A. L. Oropesa

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.

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.

Effect of long-term exposure to simazine on histopathology, hematological, and biochemical parameters in Cyprinus carpio

We investigated if residues of simazine in the natural waters would cause histological, hematological, and biochemical alterations in carps from contaminated areas in Badajoz (Spain). Some necrotic foci in kidney and liver, hepatitis, and hepatic steatosis were detected. No changes on measured hematological and biochemical parameters between fish from reference and contaminated ponds were observed. To assess if simazine exposure was the cause of these observations carps were exposed in the laboratory to simazine (45 μg/L) for 90 days. Some results obtained in the field were confirmed in laboratory, such as necrosis in kidney and liver and hepatic steatosis. Globular eosinophilic foci in kidney and a slight decrease of the hematocrit were also detected. These changes were moderate and indicative of an adaptation of the fish to the toxic stress caused by exposure to low simazine concentrations.

Effect of Long-Term Exposure to Simazine on Brain and Muscle Acetylcholinesterase Activity of Common Carp (Cyprinus carpio)

Several water‐contamination incidents with simazine have occurred in the province of Badajoz (Spain), due to its excessive use for controlling weeds in olive trees and vineyards. Simazine residues were also detected in drinking water, increasing public health concern. However, little is known on the effects that low levels of simazine pose to environment organisms. We investigated if residues of simazine in the natural waters would affect brain and muscle acetylcholinesterase activity in common carps captured in areas in which simazine residues were detected at average levels of 4.5 μg/L. Results confirmed depression on brain and muscle acetylcholinesterase activity of 20% and 29%, respectively, in carps inhabiting one of the simazine‐contaminated ponds, termed “Molinos de Matachel.” To assess the biological significance of this finding, we developed a controlled laboratory study in which carps were exposed to simazine at 45 μg/L (10‐fold that of the natural water levels) for 90 days. The results obtained in the field study were not confirmed in our laboratory experiment, since carps did not show evidence or brain or muscle acetylcholinesterase activity depression for the duration of the experiment, and therefore, we can conclude that acetylcholinesterase depression found in carps collected in “Molinos de Matachel” should be ascribed to other compounds or mixtures of xenobiotics.

Datura stramonium poisoning in horses: a risk factor for colic.

