Effects of diphenhydramine on crayfish cytochrome P450 activity and antioxidant defence mechanisms: First evidence of CYP2C- and CYP3A-like activity in marbled crayfish.

Growing evidence has reported that diphenhydramine (DPH), an ionisable antihistamine, is widely present in surface waters across the world. Relative to vertebrates studied, its impact on invertebrates, particularly concerning cytochrome P450 (CYP) metabolism and oxidative stress, remains poorly understood. In this study, we aimed to investigate the effects of 2, 20, and 200 µg/L DPH on marbled crayfish (Procambarus virginalis) after 96-h exposure. Specifically, we assessed CYP activity, antioxidant enzyme responses, and acetylcholinesterase (AChE) activity in gills, muscle, and hepatopancreas. The crayfish CYP metabolised fluorogenic CYP-metabolic substrates of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) and dibenzylfluorescein (DBF), which evidenced the activity of CYP2C and CYP3A isoforms, well known in mammalian detoxification metabolism. Both BFC and DBF dealkylations showed a positive correlation with each other but were negatively correlated to water and haemolymph DPH concentrations. Exposure to 200 µg/L DPH elicited an apparent inhibition trend, albeit not significant, in BFC- and DBF-transformation activities in crayfish. Other tested 7-benzyloxyresorufin and 7-pentoxyresorufin substrates were poorly metabolised, suggesting their relatively low activity or the lack of mammalian-like CYP1A and CYP2B isoforms in marbled crayfish. The significant modulation of antioxidant enzymes was demonstrated in gills and hepatopancreas. The exposure to DPH did not alter the activity of AChE. Integrated biomarker response version 2 showed the highest cumulative effect of DPH exposure on gills, implying that gill tissue is the most reliable matrix for evaluating DPH toxicity. Activities of glutathione peroxidase and glutathione-S-transferase were the most deviated determinants among the investigated biomarkers, providing insights into the DPH toxicity in crayfish. This study brought the first insight into utilising the fluorogenically active substrates BFC and DBF to demonstrate the CYP involvement in the detoxification metabolism in marbled crayfish. Further, our results provided information on valuable antioxidant defence mechanisms and biomarker responses for a future DPH toxicity assessment in aquatic organisms.

In Vitro Metabolic Transformation of Pharmaceuticals by Hepatic S9 Fractions from Common Carp (Cyprinus carpio).

Water from wastewater treatment plants contains concentrations of pharmaceutically active compounds as high as micrograms per liter, which can adversely affect fish health and behavior, and contaminate the food chain. Here, we tested the ability of the common carp hepatic S9 fraction to produce the main metabolites from citalopram, metoprolol, sertraline, and venlafaxine. Metabolism in fish S9 fractions was compared to that in sheep. The metabolism of citalopram was further studied in fish. Our results suggest a large difference in the rate of metabolites formation between fish and sheep. Fish hepatic S9 fractions do not show an ability to form metabolites from venlafaxine, which was also the case for sheep. Citalopram, metoprolol, and sertraline were metabolized by both fish and sheep S9. Citalopram showed concentration-dependent N-desmethylcitalopram formation with Vmax = 1781 pmol/min/mg and Km = 29.7 µM. The presence of ellipticine, a specific CYP1A inhibitor, in the incubations reduced the formation of N-desmethylcitalopram by 30-100% depending on the applied concentration. These findings suggest that CYP1A is the major enzyme contributing to the formation of N-desmethylcitalopram. In summary, the results from the present in vitro study suggest that common carp can form the major metabolites of citalopram, metoprolol, and sertraline.

Correction to: Effects of multi-component mixtures from sewage treatment plant effluent on common carp (Cyprinus carpio) under fully realistic condition.

The original version of this Article unfortunately contained an error. The authors' given and family names were transposed erroneously. It has been corrected now in this Erratum.

Effects of Multi-Component Mixtures from Sewage Treatment Plant Effluent on Common Carp (Cyprinus carpio) under Fully Realistic Condition.

