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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.

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.

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.

The effects of selected polycyclic aromatic hydrocarbons on the marine calanoid copepod Acartia tonsa were tested in laboratory short-term toxicity tests in order to facilitate risk assessment of those compounds to the marine pelagic environment. Photo-induced toxicity of pyrene was also investigated under naturally relevant UV light regimes. Lethal and sublethal effects on egg production rate, hatching and potential recruitment rate were evaluated after 48 h exposure to fluoranthene, phenanthrene and pyrene. The 48 h-median lethal concentrations (LC(50)) reducing survival by 50% were 594, 2,366 and >640 nM for fluoranthene, phenanthrene and pyrene, respectively, whilst lower concentrations induced different sublethal effects. Median effective concentrations (EC(50)) affecting the egg production rate and the recruitment rate were 433 and 385 (fluoranthene), 1,245 and 1,012 (phenanthrene) and 306 and 295 nM (pyrene), respectively. An increase in toxicity of pyrene was detected after incubation under UV light, resulting in LC(50) values of 201 nM (24 h) and 138 nM (48 h) and EC(50) values of 79 nM (egg production rate) and 41 nM (recruitment rate). Finally, a comparison between effective concentrations and worst-case environmental concentrations reported in literature indicated that pyrene may pose a threat to A. tonsa from exposure in the field, and that the risk of adverse effects is high for fluoranthene.

The toxicity of the alternative antifouling compound Sea-Nine to the early developmental stages of the sea urchin Paracentrotus lividus was investigated. The inhibition of the fertilization rate and the induction of transmissible damages to the offspring, measured as inhibition of embryonic development and larval growth, were studied by preexposure of gametes to a range of Sea-Nine concentrations. Sperm and egg exposures resulted in a significant decrease of the fertilization rate and induced a transmissible damage to the offspring. The effects of Sea-Nine throughout the embryonic development were also studied by a 48 h exposure of fertilized eggs. The larval growth was the most sensitive response tested, with toxic effects detected at 8.6 nM=2.4 microg/L (EC(10)). The inhibition of P. lividus embryonic development and larval growth was also used to study the loss of toxicity in Sea-Nine solutions exposed for 8h to direct sunlight and maintained for 28 h in dark conditions. The results showed that the toxicity of Sea-Nine solutions did not decrease but a slight increase in toxicity was observed in comparison with control solutions. The risk of Sea-Nine maximum concentrations measured in marinas around Europe to P. lividus early developmental stages was calculated and the obtained risk quotient was 5.5, indicating that adverse ecological effects of this compound are likely to occur.

The effects of medetomidine, a novel antifouling candidate, on the burrowing bivalve Abra nitida were studied. The burrowing behaviour, sediment reworking activity and faeces production were assessed after 24 h exposure of A. nitida to sublethal concentrations of medetomidine. Medetomidine caused a significant decrease in the burrowing response and in the sediment reworking activity. The median effective concentrations (EC50) were 430 nM (86 microg/l) and 4.4 nM (0.9 microg/l), respectively. No effects on the faeces production were detected. Although significant effects of medetomidine on A. nitida were registered, a reversibility of the effects was observed when 24 h-exposed animals were incubated in clean seawater and sediment for 24 h. Considerations relating to the future commercialisation of medetomidine for antifouling purposes are discussed.

This study evaluates the impact of commonly used "booster" biocides (chlorothalonil, Sea-Nine 211, dichlofluanid, tolylfluanid and Irgarol 1051) on early developmental stages of marine invertebrates of commercial and ecological relevance. Toxicity tests were conducted with embryos and larvae of the bivalve Mytilus edulis, the sea-urchin Paracentrotus lividus and the ascidian Ciona intestinalis. Toxicity was quantified in terms of the EC50 (median effective concentration) and EC10 reducing embryogenesis success, larval growth and larval settlement by 50% and 10% respectively. The EC10 and EC50 for chlorothalonil ranged from 2 to 108 and from 25 to 159 nM; for Sea-Nine 211 values were 6-204 and 38-372 nM; for dichlofluanid effective concentrations were 95-830 and 244-4311 nM; tolylfluanid yielded values between 99-631 and 213-2839 nM; and Irgarol 1051 was the least toxic compound showing values from 3145 to >25600 and from 6076 to >25600 nM. Those biocides may be ranked in the following order from highest to lowest toxicity to embryos and larvae of M. edulis, P. lividus and C. intestinalis: chlorothalonil>Sea-Nine 211>dichlofluanid=tolylfluanid>Irgarol 1051. The registered effective concentrations were compared to worst-case environmental concentrations reported in literature in order to evaluate the risk posed by these biocides to those invertebrate species. Our data support that chlorothalonil, Sea-Nine 211 and dichlofluanid predicted levels in marinas represent a threat to M. edulis, P. lividus, and C. intestinalis populations, whilst Irgarol 1051 showed no toxic effects on the biological responses tested here at worst-case environmental concentrations.

