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The pearl oyster Pinctada imbricata radiata (Leach, 1814), from the Pacific Ocean, was one of the first species to reach via Suez the Mediterranean, colonizing the eastern basin and recently spreading to the western. The species showed to be able to adapt to a wide range of climatic, hydrological, and ecological conditions. Since 2000 it reached the Strait of Messina, where is now infesting the transitional waters of the oriented natural reserve "Laguna di Capo Peloro." Due to such resistance and adaptation ability, various assays were performed. Haemocyte morpho-functional aspects were evaluated in haemolymph samples fixed with 1% and 2% glutaraldehyde for optical and electron microscopy (TEM). The following assays were carried out: cell characterization using several dyes, detection of intra- and extracellular lipids, the capability of phagocytosis using the yeast Saccharomyces cerevisiae and to produce superoxide anion (O 2- ). Detection of several enzymes, such as acid and alkaline phosphatase, arylsulfatase, chloro-acetylesterase and β-glucuronidase was also assessed. Cell count was demonstrated to be abundant with a mean of 8.263 × 106 mm 2  ± 0.935 × 106 (SD). Two main cell populations were noticed: granulocytes and hyalocytes, both competent for phagocytosis, to produce O 2- , and characterized by lipids. Based on the granule analysis, enzymatic activity was also demonstrated. The observations under TEM confirmed all the results obtained. This study supports the hypothesis that P. imbricata radiata can be usefully employed as a model organism in environmental biomonitoring. Moreover, since the species represent potential threats to native species and ecosystems, further insights into its biological adaptations in invaded ecosystems are recommended., (© 2022 Wiley Periodicals LLC.)

In the framework of investigations aimed to detect new available bioindicators in marine environment, haemolymph cells and ctenidia of the Mediterranean spiny oyster, Spondylus gaederopus, have been investigated. Haemocyte count and characterisation, phagocytosis and superoxide anion production and enzyme activity assays, have been carried out. TEM observations have been performed. After gross anatomy observations, cito-histological determinations have been carried out, especially focused on ctenidia structure and function. Main results concerned the relatively low number of circulating cells, and the rich in granules granulocytes, most of which were lysosomes. Release of lysosomal enzymes was confirmed a shared trait inside bivalves. Glycogen deposits as probable result of conversion of bacteria carbohydrates, have been detected, as well as the occurrence of both acidophilic and basophilic haemocytes. Phagocytosis, both in granulocytes and agranulocytes, has been recorded, together with the production of superoxide anion. Haemocytes were found positive to acid phosphatase, alkaline phosphatase, β-glucuronidase, chloroacetylesterase and arylsulphatase. Ctenidia showed a complex organization, including two demibranch to each ctenidium, two different kinds of lamellae filament and specialized structures as ciliated disks connecting filaments in "eutherorhabdic ctenidia". The occurrence of three different types of mucous cells in the same region of ordinary filaments has been underlined. Such features, suggesting high resistance to environmental stress and disease, allow to consider spiny oysters as promising bioindicators, although deserving of further investigations to evaluate the physiological responses to stress in controlled conditions. Present data, moreover, providing basic information on the biology of S. gaederopus, notably implement the present knowledge on the Mediterranean spiny oysters, whose under-evaluated ecological role should be carefully considered., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Continuous release of nanoparticles (NPs) into marine coastal environments results in an increased risk of exposure to complex NP mixtures for marine organisms. However, to date, the information on the effects at molecular and biochemical levels induced by the exposure to NPs, singly and as a mixture, is still scant. The present work aimed at exploring the independent and combined effects and the mechanism(s) of action induced by 7-days exposure to 1 μg/L nZnO, 1 μg/L nTiO 2 and 1 μg/L FC 60 fullerene in the Manila clam Ruditapes philippinarum, using a battery of immunological and oxidative stress biomarkers in haemolymph, gills and digestive gland. In addition, proteomics analyses were performed in gills and the digestive gland, where NP bioaccumulation was also assessed. Increased