Effects of red-light irradiation and melatonininjection on the antioxidant capacity and occurrence of apoptosis in abalones (Haliotis discus hannai) subjected to thermal stress.

High ocean temperatures caused by global warming induce oxidative stress in aquatic organisms. Melatonin treatment and irradiation using red light-emitting diodes (LEDs) have been reported to reduce oxidative stress in a few aquatic organisms. However, the effects of red LED irradiation and melatonin injection on the antioxidant capacity and degree of apoptosis in abalones, which are nocturnal organisms, have not yet been reported. In this study, we compared the expression levels of antioxidant enzymes, total antioxidant capacity, and the degree of apoptosis in abalones subjected to red LED irradiation and melatonin treatment. The results revealed that at high water temperatures (25 °C), the mRNA expression levels of the superoxide dismutase (SOD) and glutathione peroxidase (GPx) genes and the antioxidant activity of SOD decreased in abalones in the red-LED irradiated and melatonin-treated groups compared with those in abalones in the control group. Although high water temperatures induced DNA damage in the abalone samples, the degree of apoptosis was lower in the red-LED irradiated and melatonin-treated groups than in the control group. Overall, the abalones in the melatonin-treated and red-LED irradiated groups showed reduced oxidative stress and increased antioxidant enzyme levels under thermal stress compared with those in the control group. Therefore, red LED irradiation is a promising alternative to melatonin treatment, which is difficult to administer continuously for a long time, for protecting abalones from oxidative stress.

Acute toxicity of chlorpyrifos to some non-target freshwater organisms: which one is more toxic-technical grade or commercial formulation?

Chlorpyrifos is among the most widely sold organophosphates in the agriculture sector worldwide. Static bioassays were performed in the laboratory to compare the acute toxicity between the technical grade (94% a.i.) and commercial formulation (20% EC) of chlorpyrifos to four freshwater organisms: the crustacean zooplankton Cyclops viridis, the oligochaete worm Branchiura sowerbyi, the gastropod Pila globosa, and tadpole larvae of Duttaphrynus melanostictus. The recovery of actual chlorpyrifos concentrations in water after 2 h of exposure to the nominal concentrations ranged from 82.98% to 88.56%. The commercial formulation (F) of chlorpyrifos was found to be 1.94 to 2.76 times more toxic than the technical grade (T). Based on 96 h LC50 values of T and F chlorpyrifos, C. viridis was found to be most sensitive (0.56 and 0.25 µg/L) and P. globosa as most tolerant (1482 and 536 µg/L) to chlorpyrifos. Changes in LC50 values of both T and F chlorpyrifos were noted in respect of exposure hours for the three aquatic invertebrates and the tadpole larvae of the toad. In conclusion, the acute toxicity of chlorpyrifos to some non-target freshwater organisms differs between technical grade and commercial formulations.

Screening and functional verification of the target protein of pedunsaponin A in the killing of Pomacea canaliculata.

Previous study found that pedunsaponin A (PA) influenced the cytoskeleton of Pomacea canaliculata hemocytes, leading to depolarization and haemocyte destruction and eventually to snail death. In this study, we analysed the changes in protein expression by iTRAQ-mediated proteomics and identified 51 downregulated proteins. Among these, we focused on proteins related to cytoskeletal function and identified neural Wiskott-Aldrich syndrome isoform X1 (PcnWAS). The full-length PcnWAS gene contains 9791 bp and includes an open reading frame of 1401 bp that encodes 735 amino acids with a predicted molecular mass of 49.83 kD. PcnWAS exhibited a relatively distant genetic relationship with known species; the closest homologue is Biomphalaria glabrata (57%). RNA interference (RNAi) was adopted to verify the function of PcnWAS after screening the siRNA sequence with an efficiency of 97%. Interference with the gene expression of PcnWAS did not lead to snail death, but the depolarization level increased, which demonstrated that PcnWAS is an important depolarization-related protein. The results of PA treatment of snails subjected to RNAi proved that interfering with PcnWAS gene expression decreased the molluscicidal activity of PA toward P. canaliculata; snail mortality after RNAi was significantly lower (40%) than that in PA-treated snails without RNAi (54%), while the survival rate and depolarization level in haemocytes were not significant, indicating that PcnWAS is only one of the important target proteins of PA in P. canaliculata. This study lays the foundation for further exploration of the molecular mechanism by which PA kills this harmful snail.