This study characterized changes in biomarker responses in common carp (Cyprinus carpio) upon exposure to effluent water discharged from a sewage treatment plant (STP) under real conditions. Fish were exposed to contamination in Cezarka pond, which receives all of its water input from the STP in the town of Vodnany, Czech Republic. Five sampling events were performed at day 0, 30, 90, 180, and 360 starting in April 2015. In total, 62 pharmaceutical and personal care products (PPCPs) were detected in the polar organic chemical integrative sampler. Compared to a control pond, the total concentration of PPCPs was 45, 16, 7, and 7 times higher in Cezarka pond at day 30, 90, 180, and 360, respectively. The result of oxidative stress and antioxidant enzyme biomarkers indicated alterations in the liver and intestine tissues of fish from Cezarka pond at day 30 and 360, respectively. High plasma vitellogenin levels were observed in both exposed females (180 and 360 days) and males (360 days) compared with their respective controls. However, only exposed female fish had higher vitellogenin mRNA expression than the control fish in these periods. Exposed female fish showed irregular structure of the ovary with scattered oocytes, which further developed to a vitellogenic stage at day 360. Low white blood cell levels were indicated in all exposed fish. Despite numerous alterations in exposed fish, favorable ecological conditions including high availability of food resulted in a better overall condition of the exposed fish after 1 year of exposure compared to the controls.

Invasive Species Mediate Insecticide Effects on Community and Ecosystem Functioning.

Anthropogenic activities increase pesticide contamination and biological invasions in freshwater ecosystems. Understanding their combined effects on community structure and on ecosystem functioning presents challenges for an improved ecological risk assessment. This study focuses on an artificial stream mesocosms experiment testing for direct and indirect effects of insecticide (chlorantraniliprole - CAP) exposure on the structure of a benthic macroinvertebrate freshwater community and on ecosystem functioning (leaf decomposition, primary production). To understand how predator identity and resource quality alter the community responses to chemical stress, the mediating effects of an invasive predator species (crayfish Procambarus clarkii) and detritus quality (tested by using leaves of the invasive Eucalyptus globulus) on insecticide toxicity were also investigated. Low concentrations of CAP reduced the abundance of shredders and grazers, decreasing leaf decomposition and increasing primary production. Replacement of autochthonous predators and leaf litter by invasive species decreased macroinvertebrate survival, reduced leaf decomposition, and enhanced primary production. Structural equation modeling (SEM) highlighted that CAP toxicity to macroinvertebrates was mediated by the presence of crayfish or eucalypt leaf litter which are now common in many Mediterranean freshwaters. In summary, our results demonstrate that the presence of these two invasive species alters the effects of insecticide exposure on benthic freshwater communities. The approach used here also allowed for a mechanistic evaluation of indirect effects of these stressors and of their interaction on ecosystem functional endpoint, emphasizing the value of incorporating biotic stressors in ecotoxicological experiments.

Complex effects of pollution on fish in major rivers in the Czech Republic.

Monitoring the contamination level in aquatic environments and assessing the impact on aquatic life occurs throughout the world. In the present study, an approach based on a combination of biomarkers and the distribution of various industrial and municipal pollutants was used to investigate the effect of aquatic environmental contamination on fish. Monitoring was performed in ten rivers in the Czech Republic (Berounka, Dyje, Elbe, Luznice, Odra, Ohre, Otava, Sázava, Svratka, and Vltava rivers, with one or two locations in each river) at the same sites that were regularly monitored within the Czech National Monitoring Program in 2007-2011. Health status, hepatic ethoxyresorufin-O-deethylase (EROD) activity, total cytochrome P450 content, and the plasma vitellogenin concentration were assessed in wild chub (Squalius cephalus) males caught at the monitored sites. The contamination level was the highest in the Svratka River downstream of Brno. Among all measured persistent organic pollutants (POPs), polychlorinated biphenyls and dichlorodiphenyltrichloroethane and its metabolites were the major contributors of POPs in fish muscle. Elbe, Odra, and Svratka rivers were identified as the most polluted. Fish from these locations showed reduced gonad size, increased vitellogenin concentration in male plasma, EROD, and total cytochrome P450 content. These biomarkers can be used for future environmental monitoring assessments. Overall, this study improves our understanding of the relationship between human activities and pollutant loads and further contributes to the decision to support local watershed managers to protect water quality in this region.

