Sensitivity of cholinesterases and carboxylesterases to pharmaceutical products in Tinca tinca.

Discharges to the aquatic environment of pharmaceuticals represent a hazard to the aquatic organisms. Subchronic assay with 17-alpha-ethinylestradiol (EE2) and in vitro essays with pharmaceuticals of environmental concern were conducted to examine the sensitivity of tissue acetylcholinesterase (AChE) and carboxylesterase (CbE) activities of Tinca tinca to them. Subchronic exposure to 17-alpha-EE2 caused significant effects on brain, liver, and muscle CbE, but no on AChE activities. Most of the pharmaceuticals tested in vitro were considered as weak inhibitors of tissular AChE activity. Depending on the tissues, some compounds were classified as moderate inhibitors of CbE activity while other were categorized as weak enzymatic inhibitors. An opposite trend was observed depending on the tissue, while brain and liver CbE activities were inhibited, the muscle CbE activity was induced. Changes experienced on enzymatic activities after exposure to pharmaceuticals might affect the physiological functions in which these enzymes are involved. In vitro exposure to 17-alpha-EE2 in tench could be an informative, but not a surrogate model to know the effect of this synthetic estrogen on AChE and CbE activities.

Toxic and essential metal levels in the hair of red deer (Cervus elaphus) and wild boar (Sus scrofa) for monitoring the contamination in protected areas of South-Western Spain.

Contaminant monitoring in tissues of wild species can help in the knowledge not only of their health, but also of the environmental health conditions in the zones where they live. In this study, concentrations of toxic metals (As, Cd, Hg, and Pb) and an essential metal (Zn) were measured in the hair of red deer (Cervus elaphus) and wild boar (Sus scrofa) inhabiting in protected areas of South-Western Spain. Zn (69.02 ± 1.03 mg kg-1, 70.31 ± 2.22 mg kg-1), Pb (0.61 ± 0.05 mg kg-1, 0.68 ± 0.08 mg kg-1), and As (0.33 ± 0.03 mg kg-1, 0.53 ± 0.08 mg kg-1) were detected in the hair of red deer and wild boar, respectively. The other metals (Cd and Hg) were under the quantification limit (0.1 mg kg-1). No differences in the level of elements between both species were found. The levels of the detected elements in the hair showed low concentrations and similar to those observed in animals of the same species from unpolluted zones, reflecting a lack of contamination by these elements in the studied areas. Based on critical values of these elements established for organs of mammals, red deer and wild boar from the studied territories did not suffer toxicosis by the studied elements. Age-related differences in the content of As in the hair of red deer and age- and gender-related differences in the content of As in the hair of wild boar were detected, which should be considered for biomonitoring purposes. Finally, positive (Pb-As in both species) and negative (Zn-Pb in wild boar) interrelationships between elements were found.

Occurrence of Levonorgestrel in Water Systems and Its Effects on Aquatic Organisms: A Review.

Levonorgestrel is one of the active ingredients of oral contraceptives detected in the aquatic environment at concentrations in the order of ng/L. During the past decade, a wealth of new information about levonorgestrel has been produced, with several studies having reported negative effects in the reproduction and growth of aquatic organisms after exposure to this emerging contaminant of concern. In the present study, the data about its levels in water and its effects on aquatic organisms were integrated and used to perform an updated preliminary aquatic risk assessment for levonorgestrel based on the guideline for Environmental Risk Assessment of Medicinal Products for Human Use from the European Medicines Agency. The aim was to investigate if this pharmaceutical has a risk for adverse effects on aquatic organisms (i.e. for organisms residing in surface water and groundwater). The results evidenced that levonorgestrel is likely to pose an environmental risk to surface water (risk quotient >1). Based on these results, a more refined risk assessment for this pharmaceutical is needed. Besides, our findings highlight the need for investigation under the adverse outcome pathway (AOP) framework, as well as for further studies about toxicological interactions between levonorgestrel and other synthetic steroids.

Lead content in soils and native plants near an abandoned mine in a protected area of south-western Spain: an approach to determining the environmental risk to wildlife and livestock.

A study of the impact of an abandoned lead (Pb) mine ("Las Musas"), located in SW Spain, on the contamination of the surface soil and pastures in its vicinity revealed the presence of widely distributed, high levels of Pb contamination. The total Pb concentrations in soils sampled at distances from 3 to 998 m from the mine ranged between 129 and 1053 mg/kg, when it has been reported that non-polluted soils have concentrations of 29-40 mg/kg. These exceed the maximum tolerable levels in agricultural soils for the protection of environmental and human health as established in international and regional regulations. While the concentrations of potentially bioavailable Pb in the soils also surpassed the regulatory levels, the effective bioavailable fractions were low. The Pb concentrations measured in native plants ranged from 1.70 to 129 mg/kg dry weight, with Cynosurus echinatus, Philadelphus coronarius, and Fraxinus angustifolia being the species that bioaccumulated the greatest concentrations of this metal. Estimation of the environmental risk to wildlife and livestock grazing in the studied area showed no potential toxicity for these animals.

