M. Laura Martín-Díaz

Sublethal responses in caged organisms exposed to sediments affected by oil spills

This study was performed to determine sublethal responses of two invertebrate species by using field deployments in areas affected by oil spills, which are acute in the Galician Coast (NNW, Spain) and chronic in the Bay of Algeciras (SSW, Spain). The organisms employed were the crab Carcinus maenas and the clam Ruditapes philippinarum, and during 28 days the animals were exposed to contaminated sediments in cages under field conditions. Different biomarkers of exposure were determined after a 28-day period exposure: ethoxyresorufin O-deethylase (EROD), phase I detoxification enzyme, glutathione-S-transferase (GST) phase II detoxification enzyme but also implicated in oxidative stress events, glutathione peroxidase (GPX) and glutathione reductase (GR), both antioxidant enzymes. In addition, histopathological effects in target tissues of the deployed organisms were evaluated. Biomarker measurements were linked with the concentration of chemicals in the sediments in order to elucidate the type, source and bioavailability of contaminants that produce adverse effects in the bioindicator species. Results obtained in this study have shown how the application of the selected battery of biomarkers under field bioassays allows for the identification of alternative sources of stress that are not observable in laboratory experiments.

Biomarker responsiveness in different tissues of caged Ruditapes philippinarum and its use within an integrated sediment quality assessment

Biomarkers comprising activities of biotransformation enzymes (ethoxyresorufin-O-deethylase -EROD-, dibenzylfluorescein dealkylase -DBF-, glutathione S-transferase -GST), antioxidant enzymes (glutathione reductase -GR- and glutathione peroxidase -GPX), lipid peroxidation -LPO- and DNA strand breaks were analyzed in the clam Ruditapes philippinarum caged at Cádiz Bay, Santander Bay and Las Palmas de Gran Canaria (LPGC) Port (Spain). Sediments were characterized. Digestive gland was the most sensitive tissue to sediment contamination. In Cádiz Bay, changes in LPO regarding day 0 were related with metals. In LPGC Port, DBF, EROD, and GST activity responses suggested the presence of undetermined contaminants which might have led to DNA damage. In Santander Bay, PAHs were related with EROD activity, organic and metal contamination was found to be associated with GR and GST activities and DNA damage presented significant (p < 0.05) induction. R. philippinarum was sensitive to sediment contamination at biochemical level. Biomarkers allowed chemical exposure and sediment quality assessment.

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.

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.

Validation of Arenicola marina in field toxicity bioassays using benthic cages: Biomarkers as tools for assessing sediment quality

Sediment toxicity assessments using caged organisms present advantages over using laboratory and native community studies. The use of caged Arenicola marina in sediment toxicity assessments was evaluated. Lugworms were exposed in situ to sediments from coastal and port areas in Spain for seven days, and the activities of the biotransformation enzymes ethoxyresorufin O-deethylase, dibenzylfluorescein dealkylase and glutathione S-transferase, the activities of the antioxidant enzymes glutathione reductase and glutathione peroxidase and lipid peroxidation were then analyzed as biomarkers. Biomarker results and sediment physicochemical data were integrated. Cádiz Bay (SW Spain) sediments presented metal contamination that was not linked to a biochemical response. In LPGC Port (SW Spain), Pb contamination exhibited a moderate toxic potential, while PAHs, and presumably pharmaceuticals, provoked biochemical responses that efficiently prevented lipid peroxidation. In Santander Bay (N Spain), exposure to PAHs and, presumably, pharmaceuticals induced biomarker responses, but lipid peroxidation occurred nevertheless. These results indicated that caged A. marina were effective for the assessment of sediment quality and that the selected biomarkers were sufficiently sensitive to identify chemical exposure and toxicity.

The use of a kinetic biomarker approach for in situ monitoring of littoral sediments using the crab Carcinus maenas.

Caged, transplanted, intermoult, female shore crabs (Carcinus maenas) were exposed to sediments from the Port of Cadiz (SW, Spain); the Port of Huelva (SW, Spain), the Port of Pasajes (NE, Spain) and the Port of Bilbao (NW, Spain) for a period of 28 days. Organisms were sampled on days 0, 7, 14, 21 and 28 allowing examination of different biomarkers to exposure to metals and organic compounds; metallothioneins (MTs), ethoxyresorufin O-deethylase (EROD), glutathione-S-transferase (GST) and glutathione peroxidase (GPX). Sediment samples were also analyzed to determine chemical concentration of metals (As, Cd, Cr, Cu, Hg, Mn, Ni, Pb and Zn), PAHs and PCBs. Metals such as As, Cu and Zn associated with mining activities (Port of Huelva), and contaminants such as Ni, Pb, Hg and PCBs, associated with oil spills and industrial activities (Ports of Cadiz, Pasajes and Bilbao), were found to interfere in detoxification and/or anti-stress oxidative defenses in C. maenas. Positive, significant (p<0.05) induction of MTs concentration was associated with increasing sediment As, Cu, Zn and Hg concentrations. EROD activity was associated with PCBs, GST activity with Hg and GPX activity with As, Cu, Ni, Pb and Zn. The use of the parameter AR, which involves the kinetic patterns of the battery of biomarkers tested, was found to be a powerful and sensitive tool for evaluating the bioavailability and adverse effects of contaminants bound to sediments, enabling polluted and nonpolluted sites to be easily distinguished.