bioaccumulation of single NPs and the mixture was linked with increased oxidative stress and higher damage to proteins, lipids and DNA in all tissues analysed. The proteomics approach highlighted protein modulation in terms of abundance and damage (higher redox-thiol and carbonylated groups content). In particular, the modulated proteins (16 in gills and 18 in digestive gland) were mostly related to cytoskeleton and energetic metabolism. The digestive gland was the tissue more affected. For all biomarkers measured, increased detrimental effects were observed in the mixture compared to single NP exposures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Pendimethalin (PND) is a dinitroaniline preemergent herbicide widely used to control grasses and weeds. The present study aimed to evaluate the PND potential effects on the development of zebrafish early-life stages. The research focuses first on acute toxicity, followed by the integration of toxicity results through histopathology, oxidative status, and neurotoxicity evaluation of sublethal and environmentally relevant concentrations. Zebrafish larvae exposed to PND showed mortality and developed sublethal alterations including impaired fin development, lordosis, scoliosis, blood congestion, impaired blood flow, and reduced heartbeat. PND exposure (0.5 mg/L) affects musculoskeletal development leading to delayed and reduced ossification of the vertebral centra in the developing vertebral column and disruption of muscle morphology. Herbicide exposure (0.5 mg/L and 0.05 mg/L) led also to biochemical changes of antioxidant enzymes, increasing the activity of CAT, GR, and GPx, while no effects were observed on the activity of SOD and GST in zebrafish larvae. Lastly, AChE activity, a biochemical marker of neurotoxicity, was also increased in zebrafish larvae exposed to 0.5 mg/L of PND. These results confirm the developmental toxicity of PND in zebrafish early-life stages, pointing out the potential role of oxidative stress in the onset of sublethal alterations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Thiacloprid is a neonicotinoid insecticide widely exploited in agriculture and easily mobilized towards aquatic environments by atmospheric agents. However, little information about its toxicological effects on aquatic invertebrate bioindicators is available. In this study, specimens of the mussel Mytilus galloprovincialis were exposed to thiacloprid at environmental (4.5 μg L -1 ) and 100 times higher than environmental (450 μg L -1 ) concentrations for 20 days. Thiacloprid affected haemolymph biochemical parameters, cell viability in the digestive gland, antioxidant biomarkers and lipid peroxidation in the digestive gland and gills at environmentally relevant concentrations (4.5 μg L -1 ). In addition, thiacloprid exposure caused histological damage to the digestive gland and gills. Interestingly, the pesticide was detected at levels equal to 0.14 ng g -1 in the soft tissues of sentinels exposed for 20 days to 450 μg L -1 thiacloprid in seawaterμ. Due to its harmful potential and cumulative effects after long-term exposure of M. galloprovincialis, thiacloprid may pose a potential risk to nontarget aquatic organisms, as well as to human health. This aspect requires further in-depth investigation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Alternative chemicals to per- and poly-fluoroalkyl substances have recently been introduced in various industrial processes. C6O4 (difluoro{[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetic acid) is a new surfactant and emulsifier used as a replacement for perfluorooctanoic acid (PFOA). From an ecotoxicological point of view, in vitro assays are useful tools for assessing the negative effects and understanding the mechanisms of action of chemicals at the cellular level. Here, we present the results of an in vitro study in which the effects of C6O4 were evaluated-for the first time-on haemocytes of the clam Ruditapes philippinarum . Cells were exposed to three concentrations of C6O4 (0.05, 0.5, 5 μg/mL) and the effects on haemocyte viability, haemocyte morphology, differential haemocyte count, lysosomal membrane stability, superoxide anion production, acid phosphatase, and β-glucuronidase activities, as well as on the percentage of micronuclei and chromosomal aberrations were evaluated. The results demonstrated that C6O4 significantly affected haemocyte morphology, lysosomal membrane stability, hydrolytic enzyme activity, and superoxide anion production, and promoted chromosomal aberrations. To the best of our knowledge, this is the first study revealing the in vitro effects of C6O4, a substitute for PFOA, on haemocytes from a bivalve species.