Assessment of the environmental impact of an abandoned mine using an integrative approach: A case-study of the "Las Musas" mine (Extremadura, Spain).

The mine abandonment is generally associated with the release of potentially toxic metals into the environment, which may depend on metals speciation, soil properties and climate conditions. The goal of the present work was to assess the environmental impact of the abandoned Pb-Zn mine "Las Musas" (Spain) using an integrative approach. The impact on soils and surface waters was performed using: chemical parameters, quantification of potentially toxic metals (Cd, Cu, Pb and Zn), and ecotoxicological responses using lethal and sub-lethal bioassays with organisms' representative of different trophic level ((soil: Eisenia fetida (mortality and reproduction test); Latuca sativa and Lollium perenne (seedling emergence); and water: Vibrio fischeri (luminescence inhibition), Daphnia magna (immobility and reproduction test), Thamnocephalus platyurus (mortality), Pseudokirchneriella subcapitata (growth inhibition)). The results showed soils with neutral to slight alkaline pH (7.64-8.18), low electric conductivity (125-953 µS/cm) and low organic matter levels (0.20-1.85%). For most of the soil samples, Pb was the only metal which surpassed the limit proposed by the Canadian soil quality guidelines, with values ranging from 42.2 to 181.4 mg/kg. The ecotoxicological results showed that the soils with the highest levels of Pb induced a decrease on E. fetida reproduction and on L. sativa germination, indicating negative impacts on the habitat function. The analysis of the surface waters showed levels of Zn surpassing the legal limit adopted from the Water Framework Directive (37.0 to 69.0 µg/L). The ecotoxicological results highlight the importance of bioassays that evaluate the behavior of species, when assessing the risk of mining areas with non-acid soils and waters with high nutrients/organic matter concentrations and low concentrations of potentially toxic metals. The results indicated a moderate environmental risk from potentially toxic metals, at the areas analyzed around the Azuaga mine.

Ecotoxicological efficiency of advanced ozonation processes with TiO2 and black light used in the degradation of carbamazepine.

The aim of the present study was to evaluate the ecotoxicological efficiency of two advanced ozonation processes (AOzPs), the catalytic ozonation (O3/TiO2) and the photocatalytic ozonation (O3/TiO2/black light), in the remotion of carbamazepine. The ecotoxicological efficiency was assessed through the use of lethal and sublethal assays with species Vibrio fischeri and Daphnia magna. Results demonstrated that the AOzPs presented an efficiency of carbamazepine removal higher than 99% (carbamazepine < 2 µg/L) after 12 min of treatment. Relatively to ecotoxicological evaluation, application of acute assay to V. fischeri and chronic assay to D. magna allowed us to highlight that these technologies may form some transformation products that induce toxicity in the bacteria and the crustacean, once these organisms exposed to the undiluted solutions (100%) showed a decrease in the bioluminescence (vibrio) and end up dying before and during the first reproduction (daphnia). Despite that, when the chronic results obtained with the diluted solutions (50 and 25%; important to assess a more realistic scenario considering the dilution factor at the environment) were analyzed, no mortality at the mothers was observed. Compared to a carbamazepine solution (200 µg/L), diluted solutions improved of the reproduction parameters, and no toxic effects in the juvenoid system and in the embryonic development were observed. Relatively to the ecdysteroid effect of a carbamazepine solution (200 µg/L), only the photocatalytic ozonation treatment was able to remove the action of the drug. These results highlight the importance of complementing chemical analysis with ecotoxicological bioassays to assess the best technology to improve the surface water and effluent quality.

Toxic potential of the emerging contaminant nicotine to the aquatic ecosystem.

Nicotine is a "life-style compound" widely consumed by human populations and, consequently, often found in surface waters. This fact presents a concern for possible effects in the aquatic ecosystems. The objective of this study was to assess the potential lethal and sublethal toxicity of nicotine in aquatic organisms from different trophic levels (Vibrio fischeri, Pseudokirchneriella subcapitata, Thamnocephalus platyurus, and Daphnia magna). The bioassays were performed by exposing the organisms to concentrations of nicotine in a range of 0.5-1000 µg/L. Results showed that nicotine, at tested concentration, was not acutely toxic to V. fischeri and T. platyurus. On the contrary, this substance exhibited toxicity to P. subcapitata and Daphnia magna. Thus, concentrations of nicotine of 100 and 200 µg/L promoted an inhibition in the growth of P. subcapitata. In addition, a concentration of 100 µg/L nicotine acted on the reproduction of the crustacean D. magna, by decreasing the number of juveniles produced by female. On the other hand, the results showed that concentrations equal to or greater than 10 µg/L induced the production of daphnids male offspring, which may indicate that nicotine is a weak juvenoid compound of the D. magna endocrine system. Furthermore, the result showed that concentrations tested of this chemical have the capacity to revert the effect of fenoxycarb, a strong juvenoid chemical insecticide. The results of the study revealed that nicotine can induce several changes in some of the most important key groups of the aquatic compartment, which can compromise, in a short time, the balance of aquatic ecosystem. Finally, a preliminary environmental risk assessment of this stimulant was performed from the highest measured concentration in surface water and the no observable effect concentration value in the most sensitive species, i.e., D. magna. This process revealed that nicotine can produce an important risk to aquatic organisms.

