Effects of arsenic on gut microbiota and its bioaccumulation and biotransformation in freshwater invertebrate.

This study aimed to investigate the impact of arsenic stress on the gut microbiota of a freshwater invertebrate, specifically the apple snail (Pomacea canaliculata), and elucidate its potential role in arsenic bioaccumulation and biotransformation. Waterborne arsenic exposure experiments were conducted to characterize the snail's gut microbiomes. The results indicate that low concentration of arsenic increased the abundance of gut bacteria, while high concentration decreased it. The dominant bacterial phyla in the snail were Proteobacteria, Firmicutes, Bacteroidota, and Actinobacteriota. In vitro analyses confirmed the critical involvement of the gut microbiota in arsenic bioaccumulation and biotransformation. To further validate the functionality of the gut microbiota in vivo, antibiotic treatment was administered to eliminate the gut microbiota in the snails, followed by exposure to waterborne arsenic. The results demonstrated that antibiotic treatment reduced the total arsenic content and the proportion of arsenobetaine in the snail's body. Moreover, the utilization of physiologically based pharmacokinetic modeling provided a deeper understanding of the processes of bioaccumulation, metabolism, and distribution. In conclusion, our research highlights the adaptive response of gut microbiota to arsenic stress and provides valuable insights into their potential role in the bioaccumulation and biotransformation of arsenic in host organisms. ENVIRONMENTAL IMPLICATION: Arsenic, a widely distributed and carcinogenic metalloid, with significant implications for its toxicity to both humans and aquatic organisms. The present study aimed to investigate the effects of As on gut microbiota and its bioaccumulation and biotransformation in freshwater invertebrates. These results help us to understand the mechanism of gut microbiota in aquatic invertebrates responding to As stress and the role of gut microbiota in As bioaccumulation and biotransformation.

Repurposing the drug, amprolium as a novel molluscicide against the land snail (Eobania vermiculata).

Amprolium (AMP) is an organic compound used as a poultry anticoccidiostat. The aim of this work is to repurpose AMP to control the land snail, Eobania vermiculata in the laboratory and in the field. When snails treated with ½ LC50 of AMP, the levels of alkaline phosphatase (ALP), total lipids (TL), urea, creatinine, malondialdehyde (MDA), catalase (CAT), and nitric oxide (NO) were significantly increased, whereas the levels of acetylcholinesterase (AChE), total protein (TP), and glutathione (GSH) decreased. It also induced histopathological and ultrastructural changes in the digestive gland, hermaphrodite gland, kidney, mucus gland, and cerebral ganglion. Furthermore, scanning electron micrographs revealed various damages in the tegumental structures of the mantle-foot region of E. vermiculata snails. The field application demonstrated that the AMP spray caused reduced percentages in snail population of 75 and 84% after 7 and 14 days of treatment. In conclusion, because AMP disrupts the biology and physiology of the land snail, E. vermiculata, it can be used as an effective molluscicide.

Aluminum exposure alters the pedal mucous secretions of the chocolate-band snail, Eobania vermiculata (Gastropoda: Helicidae).

Aluminum (Al) is used in everyday life and present in food drugs, packaging, industry, and agriculture. Although it is the most common metal in the Earth crust, a correlation has been demonstrated between its presence and various pathologies, even serious ones, especially of a neurological type. However, there is a histological gap regarding the role Al can have in contact with the covering and secreting epithelia. The alterations of the ventral and dorsal foot mucocytes and their secretions of the snail Eobania vermiculata caused by Al were investigated in situ by histochemical and lectin-histochemical techniques. Administration to different experimental groups took place for 3 and 9 days with 50 and 200 µM of AlCl3. Several types of mucocytes were detected with a prevalent secretion of acid glycans in the foot of E. vermiculata. Sulfated glycans prevail in the dorsal region, with one type showing only fucosylated residues and another also having galactosaminylated and glycosaminylated residues. Carboxylated glycans prevail in the ventral region, with presence of galactosaminylated, glycosaminylated, and fucosylated residuals in both cells. Snails treated presented a general decrease of mucin amount in the secreting cells and affected the mucus composition. These changes could alter the rheological and functional properties of the mucus with possible implications for the health of the treated animals. RESEARCH HIGHLIGHTS: Snails were fed with Al-contaminated lettuce at different concentrations. In the foot mucocytes produced mucus with prevailing acidic glycans. In the treated resulted a reduction in the amount of mucus and an alteration of glycan composition.

