Influencia de factores endógenos (edad y sexo) en los niveles de biomarcadores de estrés oxidativo en alcatraz (Morus bassanus) / Influence of endogenous factors (age and sex) on the levels of oxidative stress biomarkers in Northern gannet (Morus bassanus)

Las aves marinas pueden ser utilizadas como bioindicadoras de las alteraciones del ambiente en que residen, identificándose en ellas distintos biomarcadores, que no informen sobre los niveles cuantitativos de contaminantes sino sobre los efectos adversos subclínicos que dichos agentes pueden causar. En el presente trabajo se han analizado los niveles de Malondialdehído (MDA) y de actividad Glutatión-S-Transferasa (GST) en muestras de hígado y riñón de 30 alcatraces (Morus bassanus) procedentes de las costas de Galicia, como posibles indicadores de estrés oxidativo en las aves. Además, se ha determinado la influencia de dos factores endógenos (sexo y edad) sobre los niveles de estos biomarcadores. En hígado los valores medios de MDA obtenidos fueron de 0,508±0,502 nmol/mg proteína, mientras que en riñón fueron de 15,67±12,18 nmol/mg proteína. La actividad GST media en hígado fue de 10,93±7,067 nmol/min/mg proteína, mucho menor que la media de los valores en riñón, que fue 62,30±26,97 nmol/min/mg proteína. En cuanto a las variaciones respecto a la edad, solo se encontraron diferencias estadísticamente significativas en riñón en la actividad GST, siendo mayor la actividad de esta enzima en alcatraces adultos que en inmaduros. Por su parte, el factor sexo no influyó en los niveles de ninguno de los biomarcadores considerados. Los resultados obtenidos apuntan a que ambos biomarcadores podrían ser de utilidad en programas de biomonitorización de contaminación ambiental centrados en el alcatraz, aunque serían necesarios estudios que consideren un mayor número de animales, incorporando un abanico más amplio de biomarcadores. (AU)

Seabirds can be used as bioindicators of changes in the environment in which they live, identifying different biomarkers in them, which do not provide information on the quantitative levels of contaminants, but on the subclinical adverse effects that these agents can cause. At the present study, the levels of Malondialdehyde (MDA) and glutathione-S-transferase (GST) activity in liver and kidney samples of 30 gannets (Morus bassanus) from the coast of Galicia have been determined, as suitable biomarkers of oxidative stress on birds. In addition, the influence of two endogenous factors (sex and age) on the levels of these biomarkers has been determined. In the liver, the mean values of MDA were 0.508 ± 0.502 nmol/mg protein, while in kidney they were 15.67 ± 12.18 nmol/mg protein. On the other hand, mean GST activity in the liver was 10.93 ± 7.067 nmol/min/mg protein, much lower than the mean activity values found in kidney, which were 62.30 ± 26.97 nmol/min/mg protein. Regarding the variations with respect to age, statistically significant differences were only found in the kidney in GST activity, the activity of this enzyme being higher in adult gannets than in immature ones. Secondly, the gender factor did not influence the levels of any of the biomarkers considered. The obtained results indicate that both biomarkers could be useful in biomonitoring programs of environmental contamination focused on the gannet, although studies that consider a larger number of animals, incorporating a broader range of biomarkers, would be necessary. (AU)

Removal efficiency for emerging contaminants in a WWTP from Madrid (Spain) after secondary and tertiary treatment and environmental impact on the Manzanares River.

The effluents from wastewater treatment plants (WWTPs) can be an important contamination source for receiving waters. In this work, a comprehensive study on the impact of a WWTP from Madrid on the aquatic environment has been performed, including a wide number of pharmaceuticals and pesticides, among them those included in the European Watch List. 24-h composite samples of influent (IWW) and effluent wastewater after secondary (EWW2) and after secondary + tertiary treatment (EWW3) were monitored along two campaigns. Average weekly concentrations in IWW and EWW2 and EWW3 allowed estimating the removal efficiency of the WWTP for pharmaceutical active substances (PhACs). In addition, the impact of EWW3 on the water quality of the Manzanares River was assessed, in terms of PhAC and pesticide concentrations, through analysis of the river water collected upstream and downstream of the discharge point. After a preliminary risk assessment, a detailed evaluation of the impact on the aquatic environment, including a toxicological study and screening of pharmaceutical metabolites, was made for the seven most relevant PhACs: sulfamethoxazole, azithromycin and clarithromycin (antibiotics), metoprolol (antihypertensive), diclofenac (anti-inflammatory/analgesic), irbesartan (antihypertensive), and the antidepressant venlafaxine. Among selected PhACs, irbesartan, clarithromycin and venlafaxine presented moderate or high risk in the river water downstream of the discharge. Albeit no acute toxicity was detected, more detailed studies should be carried out for these substances, including additional toxicological studies, to set up potential sublethal and chronic effects on aquatic organisms.