Toxicity of organic UV-filters to the aquatic midge Chironomus riparius.

Despite the frequent detection of organic ultraviolet-filters (UV-filters) in freshwater sediments, there is a lack of ecotoxicological data undermining a correct risk assessment for these emerging contaminants. The present study assessed the effects of three of the most commonly used UV-filters (benzophenone-3 - BP3; 3-(4-methylbenzylidene)camphor - 4-MBC and octocrylene - OC) on Chironomus riparius life history and biochemical responses. Standard ecotoxicological assays confirmed that all compounds impaired growth of C. riparius larvae and induced developmental effects such as delayed emergence and a reduction of imagoes weight. Concerning the biochemical responses analysed no evidences of oxidative damage in lipids or neurotoxicity (tested assessing acetylcholinesterase activity) were observed for any of the tested compounds. However, 4-MBC exposure induced a decrease in catalase activity and an increase in glutathione-S-transferase activity at 14.13mg/Kg while OC exposure caused an increase in total glutathione levels at 0.23 and 18.23mg/Kg. Exposure to all UV-filters tested, increased energy consumption on C. riparius with significant differences above 1.00mg/Kg for BP3, 0.09mg/Kg for 4-MBC and 2.13mg/Kg for OC. These results suggest that environmental relevant concentrations of UV-filters can cause deleterious effects to aquatic benthic species, such as C. riparius, and call for further research concerning effects of organic UV-filters on natural invertebrate communities and ecosystem functioning.

Assessment of thiamethoxam toxicity to Chironomus riparius.

The insecticide thiamethoxam (TMX) is a systemic neonicotinoid widely used for pest control in several agricultural crops. TMX mimics the action of acetylcholine causing uncontrolled muscular contraction eventually leading to insect death. TMX is being found in freshwater ecosystems at concentrations of up to 225µg/L. Still, chronic toxicity data for freshwater invertebrates is limited. Therefore, the aim of this study was to evaluate the acute and chronic effects (at organismal and biochemical levels) of TMX on the freshwater insect Chironomus riparius. C. riparius life history responses were significantly affected by TMX exposure, namely with a decrease in growth and delay in emergence. Concerning the biochemical responses, after a short exposure (48h) to TMX, our results showed that low concentrations of TMX significantly reduced CAT activity and LPO levels of C. riparius. No effects were observed in AChE, GST and ETS activities. Effects in terms of survival, development rates and biochemical responses of C. riparius exposed to low concentrations of TMX observed in this study suggest potential deleterious effects of this neonicotinoid on aquatic insects inhabiting freshwaters environments near agricultural areas.

Perfluoroalkyl substances in aquatic environment-comparison of fish and passive sampling approaches.

The concentrations of seven perfluoroalkyl substances (PFASs) were investigated in 36 European chub (Squalius cephalus) individuals from six localities in the Czech Republic. Chub muscle and liver tissue were analysed at all sampling sites. In addition, analyses of 16 target PFASs were performed in Polar Organic Chemical Integrative Samplers (POCISs) deployed in the water at the same sampling sites. We evaluated the possibility of using passive samplers as a standardized method for monitoring PFAS contamination in aquatic environments and the mutual relationships between determined concentrations. Only perfluorooctane sulphonate was above the LOQ in fish muscle samples and 52% of the analysed fish individuals exceeded the Environmental Quality Standard for water biota. Fish muscle concentration is also particularly important for risk assessment of fish consumers. The comparison of fish tissue results with published data showed the similarity of the Czech results with those found in Germany and France. However, fish liver analysis and the passive sampling approach resulted in different fish exposure scenarios. The total concentration of PFASs in fish liver tissue was strongly correlated with POCIS data, but pollutant patterns differed between these two matrices. The differences could be attributed to the metabolic activity of the living organism. In addition to providing a different view regarding the real PFAS cocktail to which the fish are exposed, POCISs fulfil the Three Rs strategy (replacement, reduction, and refinement) in animal testing.