Multi-stressor Effects of Ultraviolet Light, Temperature, and Salinity on Louisiana Sweet Crude Oil Toxicity in Larval Estuarine Organisms.

When oil is spilled into the environment its toxicity is affected by abiotic conditions. The cumulative and interactive stressors of chemical contaminants and environmental factors are especially relevant in estuaries where tidal fluctuations cause wide variability in salinity, temperature, and ultraviolet (UV) light penetration, which is an important modifying factor for polycyclic aromatic hydrocarbon (PAH) toxicity. Characterizing the interactions of multiple stressors on oil toxicity will improve prediction of environmental impacts under various spill scenarios. This study examined changes in crude oil toxicity with temperature, salinity, and UV light. Oil exposures included high-energy, water-accommodated fractions (HEWAFs) and thin oil sheens. Larval (24-48 h post hatch) estuarine species representing different trophic levels and habitats were evaluated. Mean 96 h LC50 values for oil prepared as a HEWAF and tested under standard conditions (20 ppt, 25 °C, No-UV) were 62.5 µg/L tPAH50 (mud snails), 198.5 µg/L (grass shrimp), and 774.5 µg/L (sheepshead minnows). Thin oil sheen 96 h LC50 values were 5.3 µg/L tPAH50 (mud snails), 14.7 µg/L (grass shrimp), and 22.0 µg/L (sheepshead minnows) under standard conditions. UV light significantly increased the toxicity of oil in all species tested. Oil toxicity also was greater under elevated temperature and lower salinity. Multi-stressor (oil combined with either increased temperature, decreased salinity, or both) LC50 values were reduced to 3 µg/L tPAH50 for HEWAFs and < 1.0 µg/L tPAH50 for thin oil sheens. Environmental conditions at the time of an oil spill will significantly influence oil toxicity and organismal response and should be taken into consideration in toxicity testing and oil spill damage assessments.

Responsiveness of metallothionein and hemocyanin genes to cadmium and copper exposure in the garden snail Cornu aspersum.

Terrestrial gastropods express metal-selective metallothioneins (MTs) by which they handle metal ions such as Zn2+ , Cd2+ , and Cu+ /Cu2+ through separate metabolic pathways. At the same time, they depend on the availability of sufficient amounts of Cu as an essential constituent of their respiratory protein, hemocyanin (Hc). It was, therefore, suggested that in snails Cu-dependent MT and Hc pathways might be metabolically connected. In fact, the Cu-specific snail MT (CuMT) is exclusively expressed in rhogocytes, a particular molluscan cell type present in the hemocoel and connective tissues. Snail rhogocytes are also the sites of Hc synthesis. In the present study, possible interactions between the metal-regulatory and detoxifying activity of MTs and the Cu demand of Hc isoforms was explored in the edible snail Cornu aspersum, one of the most common European helicid land snails. This species possesses CdMT and CuMT isoforms involved in metal-selective physiological tasks. In addition, C. aspersum expresses three different Hc isoforms (CaH ɑD, CaH ɑN, CaH ß). We have examined the effect of Cd2+ and Cu2+ exposure on metal accumulation in the midgut gland and mantle of C. aspersum, testing the impact of these metals on transcriptional upregulation of CdMT, CuMT, and the three Hc genes in the two organs. We found that the CuMT and CaH ɑD genes exhibit an organ-specific transcriptional upregulation in the midgut gland of Cu-exposed snails. These results are discussed in view of possible interrelationships between the metal-selective activity of snail MT isoforms and the synthesis and metabolism of Hc isoforms.

[Effects of copper and cadmium ions on protective enzyme activity in Oncomelania hupensis].