There is growing concern for the wide use ofperfluorooctanoic acid (PFOA) because of its toxic effects on the environment and on human health. A new compound - the so called C6O4 (perfluoro ([5-methoxy-1,3-dioxolan-4-yl]oxy) acetic acid) - was recently introduced as one of the alternative to traditional PFOA, however this was done without any scientific evidence of the effects of C6O4 when dispersed into the environment. Recently, the Regional Agency for the Protection of the Environment of Veneto (Italy) detected high levels of C6O4 in groundwater and in the Po river, increasing the alarm for the potential effects of this chemical into the natural environment. The present study investigates for the first time the effects of C6O4 on the Manila clam Ruditapes philippinarum exposed to environmental realistic concentrations of C6O4 (0.1 µg/L and 1 µg/L) for 7 and 21 days. Furthermore, in order to better understand if C6O4 is a valid and less hazardous alternative to its substitute, microbial and transcriptomic alterations were also investigated in clams exposed to 1 µg/L ofPFOA. Results indicate that C6O4 may cause significant perturbations to the digestive gland microbiota, likely determining the impairment of host physiological homeostasis. Despite chemical analyses suggest a 5 times lower accumulation potential of C604 as compared to PFOA in clam soft tissues, transcriptional analyses reveal several alterations of gene expression profile. A large part of the altered pathways, including immune response, apoptosis regulation, nervous system development, lipid metabolism and cell membrane is the same in C6O4 and PFOA exposed clams. In addition, clams exposed to C6O4 showed dose-dependent responses as well as possible narcotic or neurotoxic effects and reduced activation of genes involved in xenobiotic metabolism. Overall, the present study suggests that the potential risks for marine organism following environmental contamination are not reduced by replacing PFOA with C6O4. In addition, the detection of both C6O4 and PFOA into tissues of clams inhabiting the Lagoon of Venice - where there are no point sources of either compounds - recommends a similar capacity to spread throughout the environment. These results prompt the urgent need to re-evaluate the use of C6O4 as it may represent not only an environmental hazard but also a potential risk for human health., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Fullerene C 60 (FC 60 ), with its unique physical properties, has been used in many applications in recent decades. The increased likelihood of direct release into the environment has raised interest in understanding the biological effects of FC 60 to aquatic organisms. Nowadays, only few studies have analysed FC 60 effects and bioaccumulation in marine organisms following in vivo exposure. To provide new data about FC 60 toxicity, Ruditapes philippinarum was selected as target species to assess potential adverse effects of the contaminant. Clams were exposed for 1, 3 and 7 days to predicted environmental concentrations of FC 60 (1 and 10 μg/L) and cellular and biochemical responses were evaluated in clams' gills, digestive gland and haemolymph. The FC 60 content in gills and digestive gland was determined in all experimental conditions after 7 days of exposure. Results showed an increase in oxidative stress. In particular, a significant modulation in antioxidant enzyme activities, and changes in glutathione S-transferase activity were observed in gills. Moreover, damage to lipids and proteins was detected in FC 60 -treated (10 µg/L) clams. In digestive gland, slighter variations in antioxidant enzyme activities and damage to molecules were detected. CAT activity was significantly affected throughout the exposure, whereas damage to lipids was evident only at the end of exposure. FC 60 accumulation was revealed in both gills and digestive gland, with values up to twelve-fold higher in the latter. Interestingly, haemolymph parameters were slightly affected by FC 60 compared to the other tissues investigated. Indeed, only Single Cell Gel Electrophoresis and Neutral Red uptake assays showed increased values in FC 60 -exposed clams. Moreover, volume and diameter of haemocytes, haemocyte proliferation, and micronucleus assay highlighted significant variations in treated clams, but only in the first phases of exposure, and no changes were detected after 7 days. Our results suggested clam gills as the target tissue for FC 60 toxicity under the exposure conditions tested: the high damage detected to lipids and proteins could contribute to long-term problems for the organism., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Seawater pH lowering, known as ocean acidification, is considered among the major threats to marine environment. In this study, post-spawning adults of the sea urchin Paracentrotus lividus were maintained at three pH values (8.0, 7.7, 7.4) for 60 days. Physiological, biochemical, cellular, behavioural and reproductive responses were evaluated in males and females. Significant differences between sexes were observed, with higher ammonia excretion and lower catalase activity in males. Respiration rate (after 21 days), catalase activity in gonads and total coelomocyte count showed the same increasing trend in males and females under low pH. Ammonia excretion, gonadosomatic index and lysozyme activity exhibited opposite responses to low pH, with an increasing trend in males and decreasing in females. Results demonstrated that exposure to low pH could result in different response strategies of male and female sea urchins at a physiological, biochemical and immunological level. Reduced female gonadosomatic index under low pH suggested decreased energy investment in reproduction.

