Juan Bellas

Assessment of coastal marine pollution in Galicia (NW Iberian Peninsula); metal concentrations in seawater, sediments and mussels (Mytilus galloprovincialis) versus embryo-larval bioassays using Paracentrotus lividus and Ciona intestinalis

Sediments from three Galician Rias were tested for toxicity using sea-urchin and ascidian sediment elutriate embryo-larval bioassays. Trace metal contents in seawater, sediments and mussels were also determined and subjected to multidimensional scaling methods which grouped stations according to chemical contamination. High metal contents were found in seawater, sediments and mussels from the Ria of Pontevedra, and moderate levels were detected in the Ria of Vigo and Ria of Arousa. The results revealed that samples assessed as toxic, according to the sea-urchin and ascidian embryo-larval bioassays, were among the most polluted by trace metals. A good agreement was reported between ordination plots resulting from applying multidimensional scaling to the chemical data, and the results of the biological endpoints tested.

Integrative assessment of marine pollution in Galician estuaries using sediment chemistry, mussel bioaccumulation, and embryo-larval toxicity bioassays.

An integrative assessment of environmental quality was carried out in selected sites along the Galician coast (NW Iberian Peninsula) combining analytical chemistry of seawater and sediments, bioaccumulation in the marine mussel, and embryo-larval sediment toxicity bioassays, in order to link biological and chemical criteria for the assessment of coastal pollution. Maximum values of Hg and Cu in seawater, sediment and mussels, were found in the inner part of Ria of Pontevedra, while maximum levels of organics (polychlorinated biphenyls, hexachlorobenzene and aldrin) were found in mussels from A Coruña. Outstanding values of Cu, Pb and Zn have been found in seawater and sediment from a single site, P3, which also was the most toxic in the embryo-larval bioassays performed with four different phyla of marine organisms: mollusks, echinoderms, arthropods and chordates. Sediment quality effects range-median values provided a valuable reference to predict biological effects from sediment chemistry data, while effects range-low values were too conservative. Sediment toxicity could also be predicted by using a toxic-unit model based on published EC50 values for trace metals and mobilization factors independently obtained from measurements of metal contents in sediments and their elutriates. When chemical and toxicological data are independently used to arrange sampling sites by using non-metric multidimensional scaling, a remarkable degree of concordance between both types of configurations could be observed.

Integrated use of antioxidant enzymes in mussels, Mytilus galloprovincialis, for monitoring pollution in highly productive coastal areas of Galicia (NW Spain)

In the present work, we investigated the potential use of several antioxidant enzymes in wild mussels (Mytilus galloprovincialis) as biomarkers of marine pollution. The enzymatic activity levels of glutathione S-transferase (GST), glutathione peroxidase (GPx) and catalase (CAT) were measured in gills and digestive gland. Those enzymes participate in the cellular defense system that is involved in the adaptive response of organisms to chemical pollution. Adult mussels were collected at five sampling sites located at the Ría de Pontevedra and Ría de Vigo. Seasonal variations in the control site were observed for the CAT activity, but no significant variability was found for GST and GPx. Mussels from the most polluted sites exhibited a significantly greater GST activity compared to the control site (p<0.05) during the sampling period, whereas GPx and CAT activities have not such a marked pattern. Trace metals, PAHs, PCBs and DDT contents in mussels at sampling sites showed highly significant positive correlations with the GST activity. This study supports the use of GST as a useful biomarker for long-term pollution monitoring in marine coastal ecosystems, whilst more research is needed for GPx and CAT.

Toxicity of Hg, Cu, Cd, and Cr on early developmental stages of Ciona intestinalis (Chordata, Ascidiacea) with potential application in marine water quality assessment

The toxicity of mercury, copper, cadmium and chromium on sperm viability, fertilisation, embryogenesis and larval attachment of Ciona intestinalis was examined. Fertilisation rate (FR) showed a small decrease even at the highest metal concentration tested. The median effective concentrations (EC50) reducing rates of embryogenesis and larval attachment by 50% were 54 microg Hg/l (0.27 microM), 46 microg Cu/l (0.72 microM), 838 microg Cd/l (7.46 microM), 10,318 microg Cr/l (198 microM), and 35 microg Hg/l (0.18 microM), 34 microg Cu/l (0.54 microM) and 11,755 microg Cr/l (226 microM), respectively. Therefore, Hg is three times more toxic than Cu (on a molar basis), ca. 30 times more toxic than Cd and ca. 1000 times more toxic than Cr to early stages of C. intestinalis. Rates of larval attachment and embryogenesis were the most sensitive endpoints, although the latter is more advisable for routine assessment of seawater quality because of its greater simplicity. In addition to bivalves and sea-urchins, ascidian embryos can provide biological criteria for seawater quality standards taking into account the sensitivity of a chordate and contributing to the detection of harmful chemicals with no marked effect on the species currently in use in seawater quality bioassays.