Effects of Bacillus lincheniformis feeding frequency on the growth, digestion and immunity of Haliotis discus hannai.

To study the effects of Bacillus lincheniformis feeding frequency on the survival and growth of Haliotis discus hannai abalone, we measured the expression levels of nonspecific immune genes and monitored the anti-Vibrio parahaemolyticus immune reaction. H. discus hannai (shell length: 32.75 ± 2.63 mm, body weight: 4.91 ± 0.34 g) was selected to perform a 70 d laboratory culture experiment including a 14 d V. parahaemolyticus artificial infection experiment. The control group (C) was fed normal commercial feed every day. The M1 experimental group was given experimental feed and basal feed on alternating days until the end of the experiment. The M2 experimental group was given experimental feed for 4 d and basal feed for 3 d, and this cycle was repeated every 7 d until the end of the experiment. The M3 experimental group was given experimental feed for 2 d and basal feed for 5 d, and this cycle was repeated every 7 d until the end of the experiment. The M4 group was continuously given experimental feed for the duration of the experiment. The concentration of added B. lincheniformis in each experimental group was 105 cfu/g (according to the quantity of viable bacteria). The specific growth rate (as measured by body weight) and the feed conversion efficiency of the abalone in M1 and M2 were significantly higher than those in M4 and C (P < 0.05). The cellulose and lipase activities of abalone in M1, M2 or M4 were significantly higher than those in M3 or C (P < 0.05). The acid phosphatase, superoxide dismutase, total haemocyte counts, O2- levels generated by respiratory bursts, and the expression levels of Mn-SOD, TPx, GSTs and GSTm in abalone in the M2 group were significantly higher than those in any other feeding frequency group (P < 0.05). At the end of the V. parahaemolyticus infection, the cumulative mortality of the abalone in M2 was significantly lower than that in any other group (P < 0.05). Consequently, given the growth advantages and the enhancement of immune function, the feeding plan in which B. lincheniformis was applied for 4 d per week, and basal feed was then applied for 3 d, did not lead to a high level of immune reaction, immune fatigue or waste of resources, but increased the growth rate of individuals and their resistance to V. parahaemolyticus infection.

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.

Eobania vermiculata as a potential indicator of nitrate contamination in soil.

The effects of nitrates were analysed on the land snail Eobania vermiculata, a good bioindicator to assess the effects of certain pollutants in soil. It is known that the molluscs are very sensitive to contamination substances and can be used as sentinel organism for environmental pollution assessment. The nitrates are present in fertilizers and in food additives and their excess can not only be harmful to the environment but also dangerous for the humans. Indeed, in the mammals the nitrates are converted into nitrites and can cause a series of complications as the formation of methaemoglobin and cancers. In this study, adult organisms of E. vermiculata were exposed to soil containing 2000 mg/L of nitrates for 30 days to evaluate the stool microbiome and the histological changes at the level of the foot. Eggs of these snails were similarly treated to observe their hatching, survival and development. Histological changes were observed at level of the foot of adult snails exposed to nitrate and in their stools was evident an increase of bacteria, especially those that have a high ability to exploit nitrates and nitrogen as nutrients. Instead, the treated eggs showed changes in hatching, hypopigmentation of newborn snails and a decrease of their survival in time. The overall information obtained from these endpoints can provide important information regarding the quality of the environment. In addition, they also showed that the invertebrate organism E. vermiculata despite being a simple organism is very useful and efficient for ecotoxicological studies.

Effects of waterborne exposure to cadmium on biochemical responses in the freshwater gastropod, Bellamya aeruginosa.