OBJECTIVE: To evaluate the effects of Cu2+ and Cd2+ at different concentrations on superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activity in Oncomelania hupensis. METHODS: Cu2+- and Cd2+-containing solutions were prepared at 7 concentrations, and O. hupensis snails were exposed to the solutions for 24 h, of 15 snails in each concentration. Then, the snail body was collected following removal of the snail shell and homogenated, and the SOD, CAT and POS activities were detected in the supernatants. RESULTS: With the increase of the Cu2+ concentration, the SOD activity appeared a rise followed by a reduction in O. hupensis snails, and the CTA activity appeared a decline-rise-decline tendency, while the POD activity showed a tendency towards rise followed by decline. With the increase of the Cd2+ concentration, the SOD activity appeared a rise followed by a reduction in O. hupensis snails, and the CTA activity appeared a decline- rise- decline tendency, while the POD activity showed a decline-rise-decline tendency. CONCLUSIONS: Exposure to Cu2+ and Cd2+ at high concentrations results in a decline in the activity of SOD, CAT and POD in O. hupensis at the same time.

Ecotoxicology and environmental safety toxicity of pyrethroid cypermethrin on the freshwater snail Chilina parchappii: Lethal and sublethal effects.

The aim of the present work was to study the effect of the pyrethroid cypermethrin (CYP) on the non-target freshwater snail Chilina parchappi. Initially, the sensitivity of adult snails to CYP was evaluated via the 96-h LC50 test. Then, snails were exposed to subtethal CYP concentrations (0.1 and 10 mg/l) for 1, 4 and 10 days and the digestive glands were dissected for biomarkers analyses. Enzymatic activity of catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST), as well as total glutathione reduced (GSH) levels, were determined. Histological analyses of morphology, intracellular accumulation of lipofucsins and neutral lipids accumulation in the digestive gland were also evaluated. As compared to other molluscs, C. parchappi showed high resistance to CYP exposure evidenced by the 96-h LC50 value (44.59 mg/l). Snails exposed to sublethal CYP concentrations showed a statistically significant increase (p < 0.01) in GST (79-116%) and GPx (45-190%) activities with respect to controls. However, CAT activity showed a tendency to decrease with CYP treatment but was not statistically significantly different compared to control. Only high CYP concentration caused a statistically significant increase (p < 0.01) in GSH content (95-196%). There was evidence of structural changes in the digestive gland of snails exposed to CYP, showing a dose-dependent response. In exposed snails, some of the main symptoms included a reduction in the thickness of the epithelium, vacuolisation of the digestive cells and an increase in the number of excretory cells. Accumulation of lipofuscins (933-1006%) and neutral lipids (403%) were statistically significantly higher (p < 0.05) in snails exposed to CYP compared to control. This study showed that C. parchappii is quite tolerant to CYP exposure and that at sublethal concentrations, GSH metabolism could play a protective role against the pesticide harm in snails. Therefore, it would be interesting to study the response of this organism to other environmental stressors to assess its potential use in monitoring programs.

In vivo evaluation of oxidative stress and biochemical alteration as biomarkers in glass clover snail, Monacha cartusiana exposed to zinc oxide nanoparticles.

Oxidative stress is considered a main commonly reported mechanism of nanoparticles toxicity, so this study aimed to evaluate oxidative stress and biochemical alterations in the haemolymph and digestive gland of snail, Monacha cartusiana exposed to sublethal concentrations of zinc oxide nanoparticles (ZnONPs) for 14 days (d). The results indicated that, ZnONPs induced significant increases in lipid peroxidation (LPO) and lactate dehydrogenase (LDH) in treated animals and did not return to normal levels after recover period. A significant decline of glutathione peroxidase (GPx), glutathione-S-transferase (GST) activities, and glutathione (GSH) content in the haemolymph and digestive gland of snails was observed when compared with control. A significant increase was observed in catalase (CAT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activities of treated animals. In general, nano-materials are able to induce oxidative stress in exposed animals. The present findings indicate that, alterations of antioxidant enzyme activities, increase of LPO, LDH, and reducing of GSH content and GST, GPx activities are recognized to oxidative stress and cell damage. This species could be considered a good bioindicator to assess nano-materials exposure.

Egypt's schistosomiasis control programme in the 1980s prepared the ground for the global elimination of schistosomiasis by 2030.

A paper published in 1990 by Webbe and El Hag described the progress being made in the control of schistosomiasis with the introduction of a new medicine 'Praziquantel' to treat infected individuals, and the use of a molluscicide 'niclosamide', with which to kill the intermediate host snails. Almost 30 years from the date of publication, the situation in Egypt and indeed globally has changed out of all recognition. Almost every country in Africa has a school health programme to deworm children, each country has a national plan which includes school health, and praziquantel is now donated through the World Health Organization for countries to treat school aged children to protect them from the serious consequences of chronic schistosomiasis. As the socio-economic status in Africa improves the chances seem good for elimination of schistosomiasis as a public health problem in most areas in Africa.

