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9 pages, 5 figures, 1 table, supplementary materials https://doi.org/10.1016/j.watres.2023.120044.-- Data availability: Data will be made available on request, The purpose of this study was to investigate the occurrence of microplastics (MPs) in drinking water in Spain by comparing tap water from different locations using common sampling and identification procedures. We sampled tap water from 24 points in 8 different locations from continental Spain and the Canary Islands by means of 25 μm opening size steel filters coupled to household connections. All particles were measured and spectroscopically characterized including not only MPs but also particles consisting of natural materials with evidence of industrial processing, such as dyed natural fibres, referred insofar as artificial particles (APs). The average concentration of MPs was 12.5 ± 4.9 MPs/m3 and that of anthropogenic particles 32.2 ± 12.5 APs/m3. The main synthetic polymers detected were polyamide, polyester, and polypropylene, with lower counts of other polymers including the biopolymer poly(lactic acid). Particle size and mass distributions were parameterized by means of power law distributions, which allowed performing estimations of the concentration of smaller particles provided the same scaling parameter of the power law applies. The calculated total mass concentration of the identified MPs was 45.5 ng/L. The observed size distribution of MPs allowed an estimation for the concentration of nanoplastics (< 1 µm) well below the ng/L range; higher concentrations are not consistent with scale invariant fractal fragmentation. Our findings showed that MPs in the drinking water sampled in this work do not represent a significant way of exposure to MPs and would probably pose a negligible risk for human health, The authors acknowledge the financial support provided by Plastics Europe and that of the Spanish Government, Ministerio de Ciencia e Innovación, grants PID2020-113769RB-C21/C22., With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)

Pressures from anthropogenic activities are causing degradation of estuarine and coastal ecosystems around the world. Trace metals are key pollutants that are released and can partition in a range of environmental compartments, to be ultimately accumulated in exposed biota. The level of pressure varies with locations and the range and intensity of anthropogenic activities. The present study measured residues of trace metals in Mytilus mussel species collected from a range of locations around the world in areas experiencing a gradient of anthropogenic pressures that we classified as low, moderate, or high impact. The data showed no grouping/impact level when sampling sites in all countries were incorporated in the analysis, but there was significant clustering/impact level for most countries. Overall, high-impact areas were characterized by elevated concentrations of zinc, lead, nickel, and arsenic, whereas copper and silver were detected at higher concentrations in medium-impact areas. Finally, whereas most metals were found at lower concentrations in areas classified as low impact, cadmium was typically elevated in these areas. The present study provides a unique snapshot of worldwide levels of coastal metal contamination through the use of Mytilus species, a well-established marine biomonitoring tool, SI

Microplastics (MP) are contaminants of concern per se, and also by their capacity to sorb dissolved chemicals from seawater, acting as vehicles for their transfer into marine organisms. Still, the role ofMP as vehicles for con- taminants and their associated toxicological effects have been poorly investigated. In this work we have compared the role ofMP (high density polyethylene, HDPE, ≤22 μm) and of natural organic particles (microalgae, MA) as vehicle for chlorpyrifos (CPF), one of the most common pesticides found in river and coastal waters. We have compared the capacity of MP and MA to carry CPF. Then, the mussel Mytilus galloprovincialis has been exposed for 21 days to dissolved CPF, and to the same amount of CPF loaded onto MP and MA. The concentration of CPF inmussel' tissues and several physiological, energetics and immuneparameters have been analyzed after 7 and 21 days of exposure. Results showed similar CPF accumulation in mussel exposed to MP and to MA spiked with CPF. This revealed that MP acted as vector for CPF in a similar way (or even to a lesser extent) than MA. After 21 days of exposure mussels exposed to MP spiked with CPF displayed similar or more pronounced biological effects than mussels exposed to dissolved CPF or to MA loaded with CPF. This suggested that the combined “particle” and “organic contaminant” effect produced an alteration on the biological responses greater than that produced by each stressor alone, although this was evident only after 3 weeks of exposure., Efectos ecotoxicológicos de microplásticos en ecosistemas marinos: estudios con mejillón (Mytilus galloprovincialis), EPHEMARE, SI

There is a great concern that physical and chemical properties of microplastics (MPs) may facilitate the sorption of toxic metals and organic contaminants to the particle surface, serving as a enriched vector (Trojan horse) of contaminants to marine organisms following ingestion . Yet the extent to which sorption of contaminants onto different types of MPs enhances or mitigates the effects of these pollutants remains unclear. Apart from direct action on specific body tissues, contaminants may exert toxic effects by influencing homeostatic mechanisms, such as the immune system. Immune defence in mussels is comprised of cell-mediated and humoral mechanisms, in which hemocytes play a key role. Phagocytosis is performed by specialized cells such as macrophages, and it plays a role in the clearance of particles having a diameter greater than 0.5 μm. Particle size, shape and surface properties affect efficient entrapment and subsequent uptake by macrophages. Mussels have highly developed phagocytosis processes for the cellular internalization of microscale particles (0.1–100 μm) and they represent a suitable model for investigating the effects and modes of action of micro and nanoparticles in the cells of aquatic invertebrates. Polystyrene microspheres filtered by the blue mussel Mytilus edulis are bioaccumulated in gut and digestive tubules and subsequently translocated in hemolymph and hemocytes. The capability of microplastics to adsorb chemical pollutants from the environment is a potential risk that has motivated research. Polyethylene generally exhibits a greater sorption capacity than other plastic types. To distinguish between the potential adverse effects caused by exposure to MPs, absorbed chemicals, and their combined effects on marine life, controlled laboratory experiments are necessary. The use of freshly isolated primary cultures has the advantage that they are more differentiated than cell lines, containdifferent types of cells and are thus thought to respond more similarly to a living animal, while at the same time reducing the amount of work and animals required for in vivo testing. In this study we tested the hypothesis that the simultaneous presence of Hg and polyethylene MPs in the extracellular medium may increase the toxicity associated with Hg in an individual way, altering the immune function in mussel hemocytes. To this end we investigated the in vitro effects caused by individual and co-exposure of Hg and polyethylene MPs (size range from 15 to < 1 μm) on phagocytosis efficiency and lysosomal membrane stability in freshly primary cultures hemocytes of the Mediterranean mussel (Mytilus galloprovincialis). Postprint

The adsorption and desorption of Hg onto and from microplastics (MP) and microalgae (MA) were investigated, and fitted using pseudo-first-order and pseudo-second order kinetics models. Then, the potential role of MP as vector for the entrance and accumulation of Hg (MP-Hg) in comparison to natural pathways (via MA -MA-Hg-, and dissolved -WB-Hg-) was investigated in mussel. Mussels were exposed to a single dose of Hg (2375 ng ind−1) for 4 h. Although the clearance of MP-Hg was relevant (82 %), it was lower than that of MA (95 %) and MA-Hg (94 %). The amount of the Hg accumulated and eliminated was higher in mussels exposed to MP-Hg (1417 ng Hg) than in those exposed to MA-Hg (882 ng Hg) and WB-Hg (1074 ng Hg). However, Hg accumulation was similar in the three mussel groups (≈800 ng Hg). This was related to the fast elimination of Hg still attached to MP by MP-Hg mussels. Hg was mainly accumulated in digestive gland in MA-Hg and MP-Hg mussels, and in gills in WB-Hg mussels. Overall, the results indicated that MP facilitated the entrance of Hg in mussel but also promoted Hg elimination, which could limit the toxicological risk of Hg adsorbed onto MP.