The biochemical responses of Bellamya aeruginosa as a dominant and widespread freshwater gastropod throughout China to waterborne cadmium (Cd) were investigated to explore the impacts of exposure concentration and duration in this potential sentinel species. After the 7 days' test of dosage-mortality relationship, gastropods were exposed for either 7 days at the LC50 (1.7 mg/L), the LC10 (0.7 mg/L) and 0.02 mg/L Cd, or 28 days at 0.02 mg/L Cd. A suite of biochemical indicators including metallothionein-like protein (MTLP), reduced glutathione (GSH), catalase (CAT), contents of tissue metal (Cd, Fe, Mn, Cu, Zn), and the compartments of these metals bound to MTLP were examined. The treatment of 0.02 mg/L Cd led to the increase of Cd bound to MTLP (Cd-MTLP) levels, the decrease of GSH content, and the upregulation of CAT activity, but no induction of MTLP, indicating that the intrinsic MTLP and GSH worked together for the detoxification of Cd at the low exposure. When the exposure concentration increased, GSH was depleted severely and synthesis of MTLP was triggered, leading to a strong and significant relationship between MTLP level and Cd accumulation. At the lethal concentrations (1.7 mg/L), both MTLP induction and CAT activity were inhibited while the proportion of Cd-MTLP to total Cd were increased, suggesting more intrinsic MTLP were utilized to sequester free Cd ions. Therefore, the content of Cd-MTLP in digestive glands of B. aeruginosa was recommended as a reliable biomarker for Cd contamination.

Environmental behavior of and gastropod biomarker response to trace metals from a backwater area of Xian'nv lake.

Combined with sediment pollutant analysis, the gastropod Cipangopaludina cahayensis was chosen as an indicator organism to evaluate the environmental behavior of trace metals and the aquatic ecological risk that they present in a backwater area of Xian'nv Lake. Based on hydrological characteristics, 24 sampling sites representing the main stream (MS), tributaries (TR), lake area (LA) and lake tributaries (LT) were collected. The results revealed that cadmium (Cd) was the main pollutant and that it significantly accumulated in sediments of the research area. Based on the pollutant concentrations, the degree of Cd pollution was ranked in the following order: LA > MS > TR > LT. Several intersections between the rivers and Xian'nv Lake, including LA1, LA7 and LA 10, were observed to have higher Cd deposition. There was a significant difference in the spatial distribution of pollutants, which resulted in a higher accumulation of trace metals in the backwater area and its tributary. The Cd content in the visceral sac of C. cahayensis was positively correlated with the concentration of heavy metals in the sediment. The response of multiple antioxidant biomarkers, including superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST), as well as the glutathione (GSH) content and the level of by-products of lipid peroxidation (TBARS), in C. cahayensis revealed a potential relationship to the environmental behavior of the pollutants. By combining the different biomarkers responses, the integrated biomarker response index (IBR) corresponded well with the pollution distribution characteristics in different areas.

Cadmium Uptake, MT Gene Activation and Structure of Large-Sized Multi-Domain Metallothioneins in the Terrestrial Door Snail Alinda biplicata (Gastropoda, Clausiliidae).

Terrestrial snails (Gastropoda) possess Cd-selective metallothioneins (CdMTs) that inactivate Cd2+ with high affinity. Most of these MTs are small Cysteine-rich proteins that bind 6 Cd2+ equivalents within two distinct metal-binding domains, with a binding stoichiometry of 3 Cd2+ ions per domain. Recently, unusually large, so-called multi-domain MTs (md-MTs) were discovered in the terrestrial door snail Alinda biplicata (A.b.). The aim of this study is to evaluate the ability of A.b. to cope with Cd stress and the potential involvement of md-MTs in its detoxification. Snails were exposed to increasing Cd concentrations, and Cd-tissue concentrations were quantified. The gene structure of two md-MTs (9md-MT and 10md-MT) was characterized, and the impact of Cd exposure on MT gene transcription was quantified via qRT PCR. A.b. efficiently accumulates Cd at moderately elevated concentrations in the feed, but avoids food uptake at excessively high Cd levels. The structure and expression of the long md-MT genes of A.b. were characterized. Although both genes are intronless, they are still transcribed, being significantly upregulated upon Cd exposure. Overall, our results contribute new knowledge regarding the metal handling of Alinda biplicata in particular, and the potential role of md-MTs in Cd detoxification of terrestrial snails, in general.

Imposex Incidence in Gastropod Species from Santa Marta Coastal Zone, Colombian Caribbean Sea.

