Ricardo Beiras

The Assessment of Marine Pollution - Bioassays with Bivalve Embryos and Larvae

Tens of thousands of synthetic substances are in existence today and hundreds of new compounds are being introduced every year. Because of the complexity of the physico-chemical interactions between pollutants and the marine environment, the potential toxicity of contaminants can be assessed adequately only by means of bioassays with living organisms. From a practical point of view, a bioassay needs to be sensitive and scientifically valid, yield rapid results at moderate cost, and the organism in question must be readily available. Ecotoxicological bioassays with bivalve embryos and larvae fulfil these criteria better than most other tests. They have increasingly come into use during the past three decades and are now commonly employed to ascertain the biological effects of pure chemicals, as well as to determine the quality of effluents, coastal waters and sediments sampled in the field. There do not appear to be very great differences between bivalve species with regard to larval sensitivity to toxicants. The principal species for bioassays are oysters (Crassostrea gigas and C. virginica), and mussels (Mytilus edulis and M. galloprovincialis). Bioassays are conducted with gametes and larvae of ail ages: sperm and unfertilized eggs, embryos, young D-larvae, intermediate umboned larvae, and pediveligers towards the end ofthe pelagic period. Embryos are usually the most sensitive stage. Recent advances now also permit bioassays on metamorphosing pediveligers, a method particularly suited to investigate the effects of adsorbate-contaminated surfaces. There are various criteria for the assessment oftoxic effects, including embryogenesis success (abnormalities), larval growth, mortality, physiology (e.g. feeding or swimming activity), and metamorphosis success. Chronic toxicity studies may be carried out over periods ofseveral weeks, but larval rearing in the laboratory requires considerable effort (e.g. cultivation of algal food). The method of choice for investigations of acute toxicity and for routine monitoring studies is the embryo bioassay because it is very sensitive, relatively simple, and produces results within 24 or 48 hours. The data obtained by different investigators are often difficult to compare, however, because of differences in methodology. There is no firmly established procedure, and further simplification and standardization of techniques is required. In bioassays with a single pollutant, the effective toxic concentration may span several orders of magnitude, depending on bioassay procedures, larval stage and choice of response. Tributyl-tin (TBT) is the most toxic compound ever bioassayed with bivalve larvae, with effective concentrations (EC50) as low as a few nanograms per litre (i.e. 10−3 ppb). Heavy metals (particularly mercury, silver and copper) are next in order of toxicity, with EC50 values between a few micrograms per litre (ppb) and several hundred ppb. Chlorine and some organochlorine pesticides may also have EC50 values of less than 100 ppb, while detergents and petroleum products are generally less toxic

The physiological energetics of mussels (Mytilus galloprovincialis Lmk) from different cultivation rafts in the Ria de Arosa (Galicia, N.W. Spain)

Abstract Physiological components of growth (clearance and ingestion rates, absorption efficiencies and metabolic rates) were determined in specimens of mussels ( Mytilus galloprovincialis ) from several cultivation rafts in the Ria de Arosa (Galicia, Spain). Choice of raft location within the estuary was intended to provide an oceanic-terrestrial gradient of conditions. Experiments were performed directly on the raft, under natural conditions of food availability. Water was pumped to feeding trays from two different points (front and back) to account for the variability in seston concentration associated with the filtering activity of the hanging culture. Food concentration was found to be highly variable (0.35 to 1.01 mg POM l −1 ), the main differences being accounted for by site characteristics. Conversely, food quality (here defined as mg POM per mm 3 of packed particles) experienced minor variations (0.59 to 0.71), with the sole exception of the innermost site studied (quality 0.43). Absorption efficiencies were clearly dependent on food quality and could be adequately modelled as an exponential function of that viriable. The scope for growth (SFG) measured at the front of the raft was consistently higher than at the back and confirmed empirical evidence on growth rates. This result does not merely reflect improved feeding conditions in the input area but associated effects of increased clearance rates and absorption efficiencies exhibited by mussels from the front of the raft. On the other hand, raft positioning within the estuary appears to induce noticeable differences in the feeding behaviour of mussels, as evidenced by higher clearance rates determined in more oceanic areas.

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.

Effect of humic acids on speciation and toxicity of copper to Paracentrotus lividus larvae in seawater

The effects of humic acid (HA) on the toxicity of copper to sea urchin Paracentrotus lividus larvae were studied in chemically defined seawater. Square Wave Anodic Stripping Voltammetry (SWASV) was employed to study the complexation of copper in seawater medium. A simple complexation model assuming one ligand type and a 1:1 reaction stoichiometry successfully explained the inverse titration experiments. A conditional stability constant of 6.53+/-0.05 and a complexating capacity of 230+/-7 micromol Cu/g HA were obtained. Sea urchin bioassay tests with two endpoints, embryogenesis success and larval growth were carried out in order to study the toxicity of dissolved copper in both the presence and absence of HA. The toxicity data obtained fitted well into a logistic model, and the high sensitivity of both endpoints (EC(50) were 41.1 microg Cu/l and 32.9 microg Cu/l, respectively) encourages their use for biomonitoring. The HA had a clearly protective effect, reducing the toxicity of Cu to the sea urchin larvae. The labile copper, rather than the total copper concentrations, explained the toxicity of the Cu-HA solutions, and the Cu-HA complexes appeared as non-toxic forms. These results are in agreement with the Free Ion Activity Model, because the labile Cu concentrations in this buffered and chemically defined medium covary with the free ion activity of the Cu, validating the model to naturally occurring HA in the marine environment.

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.

Effects of dissolved mercury on embryogenesis, survival, growth and metamorphosis of Crassostrea gigas oyster larvae

The effects of mercury (Hg) concentrations ranging from 0 (control) to 1024 µg 1-1 upon embryogenesis, survival, growth and metamorphosis of Crassostrea gigas (Thunberg) oyster larvae were investigated. Embryogenesis was abnormal in 50 % of the individuals at 11 µg 1-1 . The 48 h LD50 for D·shaped, umbonate and pediveliger larvae were 33, 115 and 200 µg 1-1 respectively. The increase in LD50 was partially explained by the larval weight increase, although weight-specific tolerance to Hg was higher in smaller larvae. Growth, the most sensitive physiological process studied, was significantly retarded at 4 µg 1-1. The metamorphosis rate was significantly reduced when competent pediveligers were exposed to 64 µg 1-1 for 48 h prior to the addition of the metamorphosis inducer epinephrine. The implications of the present results for monitoring pollution by utilising different bivalve larval stages are discussed.

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.

Methodological basis for the optimization of a marine sea-urchin embryo test (SET) for the ecological assessment of coastal water quality.

The sea-urchin embryo test (SET) has been frequently used as a rapid, sensitive, and cost-effective biological tool for marine monitoring worldwide, but the selection of a sensitive, objective, and automatically readable endpoint, a stricter quality control to guarantee optimum handling and biological material, and the identification of confounding factors that interfere with the response have hampered its widespread routine use. Size increase in a minimum of n=30 individuals per replicate, either normal larvae or earlier developmental stages, was preferred to observer-dependent, discontinuous responses as test endpoint. Control size increase after 48 h incubation at 20 degrees C must meet an acceptability criterion of 218 microm. In order to avoid false positives minimums of 32 per thousand salinity, 7 pH and 2mg/L oxygen, and a maximum of 40 microg/L NH(3) (NOEC) are required in the incubation media. For in situ testing size increase rates must be corrected on a degree-day basis using 12 degrees C as the developmental threshold.