Marine bivalves including mussels Mytilus galloprovincialis are commonly used as sentinels for pollution monitoring and ecosystem health assessment in the coastal zones. Use of biomarkers to assess the pollution effects assumes that the effects of pollutants on the biomarkers exceed the natural background variability; yet this assumption has rarely been tested. We exposed mussels at different reproductive stages and nutritive states to two concentrations of a polycyclic aromatic hydrocarbon (fluoranthene, 3 and 60 μg L-1) for three weeks. Expression levels of the molecular biomarkers related to the detoxification and general stress response [cytochrome P450 oxidase (CYP450), glutathione S-transferases (GST-α; GST-S1; GST-S2), the multixenobiotic resistance protein P-glycoprotein (PgP), metallothioneins (MT10 and MT20), heat shock proteins (HSP22, HSP70-2; HSP70-3; HSP70-4), as well as mRNA expression of two reproduction-related genes, vitellogenin (Vitel) and vitelline coat lysin M7 (VCLM7)] were measured. The mussels' nutrition and reproductive state affected the baseline mRNA levels of molecular biomarkers and modulated the transcriptional responses of biomarker genes to the pollutant exposure. Thus, mussel physiological state could act as a confounding factor in the evaluation of the response of pollution through molecular biomarkers. The biomarker baseline levels must be determined across a range of physiological states to enable the use of biomarkers in monitoring programs.

International audience; The Ephemare project was supported in the period 2015–2018 by JPI Oceans, as one of 4 sister projects in the joint action on ecological aspects of microplastics. Ephemare investigated several issues concerning the ecotoxicological effects of microplastics (MPs) in marine organisms. Ephemare included 16 European Institutions from 10 Countries and was organized into seven, highly complementary Work Packages (WPs) with the aim to elucidate adsorption and release of chemicals to/from MPs, coupled with MP ingestion rates, translocation in different tissues, trophic transfer and egestion, potential toxicological effects and mechanisms of action, as well as real distributions of MPs in marine organisms from several European areas.

MPs' uptake and tissue accumulation were investigated in mussel exposed to a single dose (2.85 mg ind−1, 3 mg l−1) of a heterogeneous mixture of irregularly shaped particles of HDPE (mainly ≤10 μm), followed by a 7 days depuration period. The results showed that mussels efficiently cleared MPs from water during exposure, and that MPs were accumulated in digestive gland and gills during depuration. In digestive gland, the amount and size of the MPs accumulated decreased with time, indicating a slower processing and elimination of small MPs than of larger ones. In gills, MPs' burdens increased with time, the MPs accumulated were the smallest ones, suggesting the translocation of small MPs from the digestive system to the gills. The hazardous potential of the smaller fraction of MPs (≤4 μm) underlined that more focus should be directed towards the accumulation and effects of this fraction of MPs in the marine environment., Sí

In the marine environment, metals can be present dissolved or adsorbed to suspended particles. In the last decades a new type of particle has been introduced, microplastics (MPs). The exposure route of pollutants influences their accumulation and distribution into tissues. A pulse-chase experiment was conducted in which mussels were exposed to Hg: adsorbed onto MPs and microalgae (MA) and dissolved (WB). Mussels accumulated the same amount of Hg independently of particle, due to the Hg loading in both particles and their acceptability were similar. The highest Hg accumulation occurred in gill when the Hg exposure was through water and in digestive gland when Hg was adsorbed to particles. More than 70% of the Hg uptake through MPs was quickly eliminated due to: i) part of the cleared MPs might not really be ingested but adhered to body surfaces of mussels, ii) MPs ingested were eliminated through faeces as they are non-nutritive particles which may be rejected in stomach preventing their entry into digestive gland and iii) high affinity of Hg on surface of MPs which meant that Hg was mainly eliminated jointly to MPs. The organic nature of MA facilitates the entry of Hg into digestive gland where MA are intracellularly digested releasing the Hg adsorbed onto their surfaces. In this case, Hg may reach deeper levels by translocation of the Hg incorporated into gland towards foot and remaining tissues, a process that might occur through haemolymph. All of the Hg accumulated in WB during the exposure was internally absorbed into tissues, and later translocated from gill to gland. Although Hg elimination rate in MPs mussels was greater than in the other exposure pathways, an important amount of Hg was maintained through the depuration period, thus we cannot and should not neglect the risk of MPs as vectors for mercury., Sí

It is highly likely that phytoplanktonic organisms will interact with MPs in the ocean, and consequently with the pollutants sorbed onto their surfaces. Microalgae play an essential role in maintaining the balance of the marine ecosystem due to the fact that they are a primary producer and the base of marine trophic chains. Therefore, their fitness represents an important index in the assessment of water quality. The objectives of this study were i) to assess the toxicity of MPs and the pesticide chlorpyrifos (CPF) to the microalgae, Isochrysis galbana, clone t-ISO and ii) to ascertain whether the presence of MPs affects the toxicity of CPF. Microalgae growth rate was selected as the endpoint and a commercial virgin PE micronized powder was chosen as a micro-plastic model, with mean size ranging from 2 to 6 µm, assayed until 25 mg L−1. CPF was tested at concentrations ranging from 0 to 4 mg L−1. A constant concentration of MPs (5 mg L−1) was loaded with increasing doses of CPF (0–3 mg L−1) with a 2 h incubation period. Bioassays were performed at 20 °C, in glass tubes of 50 ml, with air and constant light and an exposure time of 72 h. Cell counts were performed using a Coulter Counter Multisizer III and HPLC was used to quantify the partition of this pollutant among MPs and water. Although microalgae growth was not impacted by MPs, growth was clearly affected by exposure to CPF from 2 mg L−1 and above, with a total growth inhibition at concentrations over 3 mg L−1. Subsequent to incubation, 80% of CPF was sorbed onto MP surfaces. Two different dose-response curves resulted from CPF bioassays depending on the presence of MP, with lower percentages of inhibition when CPF was presented through MP. Thus, the adsorption of CPF onto MP surfaces modulates the toxicity of CPF on I. galbana growth through a reduction in its toxicity, as CPF is adsorbed onto MP surfaces which are less bio-available to the algal cells.

In this study, the acute toxicity of microplastics (MPs) on unicellular organisms as marine decomposers and microalgae was assessed, by evaluating standards endpoints included in International Standard Organization (ISO) protocols. The bacteria Vibrio fischeri and the diatom Phaeodactylum tricornutum were exposed to different sizes (1–500 μm) of polyethylene MPs in order to evaluate bioluminescence inhibition and microalgal growth. No acute toxicity was found on bacteria or microalgae in an order of magnitude above environmentally relevant concentrations, suggesting that tested MPs did not affect the investigated biological processes. In conclusion, standard ecotoxicological endpoints are not sufficiently sensitive to assess the potential effects of MPs on decomposers and primary producers, conversely to nanoplastics. These findings highlight that the current approach for MP risk assessment in unicellular species should be revised, by providing alternative endpoints to be included in standardized protocols, able to monitor the fate and biological effects of MPs., Sí

Biomarkers are useful tools to assess biological effects of pollutants that are extensively used in monitoring programs to assess ecosystem health. However, they are strongly affected by mussel physiological state, especially nutritive status, which has led to the search of new biological indicators of chemical pollutants exposition. Environmental metabolomics is an approach for examining the metabolic responses (measurement of low molecular weight endogenous metabolites) of an organism to both natural and anthropogenic stressors that can occur in its environment. The aim of the present work was to assess the effect of the polycyclic aromatic hydrocarbon fluoranthene (FLU) exposure on the metabolomic profiles of mussel digestive glands under different nutritive conditions. To achieve this objective, mussels were reared, for a period of 56 days, under three different food rations in order to obtain a gradient of nutritive status (negative, zero and positive energy balance), and after that, they were exposed, during 3 weeks, to a nominal concentration of 3 μg FLU L-1. A total of 43 metabolites, including aminoacids (Ala, Val, Leu, Ile, etc.), energy metabolism related metabolites (ATP, AMP, etc.), organic osmolytes (taurine, etc.), redox metabolism (GSH, NADP+) and nucleotides, were identified and quantified in the digestive glands of the mussels. Principal Component Analysis (PCA) defined two principal components (PC1 and PC2) that explained 55.6% of the total variance, although the first component explains more than 80% of this variance, this being related to the mussel nutritive condition. The effect of the toxicant, explained by the PC2, is similar to that produced under conditions of food restriction, which masks the effect of the toxicant under these conditions. As the feeding conditions are more favorable, the toxic effect becomes more apparent. Therefore, the great influence of nutritive condition on mussel metabolome implies a handicap for the use of metabolomic biomarkers, as previously demonstrated for biochemical and other molecular biomarkers, in large-scale monitoring programs in which several food conditions coexist with pollution levels., Sí