Stimulatory effect of sesamin on hepatic cytochrome P450 activities in Atlantic salmon (Salmo salar L.) is not directly associated with expression of genes related to xenobiotic metabolism.

1. This study examined hepatic cytochrome P450 (CYP450) response to dietary sesamin in combination with different n-6/n-3 fatty acid ratios in fish diet. Over a period of 4 months, fish were fed seven different experimental diets an n-6/n-3 FA ratio of either 0.5 or 1.0 in combination with two sesamin levels: low sesamin = 1.16 g/kg feed and high sesamin = 5.8 g/kg feed. Control diets did not contain sesamin. 2. The CYP450-associated activities of ethoxyresorufin O-deethylase (EROD), 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylation (BFCOD), pentoxyresorufin O-depentylase (PROD), coumarin hydroxylase (COH), methoxyresorufin O-deethylase (MROD) and p-nitrophenol hydroxylase (PNPH) were significantly induced by dietary sesamin in a dose-related manner. 3. Expressions of the genes CYP1A1, CYP1A3, CYP3A, AhR1α, AhR2ß, AhR2δ and PXR involved in the regulation of CYP450 activities, was not the primary source of this induction.

Effects of acetone, acetonitrile, ethanol, methanol and DMSO on cytochrome P450 in rainbow trout (Oncorhynchus mykiss) hepatic microsomes.

In vitro impacts of five organic solvents on cytochrome P450 (CYP450) enzyme activity were investigated using hepatic microsomes of rainbow trout. The rates of several CYP450-mediated reactions were investigated at solvent concentrations ranging from 0.01% to 3%. The solvents greatly affected all tested reactions. In at least 0.8% ethanol, 2% methanol or acetone, 1% acetonitrile or 3% dimethyl sulfoxide (DMSO), 7-ethoxyresorufin-O-deethylase (EROD) activity decreased and at 3% acetonitrile or ethanol, it was undetected. At 3%, all tested solvents except methanol reduced 7-benzyloxy-4-trifluoromethylcoumarin-O-debenzylase (BFCOD) activity, but at low concentrations of ethanol (2% and lower) or DMSO (1% and lower), it was induced. This was not seen with the inclusion of a pre-incubation step. p-Nitrophenolhydroxylase (PNPH) activity was not affected at concentrations below 1% DMSO, and at 2% acetonitrile it was reduced, as it was above 1% methanol or 0.5% ethanol. Acetone did not affect PNPH activity with or without a pre-incubation step. In general, the degree of inhibition was similar with and without the pre-incubation step. We conclude that the concentration of organic solvent for solubilizing the substrate and inhibitor in in vitro microsomal studies should be minimized.

Contamination of fish in important fishing grounds of the Czech Republic.

The aim of this study was to compare the contamination levels of certain important fishing grounds in the Czech Republic and to assess the health risk of consuming the fish from these localities. The assessment was performed from 2006 to 2010 in 27 fishing grounds. Within this project, 707 fish from 14 different species were sampled. The concentration of selected toxic metals (Hg, Pb, Cd) and persistent organic pollutants (POPs), such as non-dioxin-like polychlorinated biphenyls (NDL-PCBs), hexachlorocyclohexane (HCH) isomers, dichlorodiphenyltrichloroethane (DDT) and its metabolites (o,p´-DDE; p,p´-DDE; o,p´-DDD; p,p´-DDD; o,p´-DDT; p,p´-DDT) and hexachlorobenzene (HCB), were analysed in the muscle tissue of the sampled fish. Atomic absorption spectrometry (AAS) was used for the analysis of toxic metals. All of the POPs were analysed using gas chromatography with an electron capture detector (GC/ECD). Common bream (Abramis brama) was chosen as a reference fish species for the comparison of fishing grounds. Mercury was found as a major pollutant in fish flesh at all of the sampling sites. Concentrations in excess of the maximum level (ML) of mercury in the muscle tissue of fish (0.5 mg kg(-1)) were registered in 32 samples. Concentrations of other monitored toxic metals in fish muscle were low, typically below the limit of quantification (LOQ). From the tested POPs, DDTs and NDL-PCBs were found as major pollutants. ML for NDL-PCBs (ICES-6) in muscle tissue of fish (0.125 mg kg(-1)) was exceeded in 7 samples. In case of tested pesticides, concentrations in excess of the MRL were not registered.