Ecotoxicological biomarkers as investigating tools to evaluate the impact of acrylamide on Theba pisana snails.

Acrylamide (ACR) is a widespread industrial chemical with recognized adverse effects not only to humans but to other organisms in the environment as well. In the present study, the ecotoxicological effects of dietary exposure to sublethal concentration (1/20 LC50) of ACR on the land snail, Theba pisana after 2 weeks of exposure and 1-week recovery with respect to oxidative stress parameters; lipid peroxidation (LPO), reduced glutathione (GSH), catalase (CAT), and glutathione-S-transferase (GST), cytogenetic parameter; deoxyribonucleic acid (DNA) content, as well as immunological parameters; cell death, phagocytosis, lysosomal membrane stability (LMS), lectins, superoxide anion (O2-) generation, phenoloxidase (PO), peroxidase (POD), and hemocyanin (Hc) were examined. The results showed that ACR significantly increased LPO level and the activity of CAT and GST, cell death, and Hc level, whereas a significant decline in DNA and GSH contents, phagocytic activity, LMS, lectins, O2- generation, POD, and PO activities compared to the controls after 2-week exposure was observed. After 1-week recovery, most of the tested parameters in exposed snails were permanent and not reversible to the control levels. This study suggests that the tested multiple parameters of T. pisana species may be used as biomarkers of ACR exposure. Besides, T. pisana snails could be used as a good sentinel organism for ACR exposure in pollution monitoring studies.

Sub-lethal effects of dimethoate alone and in combination with cadmium on biochemical parameters in freshwater snail, Galba truncatula.

The objective of the present study was to evaluate the influence of the organophosphorus pesticide dimethoate and cadmium on biochemical parameters of a freshwater snail, Galba truncatula, in laboratory conditions. In 14 days, snails were exposed to 0, 100, 200, and 400 µg L-1 of dimethoate and 0.0 and 1000 µg L-1 of cadmium chloride. The results evidenced that dimethoate induces oxidative stress and alters biochemical parameters in freshwater snails. Cadmium also induced significant changes in biochemical parameters. The combination of the dimethoate and cadmium markedly increased the effects on G. truncatula. Dimethoate and cadmium mixture caused a decrease in Acetylcholinesterase (AChE), and Glucose-6-phosphate dehydrogenase (G6PDH) activities, glycogen, and total antioxidant capacity (TAN) levels, and increased aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), glutathione peroxidase (GPx), and catalase (CAT) activities, and malondialdehyde (MDA) level in exposed snails. The increased toxicity and bioaccumulation of cadmium after the exposure to the highest concentration of dimethoate indicates a synergistic effect leading to a reduced performance of the detoxification system in the snail. Dimethoate contributed to the bioconcentration of cadmium in snails and increased its toxic effects evidenced in biomarkers of oxidative stress and cell damage.

Use of multiple endpoints to investigate the ecotoxicological effects of abamectin and thiamethoxam on Theba pisana snails.

The ecotoxicological effects of dietary exposure to sublethal concentration (1/20 LC50) of abamectin (ABM) and thiamethoxam (TMX) for two weeks exposure and one week recovery on oxidative stress parameters; lipid peroxidation (LPO), reduced glutathione (GSH), catalase (CAT) and glutathione S-transferase (GST), Deoxyribonucleic acid (DNA) damage as well as immunological parameters; cell death, phagocytosis, lysosomal membrane stability (LMS), lectins, superoxide anion (O2-) generation, phenoloxidase (PO), peroxidase (POD) and hemocyanin (Hc) of the land snail, Theba pisana were examined. The results showed that both tested compounds significantly increased DNA damage, LPO content, the activity of CAT and GST, cell death, POD activity, Hc level, whereas, significantly declined in phagocytic activity, LMS, lectins level, O2- generation, PO activity, and GSH content compared to the controls after two week exposure. After one week of recovery, the measured biochemical parameters of treated snails were slightly repaired but their levels were still less than that of the level of untreated animals. Overall, based on acute toxicity endpoints, ABM appeared to be more harmful than TMX against this animal. Indeed, the different patterns of endpoint responses could represent a useful picture to characterise exposure to these pesticides in the land snail, T. pisana. This battery of snail endpoints might be a promising option to biomonitor the health of the terrestrial ecosystem and to offer valuable insights to the pesticides toxicity mechanisms.

