G.V. Aguirre-Martínez

Are standard tests sensitive enough to evaluate effects of human pharmaceuticals in aquatic biota? Facing changes in research approaches when performing risk assessment of drugs.

Nowadays, the presence of pharmaceutical products in aquatic environments is not only common, but is also of significant concern regarding the adverse effect they may produce to aquatic biota. In order to determine the adverse effects of caffeine (CAF), ibuprofen (IBU), carbamazepine (CBZ) and novobiocin (NOV), at environmental occurring concentrations, standardized endpoints applied in current guidelines were evaluated in four organisms including bioluminescence response in Vibrio fischeri, growth inhibition in Isochrysis galbana (marine water) and Pseudokirchneriella subcapitata (fresh water) and fertilization and embryo-larval development in Paracentrotus lividus. To reach this aim bioassays were implemented by exposing organisms to water spiked with drugs dissolved in DMSO (0.001% v/v). Risk characterization was performed, calculating the environmental impact of drugs by calculating environmental concentration and predicted no effect concentration ratio (MEC/PNEC). Results indicate that acute toxicity was found above environmental concentrations in the order of mg L(-1) for bacteria bioluminescence, microalgae growth inhibition and sea urchin fertilization. However, teratogenicity was observed on sea urchin after exposure to environmental concentrations of drugs at 0.00001 mg L(-1); at this concentration CBZ and IBU were found to reduce significantly the embryo-larval development compared to controls (p<0.01). The risk calculated for selected drugs suggested they are harmless for aquatic environment except when applying the embryo-larval development endpoint. Endpoints applied in this study showed the necessity of using more sensitive responses, when assessing risk of pharmaceuticals in aquatic environments, since endpoints applied in current guidelines may not be suitable.

Yes, caffeine, ibuprofen, carbamazepine, novobiocin and tamoxifen have an effect on Corbicula fluminea (Müller, 1774)

Reports indicating the presence of pharmaceutical in fresh water environment in the ngL(-1) to µgL(-1) range are occurring with increasing frequency. It is also a fact that pharmaceuticals may produce adverse effects on aquatic organisms. Nevertheless, there is still a lack of knowledge regarding how these emergent contaminants may affect aquatic biota. The goal of this research was to evaluate the sublethal responses in Corbicula fluminea such as, general stress (lysosomal membrane stability [LMS]), biomarkers of phase I and II (etoxyresorufin O-deethylase [EROD], dibenzylfluorescein dealkylase [DBF], gluthathione-S-transferase [GST]), oxidative stress (gluthathione reductase [GR], gluthathione peroxidase [GPX], lipid peroxidation [LPO]), and biomarkers of effect (DNA damage) after 21 days of exposure to caffeine, ibuprofen, carbamazepine, novobiocin and tamoxifen at 0.1, 1, 5, 10, 15, 50µgL(-1). Environmental concentrations tested in this study caused general stress and produced changes on biomarkers tested. LMS, responses from phase I and II enzymatic activity, oxidative stress, and biomarker of effect represent important ecotoxicological information, and will provide a useful reference for the assessment of selected drugs and the effects which these compounds may have on aquatic invertebrates, using C. fluminea as a bioindicator species.

Early responses measured in the brachyuran crab Carcinus maenas exposed to carbamazepine and novobiocin: Application of a 2-tier approach

One of the main consequences of the constant input of pharmaceuticals to the aquatic environment is that biota might develop unknown chronic effects, thus affecting their health even at low concentrations. The aim of this study is to evaluate the health status of Carcinus maenas employing a 2-tier approach, after 28 days of exposure to carbamazepine (CBZ) and novobiocin (NOV) at 0.1, 1, 10 and 50µgL(-1). Lysosomal membrane stability (LMS) is employed in tier 1. In tier 2 was applied a battery of biomarkers of exposure and effect (ethoxyresorufin O-deethylase (EROD), dibenzyl flourescein dealkylase (DBF), glutathione S-transferase (GST), glutathione peroxidase (GPx), lipid peroxidation (LPO) and DNA adducts) measured in gill, hepatopancreas, muscle and gonad tissues. Results show a dose-dependent effect. LMS in crabs exposed to environmental concentrations of pharmaceuticals was significantly lower compared to controls (p<0.05), indicating their stressed status. EROD activity was induced significantly (p<0.05) in all tissues by NOV (10-50µgL(-1)). DBF activity was induced significantly (p<0.05) in gill and hepatopancreas tissues by CBZ (10-50µgL(-1)). GST activity was activated in all tissues of crabs exposed to the highest concentrations tested (p<0.05). All tissues showed induction of GPX activity after exposure to selected drugs (p<0.05). LPO was activated in gill and hepatopancreas tissues by the pharmaceuticals at 50µgL(-1) (p<0.05). Crabs exposed to NOV (50µgL(-1)) presented DNA damage in gill and hepatopancreas tissues (p<0.05). Environmental concentrations of these pharmaceuticals have a measurable effect on the biomarkers studied. The 2-tier approach applied might be a suitable tool for the assessment of sublethal responses in crabs exposed to pharmaceuticals in the marine environment.

General stress, detoxification pathways, neurotoxicity and genotoxicity evaluated in Ruditapes philippinarum exposed to human pharmaceuticals.