PLANTS are one of the most frequent causes of poisoning in horses, due to the horse’s general behaviour. These poisonings are usually associated with potentially dangerous garden refuse, including hedge clippings (for example, cherry, laurel or English yew) that are left in the vicinity of horses, or to poisonous plants, which are found mixed in their hay. However, in general, cases of poisoning are accidental rather than deliberate (Delaunois and others 1998). Horses may be induced to consume dangerous plants if, for example, little other forage is available or if they are very thirsty. There is a widespread belief that animals’ instincts protect them, but this cannot be relied on (Bamka 1999). A good knowledge of potentially dangerous plants, together with proper management of horses, pasture and hay, is the best way to avoid incidents of poisoning. Thorn apple (Datura stramonium), also referred to Jimson weed, Jamestown weed, moon flower, stinkblaar or olieboon, is well known to be poisonous to human beings and livestock, and accidental poisoning of dogs has also been described (Tostes 2002). It is native to the tropics and easily observed all around Spain; it is common in barnyards, feedlots (especially those housing pigs), cultivated fields of sunflowers, maize and lucerne, roadsides, wasteland and other disturbed habitats (Oladosu and Case 1979). D stramonium is an annual weed that grows 75 to 90 cm tall, with an erect, stout stem and spreading branches near the top, unpleasant-smelling, alternating leaves that are unevenly or sharply toothed and glabrous, a funnel-shaped corolla, and hard, prickly fruit with a whitish or purplish colour. The fruits ripen in autumn and split open to reveal numerous wrinkled, black seeds (Cooper and Johnson 1998). All parts of the plant are poisonous, especially the seeds, which accumulate higher amounts of toxic compounds (Williams and Scott 1984): the tropane alkaloids hyoscyamine, atropine and hyoscine (scopolamine). The anticholinergic substances exert a predominantly antimuscarinic effect by competitive inhibition of acetylcholine binding, resulting in a blockage of autonomic impulses at the level of the neuroeffector junctions (Schulman and Bolton 1998). The toxic effects are reported to include teratogenic effects, mild gastrointestinal clinical signs, neurological signs and death (Nelson and others 1982). However, due to the plant’s particular odour and taste, animals seldom consume enough of it to be affected, and poisoning is usually associated with the contamination of feeding stuffs with the seeds or dry plant material, rather than direct consumption (Delaunois and others 1998). This short communication describes an outbreak of D stramonium poisoning in horses in Spain as a result of the ingestion of lucerne hay grossly contaminated with dry Datura plant matter. An equine centre located in Toledo, central Spain, contained 15 horses, which were confined and typically fed barley, fodder, oat straw and lucerne hay. In September 2000, a veterinary surgeon visited the centre in response to a call for assistance; one horse was displaying a moderate colic, with signs of abdominal pain, anxiety and increased borborygmi; the horse’s temperature and hydration status were normal but its mucous membranes were slightly reddened. The clinician instigated a regimen of no food and administered 60 mg/day butylscopolamine bromide (Buscapina compositum; Boehringer Ingelheim) intravenously, and furosemide (Seguril; Aventis) intravenously, 20 mg/ampoule, five ampoules in the first dose, followed by two ampoules every hour until the horse urinated. To support liver function, the horse received 250 ml 40 mg/ml methionine (Norepar; Merial) and 20 ml 75 mg/ml betaine glucuronate (Norepar; Merial) intravenously. After a moderate walk, the horse was observed to urinate, and its general condition improved. The next morning, the horse was in a satisfactory condition, but a second horse started showing similar clinical signs. The same treatment was instituted and, as with the first horse, recovery was observed. One day later, two more horses developed the same clinical signs; these horses also received the same treatment, but only one of them seemed to improve; the other horse died after six hours, despite intravenous serum therapy being instituted. For all the affected horses, the general treatment included liver function support for three days and activated charcoal. It was observed that the horses’ recovery was better with more intense restriction of their feed, especially when the lucerne hay was eliminated from their diet. On opening the abdomen of the dead horse, general congestion of the abdominal cavity was observed, with oedematous and very gaseous content of the intestinal loops. A gastric rupture involving the greater curvature of the stomach (7 to 8 cm in length), with spillage of the gastric contents into the omentum and peritoneal cavity was observed. The gastric content was very liquid and blackish in colour. Congested kidneys and lungs were also observed; this was attributed to increased intra-abdominal pressure and respiratory distress. Inquiry revealed that all the horses had been fed on a new lot of lucerne hay three days before the clinical signs were first observed. The clinician inspected the feed, and a large amount of a desiccated plant locally called ‘cardo de castañitas’ was observed mixed in with the hay. The horses’ owner stated that all the hay had been bought from a local farm, and they were a ‘bargain, because they were the last remaining’, as indicated by the seller. A complete hay bale was sent to the authors’ laboratory, and detailed inspection revealed that it consisted of dry lucerne (6·85 kg) and a large amount of D stramonium prickly fruits and stems (7·15 kg), with occasional seeds (Fig 1). D stramonium thus made up 51 per cent of the total weight of the bale. The botanical identification of the plant confirmed the suspected diagnosis of Datura poisoning. The lot of lucerne hay was destroyed; no further cases of poisoning occurred. There are few reports of Datura species poisoning in horses; previous reports describe a subacute to chronic course of intoxication before the onset of clinical signs, with variable mortality in the affected horses (Schulman and Bolton 1998). Veterinary Record (2006) 158, 132-133

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.

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.

Antioxidant defences and lipid peroxidation in wild White Storks, Ciconia ciconia, from Spain