Short-term effects of very low dose cadmium feeding on copper, manganese and iron homeostasis: A gastropod perspective.

The available information on the interplay between low-dose cadmium intake and copper, manganese, and iron homeostasis in invertebrates is limited. We have currently studied the accumulation of these trace metals in the hepatopancreas of adult snails, Cantareus aspersus, following 14 and 28 days of exposure to low doses of dietary cadmium, up to 1 mg/kg dw (dry weight). The cadmium dose, but not the duration of exposure, had a significant effect on hepatopancreas copper deposition, the values being significantly elevated compared to controls. A significant peak in manganese levels at 14 days was found in snails administered the lowest cadmium dose. These increases occurred even in the absence of cadmium increase in the hepatopancreas. Our data suggest that low dose cadmium feeding can produce a transient disturbance in hepatopancreas copper and manganese homeostasis. Such responses may serve as early biomarkers of physiological changes occurring during the initial stages of cadmium intoxication.

Effects on tadpole snail gene expression after exposure to vinclozolin.

The fungicide vinclozolin (Vz) is an endocrine disruptor with known anti-androgenic activity in vertebrates. However, there is a lack of information about the Vz mode of action in invertebrates, although some studies have shown that this compound can produce alterations in different species. Transcriptional activity was analyzed in the freshwater snail Physella acuta in order to elucidate putative cellular processes altered by this chemical during a response. In order to identify potential molecular biomarkers, a de novo transcriptome was generated for this species that constitutes a valuable source for future studies. This data, together with some already available data, permitted the identification of several genes related to detoxification mechanisms (Cyp2u1, Cyp3a7, Cyp4f22, GSTo1, GSTt2, and MRP1), stress response (Hsp20.4, Hsp17, Hsp16.6, and Cu,Zn-SOD), the hormonal system (Estrogen Receptor and Hsp90), apoptosis (Casp3), and copper homeostasis (ATOX1). Using quantitative Real-Time polymerase chain reaction, mRNA levels of these genes were examined in snails exposed to 20 or 200 µg/L Vz for 24 h. The results showed an overall weak response, with downregulation of Hsp20.4 and no statistically significant change for the other genes. These findings suggest that P. acuta can manage the concentrations of Vz found in the environment with no relevant activation of the pathways analyzed, although additional studies are needed for longer exposure times and including other metabolic pathways. The new genes described open the range of processes that can be studied at the molecular level in toxicity tests.

Acute waterborne cadmium toxicity in the estuarine pulmonate mud snail, Amphibola crenata.

Freshwater pulmonate snails are sensitive to trace metals, but to date, the sensitivity of estuarine pulmonate snails to these important environmental toxicants is undescribed. Using the estuarine mud snail Amphibola crenata, effects of a 48-h exposure to waterborne cadmium (Cd) were investigated. The 48-h median lethal concentration (LC50) was 50.4 mg L-1, a value higher than that previously reported for any gastropod mollusc. Cadmium levels in the tissues of mud snails were highest in the viscera (digestive gland and gonad), with the foot muscle and remaining tissue compartment (kidney, mantle, remaining digestive tissues and heart) displaying significantly lower concentrations. Over a Cd exposure concentration range of 0-32 mg L-1, Amphibola exhibited reduced oxygen consumption and elevated ammonia excretion in response to increasing Cd, the latter effect likely reflecting a switch to protein metabolism. This finding was supported by a declining oxygen: nitrogen ratio (O:N) as exposure Cd concentration increased. Other energy imbalances were noted, with a decrease in tissue glycogen (an effect strongly correlated with Cd burden in the viscera and foot muscle) and an elevated haemolymph glucose observed. An increase in catalase activity in the visceral tissues was recorded, suggestive of an effect of Cd on oxidative stress. The magnitude of this effect was correlated with tissue Cd burden. The induction of antioxidant defence mechanisms likely prevented an increase in levels of lipid peroxidation, which were unchanged relative to Cd exposure concentration in all measured tissues.