The majority of plastics present in the marine environment are microplastics (MPs,5 mm). Suspension filter feeders are susceptible species to MPs ingestion. Once ingested MPs can be eliminated packed in fecal pellets, or they can be accumulated within tissues, and likely be transferred along the food web. The research on MPs is hampered by the difficulty on their quantification and the lack of standardized methodologies. Indeed, limited information exits about the capacity of marine organisms to ingest, accumulate and eliminate MPs. In this work we investigated the uptake, elimination and accumulation of MPs (irregularly shaped particles of high density polyethylene, ≤22 μm) in mussel. Mussels were exposed to two concentrations of MPs (2 and 4 mm

This study investigated whether general stress biomarkers in mussels can be applied as common first-tier biomarkers in regional biomonitoring programmes in the North Sea (including Iceland) and western Mediterranean Sea. Stress on Stress (SoS) and lysosomal membrane stability (LMS) biomarkers were analysed in resident mussels (Mytilus sp.) from 8 coastal sites and in transplanted mussels (Mytilus galloprovincialis) from two Spanish Mediterranean coastal sites. The assessment of results, as input to pollution monitoring strategies, was performed jointly for LMS and SoS data from the two regions. Contaminant body burden of the mussels was compared with biomarker results. The results demonstrated that these two general and non-expensive stress biomarkers in mussel can be applied throughout European waters, providing a cost-effective and harmonised approach to screen contaminant-related biological effects within the framework of wide-scale pollution biomonitoring programmes, such as that proposed by the European Union, i.e. the Marine Strategy Framework Directive., SI

The effect of the quality of two microalgal species on select biological and biochemical responses usedas indicators of pollution were assessed. Mussels were conditioned for 6 weeks with the diatom Chaeto-ceros neogracile and the dinoflagellate Heterocapsa triquetra, chosen for being two clearly different typesof primary production quality that differ in both biometric and biochemical characteristics. After dietaryconditioning, the mussels were exposed to a polycyclic aromatic hydrocarbon, fluoranthene (FLU), for1 week followed by 1 week of depuration. Results showed higher FLU accumulation in mussels fed onC. neogracile compared to those fed on H. triquetra. Concomitantly, a greater impact of this toxicant wasobserved in the biomarker responses of mussels fed on C. neogracile. These mussels showed an increase inthe percentage of dead hemocytes, an activation of phagocytosis and ROS production of hemocytes afterexposure. Some enzymatic activities also increased upon FLU exposure (superoxide dismutase −SOD-, catalase −CAT-, and glutathione reductases −GR-) and after depuration (glutathione-s-transferase−GST-). Results suggest that FLU exposure as well as food quality influence biomarker responses, withhigher values of SOD, CAT and GR in non-exposed mussels fed on C. neogracile. In addition, upon exposureto the same FLU concentration, GR response varied according to dietary conditioning, suggesting that dietcould act as a confounding factor in biomarker responses to pollution. Consequently, trophic conditionsshould be considered in marine pollution monitoring programs for a better interpretation of biomarker responses., SI

Biomarkers are useful tolos to assess biological effects of pollutants and have been extensively used in Monitoring programs To determine Ecosystem health. In These programs, a wide range of environmental conditions are covered and sometimes, obtained data are difficult to interpret because of natural Variables are affecting biomarker responses. Among these variables, mussel ́ ıs reproductive status has been considered one of the most changing variablesbetween sites in a monitoring survey. Thus, the main aim of this work was to identify the effect that mussel reproductive status has on biomarker responses. For that purpose, mussels sampled at two periods in the reproductive cycle (reproductive and resting stages)were conditioned to the same laboratory conditions and exposed to fluoranthene (FLU) for three weeks. Studied biomarkers covering a Wide range of organism responses were included: bioaccumulation, physi- ological rates (clearance rate −CR-,absorption efficiency− AE-,respiration rate − RR- and their integration in the scope for growth − SFG-), antioxidant enzyme activities (superoxide-dismutase − SOD-, catalase − CAT-, glutathione reductase − GR-, glutathione peroxidase − GPx-, glutathione-S-tranferase − GST-) and biochemical damage responses (lipid membrane peroxidation − LPO-). The results obtained evidenced that the levels of the biomarkers studied (RR, SOD, CAT and GPx) were higher at reproductive tan at resting stage. On the other hand, theeffect of toxicant was observed in SFG, CAT and GPx but this effect was only detected during the resting period. Moreover, there was a deterioration of mussel gonadal tissue with FLU exposure during reproductive stage. FLU accumulation in mussel tissues was also dependent of the reproductive status with higher internal concentrations during resting tan reproductive period. In conclusion, there was a strong effect of reproductive status on studied biomarkers which seems to mask the effect of FLU at reproductive stage. Te present study evidences the need to include the measure- ment of mussel biological status in marine pollution monitoring programs for a correct interpretation of biomarker data., SI

00003 ăWOS:000383930500078; International audience; The effects of polystyrene microbeads (micro-PS; mix of 2 and 6 mu m; final concentration: 32 mu g L-1) alone or in combination with fluoranthene (30 mu g L-1) on marine mussels Mytilus spp. were investigated after 7 days of exposure and 7 days of depuration under controlled laboratory conditions. Overall, fluoranthene was mostly associated to algae Chaetoceros muelleri (partition coefficient Log Kp = 4.8) used as a food source for mussels during the experiment. When micro-PS were added in the system, a fraction of FLU transferred from the algae to the microbeads as suggested by the higher partition coefficient of micro-PS (Log Kp = 6.6), which confirmed a high affinity of fluoranthene for polystyrene microparticles. However, this did not lead to a modification of fluoranthene bioaccumulation in exposed individuals, suggesting that micro-PS had a minor role in transferring fluoranthene to mussels tissues in comparison with waterborne and foodborne exposures. After depuration, a higher fluoranthene concentration was detected in mussels exposed to micro-PS and fluoranthene, as compared to mussels exposed to fluoranthene alone. This may be related to direct effect of micro-PS on detoxification mechanisms, as suggested by a down regulation of a P-glycoprotein involved in pollutant excretion, but other factors such as an impairment of the filtration activity or presence of remaining beads in the gut cannot be excluded. Micro-PS alone led to an increase in hemocyte mortality and triggered substantial modulation of cellular oxidative balance: increase in reactive oxygen species production in hemocytes and enhancement of anti-oxidant and glutathione-related enzymes in mussel tissues. Highest histopathological damages and levels of anti-oxidant markers were observed in mussels exposed to micro-PS together with fluoranthene. Overall these results suggest that under the experimental conditions of our study micro-PS led to direct toxic effects at tissue, cellular and molecular levels, and modulated fluoranthene kinetics and toxicity in marine mussels.

The potential use of digestive activities as indicators of the nutritional status in bivalves is discussed in relation to the results obtained in two clam species exposed to starvation and refeeding. Activities of some digestive enzymes (amylase, laminarinase, cellulase, and protease) were measured in juveniles of two commercially interesting species of clams, Ruditapes decussatus and Venerupis pullastra. The specimens were fed normally, being after subjected to a 15-days starvation and a further refeeding period. Samples were obtained at different moments of such feeding schedule to evaluate enzymes as well as weight (live, dry and organic) and length, in order to calculate growth rates and feeding efficiencies. Starvation led to a major decrease in clam growth as measured by dry weight and a negative growth as measured by organic weight, this coinciding with a certain degree of growth of the shell and a consumption of soft tissue. This response occurred more rapidly in R. decussatus but was of a lower magnitude than in V. pullastra. Activity of carbohydrases decreased rapidly in both species with starvation, although protease activity was maintained in R. decussatus. Recovery after the end of starvation was not similar in both species; while R. decussatus attained similar growth rates and enzyme activities to those measured prior to nutritional stress, V. pullastra only recovered 50% of its initial values. For both species of bivalves it can be concluded that digestive enzymes, and more specifically amylase, could be used as indicative of their nutritional condition.

