Carlos Gravato

Acute effects of copper and mercury on the estuarine fish Pomatoschistus microps: Linking biomarkers to behaviour ☆

The main objective of the present study was to investigate possible links between biomarkers and swimming performance in the estuarine fish Pomatoschistus microps acutely exposed to metals (copper and mercury). In independent bioassays, P. microps juveniles were individually exposed for 96 h to sub-lethal concentrations of copper or mercury. At the end of the assays, swimming performance of fish was measured using a device specially developed for epibenthic fish (SPEDE). Furthermore, the following biomarkers were measured: lipid peroxidation (LPO) and the activity of the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH), glutathione S-transferases (GST), 7-ethoxyresorufin-O-deethylase (EROD), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx). LC(50)s of copper and mercury (dissolved throughout metal concentrations) at 96h were 568 microg L(-1), and 62 microg L(-1), respectively. Significant and concentration-dependent effects of both metals on swimming resistance and covered distance against water flow were found at concentrations equal or higher than 50 microg L(-1) for copper and 3 microg L(-1) for mercury (dissolved throughout metal concentrations). These results indicate that SPEDE was efficacious to quantify behavioural alterations in the epibenthic fish P. microps at ecologically relevant concentrations. Significant alterations by both metals on biomarkers were found including: inhibition of AChE and EROD activities, induction of LDH, GST and anti-oxidant enzymes, and increased LPO levels, with LOEC values ranging from 25 to 200 microg L(-1), for copper and from 3 to 25 microg L(-1) for mercury (dissolved throughout metal concentrations). Furthermore, significant and positive correlations were found between some biomarkers (AChE and EROD) and behavioural parameters, while negative correlations were found for others (LPO, anti-oxidant enzymes and LDH) suggesting that disruption of cholinergic function through AChE inhibition, decreased detoxification capability due to EROD inhibition, additional energetic demands to face chemical stress, and oxidative stress and damage may contribute to decrease the swimming performance of fish. Since a reduced swimming capability of fish may reduce their ability to capture preys, avoid predators, and interfere with social and reproductive behaviour, the exposure of P. microps to copper and/or mercury concentrations similar to those tested here may decrease the fitness of wild populations of this species.

Biochemical effects and polycyclic aromatic hydrocarbons (PAHs) in senegal sole (Solea senegalensis) from a Huelva estuary (SW Spain).

Relations between several stress oxidative biomarkers and aromatic polycyclic hydrocarbon (PAH) concentrations have been studied in wild sole, Solea senegalensis collected in the vicinity of a petrochemical industry. Antioxidant enzyme activities in eco-toxicological studies constitute excellent markers for exposure to a large variety of pollutants. The 16 PAHs in sediment as well as oxidative damage (LPO), activity of catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione reductase (GR) and PAHs type metabolites in sole liver were analysed. Significant correlations (p<0.05) were established between some biomarkers as GST, GPx and CAT and PAHs metabolites in liver (naphthalene, pyrene and phenanthrene) and PAHs concentrations in sediments (fluoranthene, acenaphthene, anthracene and chrysene). PAHs accumulated in the sediment and organisms are inducers of antioxidant defences. GST, GPx and CAT were robust biomarkers showing correlations with both PAHs in sediments and liver PAH metabolites showing different responses to low and high molecular weight PAHs.

Genotoxicity biomarkers' association with B(a)P biotransformation in Dicentrarchus labrax L.

Dicentrarchus labrax (sea bass) were exposed during 0, 4, 8, 16, 24, 48, and 96 h to 0 and 0.1 microM benzo(a)pyrene (B(a)P), an environmental pollutant, and the following biomarkers were measured: (1) liver cytochrome P450 (P450) content and ethoxyresorufin-O-deethylase (EROD) activity as phase I biotransformation parameters, (2) liver gluthathione-S-transferase (GST) activity as a phase II biotransformation conjugation enzyme, (3) biliary and liver cytosolic B(a)P-type metabolites by fixed wavelength fluorescence detection (FF), and (4) erythrocytic micronuclei (EMN) and erythrocytic nuclear abnormalities (ENA) as genotoxicity biomarkers. Liver EROD activity (4 h), P450 content (24 h), GST activity (4, 8, and 96 h), bile (4-96 h), and liver cytosolic (4-24 h) B(a)P-type metabolites increased significantly in sea bass exposed to B(a)P as well as EMN (8-96 h) and ENA (4-96 h) frequencies. B(a)P genotoxicity is associated with increase in B(a)P-type metabolites in liver cytosol due to an impaired phase II conjugation. This increase seems to be responsible for the decrease in liver EROD and GST activities.