Oxidative stress responses of Daphnia magna exposed to effluents spiked with emerging contaminants under ozonation and advanced oxidation processes.

Integration of conventional wastewater treatments with advanced oxidation processes (AOPs) has become of great interest to remove pharmaceuticals and their metabolites from wastewater. However, application of these technologies generates reactive oxygen species (ROS) that may reach superficial waters through effluents from sewage treatment plants. The main objective of the present study was to elucidate if ROS present in real effluents after biological and then chemical (single ozonation, solar photolytic ozonation, solar photocatalytic ozonation (TiO2, Fe3O4) and solar photocatalytic oxidation (TiO2)) treatments induce oxidative stress in Daphnia magna. For this, the activity of two antioxidant enzymes (superoxide dismutase and catalase) and the level of lipid peroxidation were determined in Daphnia. The results of oxidative stress biomarkers studied suggest that D. magna is able to cope with the superoxide ion radical (O2·-) present in the treated effluent due to single ozonation by mainly inducing the antioxidant activity superoxide dismutase, thus preventing lipid peroxidation. Lethal effects (measured in terms of immobility) were not observed in these organisms after exposure to any solution. Therefore, in order to probe the ecological efficiency of urban wastewater treatments, studies on lethal and sublethal effects in D. magna would be advisable.

Characterization of plasma cholinesterase in rabbit and evaluation of the inhibitory potential of diazinon.

Several studies indicate that more than one cholinesterase form may be present in the blood of mammals. In this study the predominant plasma cholinesterase activity, the physiological cholinesterase activity as well as cholinesterase sex-dependent changes in non-exposed individuals of rabbit have been established. Plasma cholinesterase was characterized using three substrates (acetylthiocholine iodide, propionylthiocholine iodide, and S-butyrylthiocholine iodide) and three cholinesterase inhibitors (eserine sulfate, BW284C51 and iso-OMPA). The results indicated that propionylthiocholine was the preferred substrate by plasma cholinesterase followed by acetylthiocholine and butyrylthiocholine, and the predominant enzymatic activity was acetylcholinesterase. Physiological plasma cholinesterase activity was 198.9 ± 5.8 nmol/min/ml for male and 205.2 ± 5.0 nmol/min/ml for female using acetylthiocholine as substrate. Thus, sex had no significant effect on the physiological cholinesterase activity (p>0.05). In addition, the in vivo and in vitro sensitivity of plasma cholinesterase to diazinon was also investigated. In rabbits exposed to single doses of diazinon (25 or 125 mg/kg) the higher inhibitions of plasma cholinesterase were reached 9h after oral administration (53% and 87% inhibition, respectively). Cholinesterase activity significantly recovered up to values similar to pre-administration between 3 and 7d depending on the administered dose and sex of the animals. Plasma cholinesterase activity decreased to 24%, 53% and 74% of the initial activity at 9h of in vitro exposure to 1.25, 3.13 and 6.25mg/l of diazinon, respectively, and it remained steadily depressed throughout the experimental period (10d). This study has demonstrated the sensitivity of cholinesterase activity in plasma of rabbits following both in vivo and in vitro exposure to sub-lethal concentrations of diazinon.

Characterization of plasma cholinesterase from the White stork (Ciconia ciconia) and its in vitro inhibition by anticholinesterase pesticides.