The present study was aimed to evaluate differences in physicochemical characteristics (pH and temperature optima) of the main digestive enzymes (amylase, cellulase, laminarinase, and protease) in two commercially important bivalves, the intertidal clam, Ruditapes decussatus, and the subtidal clam, Venerupis pullastra, as well to assess individual variability of such activities. The assays were performed on extracts obtained from the digestive glands and crystalline styles of individuals collected in the wild and laboratory-conditioned during 1 week. In both species, maximum activity for amylase and cellulase was measured at pH 6 and that of laminarinase at pH 5.5, while pH profiles of protease activity showed important differences between them. Temperature profiles also showed interspecific variations, mainly in laminarinase. Carbohydrase activities were comparatively much higher in R. decussatus and proteolytic activities in V. pullastra. The observed differences both in physicochemical characteristics and relative importance of digestive enzymes are discussed in relation to the amount and type of food available in their respective habitats., Sí

The Mar Menor is a coastal lagoon affected by the growth of intensive agriculture and urban development in the surrounding area. Large amounts of chemical pollutants from these areas are discharged into El Albujon, a permanent water-course flowing into the lagoon. Biomarkers such as the activity of acetylcholinesterase or antioxidant enzymes have been previously tested in this lagoon demonstrating the presence of neurotoxicity and oxidative stress in clams transplanted in sites affected by the dispersion of the effluent from El Albujon. To complete this traditional toxicology work, a metabolomic profiling of these transplanted organisms has been carried out for the detection of metabolic biomarkers induced by agricultural/urban pollutants. More than 70 metabolites have been quantified using a targeting metabolomics platform based on HPLC–MS. The intracellular metabolic pattern was analyzed by PCA from the digestive gland of clams after 7 and 22 days of transplantation. Results showed a different profile of metabolite between organisms collected from control and exposed sites. At the shorter exposure time, there was an increase in several metabolites in the latter when compared with those from control sites, whereas metabolic profiling at 22 days showed that those metabolites were drastically diminished, with even lower levels than at control sites. These metabolites included: (i) 12 amino acids from the 21 proteogenic and HomoSer, (ii) osmotic protectants such as γ-butyrobetaine and taurine and (iii) nucleotides such as ITP. Regarding sulfur-containing molecules, taurine could be highlighted as a potential biomarker since its concentration was reduced by more than 30 times after 22 days of exposure, whereas the antioxidant glutathione remained constant in the organisms from both control and exposed sites. Although targeted metabolomics has been shown as an early technique of pollutant effect detection, the two-phase pattern could highlight a more complicated metabolite response to pollutants than classical biomarkers.

11 páginas, 1 figura, 3 tablas, Various diets for the spat of Ruditapes philippinarum were assessed, consisting wholly or partially of wheatgerm flour—diet A1: 100% of the daily food ration (4 mg AFDW (100 mg LW)−1) consisted of the microalga Isochrysis galbana, clone T-ISO; diet M2: 50% of the daily ration consisted of microalgae and the other 50% of flour; diet M3: 25% microalgae and 75% flour; and diet G: 100% flour. Two control diets consisting solely of microalgae were also assayed: diets A2 and A3, consisting of 50% and 25% of the daily ration, respectively. The composition of the wheatgerm used was: 18.2% of the total organic matter consisted of protein, 71.2% of carbohydrate and 10.6% of lipids, while the microalgae consisted of 27.8% protein, 38.3% carbohydrates and 33.8% lipids. The diets prepared with these two components are characterised by an increase in their carbohydrate content and a decrease in their protein and lipid content as the proportion of wheatgerm increases. The highest growth rates were obtained for the spat fed diets A1 (final dry weight, 7.12±1.15 mg individual−1) and M2 (6.72±0.30 mg individual−1),with no significant difference being observed between the two diets. The spat fed diet M3 reached a final dry weight of 4.12±0.49 mg individual−1, a figure which is greater than that obtained with the control diets A3 (1.44±0.13 mg individual−1) and A2 (2.98±0.76 mg individual−1). The growth of the spat fed exclusively wheatgerm, diet G (1.55±0.08 mg individual−1), was similar to that obtained with control diet A3. The proportion of organic matter in the spat at the end of the experimental period is related to the nutritional value of the diet, so that the spat fed the diets with the greatest nutritional value (the highest growth rates) showed higher proportions of organic matter., This study was financed by CICYT-IEO-CSIC, project MAR99-0240-CO2.

This studydealswiththeidentification andcharacterizationofbiologicalvariablesthatmayaffectsome of thebiologicalresponsesusedaspollutionbiomarkers.Withthisaim,duringthe2012musselsurveyof the SpanishMarinePollutionmonitoringprogram(SMP),attheNorth-Atlanticcoast,severalquantitative and qualitativebiologicalvariablesweremeasured(corporalandshellindices,gonadaldevelopmentand reservescomposition).Studiedbiomarkerswereantioxidantenzymaticactivities(CAT,GST,GR),lipid peroxidation(LPO)andthephysiologicalratesintegratedintheSFGbiomarker(CR,AE,RR).Sitepol- lution wasconsideredasthechemicalconcentrationinthewholetissuesofmussels. A greatgeographicalvariabilitywasobservedforthebiologicalvariables,whichwasmainlylinkedto the differencesinfoodavailabilityalongthestudiedregion.Aninverserelationshipbetweenantioxidant enzymes andthenutritionalstatusoftheorganismwasevidenced,whereasLPOwaspositivelyrelatedto nutritional statusand,therefore,withhighermetaboliccosts,withtheirassociatedROSgeneration. Mussel conditionwasalsoinverselyrelatedtoCR,andthereforetoSFG,suggestingthatmusselskeepan “ecological memory” from thehabitatwheretheyhavebeencollected.Nooverallrelationshipwasob- served betweenpollutionandbiomarkers,butasignificant overalleffectofbiologicalvariablesonboth biochemical andphysiologicalbiomarkerswasevidenced.Itwasconcludedthatwhenawiderangeof certain environmentalfactors,asfoodavailability,coexistinthesamemonitoringprogram,itdetermines a greatvariabilityinmusselpopulationswhichmasktheeffectofcontaminantsonbiomarkers, SI

This study undertakes an overall assessment of pollution in a large region (over 2500 km of coastline) of the N-NW Spanish coast, by combining the use of biochemical (AChE, GST, GPx) and physiological (SFG) responses to pollution, with chemical analyses in wild mussel populations (Mytilus galloprovincialis). The application of chemical analysis and biological techniques identified polluted sites and quantified the level of toxicity. High levels of pollutants were found in mussel populations located close to major cities and industrialized areas and, in general, average concentrations were higher in the Cantabrian than in the Iberian Atlantic coast. AChE activities ranged between 5.8 and 27.1 nmol/min/mg prot, showing inhibition in 12 sampling sites, according to available ecotoxicological criteria. GST activities ranged between 29.5 and 112.7 nmol/min/mg prot, and extreme variability was observed in GPx, showing activities between 2.6 and 64.5 nmol/min/mg prot. Regarding SFG, only 5 sites showed ‘moderate stress’ (SFG value below 20 J/g/h), and most sites presented a ‘high potential growth’ (>35 J/g/h) corresponding to a ‘healthy state’. Multivariate statistical techniques applied to the chemical and biological data identified PCBs, organochlorine pesticides and BDEs as the main responsible of the observed toxicity. However, the alteration of biological responses caused by pollutants seems to be, in general, masked by biological variables, namely age and mussel condition, which have an effect on the mussels’ response to pollutant exposure. Publicado 2

