Impact assessment of metals realeased by aluminium-based galvanic anode on the physiology of the abalone Haliotis tuberculata in controlled conditions.

To protect metal structures immersed in the sea from corrosion, the galvanic anode cathodic protection system (GACP) is often applied. However, this association leads to continuous oxidation of the galvanic anode and therefore to a release of a metal cocktail in the forms of ions or oxy-hydroxides. Therefore, the main objective of our study was to investigate the toxicity of elements released from the dissolution of an aluminium-based galvanic anode (∼95% Al, ∼5% Zn, <0.1% for In, Cu, Cd, Mn, Fe) on a grazing gastropod, the abalone Haliotis tuberculata. The present study was carried out in complement to other research currently in submission. Gastropods were exposed for 16 weeks (12 weeks of exposure and 4 weeks of decontamination phase) to 6 conditions including a control, 4 concentrations based on total aluminium level (86, 425, 1096 and 3549 µg L-1) and a trophic control, corresponding to abalones placed in non-contaminated natural seawater but fed with contaminated algae. The effects of metals on growth, glycogen levels, brix index of hemolymph, MDA levels in digestive gland and gills, hemocyte phagocytic activity, ROS production, lysosomal system and the progress of gametogenesis were investigated throughout the entire exposure allowing the realization of kinetics. The results revealed that the aluminium-based anode does not seem to have an effect on the health status of the individuals for environmentally realistic concentrations. However, in extreme conditions strong effects were reported on the growth, immune system and reproduction of abalone.

Effects on Growth of Juvenile Abalones Haliotis tuberculata Under Chronic Exposition to Metals Released from the Dissolution of an Aluminium-Based Galvanic Anode.

In the marine environment, the galvanic anode cathodic protection system (GACP) undergoes oxidation and releases metals in the forms of ions or oxy-hydroxides into the environment. The objective of the present study was to investigate the toxicity of a cocktail of metals released from the dissolution of an aluminium-based galvanic anode (~ 95% Al, ~ 5% Zn) on the abalone Haliotis tuberculata. Juveniles were exposed for 16 weeks (i.e. 12 weeks of exposure and 4 weeks of decontamination phase) and their growth, intake rate, conversion rate and metallic concentrations were monitored. A total of 6 conditions were tested: a control, 4 concentrations based on Al and a trophic control. Results showed that the mortality reached 57% for individuals exposed to 1125 µg L-1 of Al, and the abalone growth significantly decreased for an Al concentration greater than 495 µg L-1. At the highest exposure concentration, intake rate measurements revealed that the appetite of abalones was affected, supported by the large increase in the conversion rate which was indicative of a poor feed efficiency. The monitoring of metallic concentrations showed that H. tuberculata strongly bioconcentrated Al relative to zinc. The diet did not appear to be the primary pathway for metal entry. Concentrations that significantly impacted abalone growth and survival during the experiment were higher than those found in natural environment, but the bioconcentration of Al into the tissues of a primary consumer such as abalone may be a potential pathway for Al to enter food webs.

In vitro effects of glyphosate-based herbicides and related adjuvants on primary culture of hemocytes from Haliotis tuberculata.

Glyphosate-based herbicides are among the most produced and widely-used herbicides. Studies have shown that commercial formulations and adjuvants may be more toxic to non-target organisms than the active ingredients alone, but the mechanisms of action of these chemicals remain unclear. The aim of this study was to investigate the in vitro effects of glyphosate, a commercial formulation and adjuvant alone using primary culture of hemocytes from the European abalone Haliotis tuberculata, a commonly farmed shellfish. Glyphosate was found to have negligible effects on viability, phagocytic activities and lysosome stability even with very high doses (i.e. 100 mg L-1). By contrast, greater effects on viability were observed for the commercial formulation and adjuvant alone, with EC50 values of 41.42 mg L-1 and 1.85 mg L-1, respectively. These results demonstrate that the toxic sublethal effects (i.e. phagocytic activity and destabilization of lysosomal membranes) of formulated glyphosate came from adjuvants and suggest they may be related to cell and organelle membrane destabilization.

Hemocyte morphology and phagocytic activity in the common cuttlefish (Sepia officinalis).