In vitro inhibition of 7-ethoxyresorufin-O-deethylase (EROD) and p-nitrophenol hydroxylase (PNPH) activities by sesamin in hepatic microsomes from two fish species.

In the present study, we investigated the effect of sesamin on CYP1A (7-ethoxyresorufin-O-deethylase, EROD) and CYP2E1-like activities (p-nitrophenol hydroxylase, PNPH) in hepatic microsomes obtained from Atlantic salmon (Salmo salar) and common carp (Cyprinus carpio). Addition of sesamin to the incubations in a concentration range from 1 to 200 µM decreased the activities of EROD and PNPH in a concentration dependent manner. It is likely that the inhibition of EROD was mechanism-based as demonstrated by the decrease in the IC50 value from 5.9 to 3.2 µM for A. salmon and from 7.9 to 3.0 µM for common carp when 5 min pre-incubation step was included. Similarly, PNPH activity was inhibited by sesamin with a decrease in the IC50 values from 61.7 to 15.2 µM for A. salmon and from 194.3 to 20.7 µM for common carp. Thus, our results indicated that sesamin can act as a mechanism-based inhibitor of EROD and PNPH activity with similar degree of inhibition in both fish species. More importantly, the inhibition of CYP1A, in addition to being mechanism-based, was competitive with K(i) value of 5.3 µM.

Presence of UV filters in surface water and the effects of phenylbenzimidazole sulfonic acid on rainbow trout (Oncorhynchus mykiss) following a chronic toxicity test.

UV filters belong to a group of compounds that are used by humans and are present in municipal waste-waters, effluents from sewage treatment plants and surface waters. Current information regarding UV filters and their effects on fish is limited. In this study, the occurrence of three commonly used UV filters - 2-phenylbenzimidazole-5-sulfonic acid (PBSA), 2-hydroxy-4-methoxybenzophenone (benzophenone-3, BP-3) and 5-benzoyl-4-hydroxy-2-methoxy-benzenesulfonic acid (benzophenone-4, BP-4) - in South Bohemia (Czech Republic) surface waters is presented. PBSA concentrations (up to 13µgL(-1)) were significantly greater than BP-3 or BP-4 concentrations (up to 620 and 390ngL(-1), respectively). On the basis of these results, PBSA was selected for use in a toxicity test utilizing the common model organism rainbow trout (Oncorhynchus mykiss). Fish were exposed to three concentrations of PBSA (1, 10 and 1000µgL(-1)) for 21 and 42 days. The PBSA concentrations in the fish plasma, liver and kidneys were elevated after 21 and 42 days of exposure. PBSA increased activity of certain P450 cytochromes. Exposure to PBSA also changed various biochemical parameters and enzyme activities in the fish plasma. However, no pathological changes were obvious in the liver or gonads.

Multiple biomarkers responses in juvenile rainbow trout, Oncorhynchus mykiss, after acute exposure to a fungicide propiconazole.

In this study, the toxic effects of propiconazole (PCZ), a triazole fungicide present in aquatic environment, were studied in juvenile rainbow trout, Oncorhynchus mykiss, by acute toxicity test with the concentration of 5.04 mg/L (96 h LC50). Morphological indices, hematological parameters, liver xenobiotic-metabolizing response, and tissue antioxidant status were evaluated. Compared with the control group, fish exposed to PCZ showed significantly higher Leuko, PCV, MCHC, and hepatic EROD, and significantly lower MCV. CF and HSI were not significantly different among groups. SOD, CAT, GPx, and GR activities increased significantly in liver of experimental groups, but decreased significantly in gill. In general, antioxidant enzyme activity in intestine was less evident than in liver. Oxidative stress indices (levels of LPO and CP) were significantly higher in gill. Additionally, through chemometrics of all parameters measured in this study, two groups with 67.29% of total accumulated variance were distinguished. In short, the physiological and biochemical responses in different tissues of fish indicated that PCZ-induced the stressful environmental conditions. But according to PCZ residual status in the natural environment, more long-term experiments at lower concentrations will be necessary in the future. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2013.