The Mar Menor is a coastal lagoon threatened by the development of intensive agriculture in the surrounding areas. Large amounts of pesticides from these areas are discharged into El Albujón, a permanent watercourse flowing into the lagoon. We have used a multi-biomarker approach to assess the biological effects of agricultural pollution on a bivalve species. Biomarkers indicative of neurotoxicity (acetylcholinesterase, AChE), oxidative stress (catalase, CAT; glutathione reductase, GR and lipid peroxidation, LPO), phase II biotransformation of xenobiotics (glutathione S-transferase, GST) and physiological stress (scope for growth, SFG) were measured in clams transplanted to four sites of the lagoon (two reference sites and two sites affected by the dispersion of the effluent of the El Albujón), for exposure periods of 7 and 22 days. The hazards of this effluent were also examined by simultaneously measuring up to 83 contaminants (pesticides, PCBs, PAHs and others) in samples of fresh water from the watercourse mouth and seawater from the deployed sites, as well as the bioaccumulation of organochlorinated compounds and PAHs in the transplanted animals. Biomarker responses showed marked differences between reference and affected sites after 7 and 22 days. However it was only after 22 days that principal component analysis (PCA) of the biomarker responses distinguished between clams deployed in sites affected by the dispersion of the effluent of the watercourse and those from the reference sites. The chemical analysis of water showed high concentrations of pesticides close to El Albujón watercourse mouth, with the greatest input flux corresponding to the organophosphate chlorpyrifos, followed by pendimethalin and naphthalene, and at lower levels acenaphthene, terbuthylazine-desethyl and chlorpyrifos-methyl. In this regard, PCA analysis showed that the biological effects of the mixture of pesticides in caged clams after 22 days were reduced levels of AchE and SFG and increased levels of GR and phase II GST activity. An integrated biomarker response index was calculated from the combination of these biomarkers, proving useful for the assessment of the impact of agricultural pollution in caged clams., Sí

7 páginas, 3 tablas, The fatty acid composition of the Ruditages d.ecussatw spat fed on three different microalgal diets during 4 weeks was determined. The fatty acid pattern of each diet was also analysed. The diets used were Zsochrysk galbana, clone T-ISO, Tetraselmis suecica, and Phaeodactylum tricornutum. The fatty acid composition of the spat was usually well correlated with that of the diet supplied. Major differences among spat cultures were found in 14:0, 16:0, 16:ln-9, 16:ln-7, 18:ln-9, 18:2n-6, 18:3x1-3, 18:4n-3, 20:5n-3, 22:5n-6 and 22: 6n-3 fatty acids. These differences were correlated with the particular fatty acid content of each diet supplied. It has been shown that R. decussatus spat have a very low capacity to elongate and desaturate linolenic acid to n-3 PUFA, so when 20: Sn-3 or 22:6n-3 were not present in the diet, they were also absent, at least in measurable amounts, in the clams. The absence of any of the “essential” fatty acids, 20 : Sn-3 in T-IS0 or 22 : 6n-3 in Tetmselmis, did not limit spat growth, so their role as “essential” fatty acids might be a matter for discussion. Finally, the nutritive value of each diet was discussed in terms of its fatty acid composition., This study was funded by CICYT-CSIC-IEO project I + D number MAR90-082 1 X02-01.

The present study was aimed to establish the relationship between the functionality of the digestive gland and physiological rates including SFG (scope for growth) in wild mussels, Mytilus galloprovincilis. The experimental set-up consisted in the evaluation of changes in the morphology of the gland, as well as in the activity of some key digestive enzymes (amylase, laminarinase, cellulase and protease) within a broad range of SFG obtained through manipulation of food ration. The higher SFG values were correlated to an increase in both the size of the digestive gland and the activities of enzymes when expressed in relation to individual. In contrast, no clear relations were observed when the activity of enzymes was expressed in relation to soluble protein, with the exception to amylase. The higher protease activities measured in mussels showing lower SFG may reflect an initial stage of catabolic processes intended to compensate the energy deficit produced by food restriction. The potential use of parameters measured in digestive glands in studies of marine pollution was discussed.

SFG and physiological rates were measured in wild mussels from the Spanish Marine Pollution monitoring program (SMP) in order to determine seawater quality. It consists of 41 stations, covering almost 2500 km of coast, making the SMP the widest-ranging monitoring network in the Iberian Peninsula's Atlantic region. Results of the 2007 and 2008 surveys when 39 sites were sampled: (20 in 2007 and 19 in 2008, being 8 sites sampled both years) were presented. Chemical analyses were carried out to determine the relationships between physiological rates and the accumulation of toxic compounds. Data presented are the first to become available on the use of SFG as a biomarker of the marine environment on a large spatial scale (> 1000 km) along Spain's Atlantic seaboard. SFG values enable significant differences to be established between the areas sampled and between the two years surveyed. The integration of biological and chemical data suggests that certain organochlorine compounds, namely chlordanes and DDTs, may have a negative effect on SFG, although such an effect is of a lesser magnitude than that associated with certain biological parameters such as condition index and mussel age. These variables act as confounding factors when attempting to determine the effect of chemical compounds present in the marine environment on mussel SFG. Further research is therefore needed on the relation between these confounding factors and SFG in order to apply the relevant corrective strategies to enable this index to be used in monitoring programs. The effect of these confounding factors is more clearly revealed in studies that cover a wide-ranging spatial and time scale, such as those carried out within the SMP. These results do not invalidate the use of biological data in monitoring programs, but rather point to the need to analyze all the factors affecting each biological process., Sí

The energy budget of Venerupis pullastra clam seed (3 mm) fed the microalga, Isochrysis galbana T-ISO, was calculated after acclimation at 10, 15, 20 and 25°C. Physiological measurements included ingestion rate, absorption efficiency, oxygen consumption and ammonia excretion. The effect of temperature on these rates was described. Ingestion rate was directly related to temperature up to a maximum at 20°C. Further temperature increase caused a slight decrease in ingestion. Absorption efficiency was not significantly influenced by the temperature, although maximum mean values were also found at 20°C. Both respiration and ammonia excretion rates were directly related to temperature over the whole experimental range, reaching their maxima at 25°C. For all the physiological rates measured the higher slopes in the rate/temperature lines occurred in the medium range of temperatures (15–20°C). The scope for growth (SFG) was positive at all temperatures and maximum at 20°C, chiefly as a consequence of the enhanced ingestion rate which offset the concomitant elevation in metabolic rates. Both gross and net growth efficiencies are also expected to be maximum at 20°C, which is, therefore, the thermal optimum for this species under the experimental conditions applied in our study. An acclimation strategy for this low-shore species within the range of thermal variation in its natural environment (10–20°C), maximizing SFG at high temperatures by enhancing ingestion rate, despite increased metabolic costs, is proposed.

10 páginas, 3 tablas, Venerupis pullastra spat were fed for 4 weeks on diets consisting of two species of algae which were supplied in single or mixed diets. The microalgal species tested were hochrysis aff. galbana T-ISO and Tetraselmis suecica. The fatty acid pattern of the spat was basically dependent on the diet supplied. Major differences between spat cultures were detected on 14:0, 16:1n—7, 18:0, 18:1n—9, 18:3n-3, 18:4n—3, 20:5n—3 and 22:6n—3 fatty acids. These differences were correlated to those fatty acid contents in the diets supplied, with the exception of the 18:4n —3 fatty acid. This acid was not detected in the Tetraselmis diet but appeared, however, in the spat fed on this microalga. This fatty acid could be obtained from linolenic acid which was present in the Tetraselmis lipids in a high proportion. Special attention was given to the essential fatty acids, 20:5n — 3 and 22:6n — 3. While I. galbana T-ISO cells contain both fatty acids, although 20:5n — 3 in a very low proportion, T. suecica is deficient in 22:6n — 3. Similarly, the T. suecica fed spat is also deficient in 22:6n — 3. Spat fed on the mixed diet shows a fatty acid profile halfway between the two algal species. The absence of 22:6n — 3 in Tetraselmis cells may explain the lower growth rate obtained with this diet., This work was funded by CICYT-CSIC-IEO project I+D number MAR90-0821-CO2-01

Oyster (Ostrea edulis L.) seed was reared on five rations of Isochrysis galbana Parke: 10, 30, 100, 200 and 300 cells μl-1, in an open-flow system. Physiological parameters such as clearance, ingestion, absorption and respiration rates were measured, and the scope for growth (SFG) calculated from these parameters was compared with actual growth over 20 d. Actual growth was negligible at 10 cells μl-1 (daily maintenance ration=2.5% organic wt) and maximum (growth rate=13.64 d-1) at 200 cells μl-1 (daily ration=43% organic wt), which also supported the maximum gross and net growth efficiencies (K 1=50%, K 2=85%). Ingestion rate was directly correlated to cell concentration up to maximum of ∼100 cells μl-1, but further increases failed to support higher ingestions. Absorption efficiency decreased with increasing algal concentration from 95% down to 60%. Due to the costs of growth, respiration rate significantly increased when food was added above maintenance levels; however, metabolic costs associated with feeding activity were undetectable. The scope for growth calculated from these physiological parameters agreed with the long-term growth performance, validating the physiological energetics method as a valuable tool for predicting long-term growth performance under constant environmental conditions.

