Julián Blasco

The use of biomarkers to assess the impact of pollution in coastal environments of the Iberian Peninsula: a practical approach

Within the frame of the 2nd Iberian Congress of Environmental Contamination and Toxicology (University of the Basque Country, Leioa, June 1998) a workshop was held about the use of biomarkers in marine pollution monitoring. Among others, the following biomarkers received special attention: metallothionein induction, acetylcholinesterase inhibition, cytochrome P450 system induction, imposex, lysosomal enlargement and lysosomal membrane destabilisation, and peroxisome proliferation. These biomarkers can be used to evaluate exposure to and effect of different contaminants (metals, organic xenobiotics and organometallic compounds) and they can be measured using different methodological approaches (biochemistry, cytochemistry, immunochemical methods based on the use of biotechnology). Before the application of a set of biomarkers in pollution monitoring programmes, well-defined protocols of Quality Assurance have to be established to allow adequate comparison of results. It is also necessary to include analysis of standard reference materials and to obtain detailed knowledge of basal values and seasonal variations of the biomarkers in various species, as well as to integrate the information obtained with the different biomarkers. Marine bivalve molluscs such as mussels are appropriate sentinel species for most of the biomarkers proposed except for the induction of the cytochrome P450 system, which should be measured in fish, and the degree of imposex, which is a biomarker of exposure to TBT specifically measured in certain gastropod molluscs. As a result of the workshop, a battery of biomarkers of contaminant exposure and effects are proposed that could be incorporated into programmes monitoring the quality of the coastal environment in the Iberian Peninsula. These measures would be undertaken in conjunction with chemical measures of contaminant burdens in selected sentinel species.

Oxidative stress and toxicity of gold nanoparticles in Mytilus edulis

Gold nanoparticles (AuNP) have potential applications in drug delivery, cancer diagnosis and therapy, food industry and environment remediation. However, little is known about their potential toxicity or fate in the environment. Mytilus edulis was exposed in tanks to 750 ppb AuNP (average diameter 5.3 ± 1 nm) for 24h to study in vivo biological effects of nanoparticles. Traditional biomarkers and an affinity procedure selective for thiol-containing proteins followed by two-dimensional electrophoresis (2DE) separations were used to study toxicity and oxidative stress responses. Results were compared to those obtained for treatment with cadmium chloride, a well known pro-oxidant. M. edulis mainly accumulated AuNP in digestive gland which also showed higher lipid peroxidation. One-dimensional SDS/PAGE (1DE) and 2DE analysis of digestive gland samples revealed decreased thiol-containing proteins for AuNP. Lysosomal membrane stability measured in haemolymph gave lower values for neutral red retention time (NRRT) in both treatments but was greater in AuNP. Oxidative stress occurred within 24h of AuNP exposure in M. edulis. Previously we showed that larger diameter AuNP caused modest effects, indicating that nanoparticle size is a key factor in biological responses to nanoparticles. This study suggests that M. edulis is a suitable model animal for environmental toxicology studies of nanoparticles.

Role of metallothionein against oxidative stress in the mussel Mytilus galloprovincialis

Metallothionein (MT) is a sulfhydryl-rich protein involved mainly in heavy metal homeostasis and detoxification. In this study, the use of the mussel as an experimental model allowed us to test MT antioxidant properties at the molecular, cellular, and organism level. MT induction was achieved by mussel exposure to Cd (200 μg/l) in aquaria for 7 days followed by detoxification in the sea for 28 days. Cd-preexposed and nonexposed mussels were then treated with Fe (300-600 μg/l) in aquaria for 3 days. Biochemical assays on digestive gland tissue showed that treatment with Fe led to a significant increase in oxyradical production and malondialdehyde level only in mussels not preexposed to Cd. The Cd-dependent resistance to oxidative stress was ascribed to MT induction, as Cd produced no significant variation of reduced glutathione and major antioxidant enzymes. Digital imaging of isolated digestive gland cells showed lower oxyradical rise and higher viability in cells from Cd-preexposed mussels after treatments with 0.5-5 mM H2O2. Analyses on whole organisms showed that anoxic survival was lowered in mussels that had been treated with Fe, but such an effect was less pronounced in Cd-preexposed mussels compared with nonpreexposed ones. In conclusion, data suggest an antioxidant role for MT, which seems to occur through oxyradical scavenging and is able to protect both isolated cells and the entire organism from oxidative stress.