Yellow eel (Anguilla anguilla) development in NW Portuguese estuaries with different contamination levels.

The aims of the present study were to compare the health status of yellow eels (Anguilla anguilla) developing in three estuaries of the NW Portuguese coast with different levels of pollution and their physiological responses to combined effects of environmental variation and pollution. For this, a field study was performed using a multi-parameter approach, including eels condition indexes and biomarkers, water quality variables and other environmental factors. Sixteen biological parameters were assessed, namely: hepatosomatic index (LSI), Fulton’s condition index (K), lipid peroxidation (LPO), total glutathione (TG), reduced glutathione (GSH), oxidised glutathione (GSSG), GSH/GSSG, and the activity of the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH), sodium-potassium ATPase (Na+/K+-ATPase), ethoxyresorufin O-deethylase (EROD), glutathione S-transferases (GST), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR). Ten environmental factors were also measured in water: temperature, salinity, pH, phosphates, nitrates, nitrites, ammonium, silica, phenol and hardness. Globally, the biomarkers indicate exposure and toxic effects of pollutants on eels living in contaminated estuaries. The relationships between biological and environmental variables were assessed through redundancy analysis. K and LSI indexes, AChE and Na+/K+-ATPase, total glutathione levels and the antioxidant enzymes CAT, GR, and SOD where the factors most discriminating reference (Minho River estuary) from contaminated estuaries (Lima and Douro Rivers estuaries). Moreover, the most striking outcomes of pollutants exposure on biological responses were observed during winter, probably due to a joint effect of cold weather and pollution stress. Altogether, the results indicate that the development of eels in the polluted estuaries of Lima and Douro rivers is interfering with physiological functions determinant for their survival and performance. This may increase the mortality rates during the continental life-phase of the species and decrease the percentage of animals able to successfully complete their oceanic migration and, thus, reduce the contribution of each generation to the next one.

Effect of Cu-nanoparticles versus one Cu-salt: Analysis of stress biomarkers response in Enchytraeus albidus (Oligochaeta)

Abstract In the present study, the main goal was to compare the effects of ionic copper versus copper nanoparticles in Enchytraeus albidus assessing the effect at the biomarker level, testing different concentrations and exposure times. Measured parameters were lipid peroxidation (LPO), total, reduced and oxidized glutathione content (TG, GSH and GSSG), the enzymatic activity of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), glutathione S-transferases (GSTs) and cholinesterases (ChEs). Results showed that both salt- and nano-copper caused oxidative stress and damage to E. albidus, as confirmed by LPO levels, and effects could be discriminated between the copper forms. Nevertheless and despite the visible discrimination between nano and the salt form (time and exposure dependent), there was no single or a set of biomarkers that provided the best discrimination.

Dicentrarchus labrax biotransformation and genotoxicity responses after exposure to a secondary treated industrial/urban effluent.