AbstractDespite the ecological importance of the White Stork, Ciconia ciconia, and its wide distribution in southern Europe, knowledge of the impact of long-term exposure to pollution in its wild populations is still scarce. As a first step towards contributing to addressing this question, the baseline values of anti-oxidant parameters and peroxidation levels were determined in wild White Storks, and possible differences related to age were investigated. Superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, total glutathione, reduced and oxidised glutathione ratio and lipid peroxidation were determined. Superoxide dismutase activity and the level of total glutathione were statistically higher in adult compared to juvenile storks. The reduced and oxidised glutathione ratio and the lipid peroxidation level followed also the same trend, although no significant differences were found between adults and juveniles. On the contrary, catalase and glutathione peroxidase showed low activities in adults when compared to juvenile storks. Finally, glutathione reductase and glutathione S-transferase activities were similar in two groups of birds studied. Thus, it can be concluded that the period of enzymatic activity in adolescence in wild White Storks is very important in forming the organism’s response to environmental stress.ZusammenfassungAntioxidantien-Abwehr und Lipidperoxidation bei freilebenden Weißstörchen (Ciconia ciconia) in Spanien Trotz seiner ökologischen Bedeutung und weiten Verbreitung in Südeuropa weiss man nicht viel über die Auswirkungen von Langfrist-Belastungen durch Umweltverschmutzung auf freilebende Weißstorch-Populationen. Als ersten Schritt zur Untersuchung dieser Frage bestimmten wir die Normalwerte der Antioxidantien- und Peroxidations-Parameter in freilebenden Störchen und untersuchten mögliche altersabhängige Unterschiede. Konkret angeschaut wurden Superoxid Dismutase, Katalase, Glutathion-Peroxidase, Glutathion-Reduktase, Glutathion-S-Transferase, Gesamt-Glutathion, das Verhältnis von reduziertem zu oxidiertem Glutathion und Lipidperoxidation. Bei adulten Tieren lagen die Superoxid Dismutase-Aktivität sowie der Gesamt-Glutathion-Spiegel statistisch signifikant höher als bei Jungvögeln. Auch das Verhältnis von reduziertem zu oxidiertem Glutathion und die Lipidperoxidation zeigten diesen Trend, die Unterschiede zwischen adulten und jungen Tieren waren jedoch nicht signifikant. Im Gegensatz dazu zeigten Katalase und Glutathion-Peroxidase bei adulten Tieren geringere Aktivität als bei Jungstörchen. Glutathion-Reduktase und Glutathion-S-Transferase zeigten in beiden untersuchten Vogelgruppen ähnliche Aktivitäten. Aus diesen Ergebnissen kann geschlossen werden, dass für erwachsene, freilebende Weißstörche enzymatische Aktivitäten von großer Bedeutung für die Reaktion auf Umweltstress sind.

Effects of exposure to 17-alpha-ethynylestradiol on sperm quality of tench (Tinca tinca).

Alterations of sperm quality were studied in tench (Tinca tinca) exposed to sub-lethal doses of 17-alpha-ethynylestradiol-EE2-(50, 100 and 500μg/kg t.w) under semi-static conditions for 30 days. Thus, different biomarkers of sperm quality were assessed: concentration and volume of ejaculate, total number of spermatozoa, percentage of motile spermatozoa, sperm motility and percentage of live and dead spermatozoa. Sperm motility was examined by computer-assisted image analysis and the viability of spermatozoa was assessed through flow cytometry. The most relevant alterations observed were significant reductions in the reproductive parameters such as testicular somatic index, spermatozoa concentration, straight line velocity, curvilinear velocity, average path velocity and wobble in tench exposed to 50μg/kg t.w of EE2. Our study about the effects of EE2 on the sperm quality in tench provides new evidences which strengthen the fact that this synthetic estrogen is included in the list of non-monotonic dose response compounds in animal studies.

Histological alterations in the structure of the testis in tench (Tinca tinca) after exposure to 17 alpha-ethynylestradiol.

Environmental pollution with synthetic estrogens may pose a serious threat to reproduction of aquatic wildlife species. The current study describes the effects of 17α‐ethynylestradiol (EE2) on the structure of the testis in tench (Tinca tinca). Adult male tench were exposed to sublethal doses of EE2 (50, 100, and 500 μg/Kg t.w.) under semistatic conditions for a period of 30 days. The condition factor (CF), testicular somatic index (TSI), and histology (including a morphometric analysis) of the testis were examined. No consistent differences were observed in the CF of EE2‐exposed tench when compared with nonexposed fish. A significant decrease in TSI could only be observed at a 50 μg/Kg t.w. EE2 dose (p < 0.05) when compared with the control group. The histopathology of the testis was associated with loss of normal tubular structure with increased doses of exposure, decrease of tubule number, degeneration in Sertoli and Leydig cells, increase in necrotic testicular cells including formation of syncytia structures and, finally, a high incidence of fish with early primary oocytes at 100 and 500 μg/Kg t.w. EE2. These results indicate that long‐term exposure to EE2 may produce clear negative effects on testicular structure in tench.