Ecotoxicological evaluation of polycyclic aromatic hydrocarbons using marine invertebrate embryo-larval bioassays

The toxicity of polycyclic aromatic hydrocarbons (PAHs) was determined using mussel, sea-urchin and ascidian embryo-larval bioassays. Fluorescent light exposure enhanced phenanthrene, fluoranthene, pyrene and hydroxypyrene toxicity in comparison with dark conditions, but not naphthalene and fluorene toxicity. The toxicity of PAHs was inversely related to their K(OW) values following QSAR models derived for baseline toxicity of general narcotics, whereas the obtained regression using toxicity data from photoactivated PAHs significantly departed from the general narcosis model. Also, the mixture toxicity of five PAHs to the larval growth of the sea-urchin was compared with predictions derived from the concentration addition concept, indicating less than additive effects. Finally, we compared our toxicity data with worst-case environmental concentrations in order to provide a preliminary estimate of the risk to the marine environment. Naphthalene, fluorene and pyrene are not considered to pose a risk to sea-urchin, mussel or ascidian larvae, whilst phenanthrene and fluoranthene may pose a risk for mussel and sea-urchin. Moreover, a higher risk for those species is expected when we consider the photoactivation of the PAHs.

Toxicity and phototoxicity of water-accommodated fraction obtained from Prestige fuel oil and Marine fuel oil evaluated by marine bioassays

Acute toxicity and phototoxicity of heavy fuel oil extracted directly from the sunken tanker Prestige in comparison to a standard Marine fuel oil were evaluated by obtaining the water-accommodated fraction (WAF) and using mussel Mytilus galloprovincialis and sea urchin Paracentrotus lividus embryogenesis bioassays, and copepod Acartia tonsa and fish Cyprinodon variegatus survival bioassays. Aromatic hydrocarbon (AH) levels in WAF were measured by gas chromatography. Prestige WAF was not phototoxic, its median effective concentrations (EC50) were 13% and 10% WAF for mussel and sea urchin respectively, and maximum lethal threshold concentrations (MLTC) were 12% and 50% for copepod and fish respectively. Marine WAF resulted phototoxic for mussel bioassay. EC50s of Marine WAF were 50% for sea urchin in both treatments and 20% for mussel under illumination. Undiluted Marine WAF only caused a 20% decrease in mussel normal larvae. Similar sensitivities were found among sea urchins, mussels and copepods, whilst fish were less sensitive. Unlike Marine WAF, Prestige WAF showed EC50 values at dilutions below 20%, and its toxicity was independent of lighting conditions. The differences in toxicity between both kinds of fuel could not be explained on the basis of total AH content.

Ingestion of microplastics by demersal fish from the Spanish Atlantic and Mediterranean coasts.

Microplastic pollution has received increased attention over the last few years. This study documents microplastic ingestion in three commercially relevant demersal fish species from the Spanish Atlantic and Mediterranean coasts, the lesser spotted dogfish Scyliorhinus canicula, the European hake Merluccius merluccius and the red mullet Mullus barbatus. Overall 212 fish were examined, 72 dogfish, 12 hakes and 128 red mullets. The percentage of fish with microplastics was 17.5% (15.3% dogfish, 18.8% red mullets and 16.7% hakes), averaging 1.56±0.5 items per fish, and the size of the microplastics ranged from 0.38 to 3.1mm. These fish species are used currently as biomonitors for marine pollution monitoring within the Spanish Marine Pollution Monitoring Programme (SMP), and may be as well suitable candidates for monitoring spatial and temporal trends of ingested litter. The data presented here represent a baseline for the implementation of the Marine Strategy Framework Directive descriptor 10 in Spain.

Integrative assessment of coastal pollution in a Ría coastal system (Galicia, NW Spain): correspondence between sediment chemistry and toxicity.