Little is known about the immune system of cephalopods, in spite of their many highly derived characters within the molluscan clade, including a vertebrate-like high-pressure closed circulatory system. Further the economic importance of cephalopod fisheries, potential for aquaculture, and use as ecotoxicology models demand a thorough understanding of their immune system. In this study, we present a comprehensive characterization of hemocytes in the common cuttlefish Sepia officinalis. Cytological stainings, electron microscopy- and flow cytometry-observations highlight a single granulocyte population with various densities of eosinophilic granules and unstained vesicles. These hemocytes contain acid phosphatase-, lysozyme- and proPO system enzymes, and have high activity in bead phagocytosis assays. Interestingly, bead pre-incubation in plasma results in time-dependent aggregation perhaps resulting from hemocyanin-coating, and decrease in phagocytosis. This study provides the basis for understanding hemocyte-mediated immunity in the common cuttlefish, and essential background for future studies on cephalopod immunity.

Comparison of the sensitivity of seven marine and freshwater bioassays as regards antidepressant toxicity assessment.

The hazards linked to pharmaceutical residues like antidepressants are currently a major concern of ecotoxicology because they may have adverse effects on non-target aquatic organisms. Our study assesses the ecotoxicity of three antidepressants (fluoxetine, sertraline and clomipramine) using a battery of marine and freshwater species representing different trophic levels, and compares the bioassay sensitivity levels. We selected the following bioassays: the algal growth inhibition test (Skeletonema marinoi and Pseudokirchneriella subcapitata), the microcrustacean immobilization test (Artemia salina and Daphnia magna), development and adult survival tests on Hydra attenuata, embryotoxicity and metamorphosis tests on Crassostrea gigas, and in vitro assays on primary cultures of Haliotis tuberculata hemocytes. The results showed high inter-species variability in EC50-values ranging from 43 to 15,600 µg/L for fluoxetine, from 67 to 4,400 µg/L for sertraline, and from 4.70 µg/L to more than 100,000 µg/L for clomipramine. Algae (S. marinoi and P. subcapitata) and the embryo-larval stages of the oyster C. gigas were the most sensitive taxa. This raises an issue due to their ecological and/or economic importance. The marine crustacean A. salina was the least sensitive species. This difference in sensitivity between bioassays highlights the importance of using a test battery.

Comparative effects of trace metal elements released from dissolution of aluminum-based galvanic anodes, aluminum chloride, zinc chloride, and their mixture on the development of the Pacific oyster D-larvae, Crassostrea gigas.

As the most abundant metal in the earth's crust, aluminum (Al) is used in many sectors, and nowadays, there is an increase in anthropogenic releases to aquatic ecosystems. This is particularly true in the context of corrosion protection systems involving galvanic anodes, which are mostly made of Al. Corroded instead of the steel structures they protect, galvanic anodes are described as sacrificial anodes. In contact with seawater, they undergo oxidation and release various metals in the form of ions or oxy-hydroxides into the marine environment, mainly Al and zinc (Zn). Several studies agree that Al increases the incidence of abnormal development in bivalve larvae from 150 µg L-1 which is close to the highest Al concentrations recorded in coastal waters. Therefore, we studied the impact of the cocktail of metals released by aluminum-based galvanic anodes on the development of Crassostrea gigas larvae, which we compared to the effects of aluminum chloride hexahydrate and zinc chloride alone and their mixture. The anode solution was realized thanks to an experimental device simulating the dissolution of a galvanic anode in the marine environment in order to reproduce the cocktail of metal species. We calculated an EC50 of 193.55 µg L-1 and 100.05 µg L-1 for Al and Zn chloride alone, respectively, and we highlighted an EC50 of 190.22 µg L-1 for the galvanic anode based on Al concentration. The mixture of the two metals in their chloride form resulted in the observation of additive and synergistic effects, which underlines the importance of considering the cocktail effect in ecotoxicological studies.

Aluminium-based galvanic anode impacts the photosynthesis of microphytobenthos and supports the bioaccumulation of metals released.