The aim of this study was to assess environmental quality at some of the sites most severely affected by the Prestige oil spill off 2 years after the spillage (April and November 2004). For this purpose analyses of polycyclic aromatic hydrocarbons (PAH) and several biochemical (antioxidant enzymes catalase, glutathione reductase, glutathione peroxidase, superoxide dismutase and DT-diaphorase and lipid peroxidation) and physiological [scope for growth (SFG)] biomarkers were determined on wild mussel populations (Mytilus galloprovincialis) collected at four points along the Costa da Morte and compared with those of a reference site not affected by the oil spill. Results showed that PAH contents had markedly decreased 17 months after the accident, although they were higher in April than in November, when they showed values similar to background levels reported for this area. Nevertheless, the predominance of chrysene on PAH profiles, similarly to findings obtained immediately after the spill, indicated the Prestige as their main source. In spite of the low PAH levels recorded, antioxidant activity levels (explained through the integrated antioxidant response-IAR) were higher in the Costa da Morte than at the reference site either in April and November. In April IAR seems to be related to PAH levels found 3 months after the accident (February 2003), suggesting the persistence in the environment of oxidative stress-producing components from the spill. However, evidence of oxidative stress was not reflected at physiological level by scope for growth, with only very slight differences being observed between values from the reference site and those from Costa da Morte sites. In conclusion, although 2 years after the spill PAHs bioaccumulated by mussels from the Costa da Morte had decreased to background levels, biochemical parameters showed signals of oxidative stress in mussels from this area. However, SFG reflected a good health status for the mussel populations studied and did not reveal evidence of physiological disturbance either 17 or 24 months after the Prestige spill.

9 páginas, 3 tablas,3 figuras, Adult specimens of two species of clams, Ruditapes decussatus and Venerupis pullastra, were starved for 84 days to determine the effect of starvation on their metabolism. The energy cost of metabolism in starvation was assessed using the oxygen consumption rates (respiration rates) of the clams and assessing the type of fuel used to provide this energy by analyzing their biochemical composition. Respiration rates decreased in both species after 15 days of starvation and remained at basal levels throughout the rest of the experimental period. Both during the first period and also after the metabolism had slowed down, the amount of energy consumed in respiration was higher in V. pullastra than in R. decussatus. The energy needed to maintain vital functions in both species is obtained from catabolism of body components, with a reduction in dry mass of the specimens, and consequently their energy content, in both species. This reduction was greater in V. pullastra given that energy demand is higher in this species. In both species carbohydrates made the largest contributions to energy output, followed by lipids in males and proteins in females. However, the energy contribution of each biochemical component differs according to species: R. decussatus obtains its energy from the catabolism of carbohydrates and proteins in equal proportion, while V. pullastra obtains it from proteins and lipids. In both species, albeit to a greater extent in R. decussatus, we observed that female specimens conserved their lipids until the later stages of the period of starvation (day 70), after which they started to metabolise their lipid components more intensely. The interspecific differences are interpreted in relation to the different habitat occupied by the two species, Funding for this research was provided by Comisión Interministerial de Ciencia y Tecnología, Spain, project MAR99- 0240-CO2.

9 páginas, 4 tablas, This study investigates the influence of the lipid composition of microalgal and commercial flour diets on the lipid classes and fatty acids of Ruditapes philippinarum spat. Aspects of the nutritional role of the diets and the feeding ration are discussed with regard to previously published spat growth data. Our results demonstrated that clams showed the best growth rates when fed with diets that supplied a larger quantity of lipids, further characterised by a high content of phospholipids and triacylglycerols. We observed a significant correlation between the amount of triacylglycerols (r = 0.929, p < 0.05) and phospholipids (r = 0.781, p < 0.05) supplied and spat growth. In addition, R. phillipinarum spat reached the highest percentages of triacylglycerols (about 12%) and the lowest percentages of phospholipids (about 60%) and sterols (about 4%) with these growth-promoting diets. Spat fed with the other diets and/or rations showed lower growth rates and significantly lower quantities of triacylglycerols. In the present study, the alternative essentiality of 20:5n − 3 and/or 22:6n − 3 is confirmed. The diminishing supply of 22:6n − 3 without an increase of 20:5n − 3 has an effect on the growth of the spat. The dietary composition of fatty acids influenced the fatty acid profiles in bivalves. The results of our study would suggest that R. philippinarum is incapable of transforming 18:3n − 3 to 20:5n − 3 and then to 22:6n − 3. Accordingly, although diets contained 18:3n − 3, the spat reflected the low content of 20:5n − 3 of the diets. With respect to 22:6n − 3, given that this fatty acid is present in high quantities in Isochrysis, the spat content of this fatty acid was relative to its content in the diet. This study showed that clams with the lowest growth rates presented an increase in 20:4n − 6. In the spat fed with the microalgal diets, 18:2n − 6 scarcely reached 2% whereas in spat fed with wheat germ 18:2n − 6 amounted to 18% of the total fatty acids. This fatty acid, by means of elongation, transforms to 20:2n − 6, which also appeared in important quantities in the lipids of the spat fed totally or partially by wheat germ. We note that 20:2n − 6 did not originate from the diet since it is absent in the microalgae and the flour. The desaturation of 20:2n − 6 to 20:3n − 6 has not been observed (low levels of 20:3n − 6 in all cases) and therefore it can be assumed that the observed levels of 20:4n − 6 were diet-related, This work was funded by MEC. AGL2004-07023-C02-02/ACU.

This study evaluates the risk posed by trace metals on the culture of bivalves in the Galician coast (NW Iberian Peninsula), which depends on collection of natural seed and larval rearing with natural seawater in hatcheries. With this aim, toxicity tests were carried out with embryos of the commercial bivalves Ruditapes decussatus and Mytilus galloprovincialis, and the toxicity of Hg, Cu, Zn, Cd and Pb was quantified in terms of median effective concentration (EC 50 ) and toxicity threshold, using embryogenesis success as endpoint. The EC 50 values ranged from 4 to 10 µg/l for Hg and Cu, from 100 to 300 µg/l for Zn and Pb, and from 400 to 2000 µg/l for Cd. The toxicity of copper and zinc combinations could be predicted using a strictly additive model. Effective concentrations found were compared to measured metal concentrations in the Galician Rias, and to local and international seawater quality criteria, in an attempt to evaluate the risk posed by metals to these commercial species. Zinc and especially copper pollution were found to represent a serious threat in certain areas. Furthermore, local seawater quality criteria recently implemented are, in the case of copper and zinc, two orders of magnitude too high to offer any protection for these sensitive early life stages of bivalves.