Determining contamination sources in marine sediments using multivariate analysis

Abstract The problem of identifying sources of anthropogenic pollutants in a local marine environment has been intensively studied during the last two decades. An approach is descibed for tracing anthropogenic pollutant sources, and for characterizing various geochemical and chemical processes related to contamination by anthropogenic heavy metals and surfactants in the marine coastal environment, which uses a multivariate technique of principal components analysis (PCA). This approach was applied to study two selected littoral ecosystems in the Gulf of Cadiz. The multivariate analysis approach can be used successfully for contamination source assessments. From this information, it was possible to identify current superficial sources of contamination and it might be possible to determine the areas where further toxicological testing is required.

Bioavailability of heavy metals bound to estuarine sediments as a function of pH and salinity values

Abstract The role of two key-variables such as pH and salinity in chemical speciation and bioavailability of heavy metals originated by mining activities bound to sediments was evaluated under estuarine conditions. Two sediment samples collected in two estuaries in southern Spain (Ría of Huelva and Guadalquivir estuary) together with dilution of toxic mud from the Aznalcóllar mining spill (April, 1998) were used to determine their chemical speciation and bioavailability at different pH (6.5, 7.5, 8.5) and salinity (10, 20, 35) values using the estuarine clam Ruditapes philippinarum. The chemical speciation was established by means of measurements of the mobilization of heavy metals from sediments to waters and determining in it pH, salinity, alkalinity and heavy metal concentration. The geochemical model MINTEQA2 was used to establish the thermodynamic species in the assays. To assess the bioavailability of the heavy metals the concentration of metallothioneins in the clam tissues and the mortality of this organism was measured at different pH and salinity values. The influence of both salinity and pH was detected in the chemical behavior of metals and in their associated biological responses established by metallothioneins and the percentage of mortality. At low values of both variables (pH=6.5 and S=10), the biological effects were highest, and it was related to the free ion Zn2+.

Heavy metals in organisms of the River Guadalquivir estuary: possible incidence of the Aznalcóllar disaster

On the 25 April 1998, the giant holding pool of the Aznalcóllar mine, owned by the Canadian-Swedish Company Boliden Ltd burst its banks; 5 million cubic meters (6.7 million cubic yards) of sludge poured into the nearby River Guadiamar, which is a tributary of the River Guadalquivir (the main river of the south Iberian Peninsula). Within hours of the accident, approximately 2.5 Hm3 of acidic water with high concentrations of metals, especially Zn, had entered the River Guadalquivir. A biomonitoring program has been carried out to evaluate the incidence of this spill on the fauna of the River Guadalquivir. Six sampling stations within the estuary of were established and six species of molluscs (Crassostrea angulata, Scrobicularia plana), crustaceans (Palameon longirostris, Uca tangeri, Melicertus kerathurus) and fish (Liza ramada) were chosen for analysis. In the Portuguese oyster, Crassostrea angulata, Cd and Cu concentrations were above the maximum allowed by the Spanish legislation for bivalves for human consumption (60 micrograms.g-1 wet wt. for Cu and 1.0 microgram.g-1 wet wt. for Cd). The crustaceans Palaemon longirostris and Uca tangeri, also showed values above the legal limits for Cu (20 micrograms.g-1 wet wt.). An increase of the Zn levels was observed, as Zn concentrations in C. angulata were higher than those reported 30 years ago. The heavy metal concentrations in some of the organisms collected in the River Guadalquivir were higher than in the Bay of Cadiz or the Mediterranean Sea (clean sites), showing that this estuary was subjected to a heavy metal load. One month after the accident, concentrations of Zn were higher in C. angulata and in Palaemon longirostris compared with levels recorded on the 25 April. Nevertheless, some organisms did not show a clear tendency during the sampling period, probably due to the fluctuations of environmental (salinity, temperature) and physiological variables that affect bioaccumulation.

Heavy metal fluxes at the sediment–water interface of three coastal ecosystems from south-west of the Iberian Peninsula

Concentrations of the heavy metals Cr, Cu, Fe and Mn were measured in sediments and porewater samples collected in three coastal ecosystems southwest of the Iberian Peninsula: the Odiel and Barbate River Salt Marshes and the Bay of Cadiz. Both the sediment and the porewater metal concentrations in the Odiel River Salt Marshes are higher than the values found in the Bay of Cadiz and Barbate River Salt Marsh, particularly for copper, a metal associated with mining activity. In porewater, the profiles were not the same as those in the solid phase and reflect the different behaviours of the elements in relation to the redox conditions. The heavy metals Cr and Cu show a typical enrichment in the porewater of the oxic zone. The heavy metals Mn and Fe show an increase in the porewater at the depths where the maximum nitrate and phosphate concentrations occur, respectively. Significant differences between background levels for each heavy metal in the various studied zones exist. Iron and Cu showed larger background levels in the Odiel River Salt Marshes than those in the Cadiz Bay and the Barbate River Salt Marshes. In the Bay of Cadiz the background levels are also high, particularly for Cr. At the Odiel River Salt Marshes the diffusive flux of Cu is high (1.3-230.1 microg cm(-2) year(-1)), which suggests that the Odiel River Salt Marshes are subject to strong contamination by Cu, which is presumably introduced to the sediment in particulate form. In the Bay of Cadiz, Cr is the only metal with positive diffusive flux (2.15 microg cm(-2) year(-1)). It is higher than those obtained in other coastal ecosystems including the Odiel River Salt Marshes. The positive diffusive flux of Cr has been associated with the input of this metal by the naval industry and the manufacturing of car and aircraft components.