The effects of the new antifouling compound zinc pyrithione (Zpt) on the embryonic development of sea urchin (Paracentrotus lividus) and mussel (Mytilus edulis) were investigated in laboratory toxicity tests. The median effective concentrations (EC50) were 7.7 nM for sea urchin embryos and 8 nM for mussel embryos. Toxic effects of Zpt on the larval growth of the sea urchin were detected at 0.5 nM. Predicted environmental concentrations of Zpt in pleasure craft harbours are higher than the predicted no effect concentrations for sea urchin and mussel embryos, indicating that Zpt may pose a threat to those species from exposure in the field.

This study investigated the toxic effects of the insecticides lindane and chlorpyrifos, the herbicide diuron, the organometallic antifoulant tributyltin (TBT), and the surfactant sodium dodecyl sulfate (SDS) on the early life stages of Paracentrotus lividus (Echinodermata, Euechinoidea), Ciona intestinalis (Chordata, Ascidiacea), Maja squinado and Palaemon serratus (Arthropoda, Crustacea) in laboratory acute toxicity tests. The assays studied embryogenesis success from fertilized egg to normal larvae in P. lividus (48 h incubation at 20 degrees C) and C. intestinalis (24 h incubation at 20 degrees C), and larval mortality at 24 and 48 h in M. squinado and P. serratus. For P. lividus, the median effective concentrations (EC50) reducing percentages of normal larvae by 50% were: 350 microg l(-1) for chlorpyrifos, 5500 microg l(-1) for diuron, 4277 microg l(-1) for SDS, and 0.309 microg l(-1) for TBT. For C. intestinalis, the EC50 values affecting embryogenesis success were 5666 microg l(-1) for chlorpyrifos, 24,397 microg (l-1) for diuron, 4412 microg l(-1) for lindane, 5145 microg I(-1) for SDS, and 7.1 microg l(-1) for TBT. The median lethal concentrations (LC50) for M. squinado larval survival were 0.84 microg l(-1) (24 h) and 0.79 microg l(-1) (48 h) for chlorpyrifos, 2.23 microg(l(-1) (24 h) and 2.18 microg l(-1) (48 h) for lindane, and 687 microg l(-1) (48 h) for SDS. For P. serratus the LC50 values obtained were 0.35 microg l(-1) (24 h) and 0.22 microg l(-1) (48 h) for chlorpyrifos, 3011 microg l(-1) (24 h) and 3044 microg l(-1) (48 h) for diuron, 5.20 microg l(-1) (24 h) and 5.59 microg l(-1) (48 h) for lindane, and 22.30 microg l(-1) (24 h) and 17.52 microg l(-1) (48 h) for TBT. Decapod larvae, as expected, were markedly more sensitive to the insecticides than sea urchins and ascidians, and SDS was the least toxic compound tested for these organisms. Lowest observed effect concentrations (LOEC) of TBT for sea urchin and ascidian embryos, chlorpyrifos and lindane for crustacean larvae, and SDS, were similar to those found in many coastal areas indicating that there would be a risk to invertebrate embryos and larvae from exposure in the field to these pollutants.

This study investigated the toxicity of zinc pyrithione (Zpt) on the early stages of development of the ascidian Ciona intestinalis. Larval morphological abnormalities were studied after the exposure of C. intestinalis embryos at different stages of development. The median effective concentrations (EC50) ranged from 226-590 nM. The larval settlement stage was the most sensitive to Zpt. Toxic effects of Zpt on larval settlement were detected at 9 nM (EC10). The inhibition of C. intestinalis embryonic development was also used to study the loss of toxicity in Zpt solutions exposed to direct sunlight and laboratory UV light. The results showed that the toxicity of Zpt solutions decreased but did not disappear after 4 h exposure to direct sunlight (EC50 = 484 nM) or UV light (EC50 = 453 nM), compared to control Zpt solutions prepared in dark conditions. On the basis of the present data, predicted no effect concentrations of Zpt to C. intestinalis larvae are lower than predicted environmental concentrations of Zpt in certain polluted areas and therefore, may pose a risk to C. intestinalis populations.

Sublethal effects of medetomidine, a new generation antifouling compound, on lumpfish (Cyclopterus lumpus L.) and cod (Gadus morhua L.) larvae were examined. The effects on respiration rate and on colour adaptation of newly hatched larvae were assessed after 24-96 h exposure. Exposure of lumpfish larvae to the experimental concentrations resulted in a significant decrease in respiration rate (Lowest Observed Effect Concentration (LOEC) = 5-10 nM) and in the percentage of dark larvae (LOEC = 4 nM). However, no effects on respiration rate of cod larvae were detected. In addition to lumpfish larvae being affected at low concentrations of medetomidine, a reversibility of the effects was observed when 96 h-exposed larvae were incubated in clean seawater for 24-48 h. Considerations relating to the future commercialisation of medetomidine for antifouling purposes are discussed.

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.

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.