7 páginas, 1 figura, 2 tablas, The feeding behavior and the efficiency of the absorption of biochemical components in the diet of specimens of two species of clams, R adit apes decussatus and Ycncruph pullastra fed on natural and carbohydrate-rich diets were studied. Both the natural diet, which consisted of the microalga Isochnsis aff, galhana, clone T-ISO and ashed sediment, and the carbohydrate-rich diet, which consisted of microalgae and corn starch as organic ingredients, and ashed sediment as the inorganic component, were assayed at a concentration of total particulate matter close to 1 mg TPM L _l , and a concentration of particulate organic matter of approximately 0.6 mg POM L _l , which arc similar conditions to those found in the Galician Rias. The feeding behavior of both species for each diet is described with reference to the clearance and ingestion rates, whereas the absorption of the biochemical components of the two diets was determined by biochemical analysis of the diet and the resulting feces. Both ingestion and absorption rates were higher for V. pullastra when the clams were fed on a natural diet. Enriching the diet with carbohydrates led to a notable increase in the ingestion and absorption rates in both species, although this increase was greater in R. decussatus than in V. pullastra, and in consequence the energy absorbed from the carbohydrate-rich diet was greater in the case of R. decussatus. The energy absorbed by R. decussatus fed on this latter diet was three times greater than that absorbed on the natural diet, allowing it to maintain similar rates of protein absorption for both diets. However, in the case of V. pullastra, the amount of total energy absorbed that derived from proteins is 50% lower in the carbohydrate-rich diet than in the natural diet. The energy absorbed from carbohydrates in the carbohydrate-rich diet was greater for R. decussatus than for t' pullastra. The contribution of lipids to the total energv absorbed was found to be almost double in R. decussatus fed on the carbohydrate-rich diet, in comparison with the natural diet, although in V. pullastra this contnbution was lower. Thus, the effect of diet on the feeding behavior of both species, i.e., the increase in the ingestion rate and the corresponding increase in the absorption rate, allows /?. decussatus to compensate for the nutritional deficiencies of the carbohydrate-rich diet, whereas in the case of V. pullastra it does not appear to be sufficient for the clams to maintain the same protein absorption rate as on the natural diet. These results are discussed in relation to the possible existence of major differences in the metabolism of the two species of clams, differences which would be connected to the habitats in which they live., Funding for this research was provided by Comision Interministerial de Ciencia y Tecnología, Spain, project MAR99-0240- C02.

Samples of Musculista senhousia, 20 mm in length, were gathered from San Diego Bay and fed on three different food concentrations: 0.2, 0.4 and 0.8 mg AFDW L m 1, and their energy balance studied. Food concentration ranges were selected to reflect those observed in the field where the invasive mussel M. senhousia co-occurs with the angiosperm, Zostera marina. The lowest and intermediate concentrations correspond to concentrations described in and over large eelgrass patches, respectively. The upper concentration is equivalent to that occurring during the spring phytoplankton blooms. Ingestion rates varied between 81.6 µg AFDW h m 1 for the mussels fed on the lowest concentration, 191.2 µg AFDW h m 1 when the concentration rose to 0.4 mg AFDW L m 1, and 164.0 µg AFDW h m 1 for the highest concentration. Food concentration had a significant effect on absorption efficiency, which was approximately 70% at the intermediate and upper concentrations, but rose to over 80% at the lowest concentration. The food absorption rate varied with food concentration in a manner similar to that of the ingestion rate, i.e., with a maximum value of 142.34 µg AFDW h m 1 for the mussels fed on the intermediate concentration, this being higher than the values for both the highest concentration (114.72 µg AFDW h m 1) and the lowest (65.89 µg AFDW h m 1). Respiration rates were 54 µg O 2 h m 1 for the mussels fed on the lowest concentration and 74 µg O 2 h m 1 for those fed on the intermediate concentration. These results show a close relationship between respiration and ingestion rates, which can be fitted to the following equation: RR = e (m 0.42 + 0.10 IR) (r = 0.5703, R 2 = 32.53, p 0.05), whilst SFG for the lowest food concentration was only 0.69 J h m 1. The physiological rate with the greatest effect on growth was the absorption rate. Its relation to SFG is described by the equation SFG = 0.67 + 0.90AR (r = 0.977, R 2 = 95.56%, p, Sí

15 páginas, 3 tablas, 1 figura, Trials were carried out for different growth diets, consisting wholly or partially of wheatgerm flour, for the spat culture of the little-neck clam, Ruditapes decussatus (L.). The following diets were tested: diet A, consisting of 100% of the daily food ration of the microalga Isochrysis galbana, clone T-ISO; diet B, consisting of 50% microalgae and 50% wheatgerm; diet C, consisting of 25% microalgae and 75% wheatgerm; and diet D, consisting of 100% wheatgerm. Diet A was taken as the reference diet and two control diets were added: suboptimal algal diet B, consisting of 50% of the daily ration of microalgae, and suboptimal algal diet C, consisting of 25% microalgae. Spat growth was determined by live weight, dry weight, organic content and length. Biochemical analyses of the diets and the spat were carried out at the beginning and the end of the 35-day experimental period. The growth achieved when 50% of the daily ration of microalgae was replaced by wheatgerm was similar to that obtained with control diet A, consisting of 100% microalgae. As regards organic matter, the growth of the spat fed on this diet, diet B, was double that shown by the spat fed on suboptimal algal diet B. When 75% of the microalgae were replaced by wheatgerm the growth rates decreased, being only equivalent to 60% of those for reference diet A. Nevertheless, the percentage of organic matter of this spat, indicative of the index of condition, was comparable to that of the spat fed on the reference diet. The total replacement of microalgae by wheatgerm produced a considerable decrease in spat growth, 20% of that obtained with reference diet A. The percentage of organic matter in the spat fed on the extreme diets, diet D and suboptimal diets B and C, was observed to be significantly less than that found in the spat fed on the other diets. Another characteristic of the diets tested was a decrease in protein and lipid content, together with an increase in that of carbohydrates, which corresponded to the extent to which microalgae were replaced by wheatgerm. The biochemical composition of the spat reflects a limitation in the lipid content of diets C and D, reflected in a lower lipid growth rate in the spat compared to that of other body matter., This study has been financed by CICYT-CSIC-IEO project AGF95-1003-C02.

10 páginas, 6 tablas, The influence of the lipid composition of microalgal diets and cornstarch on the lipid classes and fatty acids of Ruditapes decussatus spat was studied. These aspects of the nutritional value of the diets are discussed in relation to the growth of the spat. Cornstarch is rich in carbohydrate, but is deficient in protein and lipids. The microalgal cells contains lipid as well as protein and carbohydrate. Tahitian Isochrysis cells contain phospholipids and triacylglycerols as majority lipids, whereas in the cornstarch particles the more abundant lipids are free fatty acids and phospholipids.The n-3 fatty acids were the most abundant acids in the microalgae whereas the n-6 fatty acids were in the cornstarch. The n-3 PUFA were not detected in cornstarch. Diets with a lipid composition high in phospholipids and triacylglycerols and in essential fatty acids produce good growth rates in spat of R. decussatus. However these elements alone are not sufficient to explain the quality of the diet for R. decussatus. In fact, the lipid content of the diet affects growth, but does not explain it totally and it is the carbohydrates content that explains the differences in growth found when we use diets with the same composition in lipids., This work was funded by CICYT-CSIC.IEO projet I D number AGF95-1003-CO2.

9 páginas, 6 tablas, The influence of both the lipid composition of microalgal diets and commercial flours on the lipid classes and fatty acids of Ruditapes decussatus spat was studied. These aspects of the nutritional value of the diets were discussed in relation to the growth of the spat. Four diets were tested; Diet A, composed of 100% of the daily food ration of microalgae; Diet B, composed of 100% of wheatgerm; Diet C, composed of 50% of microalgae and 50% of wheatgerm; and Diet D, composed of 25% of microalgae and 75% of wheatgerm. The microalgal cells present a higher lipid content than that for wheatgerm. Tahitian Isochrysis cells have phospholipids and triacylglycerols as majority lipids, whereas in the wheatgerm particles, the lipids more abundant are triacylglycerols. Fatty acid content was higher in the microalgal cells than in the wheatgerm particles. The n-3 fatty acids were the most abundant acids in the microalgae, whereas the n-6 fatty acids were in the wheatgerm. The n-3 PUFA were not detected in wheatgerm. Phospholipids were the main lipids present in the clam spat, followed by triacylglycerols. Other lipid classes, detected in significantly lower amounts, included free fatty acids, sterols, and sterol ester 1 waxes. The composition of fatty acids in the spat was influenced by the fatty acid composition of the diet. Highest spat growth rates were observed with those diets that present a higher phospholipid/triacylglycerol relation. A negative correlation in the relation n-6/n-3 vs. growth has also been observed, with better growth rates in diets with a lower ratio. If the fatty acid 20:5n-3 and 22:6n-3 considered ‘‘essential’’ for marine animals were not present in the diet, they were not present in the spat either. Desaturation and elongation capabilities of R. decussatus spat were also discussed. comp biochem physiol 119A;1:369–377, 1998, This work was funded by CICYT-CSIC.IEO project I 1 D number AGF95-1003-CO2.