Elutriate embryo-larval bioassays with sea-urchins (Paracentrotus lividus) and ascidians (Ciona intestinalis) were conducted concurrently with trace metal analyses as part of an integrative assessment of sediment pollution at Ría de Pontevedra (Galicia, NW Spain). High metal contents in sediments were found in localised areas from the inner part of the estuary indicating a clear anthropogenic influence. In particular, very high Cu, Zn and Pb levels were found at sites P2 and P3, which were also the most toxic to the embryo-larval bioassays. Sediment quality guidelines were used to help in the ecological interpretation of sediment chemistry data and to identify pollutants of concern. Cu and Zn in P3 were consistently above the effects range median (ERM) values, which seem to be good predictors of toxicity to sea-urchin and ascidian embryos. A toxic unit approach, based on published EC(50) values and metal levels in elutriates, was used to assess the harmful ecological effects associated to sediment chemistry. Toxicity detected in P3 may be explained on the basis of the toxic unit model; however, the high toxicity detected at P2 may be attributable not only to the metals quantified in the analyses but also to unmeasured organic pollutants. Multidimensional scaling applied independently to the toxicology and chemistry data resulted in a good agreement between both types of configurations. Moreover, the Mantel test revealed a significant correlation (r(M)=0.481; p=0.019) between metal concentrations and toxicity data profiles, supporting the correspondence between configurations.

Practical procedures for selected biomarkers in mussels, Mytilus galloprovincialis--implications for marine pollution monitoring.

Biomarkers are required to assess the biological effects of pollutants on marine organisms in order to monitor ecosystem status, but their use is often limited by their strong variability due to environmental and/or intrinsic biological factors. Accordingly, the main aim of this work was to set up practical procedures for a battery of widely used biomarkers in mussels (Mytilus galloprovincialis). Antioxidant enzymes (catalase [CAT] and glutathione peroxidase [GPx]), a phase II detoxification enzyme (glutathione S-transferase [GST]) and a neurotransmitter catabolism enzyme (acetylcholinesterase [AChE]), were considered. Several relevant aspects were studied in order to obtain a more realistic interpretation of biomarker responses, including the calculation of the minimum sample size required to estimate the population mean with a fixed error margin, the selection of the specific organ or tissue where the enzymatic activity is higher for each biomarker, and the influence of tidal height and temperature on the basal enzymatic activity. GST and CAT activities needed a minimum sample size of 12, whereas for GPx and AChE activities a minimum sample size of 14 was required. The gills were the organ with higher GST, GPx and AChE enzymatic activities, whereas the digestive gland showed the highest CAT activity. Also, the low inter-tidal was the recommended tide level whilst no significant effect of temperature was observed on GST, GPx and CAT, and no clear pattern could be identified for AChE. The implications for environmental monitoring are discussed.

A standardisation of Ciona intestinalis (Chordata, Ascidiacea) embryo-larval bioassay for ecotoxicological studies

A standardisation of the ascidian Ciona intestinalis embryo-larval bioassay for marine pollution assessment has been developed. The minimum percentage of embryogenesis success was established to assess the quality of the biological material used; minimum sample size and number of replicates per treatment were also estimated. The suitability of artificial and natural seawater for the incubation of ascidian embryos and larvae was compared, and the optimum conditions of temperature, salinity, pH, density of embryos in the vials and the sperm/egg ratio were investigated. On the basis of the 10th percentile of the distribution of larval abnormalities, we proposed a threshold of 50% normal larvae in the control in order to consider the test of acceptable biological quality. According to our results n=5 is a sufficiently high replication to detect 5% differences among treatment means with a power of P=90% and alpha=0.05, and a sampling size >/=222 allows a 95% confidence in the estimate with an error of 0.05. Egg density did not affect larval development within the range 1-20 eggs/ml, and the optimum sperm/egg ratio which fertilise 100% of the eggs was 3000-30,000 sperm/egg (i.e. 10(8)-10(7) sperm/ml). There were not significant differences between the two water types tested, and the optimum tolerance ranges were 18-23 degrees C temperature, 34-42 ppt salinity (42 ppt was the highest salinity tested), and 7.4-8.8 pH. The median effective concentration (EC(50)) of copper (Cu) causing a 50% reduction of normal hatched larvae was 54.2 microg/l (0.85 microM), which shows a sensitivity of this species similar to the commonly used bivalve and sea-urchin tests. The ascidian embryo-larval bioassay is an accurate, reliable, simple and rapid method that can be used in ecotoxicological studies.