Imposex is a phenomenon widely associated with environmental exposure to organotin compounds which were quite common components of antifouling paints applied on boats and ship hulls. Here we study the incidence of imposex in neogastropods and its relation with water quality and maritime traffic in the coastal strip of Santa Marta, Colombia. Imposex was determined via specialized indexes and related to the organisms' size, somatic conditions, variables of water quality and maritime traffic, in a space-time assessment. There was evidence of imposex in five species Plicopurpura patula, Vasula deltoidea, Stramonita haemastoma, S. floridana, and Gemophos auritulus. Purpura patula and Vasula deltoidea species were found in all sampling sites. The results have proved that imposex is highly influenced by the maritime traffic variable, with greater prevalence during the dry season, and with P. patula being more sensitive than V. deltoidea.

The similarity of crawling mechanisms in aquatic and terrestrial gastropods.

Crawling gastropods are unique models for studying the functioning of smooth muscles and ciliated epithelia, since they cover the foot sole and are involved in locomotion, allowing for direct investigation. Two types of crawling are known: creeping by muscular waves in terrestrial gastropods such as Helix and сiliary gliding in aquatic gastropods such as Lymnaea. It was found that the smooth muscles that underlie the ciliated epithelium in Lymnaea are involved in gliding and contribute significantly to fast crawling. Thus, the locomotor apparatus is fundamentally the same in both snails and the difference between crawling reflects an adaptation to a habitat. The control of crawling speed is also the same. Tonic contraction, relaxation, and rhythmic contractions are involved in this control. During a locomotor episode, the sole length and crawling speed spontaneously change and directly correlate with each other via the contraction force of the muscle cells in the locomotory waves. Dopamine, unlike ergometrine, decreases the sole length and crawling speed. Serotonin stimulates, increases crawling and determines the number of muscle cells involved in the locomotory waves for each locomotor episode. This control (taking into account heterogeneity) apparently might exist in any other phasic smooth muscle, including vertebrates.

Mannan oligosaccharide increases the growth performance, immunity and resistance capability against Vibro Parahemolyticus in juvenile abalone Haliotis discus hannai Ino.

This trial was conducted to investigate the effect of mannose oligosaccharides (MOS) on the growth performance, antioxidation, immunity and disease resistance of Vibro Parahemolyticus in juvenile abalone Haliotis discus hannai Ino. Four formulated diets were produced to contain 0.00 g/kg, 0.40 g/kg, 0.80 g/kg and 1.60 g/kg Actigen®, with functional ingredients of MOS, respectively. Accordingly, the experimental diets were named as A0, A4, A8 and A16. After 120-days feeding trial, the best growth performance was observed in A8 group (P < 0.05) and there was no significant difference in A0, A4 and A16 groups. With the increase of dietary MOS, the activity of the total antioxidant capacity in hepatopancreas is increasingly elevated (P < 0.05) while no significant difference was observed on activity of glutathione S-transferase (P > 0.05). The activities of superoxide dismutase and glutathione peroxidase were firstly increased and then decreased, with the highest values in A8 group (P < 0.05). Immune-related parameters were significantly affected by dietary MOS inclusion. Specifically, the activities of alkaline phosphatase and acid phosphatase in hepatopancreas and serum of abalone fed diets containing MOS were significantly higher than those of control A0 group (P < 0.05). Moreover, the highest values of both enzymes were observed in hepatopancreas of A8 group but in serum of A16 group, respectively. The lysozyme activities in hepatopancreas and serum of A4 group were significantly higher than those of other groups (P < 0.05) and there was no significant difference in A0, A8 and A16 groups (P > 0.05). The activities of cytophagy and respiratory burst in serum of abalone were not significantly affected by dietary MOS content (P > 0.05). The mRNA levels of focal adhesion kinase and integrin-linked kinase were gradually elevated with the increase of dietary MOS, with the highest value recorded in A16 group (P < 0.05). The gene expression of caspse-3 in A8 group was dramatically higher than those of other groups (P < 0.05) and there was no significant difference in A0, A4 and A16 groups (P > 0.05). The mRNA level of nuclear factor-κB was not significantly affected by dietary MOS (P > 0.05). During 56 h of V. Parahemolyticus challenge period, the accumulated mortality rate of abalone fed diets containing MOS were significantly lower than that of control A0 group in each time point (P < 0.05). Overall, the lowest rate was happened in A8 group (P < 0.05). In conclusion, MOS inclusion in diet has obviously positive effect on growth, immunity and disease resistance capability of abalone, with the optimal level of Actigen® at 0.80 g/kg in diet.