Biochemical and molecular alterations in freshwater mollusks as biomarkers for petroleum product, domestic heating oil.

To investigate the effect one of the oil products, domestic heating oil (DHO), on freshwater mollusks, Unio tigridis and Viviparous bengalensis were exposed to three DHO concentrations for each species (5.8, 8.7, and 17.4 ml L-1 for mussels; 6.5, 9.7, and 19.5 mlL-1 for snails, respectively). Antioxidant enzymes (superoxide dismutase, catalase), malondialdehyde, acetylcholinesterase and DNA damage in both species tissues were monitored over 21 days. The results showed that both antioxidant enzymes concentration (SOD and CAT) increased in the lowest DHO concentrations (5.8, and 8.7 ml L-1), and then decreased in the highest concentration (17.4 ml L-1) as the same pattern for Unio tigridis, but this not occurred for Viviparous bengalensis. MDA values recorded significantly increased compared to control. No reduction was observed in AChE concentrations in soft tissues of both mollusks may due to that DHO was a non-neurotoxicant to Unio tigridis and Viviparous bengalensis. The results of DNA damage parameters were showed significant differences (p≤ 0.05) between control and DHO concentrations except lowest concentration for each parameter measured in digestive gland of Unio tigridis. As well as, these significant differences were recorded between control and three concentrations of DHO exposure for comet length, and tail length parameters, and between control and highest oil concentration for tail moment in Viviparous bengalensis. DHO has the ability to prevent the reproduction of Viviparous bengalensis snail relation to control, that is what we considered strong evidence of the toxicity properties of DHO on the reproductive status of this species of snails. SOD, CAT, and MDA were useful biomarkers for evaluating the toxicity of DHO in mussel and snails, and comet assay was a good tool to assess the potential genotoxicity of DHO.

Cadmium bioaccumulation and antioxidant enzyme activity in hepatopancreas, kidney, and stomach of invasive apple snail Pomacea canaliculata.

The acute toxicity of Cd was tested, and metal bioaccumulation in tissue was determined for the alien invasive species Pomacea canaliculata and its native competitor Sinotaia quadrata under experimental conditions. The invasive species was more tolerant to Cd toxicity than native species, for which the LC50 values were 4.26, 2.24, and 1.98 mg/L at exposure times of 48, 72, and 96 h, respectively, approximately three times higher than those of the native snails. The viscera accumulated the highest Cd concentration, followed by the foot and shell in both species. Metal concentrations in the above three tissues of P. canaliculata were much higher than those of S. quadrata, irrespective of Cd dose and exposure time. For P. canaliculata, the highest concentration of metal was further observed in the hepatopancreas (0.64-3.98 mg/g) followed by the kidney (0.067-3.78 mg/g), with lowest levels in the stomach (0.062-1.53 mg/g). Among the five antioxidant enzymes, the most responsive enzymes were CAT, ALP, and GST in the hepatopancreas; CAT, POD, and GST in the kidney; and POD in the stomach of exposed animals. These results, demonstrating a high Cd tolerance, may partly explain the ability of P. canaliculata to displace S. quadrata in Cd-contaminated habitat. The Cd was accumulated mainly in the hepatopancreas and kidney of invasive species, which changed the activity of antioxidant enzymes allowing the animals to cope with the toxicity. Graphical abstract Cadmium bioaccumulation and antioxidant enzyme activity in the invasive Pomacea canaliculata.

Feeding Behavior of an Aquatic Snail as a Simple Endpoint to Assess the Exposure to Cadmium.

One of the aims of ecotoxicology is the assessment of the effects of chemicals on the ecosystems. Bioassays assessing lethality are frequently used in ecotoxicology, however they usually employ supra-environmental toxic concentrations. Toxicity tests employing behavioral endpoints may present a balance between simplicity (i.e., laboratory bioassays) and complexity (i.e., relevant ecological effects). The aim of this study was to develop a feeding behavioral bioassay with the aquatic snail, Potamopyrgus antipodarum, which included a 2 days exposure to cadmium, followed by a 9 days post-exposure observational period. Several behavioral feeding endpoints were monitored, including percentage of actively feeding animals, percentage of animals in food quadrants and a mobility index. The percentage of actively feeding animals was reduced by the four cadmium treatments (0.009, 0.026, 0.091 and 0.230 mg Cd/L) with the stronger effect in the highest concentration. The two highest cadmium concentrations significantly reduced the percentage of animals in food quadrants and the mobility index. Therefore, the percentage of actively feeding animals was the most sensitive endpoint to cadmium toxicity as the four cadmium concentrations caused a significant decrease in this endpoint. It is concluded that feeding behavior is a useful endpoint to detect the exposure of aquatic snails to cadmium.