The present research work was designed to study Dicentrarchus labrax (sea bass) biotransformation and genotoxicity responses to the soluble fraction of a secondary treated industrial/urban effluent (SF-STIUE) discharged through a submarine pipe outlet into the Aveiro coastal area. Sea bass was exposed for 4, 8, 16, 24, 48, and 96 h to 0%, 0.1%, and 1% SF-STIUE and the following biological responses were measured: (1) liver cytochrome P450 (P450) content and ethoxyresorufin-O-deethylase (EROD) activity, as phase I biotransformation parameters; (2) liver gluthathione S-transferase (GST) activity as a phase II conjugation enzyme; (3) biliary and liver cytosol naphthalene (Naph)- and benzo(a)pyrene (B(a)P)-type metabolites, by fixed wavelength fluorescence detection (FF); (4) liver DNA strand breaks, erythrocytic micronuclei (EMN), and erythrocytic nuclear abnormalities (ENA) as genotoxicity parameters. Both SF-STIUE dilutions (0.1% and 1%) failed to significantly increase liver EROD activity, despite a significant increase of liver P450 at 16 and 48 h exposure to 0.1%. Liver GST activity increased significantly at 4h of sea bass exposure to 1% SF-STIUE, being inhibited at 96 h of exposure to this SF-STIUE dilution. Naph- and B(a)P-type metabolite contents were not significantly increased in bile. However, Naph-type metabolite contents increased significantly in liver cytosol at 4h exposure to 1% SF-STIUE, and at 24h exposure to 0.1% and 1% SF-STIUE. Furthermore, B(a)P-type metabolites increased significantly in liver cytosol at 4h exposure to 1% SF-STIUE, and 16 h exposure to 0.1% and 1% SF-STIUE. EMN and ENA frequencies increased significantly at 4, 8, 16, 24, 48, and 96 h exposure to 0.1% and 1% SF-STIUE. Liver DNA integrity decreased significantly at 96 h of sea bass exposure to 1% SF-STIUE. The STIUE discharged into Aveiro coastal area is of great ecotoxicological concern due to its genotoxic potential.

Challenges in assessing the toxic effects of polycyclic aromatic hydrocarbons to marine organisms: A case study on the acute toxicity of pyrene to the European seabass (Dicentrarchus labrax L.)

The acute toxicity (96 h) of pyrene (PY) to European seabass (Dicentrachus labrax) juveniles assessed in a semi-static bioassay (SSB) with medium renewal at each 12h, and in a static bioassay (SB) without medium renewal was compared in laboratorial conditions (water PY concentrations: 0.07-10 mg L(-1)). Main findings in the SSB that assessed mainly the toxicity of PY and its metabolites were: increased levels of bile PY metabolites in good agreement with the profile of lipid peroxidation levels (LPO) in exposed fish relating PY exposure and oxidative damage; increased levels of PY-type compounds in the brain indicating their ability to cross the blood-brain barrier; increased levels of these substances in liver and muscle which are edible tissues for humans thus raising concern on potential adverse effects on consumers of fish from PY contaminated areas; a significant inhibition of glutathione S-transferase activity suggesting its involvement in PY detoxication as toxicant scavenger; finally, an almost complete impairment of the swimming velocity at all the PY concentrations linking sub-individual to higher population level effects. In the SB, where the overall toxicity of PY, its metabolites and environmental degradation products was evaluated, 19% and 79% of PY decay in test media was found at 12 and 96 h, respectively. In general, the effects were similar to those of SSB but with significant effects being induced at higher PY concentrations indicating that the parental compound is more toxic than its environmental degradation products. The other main differences relatively to the SSB were: increased levels of PY-type substances in the liver suggesting more accumulation in this organ. Therefore, these findings highlight the need of carefully considering experimental design options when assessing the toxicity of readily degradable substances to marine fish, and stress the importance of taking into consideration the toxicity of environmental degradation products in addition to toxic effects of the parental substance and its metabolites for marine ecological risk assessment.

Comparative study about the effects of pollution on glass and yellow eels (Anguilla anguilla) from the estuaries of Minho, Lima and Douro Rivers (NW Portugal).

The health status of eels (Anguilla anguilla) developing in three estuaries of the NW Portuguese coast with different types and levels of pollution was compared in relation to morphometric parameters, Fulton condition index (F index) and several biomarkers. Relatively to the reference population, glass eels from the Lima estuary had lower weight and length, cholinesterase (ChE) and lactate dehydrogenase (LDH) inhibition, and lower levels of some anti-oxidant parameters, while yellow eels also showed a decreased F index, and increased Na(+)/K(+)-ATPase and lipid peroxidation (LPO) levels. Relatively to the reference population, glass eels from the Douro estuary had increased Na(+)/K(+)-ATPase and glutathione-S-transferase activities and LDH inhibition, while yellow eels also had ChE inhibition and increased LPO. Overall, these results indicate that eels from polluted estuaries showed a poor health status than those from a reference estuary, and that adverse effects become more pronounced after spending several years in polluted estuaries.