Gastropods and Insects Prefer Different Solanum dulcamara Chemotypes.

Solanum dulcamara (Bittersweet nightshade) shows significant intraspecific variation in glycoalkaloid (GA) composition and concentration. We previously showed that constitutive differences in overall GA levels are correlated with feeding preference of the grey field slug (GFS; Deroceras reticulatum). One particularly preferred accession, ZD11, contained low GA levels, but high levels of previously unknown structurally related uronic acid conjugated compounds (UACs). Here we test whether different slug species as well as insect herbivores show similar feeding preferences among six S. dulcamara accessions with different GA chemotypes. In addition, we investigate whether slug feeding can lead to induced changes in the chemical composition and affect later arriving herbivores. A leaf disc assay using greenhouse-grown plants showed that three slug species similarly preferred accessions with low GA levels. Untargeted metabolomic analyses showed that previous slug feeding consistently increased the levels of N-caffeoyl-putrescine and a structurally related metabolite, but not the levels of GAs and UACs. Slug-induced responses only affected slug preference in one accession. A common garden experiment using the same six accessions revealed that ZD11 received the highest natural gastropod feeding damage, but suffered the lowest damage by specialist flea beetles. The latter preferred to feed on accessions with high GA levels. Our study indicates that different selection pressures imposed by generalist gastropods and specialist insects may explain part of the observed chemical diversity in S. dulcamara.

Protection of dietary selenium-enriched seaweed Gracilaria lemaneiformis against cadmium toxicity to abalone Haliotis discus hannai.

Seaweed Gracilaria lemaneiformis is the main dietary source of the abalone mariculture industry in China. In this study, we examined the protection of selenium (Se)-enriched G. lemaneiformis against cadmium (Cd) toxicity in the abalone, Haliotis discus hannai, using various indices including metal concentration (Se and Cd), growth rate, GPx enzymatic antioxidants, and metallothionein (MT) concentration over a period of 28 days of exposure. The growth rates and Se contents increased significantly in abalones fed with Se-enriched G. lemaneiformis, while the toxicity of Cd was reduced. Seven to 12 days Cd exposure to the Se-enriched G. lemaneiformis not only affected GPx activity but the MT levels fluctuated irregularly. MT concentrations increased after 3 days exposure and then gradually decreased to the control level after Day 7. There were statistically significant positive correlations between MT levels, GPx activity and Se concentrations, and negative relationships between MT levels, GPx activity and Cd levels in abalones. These findings suggest that Se-enriched Gracilaria protects abalone against Cd toxicity. The possible mechanism is the induction of MT with a concomitant increased capacity of GPx enzymatic antioxidants.

Effect of the earthquake-tsunami (Chile, 2010) on toxic metal content in the Chilean abalone mollusc Concholepas concholepas.

Toxic metal content were measured in samples of mollusc Concholepas concholepas obtained from the Chilean coast. Samples were collected during two periods, one before and one after the earthquake-tsunami which occurred in the Maule Region, Chile, February 27th, 2010 as a result of an earthquake with a magnitude of 8.8. Quantification of toxic metals in samples of hepatopancreas and muscle tissue was performed using flame atomic absorption spectroscopy. The analytical methodology was validated with certified reference material. The content means measured in muscle tissue for January 2010 were Cu: 10.3; Cr: 0.7; Cd: < 0.1; Mn: 3.6 and Zn: 38.6 µg g-1 (dry weight). For October 2014, the means were Cu: 8; Cr: 2.4; Cd: < 0.1; Mn: 5.6 and Zn: 45.4 µg g-1 (dry weight). In hepatopancreas tissue, the content means were Cu: 14.8; Cr: 2.4; Cd: 246.2; Mn: 4.4 and Zn: 1552.9 µg g-1 (dry weight). For October 2014, the means were Cu: 53.7; Cr: 3.5; Cd: 118; Mn: 13.4 and Zn: 1352.3 µg g-1 (dry weight). Cd, Cr, Cu, Mn and Zn content in the samples of hepatopancreas were generally higher than those recorded in muscle tissue however they were not always statistically significant. Statistical analysis showed that Cu and Mn content in the post-tsunami period increased in the hepatopancreas tissue. The concentrations of Cd, Cr, Cu, Mn, and Zn measured in the muscular tissue (edible part) of the species Conchalepas concholepas, were lower than the maximum contents allowed by the current legislation (FAO/WHO, 2004; EU, 2001) and its consumption is not a risk to human health.