Ensayo estandarizado (ISO 17512) de comportamiento de evitación de suelos contaminados. Efecto del pesticida dimetoato sobre la lombriz de tierra Aporrectodea caliginosa / Standardized avoidance test (ISO 17512) for contaminated soils. Effect of the pesticide dimethoate on the earthworm Aporrectodea caliginosa

La creciente preocupación por el bienestar animal ha propiciado el rechazo por parte de la sociedad a la utilización de animales en experimentación. Por este motivo, actualmente se están desarrollando métodos alternativos que cumplan con el principio de las 3Rs (reemplazar, reutilizar y refinar). Muchos de estos métodos se basan en la utilización de organismos inferiores, como las lombrices de tierra. El presente estudio desarrolla el ensayo estandarizado de la Organización Internacional de Normalización (ISO) 17512-1:2008, en el que se evalúa la toxicidad producida por el plaguicida organofosforado dimetoato en el suelo, utilizando la capacidad de repulsión o evitación que presentan las lombrices de tierra pertenecientes a la especie Aporrectodea caliginosa. Las concentraciones seleccionadas han sido 0,06, 0,3 y 1,5 mg/kg de suelo, ensayada cada una de ellas por triplicado, empleando 10 lombrices por cada réplica. Los resultados reflejan la capacidad de las lombrices de tierra de la especie Aporrectodea caliginosa para evitar suelos contaminados con el formulado comercial de dimetoato. Por otra parte, se confirma la tendencia de las lombrices a evitar el suelo contaminado a medida que aumenta la concentración de plaguicida ensayada, con significación estadística a la dosis más alta empleada (1,5 mg/kg). (AU)

The increasing concern about animal wellness has induced the repulse of society to the use of animals in research. For this reason, alternative methods which comply with the 3Rs principle (Replace, Reuse and Refine) are currently being developed. Many of these methods are based on the use of lower organisms on the evolutionary scale, such as earthworms. The present study develops the standardized test of the International Organization of Standardization (ISO) 17512-1:2008, in which the toxicity produced by the pesticide organophosphate dimethoate in the soil is evaluated, using the capacity of repulsion or avoidance that presents the earthworms Aporrectodea caliginosa. The assayed concentrations were 0.06, 0.3 and 1.5 mg/kg of soil, each one tested in triplicated, using 10 earthworms for each repetition. The results reflect the ability of earthworms of the species Aporrectodea calliginosa to avoid soils contaminated with the commercial formulation of dimethoate. On the other hand, the tendency of worms to avoid contaminated soils is confirmed as the concentration of pesticide increases, with statistical significance at the highest concentration used (1.5 mg/kg). (AU)

Ensayo estandarizado (ISO 17512) de comportamiento de evitación de suelos contaminados II. Respuesta de distintos biomarcadores tras la exposición a dimetoato en la lombriz de tierra Aporrectodea caliginosa / Standardized avoidance test (ISO 17512) for contaminated soils II. Response of different biomarkers after exposure to dimethoate in earthworm Aporrectodea caliginosa