Pesticides can enter aquatic environments potentially affecting non-target organisms. Unfortunately, the effects of such substances are still poorly understood. This study investigated the effects of the active neonicotinoid substance thiacloprid (TH) and the commercial product Calypso 480 SC (CA) (active compound 40.4% TH) on Mytilus galloprovincialis after short-term exposure to sublethal concentrations. Mussels were tested for seven days to 0, 1, 5 and 10 mg L -1 TH and 0, 10, 50 and 100 mg L -1 CA. For this purpose, several parameters, such as cell viability of haemocytes and digestive cells, biochemical haemolymph features, superoxide dismutase (SOD) and catalase (CAT) enzymatic activity of gills and digestive gland, as well as histology of such tissues were analysed. The sublethal concentrations of both substances lead to abatement or completely stopping the byssal fibres creation. Biochemical analysis of haemolymph showed significant changes (P < 0.01) in electrolytes ions (Cl - , K + , Na + , Ca 2+ , S-phosphor), lactate dehydrogenase (LDH) enzyme activity and glucose concentration following exposure to both substances. The TH-exposed mussels showed significant imbalance (P < 0.05) in CAT activity in digestive gland and gills. CA caused significant decrease (P < 0.05) in SOD activity in gills and in CAT activity in both tissues. Results of histological analyses showed severe damage in both digestive gland and gills in a time- and concentration-dependent manner. This study provides useful information about the acute toxicity of a neonicotinoid compound and a commercial insecticide on mussels. Nevertheless, considering that neonicotinoids are still widely used and that mussels are very important species for marine environment and human consumption, further researches are needed to better comprehend the potential risk posed by such compounds to aquatic non-target species., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Glyphosate-based herbicides (GBHs) occur in aquatic ecosystems at concentrations of hundreds of micrograms per liter. As formulation adjuvants are suspected to be endocrine-disrupting chemicals, we assessed the effects of the recent GBH formulation Roundup® Power 2.0 on vitellogenin (VTG) in Mytilus galloprovincialis. Mussels were exposed for 7, 14, and 21 days to two concentrations of the commercial formulation, corresponding to 100 and 1000 μg/L of glyphosate. The expression of the vtg gene in gonads of females and males, as well as the levels of alkali labile phosphates (ALP) in gonads and non-gonadal tissues from the two sexes were measured. No significant alterations were observed in vtg expression values during the exposure. Conversely, a significant reduction in gonadal ALP levels was observed in females exposed for 21 days and in males exposed for 7 days. In addition, ALP levels increased significantly in gonads from males exposed for 21 days to the two concentrations of Roundup®. As for non-gonadal tissues, ALP levels did not change significantly in females, whereas ALP levels decreased significantly in non-gonadal tissues from males exposed for 21 days to the lowest concentration tested. An overall statistically significant difference in ALP levels was found between females and males. Although preliminary, our study suggests that GBH can affect reproduction-related parameters in mussels.