14 páginas, 5 tablas, 1 figura, Research was carried out into the effect of phytoplankton, cornmeal and cornstarch diets on growth and biochemical composition of the seed of the little-neck clam, Ruditapes decussatus. The seed of R. decussatus, fed on daily rations of Isochrysis galbana organic weight. of 0.5 and 1% of live weight of the seed, showed an improvement in growth rate when cornstarch, which is 99% carbohydrate, was added to these diets. Thus in the case of a daily ration of 0.5%, daily growth rates increased by between 33.5 and 32.3%, depending on whether we are referring to organic weight, dry weight or live weight, when 1.5% cornstarch was added. In the case of a ration of 1% I. galbana, the addition of another 1% cornstarch lead to an improvement in daily growth rates, depending on the different weight class in question, of between 14.1 and 15.5%. When compared to a daily ration consisting of 2% phytoplankton, which was considered to be the optimal ration for growth in the seed of these clams, the replacement of half the quantity of I. galbana by a quantity of cornstarch of equivalent weight gave a growth rate in terms of organic weight of 87.9% that of the phytoplankton diet, while the rates for dry weight and live weight were 89.6 and 87.9%, respectively. These results improved noticeably when cornmeal, consisting of 10% protein and 90% carbohydrate, was used instead of cornstarch. In the case of a 2% phytoplankton diet, if we substituted an equivalent quantity of cornmeal for 50% of the phytoplankton, the growth rate in organic matter was the same 99.0%. as those for the diet consisting of phytoplankton alone, while growth rates in dry weight and live weight were 6.2 and 5.9% higher, respectively, than those of the phytoplankton diet. It would therefore appear that cornmeal and to a lesser extent cornstarch. can be successfully used as a partial substitute for phytoplankton in diets for the seed of R. decussatus and its use in hatcheries and nurseries devoted to the culture of this species would lead to a considerable reduction of production costs, This study was funded by CICYT-IEO-CSIC project IqD number AGFC-1003-CO2-01.

13 páginas, 3 figuras, 3 tablas, The nutritional quality of several microalgal diets used in the seed culture of the little-neck clam, Rudifupes decussatus, was evaluated in the present study. The live diets tested were Isochrysis galbana, clone T-ISO, Tetraselmis suecica and Phaeodactylum tricornutum. Criteria used in the evaluation of the diets were acceptability, digestibility, growth and biochemical composition. The highest growth rate was registered in the seed fed the T. suecicu diet, followed by the seed fed I. galbana, while the lowest one was obtained in the seed fed P. tricornutum. Food ingestion rates were higher in the seed fed Tetruselmis. This fact would partly explain the higher growth observed with this diet. The limited nutritional value of Phaeodactylum could be related to the low digestibility of its cell wall, the degree of acceptability of this diet being similar to or even higher than that of the other two. The low protein content of Tetraselmis seems to indicate that R. decussatus shows a low requirement for proteins, which can be spared by carbohydrate, an abundant component in the cells of Tetraselmis. Lipid needs of this bivalve also seem to be rather low., This study has been financed by CICYT-CSIC-IEO I + D project MAR90-0812-C02-01.

Ruditapes decussatus (L.) seed, 1.6 mm in length, was reared in an open-flow system for 28 days at different concentrations of food: 15, 50, 100, 200 and 300 Isochrysis galbana, clone T-ISO, cells μL−1. Rates of ingestion, absorption, and respiration were measured during the experiment and used to calculate the energy balance. Rate of ingestion and absorption efficiency were the primary physiological processes governing seed growth. Ingestion rates increased with food concentration, up to a maximum of 100 cells μL−1. Greater increases in concentration resulted in a decrease in ingestion. Clearance rates were inversely correlated to food concentration. Absorption efficiency, estimated using the Conover method, also decreased with increasing concentrations of food. Respiration rates were independent of food concentration. Potential growth rates were lower than actual growth rates for each of the concentrations tested, although the effect of food concentration on actual and potential growth was equal, with maximum growth occurring at a concentration of 50 cells μL−1. Thus, SFG (‘scope for growth’) is a good qualitative estimator of actual growth, but a poor quantitative estimator.

Young post-metamorphic oysters, Ostrea edulis L., previously reared at 20 °C, were exposed to 14 °C (cold treatment), 20 °C (control) and 26 °C (warm treatment) for 3 wk. Algal (Isochrysis galbana Parke) ingestion rate (IR), absorption efficiency (AE), oxygen consumption (ifVo2) and ammonia excretion (VNH4) were measured at the beginning, halfway and at the end of the experimental period. These rates were integrated in the energy budget and the net energy gain available for biomass increase, or scope for growth (SFG), was calculated. All the physiological parameters, including AE, were significantly reduced in oysters acutely exposed to the cold treatment. However, following prolonged cold exposure an increase in IR and a slight decrease in Vo2 allowed compensation of the SFG, which in the long-term achieved similar levels to control. After acute exposure to the warm treatment, oysters showed slightly enhanced IR and VNH4 but no major changes in the other physiological parameters, which yielded a slightly increased SFG. In the long-term, IR markedly increased in warm acclimated oysters which, coupled with sustained metabolic costs, allowed an almost three times increase in the SFG. Different adaptive strategies of acclimation described for bivalves are discussed, under the common aim of SFG maximization following long-term exposure to a temperature change; and peculiarities of fast-growing juveniles are evidenced. It is concluded that juvenile oysters are able to respond in the long-term to either a decrease or increase of temperature, regulating the physiological rates integrated in the energy balance in a manner that renders an overall improvement in the net energy available for growth.

9 pages, 8 figures, 7 tables., Clam spat Venerupis pdlastra were fed for 4 wk on diets of 2 species of microalgae supplied singly or mixed. Microalgae were Isochrysis galbana and Tetraselmis suecica. species normaliy used in bivalve hatcheries. Observed growth responses were high for mixed and I. galbana diets and intermediate for the T suecica diet. A slightly higher growth rate was obtained with the mixed diet than with the I. galbana diet. Differences in growth rate were explained by differences in rates of ingestion and digestibility and in the biochemical composition of diets. The intermediate growth rate for spat fed on T. suecica was related to lower ingestion rates, lower assimilation efficiencies and lower dietary protein and lipid content. Spat biochemical composition was correlated with growth indices. Lower growth indices were related to lower protein and higher lipid content of spat. The high growth response to the mixed diet was not explained by the factors considered., This work was funded bv CICYT-CSIC-IEO project I+D number MAR90-0821-C02-01.

Seed of the clam Venerupis pullastra were reared with five different rations of Isochrysis galbana: 15, 30, 100, 200 and 300 cells μl−1, in an open-flow system. Physiological parameters such as clearance, ingestion and absorption rates were recorded, and energy balances calculated from these parameters were compared with the long-term growth performance of the seed. High food concentrations inhibited clearance rate but ingestion could not be regulated, being higher with higher food levels. Absorption efficiency decreased moderately at high I. galbana concentrations. Nevertheless, absorption rate was directly correlated with ingesta. Estimated metabolic rate was at a routine level for medium and high food rations and markedly lower at poor food rations. Growth performance was insignificant at 15 cells μl−1 (daily maintenance ration = 2% organic weight), optimal at 100 cells μl−1 (K1 = 46%) and maximal at 300 cells μl−1 (growth rate = 11.9 day−1), in agreement with the physiological energetics measurements. Growth rates of V. pullastra seed were similar to those reported for other species, such as Ruditapes philippinarum, regarded as a fast growing member of the Veneridae.