Very few studies have looked at the potential biological effects of degradation products of galvanic anodes particularly on primary producers which are central to food webs in marine ecosystems. The galvanic anode cathodic protection system (GACP) is widely used to protect submerged metallic structures from corrosion. Aluminium (Al) and zinc (Zn) are the main constituents of galvanic anodes and are therefore released in the marine environment by oxidation process to form ions or oxy-hydroxides. The main objective of our study was to evaluate the effects of the metals released from an aluminium-based galvanic anode on microphytobenthos performance in term of biofilm growing through the analysis of photosynthetic parameters, the determination of chlorophyll and extracellular polymeric substances (EPS). The bioaccumulation of Al and Zn were measured in the microphytobenthic compartment collected at the surface of polyvinyl chloride (PVC) plates exposed during 13 days to seawaters enriched in different concentrations of metals released from dissolution of one anode. Determination of bioconcentration factors confirmed that the microphytobenthos has incorporated Al. A significative effect was observed on the Chl a concentration for the higher tested concentration ([Al] = 210.1 ± 60.2 µg L - 1; [Zn] = 20.2 ± 1.4 µg L - 1). The seawater exposed to the anode affected the MPB productivity (ETRIImax) with consequences on acclimatation light (Ek), absorption cross section of PSII (σPII), Fv/Fm and NPQ. Regarding the EPS production, the anode degradation presented an impact on high and low molecular weight of both carbohydrates and protein fractions of microphytobenthos suggesting that EPS play an essential role in sequestering metal contaminants to maintain the integrity of the biological membranes and the functionality of the cellular organelles. The accumulation of Al released by GACP in microphytobenthos cells could lead to physiologic problems in photosynthetic organisms.

Impact of a sacrificial anode as assessed by zinc accumulation in different organs of the oyster Crassostrea gigas: results from long- and short-term laboratory tests.

Sacrificial anodes made of zinc are currently used in marine environments to mitigate marine corrosion as part of CP systems of immerged metallic structures. The aim of this work was to study zinc bioconcentration in the oyster Crassostrea gigas by performing two in vivo tests during different time periods and at different zinc concentrations. The first test was conducted during a period of 10 weeks at a concentration of 0.53 ± 0.04 mg Zn L(-1) to simulate long-term exposure, and a second test was conducted during a 168-hour period at a concentration of 10.2 ± 1.2 mg Zn L(-1) to reproduce short-term exposure. In these experiments, the zinc source was an electrochemical device that included a sacrificial anode to mimic the in situ conditions. During the first 14 days of the long-term experiment, digestive glands of C oysters exhibited bioaccumulation of zinc that varied according to the oysters' reproductive cycle. Both a bioconcentration factor (BCF) of ≤ 13,397 and a zinc accumulation percentage of +297% of zinc occurred in this organ after 10 weeks. The results obtained from the short-term test showed a lower BCF of 405 but a faster bioaccumulation of zinc (starting from the first day) in the same organ. No mortality was observed in long-term assay, but 81.8% of the oysters died at the end of the short-term assay. These results demonstrate the great capacity of C. gigas to accumulate zinc released from the anode, especially when low concentrations are released, as in the case of anode dissolution used as CP. This study confirmed the necessity to monitor this zinc-contamination source in marine environments in relation to the usual oyster consumption by humans (especially in France). No implication for human health of this zinc-contamination source was demonstrated until now, and this was not the purpose of this study; however, zinc remains one of the most abundant nutritionally essential elements in the human body that may affect the human immune system at high-level uptake.

Effects of chronic exposure of metals released from the dissolution of an aluminium galvanic anode on the Pacific oyster Crassostrea gigas.

Among the anthropogenic sources releasing metallic species into the marine environment, the galvanic anode cathodic protection system (GACP) is widely used to protect submerged metallic structures from corrosion. Galvanic anodes are an alloy of metals of which the main component is aluminum or zinc. Very few studies were performed to study their potential biological effects. We investigated the chronic toxicity of an aluminum-based galvanic anode on the Pacific oyster, Crassostrea gigas. Oysters were exposed for 84 days to three concentrations of aluminum (50, 100 and 300 µg L-1) obtained with an electrochemical experimental device simulating the dissolution of a galvanic anode. At different exposure times, we studied a battery of biomarkers of the immune system, reproductive parameters and the metabolic state of the oysters. Results demonstrated a sensitivity of oysters at the highest concentration and some biological effects were observed especially for the malondialdehyde content in the digestive gland after 84 days of exposure. In addition to these biomarkers, the bioaccumulation of the different metals composing the anode was measured in oysters' tissues. Bivalves bioaccumulated more zinc than aluminum, even if aluminium was present in greater concentrations during exposures. Moreover, exposure time did not influence the bioaccumulation of aluminum in contrast to zinc.

Effect of in vitro exposure to zinc on immunological parameters of haemocytes from the marine gastropod Haliotis tuberculata.