A battery of biomarkers was evaluated on Ruditapes philippinarum exposed during 14 days to caffeine, ibuprofen, carbamazepine and novobiocin (0.1, 1, 5, 10, 15, and 50µgL(-1)). The battery included general stress (lysosomal membrane stability - LMS) analysed in the hemolymph, and biochemical biomarkers analysed in digestive gland tissues including: biomarkers of phase I (etoxyresorufin O-deethylase - EROD, dibenzylfluorescein dealkylase - DBF), phase II (gluthathione-S-transferase - GST), oxidative stress (gluthathione reductase - GR, gluthathione peroxidase - GPX, lipid peroxidation - LPO), neurotoxicity (acetylcholinesterase activity - AChE), and genotoxicity (DNA damage). Pharmaceuticals tested induced the sublethal responses (even at the environmental range 0.1µgL(-1)). At this low concentration; caffeine, ibuprofen and carbamazepine decreased the LMS significantly compared with controls (p<0.05). The four compounds induced significantly the detoxification metabolism and oxidative stress (p<0.05). Neurotoxicity was noticed in clams exposed to caffeine and carbamazepine (p<0.05). Ibuprofen, carbamazepine and novobiocin produced genotoxic effects (p<0.05). Results from this research validate the use of biomarkers when assessing the effects of pharmaceuticals within a marine environmental risk assessment framework, using as a laboratory bioassay model the species R. philippinarum.

Toxicidad no específica en sedimentos portuarios, una aproximación al contenido de contaminantes críticos: an approach to the content of critical pollutants

Se analizo la calidad de sedimentos de cuatro puertos chilenos con diferentes actividades de cabotaje, en funcion del contenido de materia organica (MOT), hidrocarburos aromaticos policiclicos (HAPs), metales traza (Cd, Pb y Cu) y toxicidad no especifica. MOT se analizo por ignicion, metales por polarografia, HAPs por GC-MS y la toxicidad no especifica aplicando la tecnica de fecundacion (USEPA 1988). Los puertos de Iquique, San Vicente y Talcahuano presentaron sedimentos fangosos con alta concentracion de MOT (ca. 12%). San Vicente presento las mayores concentraciones de HAPs (18.930 µg kg-1 de benzo(b)fluoranteno; 10.820 µg kg-1 de benzo(a)antraceno y 8.550 µg kg-1 de benzo(a)pireno) e Iquique las mayores concentraciones de metales (20 µg g-1 Cd; 370 µg g-1 Pb and 514 µg g-1 Cu). Puerto Patache mostro sedimentos con arena fina, bajos contenidos de MOT, metales y HAPs. Los ensayos de toxicidad indicaron diferencias significativas respecto de los controles. El indice de contaminacion urbana e industrial (ICUI) referido al contenido de metales, revelo como mas contaminados a Iquique y Talcahuano; en cambio el indice de adicion de HAPs a San Vicente (IA HAPs), al igual que la toxicidad. Se postula que sinergias entre los contaminantes, mas que los contenidos de cada uno de ellos, serian los responsables de la toxicidad de los sedimentos.

The effects of human drugs in Corbicula fluminea. Assessment of neurotoxicity, inflammation, gametogenic activity, and energy status

The constant release of pharmaceuticals products to aquatic environment even at low concentrations (ng L-1 to µg L-1) could lead to unknown chronic effects to non-target organisms. The aim of this study was to evaluate neurotoxic responses, inflammation, gametogenic activity and energy status on the fresh water clam C. fluminea after exposure to different concentrations of caffeine (CAF), ibuprofen (IBU), carbamazepine (CBZ), novobiocin (NOV) and tamoxifen (TMX) for 21 days under laboratory conditions. During the assay, water was spiked every two days with CAF (0; 0.1; 5; 15; 50µgL-1), IBU (0; 0.1; 5; 10; 50µgL-1), CBZ, NOV, and TMX (0.1, 1, 10, 50µgL-1). After the exposure period, dopamine levels (DOP), monoamine oxidase activity (MAO), arachidonic acid cyclooxygenase activity (COX), vitellogenin-like proteins (VTG), mitochondrial electron transport (MET), total lipids (TLP), and energy expenditure (MET/TLP) were determined in gonad tissues, and acetyl cholinesterase activity (AChE) was determined in digestive gland tissues. Results showed a concentration-dependence response on biomarkers tested, except for MAO. Environmental concentrations of pharmaceuticals induced significant changes (p < 0.05) in the neurotoxic responses analyzed (CAF, CBZ and NOV increased DOP levels and CBZ inhibited AChE activity), inflammation (CAF induced COX), and energy status (MET and TLP increased after exposure to CBZ, NOV and TMX). Responses of clams were related to the mechanism of action (MoA) of pharmaceuticals. Biomarkers applied and the model organism C. fluminea constituted a suitable tool for environmental risk assessment of pharmaceutical in aquatic environment.

Applicative implications of Carcinus maenas and Ruditapes philippinarum in biomonitoring studies after oil spills

Biomarkers have been tested in order to address the most suitable battery for determining adverse effects of crude oil spills on marine invertebrates. An oil spill with increasing degrees of severity was simulated by mixing crude oil (0%, 0.5%, 2%, 8%, 16%, 32%) with sediment. Carcinus maenas and Ruditapes philippinarum were exposed to this sediment for seven days with the aim of comparing their applicability in biomonitoring studies. Four biomarkers including ethoxyresorufin O-deethylase (EROD), glutathione S-transferase (GST), glutathione peroxidase (GPx) and lipid peroxidation (LPO) were analysed in gill and digestive gland tissues of clams; and in gill and hepato-pancreas tissues of crabs. EROD, GST and GPx enzymatic activities were significantly induced in gill and digestive gland tissues of clams when increasing oil concentrations (p<.01). In crabs all the biomarkers were significantly activated in gill tissues, whereas EROD and LPO activities were induced only in hepato-pancreas tissues (p<.01). Gill and digestive gland in clams and gill in crabs were found to be the most reliable tissues for analysis of biomarkers. The biomarkers selected are thus considered suitable for assessing toxicity of sediments after a marine crude oil spill accident. Both species were found to be sensitive and suitable for biomonitoring purposes.