Effect of lead on ALA-D activity, metallothionein levels, and lipid peroxidation in blood, kidney, and liver of the toadfish Halobatrachus didactylus

The effects of lead (Pb) on ALA-D activity, metallothionein (MT) levels, and lipid peroxidation in liver, kidney, and blood of the toadfish Halobatrachus didactylus were investigated. A time-course experiment was performed with sampling on days 0, 2, 5, and 7 following intraperitoneal Pb injection. This indicated a rank order for lead concentration of kidney > liver > blood in fish exposed to Pb. No significant variation of ALA-D activity was observed in liver and kidney while in blood, a slight decrease of ALA-D activity was found but this was not attributed to acute metal stress. Hepatic and renal MT levels were both affected in different ways by metal uptake. The progressive decrease of MDA concentration in the liver and the lack of a clear induction in kidney suggested the hypothesis that Pb is not a good inductor of lipid peroxidation. The histological and histochemical results demonstrated degenerative effects of lead accumulation on the tissues and the activation of lysosomal responses to induced stress.

Sunscreen Products as Emerging Pollutants to Coastal Waters

A growing awareness of the risks associated with skin exposure to ultraviolet (UV) radiation over the past decades has led to increased use of sunscreen cosmetic products leading the introduction of new chemical compounds in the marine environment. Although coastal tourism and recreation are the largest and most rapidly growing activities in the world, the evaluation of sunscreen as source of chemicals to the coastal marine system has not been addressed. Concentrations of chemical UV filters included in the formulation of sunscreens, such as benzophehone 3 (BZ-3), 4-methylbenzylidene camphor (4-MBC), TiO2 and ZnO, are detected in nearshore waters with variable concentrations along the day and mainly concentrated in the surface microlayer (i.e. 53.6–577.5 ng L-1 BZ-3; 51.4–113.4 ng L-1 4-MBC; 6.9–37.6 µg L-1 Ti; 1.0–3.3 µg L-1 Zn). The presence of these compounds in seawater suggests relevant effects on phytoplankton. Indeed, we provide evidences of the negative effect of sunblocks on the growth of the commonly found marine diatom Chaetoceros gracilis (mean EC50 = 125±71 mg L-1). Dissolution of sunscreens in seawater also releases inorganic nutrients (N, P and Si forms) that can fuel algal growth. In particular, PO4 3− is released by these products in notable amounts (up to 17 µmol PO4 3− g−1). We conservatively estimate an increase of up to 100% background PO4 3− concentrations (0.12 µmol L-1 over a background level of 0.06 µmol L-1) in nearshore waters during low water renewal conditions in a populated beach in Majorca island. Our results show that sunscreen products are a significant source of organic and inorganic chemicals that reach the sea with potential ecological consequences on the coastal marine ecosystem.

Exposure of the blue mussel, Mytilus edulis, to gold nanoparticles and the pro-oxidant menadione

Relatively little is known about how gold nanoparticles (GNP) might interact in vivo with marine organisms. Mytilus edulis was exposed (24h) to approximately 15 nm GNP, menadione and both compounds simultaneously (GNP/menadione). GNP was detected by inductively coupled plasma-optical emission spectroscopy mainly in digestive gland of samples exposed to GNP though not GNP/menadione, perhaps due to impaired feeding. Thioredoxin reductase activity and malondialdehyde levels were determined in all tissues. Thioredoxin reductase inhibition was detected only in digestive gland exposed to menadione whilst malondialdehyde levels did not vary in response to treatment in all tissues. GNP caused a decrease in the reduced/oxidized glutathione ratio in digestive gland, but no difference was found in other tissues or for other treatments. One dimensional electrophoresis of proteins containing thiol groups was performed in all tissues and revealed a reduction in protein thiols for all treatments in digestive gland. Two dimensional electrophoresis of digestive gland extracts, from GNP and control groups, showed decreased levels of thiol proteins in response to GNP which we attribute to oxidation. Our results suggest that GNP causes a modest level of oxidative stress sufficient to oxidize thiols in glutathione and proteins but without causing lipid peroxidation or induction of thioredoxin reductase activity.