Environmental pollutants such as heavy metals exert immunotoxic effects on aquatic organisms. The immune defence of molluscs is comprised of cell-mediated and humoral mechanisms, in which haemocytes play a key role. In this study, a model based on primary cultured haemocytes from the gastropod mollusc Haliotis tuberculata was established to investigate the effects of zinc in vitro. Cells were exposed for 24 h to ZnCl(2) concentrations of 0, 10, 100 or 1000 microM. The effects of zinc on haemocyte parameters were investigated using morphological, spectrophotometric and flow cytometry analysis. Immunotoxicity was reflected by a significant decrease in the number of viable haemocytes (LC(50)(24 h) = 314 microM). Moreover, the cell area was dramatically reduced, and the percentage of rounded cells increased with increasing zinc concentrations. Exposure to 1000 muM zinc induced a significant reduction in acid phosphatase activity, phagocytic activity and reactive oxygen species production in haemocytes. However, several haemocyte parameters increased significantly after 24 h of zinc exposure. In response to a 1000 microM exposure, the phenoloxidase level was 26-fold higher than that of the control, and non-specific esterase activity was increased by 69% above that of the control. These results suggest a relationship between zinc exposure and alterations in the functional responses of haemocytes from H. tuberculata.

Regulation of Extracellular Matrix Synthesis by Shell Extracts from the Marine Bivalve Pecten maximus in Human Articular Chondrocytes- Application for Cartilage Engineering.

The shells of the bivalve mollusks are organo-mineral structures predominantly composed of calcium carbonate, but also of a minor organic matrix, a mixture of proteins, glycoproteins, and polysaccharides. These proteins are involved in mineral deposition and, more generally, in the spatial organization of the shell crystallites in well-defined microstructures. In this work, we extracted different organic shell extracts (acid-soluble matrix, acid-insoluble matrix, water-soluble matrix, guanidine HCl/EDTA-extracted matrix, referred as ASM, AIM, WSM, and EDTAM, respectively) from the shell of the scallop Pecten maximus and studied their biological activities on human articular chondrocytes (HACs). We found that these extracts differentially modulate the biological activities of HACs, depending on the type of extraction and the concentration used. Furthermore, we showed that, unlike ASM and AIM, WSM promotes maintenance of the chondrocyte phenotype in monolayer culture. WSM increased the expression of chondrocyte-specific markers (aggrecan and type II collagen), without enhancing that of the main chondrocyte dedifferentiation marker (type I collagen). We also demonstrated that WSM could favor redifferentiation of chondrocyte in collagen sponge scaffold in hypoxia. Thus, this study suggests that the organic matrix of Pecten maximus, particularly WSM, may contain interesting molecules with chondrogenic effects. Our research emphasizes the potential use of WSM of Pecten maximus for cell therapy of cartilage.

Identification of cDNAs encoding HSP70 and HSP90 in the abalone Haliotis tuberculata: Transcriptional induction in response to thermal stress in hemocyte primary culture.

Heat-shock proteins are a multigene family of proteins whose expression is induced by a variety of stress factors. This work reports the cloning and sequencing of HSP70 and HSP90 cDNAs in the gastropod Haliotis tuberculata. The deduced amino acid sequences of both HSP70 and HSP90 from H. tuberculata shared a high degree of homology with their homologues in other species, including typical eukaryotic HSP70 and HSP90 signature sequences. We examined their transcription expression pattern in abalone hemocytes exposed to thermal stress. Real-time PCR analysis indicated that both HSP70 and HSP90 mRNA were expressed in control animals but rapidly increased after heat-shock.

Seasonal changes in mRNA encoding for cell stress markers in the oyster Crassostrea gigas exposed to radioactive discharges in their natural environment.