Toxicological effects of diclofenac on signal crayfish (Pacifastacus leniusculus) as related to weakly acidic and basic water pH.

The widespread use and continuous discharge of pharmaceuticals to environmental waters can lead to potential toxicity to aquatic biota. Pharmaceuticals and their metabolites are often complex organic and environmentally persistent compounds that are bioactive at low doses. This study aimed to investigate the effects of diclofenac (DCF) on the antioxidant defence system and neurotoxicity biomarkers in signal crayfish (Pacifastacus leniusculus) under weakly acidic and basic conditions. Crayfish were exposed to 200 µg/L of DCF at pH 6 and 8 for 96 h and subsequently underwent the depuration phase for 96 h. Gills, hepatopancreas, and muscle were sampled after the exposure and depuration phases to assess the toxicological biomarker responses of DCF in crayfish by evaluating lipid peroxidation (LPO) levels, activities of antioxidant enzymes and acetylcholinesterase. After the exposure phase, the hemolymph DCF concentration was detected one order higher at pH 6 than at pH 8. The DCF was subsequently fully eliminated from the hemolymph during the depuration phase. Our results showed that DCF caused alteration in the activities of six of the seven tested biomarkers in at least one crayfish tissue. Although exposure to DCF caused imbalances in the detoxification system on multiple tissue levels, it was regenerated to a balanced state after the depuration phase. Integrated biomarker response (IBRv2) showed that the highest toxicological response to DCF exposure was elicited in the gills, whereas the hepatopancreas was the highest-responding tissue after the depuration phase. Exposure to DCF at pH 6 caused higher toxicological effects than at pH 8; however, crayfish antioxidant mechanisms recovered more quickly at pH 6 than at pH 8 after the depuration phase. Our results showed that water pH influenced the toxicological effects of DCF, an ionisable compound in crayfish.

Insights into environmental caffeine contamination in ecotoxicological biomarkers and potential health effects of Danio rerio.

Caffeine (CAF) exposures have been shown to cause several pharmacological and biological effects in target and non-target organisms. Although there are already several ecotoxicological studies with CAF in non-target organisms, they are focused on marine organisms, with relevant concentrations in these ecosystems, therefore, less ecologically relevant to freshwater ecosystems (the main ecoreceptor of this type of anthropogenic contaminant). The present study aimed to assess the chronic effects (28 days) of sub-lethal and environmentally relevant concentrations of CAF (0.16, 0.42, 1.09, 2.84, 7.40, 19.23, and 50 µg/L) in Danio rerio. Biochemical endpoints as biomarkers of antioxidant defense, biotransformation, lipid peroxidation, energy sources, and neurotransmission were assessed. CAF exposure induced alterations in antioxidant defenses (superoxide dismutase and glutathione reductase activities, and glutathione content) preventing lipid peroxidation. Lactate dehydrogenase activity decreased in all the concentrations tested, while acetylcholinesterase activity was only affected by the highest concentrations tested (19.23 and 50 µg/L). We also utilized a multi-biomarker approach (Integrated Biomarker Response version 2, IBRv2) to investigate the effects of CAF in the dispersion scope of individual biochemical responses of D. rerio. IBRv2 showed that the concentration of 50 µg/L promotes the highest stress. However, the results showed that CAF induced disturbances in the metabolic pathways studied in D. rerio. These results demonstrated the toxic effects of CAF on freshwater fish, compromising their physiological functions and evidencing the need for monitoring the residues of CAF released into the inland aquatic environments. Furthermore, this research evidence that phylogenetically and physiologically different species may present different biological responses with concern for ecologically relevant environmental conditions. In this sense, the present study generated ecotoxicologically relevant data, that can be considered by environment regulators, since the here-endpoints evaluated showed sensitivity and consistency in the evaluation of caffeine risks in freshwater environments.

Toxicity assessment of binary mixtures of BP3 with 4-MBC (UV-filters), and BP3 with DEET (insect repellent) using the aquatic midge Chironomus riparius.