In situ evaluation of sediment toxicity in Guadalete Estuary (SW Spain) after exposure of caged Arenicola marina

A 7 day in situ assay to assess sediment toxicity was carried out per replicate in two sites from Guadalete Estuary (Guad1 and Guad2) (South‐West, Spain) using the polychaete Arenicola marina (n = 20) in benthic cages. After the exposure period, a battery of biomarkers were determined to asses the sediment toxicity. Five of them were exposure biomarkers [ethoxyresorufin O‐deethylase (EROD), dibenzylfluorescein (DBF) dependant CytP450, gluthation reductase (GR), and gluthation‐S‐transferase (GST) activities] and one of them was an effect biomarker [lipid peroxidation (LPO)]. Toxicity due to metals bound to the sediment was found in Guad2. Metals provoke the inhibition of EROD, DBF, and GR and the induction of GST, which is showed to carry out antioxidative stress defense. Toxicity caused by metals and also PAH was detected in Guad1. The presence of these chemicals leads to GST induction in and presumably in consequence, to LPO inhibition. GST activity resulted to be the main protection mechanism against oxidative stress in Arenicola marina. We suggest further research in Guadalete Estuary focusing on the detection and toxicity assessment of pharmaceutical and personal care products.

Response of gene expression in zebrafish exposed to pharmaceutical mixtures: Implications for environmental risk

Complex mixtures of pharmaceutical chemicals in surface waters indicate potential for mixture effects in aquatic organisms. The objective of the present study was to evaluate whether effects on target gene expression and enzymatic activity of individual substances at environmentally relevant concentrations were additive when mixed. Expression of zebrafish cytochrome P4501A (cyp1a) and vitellogenin (vtg) genes as well as activity of ethoxyresorufin-O-deethylase (EROD) were analyzed after exposure (96h) to caffeine-Caf, ibuprofen-Ibu, and carbamazepine-Cbz (0.05 and 5µM), tamoxifen-Tmx (0.003 and 0.3µM), and after exposure to pharmaceutical mixtures (low mix: 0.05µM of Caf, Ibu, Cbz and 0.003µM of Tmx, and high mix: 5µM of Caf, Ibu, Cbz and 0.3µM of Tmx). Pharmaceuticals tested individually caused significant down regulation of both cyp1a and vtg, but EROD activity was not affected. Exposure to low mix did not cause a significant change in gene expression; however, the high mix caused significant up-regulation of cyp1a but did not affect vtg expression. Up-regulation of cyp1a was consistent with induction of EROD activity in larvae exposed to high mix. The complex mixture induced different responses than those observed by the individual substances. Additive toxicity was not supported, and results indicate the need to evaluate complex mixtures rather than models based on individual effects, since in environment drugs are not found in isolation and the effects of their mixtures is poorly understood.

Immune- and Pollution-mediated DNA Damage in Two Wild Mya arenaria Clam Populations

In aquatic environments, genotoxicity results from the effects of pollution combined with the inflammatory response triggered by the immune system. Indeed, the production of nitrosylated DNA and proteins are though to arise from the production of peroxinitrite during phagocytosis and inflammation. The purpose of this study was to examine new DNA biomarkers that differentiate between immune- and pollution-mediated genotoxicity in wild clam populations. Intertidal clam populations were sampled and analyzed for gonadal DNA strand breaks, DNA nitrosylation and xanthine oxidoreductase (XOR) activity (purine salvage pathway). The clam weight-to-shell-length ratio, the gonado-somatic index (GSI), age status, lipid peroxidation, xenobiotic conjugation activity (glutathione S-transferase (GST) and phagocytic activity were examined to shed light on their relationships with the observed genotoxic endpoints. XOR activity and DNA strand breaks were generally elevated at polluted sites and correlated significantly with clam weight-to-shell-length ratios and DNA nitrosylation. DNA nitrosylation was also higher at some sites and correlated significantly with phagocytic activity and with DNA strand breaks. This study showed that DNA strand breaks were associated with both immune-and pollution-mediated effects. This suggests that there is a loss of DNA repair capacity due to the combined effects of aging, pollution and immune response in wild clam populations that are impacted by anthropogenic activity.