Acute effects of deltamethrin on swimming velocity and biomarkers of the common prawn Palaemon serratus

The main purpose of the present study was to investigate the effects of deltamethrin on biomarkers and behavior of Palaemon serratus (common prawn), since this attempt to link different levels of biological organization will allow determining which biomarkers might be ecologically relevant and will be useful to complement the information about the effects of pesticides by using behavioral parameters. Therefore, parameters of liver antioxidant status, energy metabolism and neurotransmission were determined in different tissues of the common prawn and used to assess the effects at sub-individual level, whereas swimming velocity was used to assess the effects at the individual level. It was also investigated if the swimming velocity can be used as an endpoint in ecotoxicology bioassays and if it can be as sensitive as biomarker endpoints. Swimming velocity was significantly reduced in prawns exposed to deltamethrin, showing a lowest observed effect (LOEC) of 0.6 ng L(-1). Eye acetylcholinesterase (AChE) activity was significantly increased in prawns exposed to 0.6, 1.2 and 2.4 ng L(-1) deltamethrin, whereas muscle cholinesterase (ChE) activity was significantly increased in prawns exposed to 19 and 39 ng L(-1). On the other hand, lactate dehydrogenase (LDH) activity was significantly increased in muscle of prawns exposed to 0.6, 1.2, 2.4, 4.9 ng L(-1) deltamethrin, showing that organisms were requiring additional energy, but probably using it for detoxification processes rather than locomotion, since swimming velocity was inhibited. Glutathione S-transferase (GST) activity was significantly increased in the digestive gland of common prawn exposed to 19 and 39 ng L(-1) deltamethrin. Catalase (CAT) activity was significantly increased in digestive gland of prawn exposed to 19 ng L(-1) deltamethrin. However, CAT activity decreased in digestive gland of prawn exposed to 39 ng L(-1), suggesting an antioxidant defense system failure concomitant with high levels of lipid peroxidation. Thus, global results showed that decreased swimming velocity was not associated with cholinesterase inhibition. In fact, the impairment of swimming velocity may be due to allocation of energy for detoxification and antioxidant protection instead of swimming activity. The present study showed that swimming velocity could be used as an ecologically relevant tool and a sensitive endpoint to assess and complement the study of pesticide effects on marine organisms.

Effects of carbofuran on the sea bass (Dicentrarchus labrax L.): Study of biomarkers and behaviour alterations

The objective of this study was to investigate the acute effects of the pesticide carbofuran on the sea bass (Dicentrarchus labrax) using parameters at different levels of biological organisation (swimming behaviour and several biomarkers) and possible relationships between alterations found in different effect criteria. In a bioassay, sea bass juveniles were individually exposed to different doses of carbofuran (31, 63, 125 and 250 μg/L) for 96 h. At the end of the bioassay, the swimming performance and 11 biomarkers were determined. Biomarkers were: hepatosomatic index (HSI), lipid peroxidation (LPO), reduced glutathione and the activities of the enzymes ethoxyresorufin O-deethylase (EROD), glutathione S-transferases, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, brain acetylcholinesterase (AChE) and muscle cholinesterases (ChE). After 96 h of exposure, carbofuran induced a decrease of the swimming velocity and inhibition of EROD activity at all concentrations tested, and inhibition of muscle ChE and brain AChE activities at 250 μg/L. No relevant alterations in any of the other tested parameters were found. These results show that carbofuran induced adverse effects on fish by interfering with neurofunction, capability of detoxication and swimming velocity. In addition, positive and significant correlations between the swimming velocity and (i) brain AChE activity, (ii) muscle ChE activity and (iii) EROD activity suggest that the inhibition of these enzymes may somehow be related to the behavioural changes observed. Since these functions are determinant for the survival and performance of the fish in the wild, the findings of the present study suggest that adverse effects may occur in populations exposed to carbofuran if a sufficient number of animals is affected.