En la primera parte del presente estudio, publicado en 2021, se mostraron los resultados obtenidos al aplicar el ensayo estandarizado de la Organización Internacional de Normalización (ISO) 17512-1:2008. En dicho trabajo se evaluó la toxicidad producida por el plaguicida organofosforado dimetoato en el suelo, utilizando la capacidad de repulsión o evitación que presentan las lombrices de tierra de la especie Aporrectodea caliginosa. En esta segunda parte, para evaluar los efectos subletales producidos por el dimetoato sobre las lombrices, se han determinado como biomarcadores la actividad de la enzima acetilcolinesterasa (AChE, cuya inhibición es el principal mecanismo de acción de este tipo de compuestos) y la actividad glutatión S-transferasa (GST, que comúnmente se activa como mecanismo de detoxificación de plaguicidas). Las concentraciones seleccionadas han sido 0,06, 0,3 y 1,5 mg/kg de suelo, ensayada cada una de ellas por triplicado, empleando 10 lombrices por cada réplica.La actividad AChE se inhibió significativamente en la exposición a las tres concentraciones empleadas. No se ha producido aumento de la actividad GST que, por el contrario, aparece significativamente inhibida a la concentración más alta de plaguicida ensayada. La falta de una inducción de la actividad GST puede deberse a que el mecanismo de detoxificación no se ha puesto en marcha por el corto tiempo de exposición al plaguicida. Estos resultados reflejan la sensibilidad de la AChE como biomarcador de la contaminación de los suelos con organofosforados. (AU)

In the first part of this study, published in 2021, the results obtained when applying the standardized test of the International Organization of Standardization (ISO) 17512-1:2008 were shown. In this study, the toxicity produced by the pesticide organophosphate dimethoate in the soil was evaluated, using the capacity of repulsion or avoidance that presents the earthworms Aporrectodea caliginosa. To evaluate the sublethal effects produced by dimethoate on worms, the activities of the acetylcholinesterase enzyme (AChE, the inhibition of which is the main mechanism of action of this type of compound) and glutathione S-transferase enzyme (GST, which is commonly activated as a pesticide detoxification mechanism) have been determined. The commercial product called DIMAFID40, whose active substance is the organophosphate pesticide dimethoate, was used. The selected concentrations were 0.06, 0.3 and 1.5 mg / kg of soil, each one tested in triplicate, using 10 worms per replicate.The AChE activity was significantly inhibited after exposure to the three assayed concentrations. With respect to GST activity, it was not increased but significantly inhibited after exposition to the highest concentration of pesticide. The non-appearance of an induction of GST activity may be due to the fact that the detoxification mechanism has not been started because the time of exposure to the pesticide has been too short. These results reflect the sensibility of AChE as a biomarker of the contamination in soils with organophosphate. (AU)

Comparative study of esterase activities in different tissues of marine fish species Trachurus trachurus, Merluccius merluccius and Trisopterus luscus.

Pesticides are one of the most frequently anthropogenic xenobiotics detected in water. Among these, the organophosphorus pesticides (OPs) are very widely used in agriculture due to their broad spectrum of activity and their low price, but they also have high potent effects as neurotoxic compounds in non-target organisms. The aim of this study was to evaluate biomarkers acetylcholinesterase (AChE), butyrylcholinesterase (BChE), propionylcholinesterase (PChE) and carboxylesterase (CbE) in the representative Atlantic fish species Trachurus trachurus, Merluccius merluccius and Trisopterus luscus from "Rías Gallegas", a traditional Spanish fishing area. These esterase activities were evaluated in the brain, muscle and liver to determine the most adequate tissue to measure such enzymatic activities. The sensitivity of AChE and CbE activities from different tissues the widely used organophosphorus insecticide chlorpyrifos (CP), and its toxic metabolite (CP-oxon) was also tested. AChE activity was predominant in all tissues of the analysed species (particularly in brain constituting from 78.33%, 89.83% and 88.43% of total ChEs in Trachurus trachurus, Merluccius merluccius and Trisopterus luscus, respectively). Under in vitro exposure, esterases were shown to be highly sensitive to CP and especially to CP-oxon. Moreover, a similar effect observed on AChE and CbE activities could suggest that CbE activity might contribute efficiently against the toxic effects of CP, especially in muscle and the liver. The presence of BChE, PChE and upper CbE activities in muscle and the liver and their OP-sensibilities can be used to study their function in the pesticide biochemical detoxification pathways with a prominent role as a safeguarding mechanism against pesticide toxicity.