The North Cotentin area (Normandy, France) hosts several nuclear facilities among which the AREVA reprocessing plant of La Hague is responsible for controlled discharges of liquid radioactive wastes into the marine environment. The resulting increase in radioactivity is very small compared to natural radioactivity. However, concerns about environment protection prompted the scientific community to focus on the effects of the chronic exposure to low concentrations of radionuclides in non-human biota. This study contributes to the evaluation of the possible impact of radioactive discharges on the oyster Crassostrea gigas in the field. Real-time polymerase chain reaction was used to quantify the expression levels of genes involved in cell stress in the oyster. They included members of the heat shock protein family (Hsp70, Hsc72, Hsp90), superoxide dismutase (SOD) and metallothionein (MT). Times series measurements were built from periodic samplings in the natural environment in order to characterize the natural variability as well as possible seasonal fluctuations. The genes studied exhibited a general seasonal expression pattern with a peak value in winter. The data inversely correlated with seawater temperature and the nature of the relationship between gene expression and temperature is discussed. In parallel, oysters were collected in four locations on the French shores, exposed or not to radioactive liquid wastes from the nuclear facilities hosted in the North Cotentin. The comparison of data obtained in the reference location on the Atlantic coast (not exposed) and data from oysters of the English Channel (exposed) gave no evidence for any statistical difference. However, because of the complexity of the natural environment, we cannot rule out the possibility that other parameters may have masked the impact of radioactive discharges. This dense set of data is a basis for the use of the expression levels of those genes as biomarkers to address the question of the possible effects of chronic exposure of the oyster to low concentrations of radionuclides in controlled laboratory experimental conditions.

Responses of primary cultured haemocytes derived from the marine gastropod Haliotis tuberculata to an industrial effluent exposure.

This study assessed the responses of primary cultured haemocytes from the marine gastropod Haliotis tuberculata exposed to the increasing concentrations of industrial effluent (0, 0.5, 1, 10, 15 and 20%) discharged into the Tunisian coastal area. Analyses showed the presence of metals such as cadmium (Cd), chromium (Cr), copper (Cu), manganese (Mn), zinc (Zn), nickel (Ni) and lead (Pb) in the effluent. The effects of this mixture of pollutants on abalone haemocyte parameters were reflected by a significant decrease of cell viability, phagocytotic activity and reactive oxygen species (ROS) production as well as morphological and lysosomal membrane alterations. Thus, these results indicated that our primary culture system represents a suitable in vitro model for monitoring of anthropogenic contaminants in aquatic environments.

Sub-lethal effects of a glyphosate-based commercial formulation and adjuvants on juvenile oysters (Crassostrea gigas) exposed for 35days.

Glyphosate-based herbicides include active matter and adjuvants (e.g. polyethoxylated tallow amines, POEAs). In addition to a previous investigation on the effect of glyphosate on oysters, the aim of the present study was to investigate the effects of sub-chronic exposures (35days) to three concentrations (0.1, 1 and 100µgL-1) of Roundup Express® (REX) and POEAs on oysters belonging to the same age group. Low mortality rates were calculated, and only few significant differences (i.e. shell length) between exposure conditions were observed at a given date. However, when comparing the biomarker's temporal variations, some different patterns (e.g. condition index, reproduction, parameters of oxidative stress) were observed depending on the molecules and concentrations. These results suggest that a longer exposure to an environmental concentration (0.1µgL-1) of REX and POEAs could induce harmful effects on oysters.

Shell extracts of the edible mussel and oyster induce an enhancement of the catabolic pathway of human skin fibroblasts, in vitro.

Mollusc shells are composed of more than 95% calcium carbonate and less than 5% organic matrix consisting mostly of proteins, glycoproteins and polysaccharides. In this study, we investigated the effects of matrix macromolecular components extracted from the shells of two edible molluscs of economic interest, i.e., the blue mussel Mytilus edulis and the Pacific oyster Crassostrea gigas. The potential biological activities of these organic molecules were analysed on human dermal fibroblasts in primary culture. Our results demonstrate that shell extracts of the two studied molluscs modulate the metabolic activities of the cells. In addition, the extracts caused a decrease of type I collagen and a concomitant increase of active MMP-1, both at the mRNA and the protein levels. Therefore, our results suggest that shell extracts from M. edulis and C. gigas contain molecules that promote the catabolic pathway of human dermal fibroblasts. This work emphasises the potential use of these shell matrices in the context of anti-fibrotic strategies, particularly against scleroderma. More generally, it stresses the usefulness to valorise bivalve shells that are coproducts of shellfish farming activity.

Sub-chronic exposure to fluoxetine in juvenile oysters (Crassostrea gigas): uptake and biological effects.