Aging and retinoid X receptor agonists on masculinization of female Pomacea canaliculata, with a critical appraisal of imposex evaluation in the Ampullariidae.

Ampullariidae are unique among gastropods in that females normally show a primordium of the copulatory apparatus (CApp). The aims of this study were (a) to quantitatively evaluate the development and growth of the female CApp with age; (b) to compare the effects of RXR and PPARγ agonists in adult females of known age and (c) to explore the effect of masculinizing RXR agonists on the expression of RXR in the CApp. It was found that the CApp grows and develops with age. A significant increase in penile sheath length (PsL) and also in a developmental index (DI) was observed in 7-8 months old females, as compared with 4-5 months old ones. A reported endogenous agonist of RXR, 9-cis retinoic acid (9cis-RA), as well as two organotin compounds, tributyltin (TBT) and triphenyltin (TPT) which have been also reported to bind to RXR, were injected and its masculinizing effects were measured. Also, the effect of a PPARγ agonist, rosiglitazone, was studied. All studied RXR agonists, but not the PPARγ agonist, were effective in increasing PsL, penile length (PL) and DI. Finally, the expression of the RXR in the CApp was studied (Western blot) in control, TBT, TPT, and 9cis-RA treated females. A significantly increased expression of RXR was only observed after 9cis-RA treatment. It is concluded that (a) development and growth of the CApp is significantly affected by female age; (b) reported RXR agonists, but not a PPARγ agonist, cause female masculinization of young females. An appraisal of previous studies of female masculinization in the Ampullariidae has also been made and it is emphasized that the masculinizing effect of aging should be considered, particularly when interpreting field data.

Laboratory-to-field extrapolation: Increase in carbamazepine toxicity in a higher tier, multiple-stress experiment.

The toxicity and environmental risk of chemicals, such as the antiepileptic drug carbamazepine (CBZ), is commonly assessed using standardized laboratory tests and laboratory-to-field extrapolation. To investigate the toxicity of CBZ to aquatic key organisms in a more complex and environmentally relevant scenario, we conducted a 32-day multiple-stress experiment in artificial indoor streams. We exposed the non-biting midge Chironomus riparius, the blackworm Lumbriculus variegatus, and the New Zealand mud snail Potamopyrgus antipodarum to 80 and 400 µg CBZ/L in six artificial indoor streams. In addition to hydraulic stress, species' interaction, and low organic content in the sediment, organisms were co-exposed to the herbicide terbutryn (TBY) as a second chemical stressor at a concentration of 6 µg/L. The exposure to CBZ under multiple stress conditions resulted in a 10- to more than 25-fold higher toxicity in C. riparius and P. antipodarum when compared to a previous, standardized laboratory experiment. The co-exposure to TBY enhanced the adverse effects of CBZ on snails (reduced production of embryos). This effect was additive as the single exposure to TBY also reduced the reproduction of snails, most likely through the reduction of biofilm biomass. The emergence of C. riparius declined at a CBZ concentration of 400 µg/L (without the co-exposure to TBY) and at 80 µg/L in combination with TBY. The difference in sensitivity between laboratory and indoor stream experiments is indicative of a potential underestimation of risk when toxicity data are extrapolated to field conditions. The present results suggest the inclusion of non-chemical and chemical stressors in environmental hazard and risk assessments.

Ecotoxicological characterization of the antiepileptic drug carbamazepine using eight aquatic species: baseline study for future higher tier tests.