Blood plasma cholinesterase (ChE) activity is a sensitive biomarker of exposure to organophosphorus (OP) and carbamate (CB) insecticides in vertebrates. Several studies indicate that more than one ChE form may be present in blood of birds. In this study the predominant ChE activity (acetylcholinesterase - AChE- or butyrylcholinesterase - BChE-), the range of ChE activity as well as ChE age-dependent changes in non-exposed individuals of White stork (Ciconia ciconia) have been established. The in vitro sensitivity of ChE to OP and CB insecticides such as paraoxon-methyl, carbofuran and carbaryl was also investigated. Plasma ChE was characterised using three substrates (acetylthiocholine iodide, propionylthiocholine iodide, and S-butyrylthiocholine iodide) and three ChE inhibitors (eserine sulphate, BW284C51 and iso-OMPA). The results indicated that propionylthiocholine was the preferred substrate by plasma cholinesterase followed by acetylcholine and butyrylcholine and the predominant enzymatic activity in plasma of White storks was BChE. Normal plasma BChE activity in White stork was 0.32±0.01µmol/min/ml for adults and 0.28±0.03µmol/min/ml for juveniles. So, the age had no significant effect on the range of BChE activity. The study on the in vitro inhibitory potential of tested anticholinesterase pesticides on plasma ChE activity revealed that paraoxon-methyl is the most potent inhibitor followed by carbofuran and finally by carbaryl. The percentage of in vitro plasma ChE inhibition was observed to be similar between adults and juveniles.

Screening risk assessment tools for assessing the environmental impact in an abandoned pyritic mine in Spain.

This paper describes a new methodology for assessing site-specific environmental impact of contaminants. The proposed method integrates traditional risk assessment approaches with real and variable environmental characteristics at a local scale. Environmental impact on selected receptors was classified for each environmental compartment into 5 categories derived from the whole (chronic and acute) risk assessment using 8 risk levels. Risk levels were established according to three hazard quotients (HQs) which represented the ratio of exposure to acute and chronic toxicity values. This tool allowed integrating in only one impact category all the elements involved in the standard risk assessment. The methodology was applied to an abandoned metal mine in Spain, where high levels of As, Cd, Zn and Cu were detected. Risk affecting potential receptors such as aquatic and soil organisms and terrestrial vertebrates were assessed. Whole results showed that impact to the ecosystem is likely high and further investigation or remedial actions are necessary. Some proposals to refine the risk assessment for a more realistic diagnostic are included.

Glutathione and malondialdehyde levels in common carp after exposure to simazine.

Several water contamination incidents with simazine have occurred in the province of Badajoz (Spain). Simazine residues were also detected in drinking water, increasing public health concern. Since little information was found concerning the effects of sublethal concentrations of simazine on aquatic organisms, we investigated some oxidative stress biomarkers in tissues of carps from contaminated waters. Results confirmed an increase in tissue reduced glutathione and malondialdehyde levels in carps inhabiting one of the contaminated ponds. To assess the biological significance of this finding, a laboratory study in which carps were exposed to simazine at 45µgL(-1) (10-fold that of the natural water levels) for 90 days was developed. The results obtained in the field study were not confirmed in the laboratory exposure, where no differences were seen between the control and the exposure groups. Therefore, carps did not suffer oxidative stress phenomena by the presence of the simazine, at concentrations found in both studies.

Acetylcholinesterase activity in seabirds affected by the Prestige oil spill on the Galician coast (NW Spain).

In November 2002, the tanker Prestige broke in two and sank at the bottom of the ocean spilling about 70,000 t of fuel oil, which reached the coast of Galicia. It was considered the largest spill in maritime history, greatly affecting marine and related avian species. The spilled fuel oil contained high concentrations of polycyclic aromatic hydrocarbons (PAHs). Many species were affected and were found dead, although ongoing research is still being carried out on the sublethal effects. In this sense, little is known about the action of PAHs on Cholinesterase activity in seabirds. Consequently, the purpose of this study was to provide more information on the neurotoxicity of fuel oil on the seabirds most affected by the Prestige accident: common guillemot, Atlantic puffin and razorbill. On the other hand, data on normal values of acetylcholinesterase (AChE) activity were obtained to supply non-exposed values in seabirds. The oil spill produced a clear inhibitory effect on brain AChE activity in common guillemot (16%, p< or =0.01) and razorbill (22%, p< or =0.01), but not in Atlantic puffin (4%). Physiological levels of brain AChE, expressed in nmol acetylcholine hydrolysed min(-1) mg(-1) protein were similar in non-exposed common guillemot (388.6+/-95.0) and Atlantic puffin (474.0+/-60.7), however, razorbill values were higher (644.6+/-66.9).

Heavy metal and arsenic content in seabirds affected by the Prestige oil spill on the Galician coast (NW Spain).

Seabirds are top consumers in marine foodchains which offer opportunities to detect and assess the toxicological effects of different inorganic elements on the marine ecosystem. In order to provide baseline data concerning trace element levels in seabird species from NW Spain, zinc, copper, arsenic, chromium, lead, cadmium and mercury concentrations were analyzed in liver of three different seabird species (common guillemot, Atlantic puffin and razorbill) affected by the Prestige oil spill in September 2002 on the Galician coast. In general, with the exception of mercury, levels of all the analyzed elements were similar or lower in comparison with those reported for the same species in other Atlantic areas, and did not exceed levels indicative of increased environmental exposure.