The bioconcentration potential of fluoxetine (FLX) and its biological effects were investigated in juvenile Pacific oyster exposed for 28 days to environmentally relevant concentrations of FLX (1 ng L(-1), 100 ng L(-1) and up to 10 µg L(-1)). FLX bioaccumulated in oyster flesh resulting in 28-day bioconcentration factors greater than 2,000 and 10,000 by referring to wet and dry weights, respectively. Nevertheless, FLX did not induce oyster mortality, delayed gametogenesis, or lead to adverse histopathological alterations. At the two highest concentrations, despite non-optimal trophic conditions, FLX stimulated shell growth but only in a transient manner, suggesting a role of serotonin in the regulation of feeding and metabolism in bivalves. Those high concentrations seemed to drive bell-shaped responses of catalase and glutathione S-transferase activities throughout the exposure period, which may indicate the activation of antioxidant enzyme synthesis and then an enhanced catabolic rate or direct inhibition of those enzymes. However, no clear oxidative stress was detected because no strong differences in thiobarbituric acid-reactive substance (TBARS) content (i.e. lipid peroxidation) were observed between oyster groups, suggesting that cellular defence mechanisms were effective. These results demonstrate the importance of considering additional biomarkers of oxidative stress to obtain a comprehensive overview of the FLX-induced changes in marine bivalves exposed under realistic conditions. Considering the battery of biomarkers used, FLX appears to induce little or no effects on oyster physiology even at a concentration of 10 µg L(-1). These results do not confirm the lowest observed effect concentration (LOEC) values reported by some authors in other mollusc species.

Does a short-term exposure to cadmium chloride affects haemocyte parameters of the marine gastropod Haliotis tuberculata?

In this study, a model based on primary cultured haemocytes from the gastropod mollusc Haliotis tuberculata was established to investigate the effects of cadmium chloride in vitro. Cells were exposed for 24 h to CdCl2 concentrations of 0, 1 and 100 µg ml(-1). The effects of cadmium on haemocyte parameters were investigated using morphological, spectrophotometric and flow cytometry analysis. Results showed that cadmium has no significant effects on cell viability and phagocytotic activity under the tested conditions. However, haemocytes became more rounded after cadmium exposure, which could explain the significant decrease of cell area beginning at 1 µg ml(-1) of CdCl2.

Effects of subchronic exposure to glyphosate in juvenile oysters (Crassostrea gigas): From molecular to individual levels.

Glyphosate-based herbicides are extensively used and can be measured in aquatic ecosystems, including coastal waters. The effect of glyphosate on non-target organisms is an issue of worldwide concern. The aim of this study was to investigate the effects of subchronic exposure to glyphosate in juvenile oysters, Crassostrea gigas. Yearling oysters were exposed to three concentrations of glyphosate (0.1, 1 and 100µgL(-1)) for 56days. Various endpoints were studied, from the individual level (e.g., gametogenesis and tissue alterations) to the molecular level (mRNA quantification), including biochemical endpoints such as glutathione-S-transferase (GST) and catalase activities and malondialdehyde content. No mortality and growth occurred during the experiment, and individual biomarkers revealed only slight effects. The levels of gene expression significantly increased in oysters exposed to the highest glyphosate concentration (GST and metallothioneins) or to all concentrations (multi-xenobiotic resistance). These results suggested an activation of defence mechanisms at the molecular level.

Effects of acute exposures to mecoprop, mecoprop-p and their biodegradation product (2-MCP) on the larval stages of the Pacific oyster, Crassostrea gigas.

Studies have shown that pesticides are sometimes detected at rather high levels in seawater and it has been suggested that these chemical compounds could act as additional stress factor for oysters cultured in coastal environments. The effects of pesticides on marine molluscs could be particularly harmful in the early stages which correspond to critical life stages. This study aimed to assess the effects of mecoprop, mecoprop-p and their degradation compound 2-methyl-4-chlorophenol on two larval stages of Crassostrea gigas. Embryotoxic effects were assessed on veliger larvae after 36 h exposures, and both percentages of normal larvae and types of abnormalities were taken into account. The effects of the three substances were evaluated on 21-day-old pediveliger larvae by calculating metamorphosis rates after 24h exposures. The results of the embryotoxicity assay indicated that 2-methyl-4-chlorophenol was more toxic (EC50: 10.81 mg L(-1)) than its parent compounds (EC50 mecoprop: 42.55 mg L(-1); EC50 mecoprop-p: 78.85 mg L(-1)). Mecoprop in particular injured shell formation with an increase of shell abnormalities following herbicide concentrations. The active substances were not toxic to metamorphosis processes, but 2-MCP was revealed to be more toxic to the success of metamorphosis (EC50: 7.20 mg L(-1)) than to embryo-larval development. However, the toxic concentrations were several orders of magnitude higher than environmental concentrations.