Ecotoxicological effects of the antiepileptic drug carbamazepine (CBZ) were investigated in one primary producer (Desmodesmus subspicatus) and seven invertebrate species (Daphnia magna, Daphnia pulex, Ceriodaphnia dubia, Gammarus fossarum, Potamopyrgus antipodarum, Lumbriculus variegatus and Chironomus riparius) using OECD and US EPA guidelines for chronic toxicity testing. The present data set was used to conduct a hazard assessment for CBZ including confirmatory data. While most of our results were in accordance with previous studies, published effect data for C. dubia and D. pulex could not be confirmed, even though they have previously been considered to be the most sensitive invertebrate species to CBZ exposure. The non-biting midge, C. riparius, was the most sensitive test organism in the present study. From an EC10 of 406 µg/L and a no observed effect concentration (NOEC) of 400 µg/L, a predicted no effect concentration (PNEC) of 8 µg/L was calculated. With regard to realistic predicted and measured environmental concentrations, the environmental risk can be considered as low for CBZ when the assessment is based on laboratory-based effect data. To conduct a refined and more realistic assessment, this study provides foundational data for two future, higher tier studies: one multiple-stressor experiment and one mesocosm study.

Assessment of reproductive disorder (imposex) induced by tributyltins in marine gastropods.

Imposex is a genital disorder characterized by imposition of male sexual characteristics in female gastropods due to exposure to tributyltin (TBT). TBT is used as biocidal agent in antifouling paints, applied on the ship hulls and marine submerged structures such as fishing gears and buoys. In the present study bioassay experiment was carried out to determine imposex inductive and endocrine disruptive effect of TBT in two species of gastropods of genus Thais. In this experiment normal specimens of T. bufo and T. rudolphi were exposed to three different concentrations (100, 500 and 1000ngl-1) of TBTCl for four weeks in laboratory and at the end of experiment level of free testosterone and TBT body burden was estimated by radioimmunoassay and gas chromatograph coupled with a flame photometric detector respectively. In both tested species exposed to 500 and 1000ngl-1 of TBT imposex stages developed, while in 100ng l-1 and control groups showed no imposex condition. Elevation of free testosterone level in imposex females has also been observed. These observations indicate that the TBT act as potential imposex inducer and endocrine disruptor in the targeted gastropod species and these species can be used as sensitive biomonitoring tool for TBT contamination.

The effects of feeding Lactobacillus pentosus on growth, immunity, and disease resistance in Haliotis discus hannai Ino.

To study the effects of probiotic-added food on the survival and growth of abalone (Haliotis discus hannai Ino), the expression levels of nonspecific immune genes and the anti-Vibrio parahaemolyticus infection were examined. During an 8-week culturing experiment in an indoor aquarium and a 2-week V. parahaemolyticus artificial infection experiment, the control group was fed with untreated food once a day, while the experimental groups (L1, L2 and L3) were fed with Lactobacillus pentosus added food. The concentration of probiotics in the experimental food was 103 cfu/g (L1), 105 cfu/g (L2) and 107 cfu/g (L3), respectively. The results showed that the survival rate, shell length-specific growth rate, and the food conversion rate (FCR) of abalones in L1 and L2 were significantly higher than the control group. The food intake of abalones in L3 was significantly lower than that in L1, L2 and the control group, but there was no significant difference in FCR identified between L1, L2 and L3. In the L. pentosus-added groups, the total number of blood lymphocytes, lysozyme activity, acid phosphatase, superoxide dismutase, and expression levels of Mn-superoxide dismutase (Mn-SOD) and thioredoxin peroxidase (TPx) were significantly higher than the control group, while the malondialdehyde (MDA) content was significantly lower than the control group. The phagocytic activity of blood lymphocytes, catalase activity and the expression levels of heat shock protein 70 (HSP70) of abalones in the control group were significantly lower than that in L1 and L2, but there was no significant difference when compared with L3. The levels of O2-, NO produced by respiratory burst of blood lymphocytes and the expression levels of catalase (CAT) in L1 and L2 were significantly higher than both L3 and the control group. Seven days after infection with V. parahaemolyticus, all abalones in the control group were dead. After 14 days the cumulative mortality rate of abalones in the L. pentosus-added groups was significantly lower than that in the control group. Therefore, the 103 cfu/g and 105 cfu/g L. pentosus-added food not only promoted food intake and growth of abalones, but also improved their non-specific immunity and reduced V. parahaemolyticus infection, indicating that this strain is a good potential candidate for probiotic added food in the aquaculture industry.