Personal care products have various organic ultraviolet filters (UV filters) in their composition to increase protection against ultraviolet radiation. Some of these products also contain insect repellents in their formulations. Consequently, these compounds reach freshwater ecosystems, exposing aquatic organisms to a cocktail of anthropogenic contaminants. In this study, the joint effects of two most frequently detected UV filters (Benzophenone - 3 (BP3) and Enzacamene (4-MBC)) and joint effects of BP3 combined with an insect repellent (N, N diethyl-3-methylbenzamide - DEET) were evaluated using life-history traits of the aquatic midge Chironomus riparius such as emergence rate, time to emergence and imagoes body weight. The results showed synergistic effects between BP3 and 4-MBC for C. riparius emergence rate. Regarding the effects of BP3 and DEET mixture, our analysis suggests synergism in the case of males but antagonism in the case of females' time to emergence. Our results imply that the effects of UV filters present in sediments within chemical mixtures are complex and that the evaluation of effects using different life-history traits can yield different patterns of responses. This study demonstrates the importance of assessing the combined effects of pollutants used/found concomitantly in aquatic systems for a more accurate risk assessment, as individual chemical testing can underestimate the toxicity of organic UV filters.

Tolbutamide hydroxylation by hepatic microsomes from Atlantic salmon (Salmo salar L.).

Metabolic transformations of two substrates for human cytochrome P450 (CYP450) 2C9, tolbutamide and diclofenac, were investigated in hepatic microsomes from Atlantic salmon (Salmo salar L.). Tolbutamide hydroxylation followed Michaelis-Menten kinetics. Mean apparent Michaelis-Menten constant (K(m)) and maximum reaction velocity (V(max)) values for 4-hydroxytolbutamide (TBOH) formation were 0.09 ± 0.031 mM and 49.5 ± 6.03 pmol/min/mg, respectively. Addition of sulfaphenazole, an inhibitor for mammalian CYP2C9, in a range from 1 to 200 µM decreased formation of TBOH in a concentration-dependent manner, but not to 50%. Neither fluconazole, an inhibitor of human CYP2C9, nor ketoconazole, inhibitor of CYP1A and CYP3A in fish, affected TBOH formation. In contrast ellipticine, an inhibitor of CYP1A in fish inhibited TBOH formation with the IC(50) value of 12.1 µM. The rate of TBOH formation was competitively inhibited by 100 µM of sesamin in the incubations, but the degree of inhibition did not increase with increased sesamin concentration. Ethoxyresorufin hydroxylase (EROD) activity was inhibited by tolbutamide in a non-competitive manner (inhibition constant K(i) = 218 µM). Our data suggest that tolbutamide is metabolized by salmon microsomes with formation of TBOH. CYP1A might be involved in this reaction as suggested by decreased TBOH formation in the presence of ellipticine and decreased EROD activity in the presence of tolbutamide. Incubation of diclofenac with the microsomes yielded no metabolite formation, suggesting that salmon does not possess diclofenac-metabolizing activity.

Integrated biomarker response in signal crayfish Pacifastacus leniusculus exposed to diphenhydramine.

Diphenhydramine (DPH) is a pharmaceutical with multiple modes of action, primarily designed as an antihistamine therapeutic drug. Among antihistamines, DPH is a significant contaminant in the environment, frequently detected in surface waters, sediments, and tissues of aquatic biota. In the present study, signal crayfish Pacifastacus leniusculus was used as a model organism because of their prominent ecological roles in freshwater ecosystems. The biochemical effects were investigated in crayfish exposed to the environmental (low: 2 µg L-1), ten times elevated (medium: 20 µg L-1), and the sublethal (high: 200 µg L-1) nominal concentrations of DPH in water for 96 h. Lipid peroxidation, antioxidant enzyme activities, and acetylcholinesterase activity were assessed as toxicological biomarkers in crayfish hepatopancreas, gills, and muscles. Low and medium DPH exposure caused imbalances only in glutathione-like enzyme activities. Integrated biomarker response showed the absolute DPH toxicity effects on all tested tissues under high exposure. This study identified that high, short-term DPH exposure induced oxidative stress in crayfish on multiple tissue levels, with the most considerable extent in muscles.