) was studied. Tested prey species were midge larvae (Chironomus riparius), zebra mussels (Dreissena polymorpha) and oligochaetes (Tubifex tubifex). The Cd load of the larvae did not affect the Cd AE in the carp. The Zn AE however, was negatively related to the Zn load of the prey. Food quantity and starvation of the carp did not significantly affect the Cd AE. For Zn, a significant decrease in AE was found when carp were fed ad libitum. Decreasing the temperature from 25 degrees C to 15 degrees C did not influence the Cd AE, while for Zn a significant decrease of the AE was measured. Carp assimilated Cd from both zebra mussels and oligochaetes with a significantly lower efficiency in comparison to the midge larvae, although Zn AEs was prey independent.