Comparative Toxicity of Two Chemical Dispersants and Dispersed Oil in Estuarine Organisms.

Chemical dispersants can be a useful tool to mitigate oil spills. This study examined potential risks to sensitive estuarine species by comparing the toxicity of two dispersants (Corexit® EC9500A and Finasol® OSR 52) individually and in chemically enhanced water-accommodated fractions (CEWAFs) of Louisiana Sweet Crude oil. Acute toxicity thresholds and sublethal biomarker responses were determined in seven species (sheepshead minnow, grass shrimp, mysid, amphipod, polychaete, hard clam, mud snail). Comparing median lethal (LC50) values for the dispersants, Finasol was generally more toxic than Corexit and had greater sublethal toxicity (impaired embryonic hatching, increased lipid peroxidation, decreased acetylcholinesterase activity). The nominal concentration-based mean LC50 for all species tested with Corexit was 150.31 mg/L compared with 43.27 mg/L with Finasol. Comparing the toxicity of the CEWAFs using the nominal concentrations (% CEWAF), Corexit-CEWAFs appeared more toxic than Finasol-CEWAFs; however, when LC50 values were calculated using measured hydrocarbon concentrations, the Finasol-CEWAFs were more toxic. There was greater dispersion efficiency leading to greater hydrocarbon concentrations measured in the Corexit-CEWAF solutions than in equivalent Finasol-CEWAF solutions. The measured concentration-based mean LC50 values for all species tested with Corexit-CEWAF were 261.96 mg/L total extractable hydrocarbons (TEH) and 2.95 mg/L total polycyclic aromatic hydrocarbons (PAH), whereas the mean LC50 values for all species tested with Finasol-CEWAF were 23.19 mg/L TEH and 0.49 mg/L total PAH. Larval life stages were generally more sensitive to dispersants and dispersed oil than adult life stages within a species. These results will help to inform management decisions regarding the use of oil-spill dispersants.

High-level dietary cadmium exposure is associated with global DNA hypermethylation in the gastropod hepatopancreas.

5-methylcytosine (5mC) is a key epigenetic mark which influences gene expression and phenotype. In vertebrates, this epigenetic mark is sensitive to Cd exposure, but there is no information linking such an event with changes in global 5mC levels in terrestrial gastropods despite their importance as excellentecotoxicological bioindicators of metal contamination. Therefore, we first evaluated total 5mC content in DNA of the hepatopancreas of adult Cantareus aspersus with the aim to determine whether this epigenetic mark is responsive to Cd exposure. The experiment was conducted under laboratory conditions and involved a continuous exposure, multiple dose- and time-point (14, 28, and 56 days) study design. Hepatopancreas cadmium levels were measured using Flame Atomic Absorption Spectrometry and the percentage of 5-mC in samples using an ELISA-based colorimetric assay. Snail death rates were also assessed. Our results, for the first time, reveal the presence of 5mC in C. aspersus and provide evidence for Cd-induced changes in global 5mC levels in DNA of gastropods and mollusks. Although less sensitive than tissue accumulation, DNA methylation levels responded in a dose- and time-dependent manner to dietary cadmium, with exposure dose having a much stronger effect than exposure duration. An obvious trend of increasing 5mC levels was observed starting at 28 days of exposure to the second highest dose and this trend persisted at the two highest treatments for close to one month, when the experiment was terminated after 56 days. Moreover, a strong association was identified between Cd concentrations in the hepatopancreas and DNA methylation levels in this organ. These data indicate an overall trend towards DNA hypermethylation with elevated Cd exposure. No consistent lethal effect was observed, irrespective of time point and Cd-dosage. Overall, our findings suggest that the total 5mC content in DNA of the hepatopancreas of land snails is responsive to sublethal Cd exposure and give new insights into invertebrate environmental epigenetics.