Desarrollo del ensayo de toxicidad estandarizado OECD nº 207 en lombriz de tierra expuesta al pesticida organofosforado Dimetoato / Development of the OECD standardized toxicity test no. 207 on earthworms exposed to the organophosphorus pesticide Dimethoate

La importancia de los estudios toxicológicos sobre suelos es evidente, y el desarrollo y uso de los ensayos estandarizados para llevarlos a cabo es un campo en constante crecimiento. En el presente trabajo se ha procedido a realizar el ensayo de toxicidad aguda con lombrices de tierra Eisenia foetida de acuerdo con el protocolo estandarizado de la OECD nº 207. La sustancia activa de elección ha sido el insecticida organofosforado dimetoato. A partir de la concentración recomendada por el fabricante del formulado comercial DIMAFID40, y siguiendo el protocolo arriba indicado, se ha realizado una exposición a 5 concentraciones diferentes del pesticida (0,001%; 0,01%; 0,1%; 1%; 10%), estableciéndose además un grupo control. Como primer resultado merece destacar que, a mayor dosis de ensayo del producto, mayor ha sido el porcentaje de mortalidad en los animales, observándose además mayores cambios fisio-patológicos en los animales expuestos. Dichos cambios no fueron evidentes en todos los grupos de animales, si bien fueron progresivos y cada vez más claros a medida que aumentaba la concentración de pesticida. La Concentración Letal Media (CL50) del ensayo coincidió con la concentración recomendada por el fabricante, estableciéndose en 0,1%. Por su parte, el Nivel sin efecto observable (NOEL), de gran relevancia ambiental, se alcanzó a una concentración diez veces inferior a la concentración recomendada por el fabricante

It is evident the relevance of toxicological studies on soils, and the development and use of standardized tests to carry them out is a field in constant growth. In the present work, the acute toxicity test with Eisenia foetida earthworms has been performed according to the standardized protocol of the OECD nº 207. The active substance of choice has been the organophosphate insecticide Dimethoate. Starting from the concentration recommended by the manufacturer of the commercial formulation DIMAFID40, an exposure to 5 different concentrations of the pesticide (0.001%; 0.01%; 0.1%; 1%; 10%) was performed, also establishing a control group. As a first result, it is worth mentioning that, the higher the test dose of the product, the higher the percentage of mortality in the animals. Furthermore, major physical-pathological changes were observed in exposed animals. These changes were not evident in all groups of animals, although they were progressive and clearly increasing as the concentration of pesticide raised. The lethal Media Concentration (LC50) of the trial coincided with the concentration recommended by the manufacturer, being set at 0.1%. Moreover, the level with no observed effect (NOEL), of great environmental relevance, was reached at a concentration ten times lower than the concentration recommended by the manufacturer

Avoidance behaviour response and esterase inhibition in the earthworm, Lumbricus terrestris, after exposure to chlorpyrifos.

The avoidance response of earthworms to polluted soils has been standardised using a simple and low-cost test, which facilitates soil toxicity screening. In this study, the avoidance response of Lumbricus terrestris was quantified in chlorpyrifos-spiked soils, depending on the pesticide concentration and exposure duration. The inhibition of acetylcholinesterase (AChE) and carboxylesterase (CbE) activities was also determined as indirect measures of pesticide bioavailability. The effects of different chlorpyrifos concentrations were examined in a standardised test (two-chamber system) with 0.6, 3 and 15 mg/kg chlorpyrifos. A modification of the test involved a pre-exposure step (24, 48 or 72 h) in soils spiked with 15 mg/kg. In both protocols, earthworms were unable to avoid the contaminated soils. However, the esterase activities showed that all earthworms were exposed to chlorpyrifos. Acetylcholinesterase activity did not change in earthworms in the standardised behavioural test (0.58 ± 0.20 U/mg protein, mean ± SD; n = 72), whereas the CbE activity was significantly inhibited (62-87 % inhibition) in earthworms exposed to 3 and 15 mg/kg. In the modified test, earthworms had greatly inhibited AChE activity (0.088 ± 0.034 U/mg protein, n = 72), which was supported by reactivation of the inhibited enzyme activity in the presence of pralidoxime (2-PAM). Similarly, the CbE activity was significantly inhibited in earthworms with all treatments. This study suggests that the avoidance behaviour test for organophosphorus-contaminated soils could be supported by specific biomarkers to facilitate a better understanding of pesticide exposure and toxicity during this test.