Assessment of the effects of chemical mixtures is a very important objective of the ecotoxicological risk assessment. This study was aimed at evaluating for the first time the effects of a mixture of glyphosate and its main breakdown product aminomethylphosphonic acid (AMPA) on various biomarkers in the mussel Mytilus galloprovincialis. Mussels were exposed for 7, 14 and 21 days to either 100 µg/L of glyphosate, 100 µg/L of AMPA or a mixture of both (100 + 100 µg/L). Various haemocyte parameters, such as total haemocyte counts, haemocyte diameter and volume, haemocyte proliferation, haemolymph lactate dehydrogenase activity and haemocyte lysate acid phosphatase activities were measured. In addition, the effects of exposure on the activity of antioxidant enzymes, acetylcholinesterase and glutathione-S-transferase were evaluated in gills and digestive gland from mussels. On the whole, this study demonstrated that the variables considered in the experimental plan, namely treatment, exposure time and their interaction, affect significantly biomarker responses in M. galloprovincialis. The effects of the mixture were comparable to those of the individual compounds, whereas their synergistic effects were occasionally observed, under the experimental conditions tested at least.

The combined effects of seawater acidification and the non-steroidal anti-inflammatory drug diclofenac on haemocyte parameters of the mussel Mytilus galloprovincialis and the clam Ruditapes philippinarum were investigated for the first time. Animals were maintained for one week (T0) in natural pH condition (8.1) and two reduced pH values (pH -0.4 units and pH -0.7 units). Bivalves were then exposed for additional 14 days (T1 and T2) to the three experimental pH values in both the presence and absence of environmentally realistic concentrations of diclofenac (0.05 and 0.50 μg/L). To assess potential impairment in immunosurveillance, haemocyte parameters (total haemocyte count, haemocyte volume and diameter, Neutral Red uptake, haemocyte proliferation and lysozyme activity) were measured after 7, 14 and 21 days of exposure to differing pH value or pH/diclofenac combinations. In both species, pH affected the whole haemocyte data set at all sampling times, influencing most of the parameters measured at T0 and T1 in clams, and at T2 in mussels. Conversely, in both species diclofenac affected the overall haemocyte response at T2 only. However, in R. philippinarum a higher number of haemocyte parameters were significantly influenced even at T1. A significant interaction between pH and diclofenac was mainly evident in mussels, affecting haemocyte size and lysozyme activity at both T1 and T2. Overall, the results obtained demonstrated that the experimental conditions tested can alter markedly haemocyte parameters in marine bivalves., (Copyright © 2019. Published by Elsevier Inc.)

This study was aimed at evaluating the effects of glyphosate on haemocyte parameters of the clam Ruditapes philippinarum. Clams were exposed for 7 days to differing glyphosate concentrations (10, 100 and 1000 μg/L) and various haemocyte parameters were measured, such as total haemocyte count (THC), haemocyte diameter and volume, haemocyte proliferation, haemolymph lactate dehydrogenase activity, haemocyte lysate lysozyme and acid phosphatase activities. Glyphosate reduced significantly THC values, while increased both diameter and volume of haemocytes. Exposure to the highest herbicide concentration increased significantly haemocyte proliferation. No significant effects on haemolymph lactate dehydrogenase and haemocyte lysate lysozyme activities were observed, whereas haemocyte lysate acid phosphatase activity resulted significantly increased in clams exposed at 100 and 1000 μg/L. On the whole, this study demonstrated that glyphosate influenced significantly haemocyte parameters in R. philippinarum., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The effects of the breakdown products of herbicides on aquatic species are largely unknown. In a recent study, we evaluated the effects of glyphosate on the mussel Mytilus galloprovincialis. This study was designed to evaluate for the first time the impact of aminomethylphosphonic acid (AMPA) - the main breakdown product of glyphosate - on cellular and biochemical parameters of the mussel Mytilus galloprovincialis. Bivalves were exposed for 7, 14 and 21 days to 1, 10 and 100 μg/L of AMPA and various biomarkers were measured in haemolymph (total haemocyte counts, haemocyte diameter and volume, haemolymph pH, haemocyte proliferation, haemolymph lactate dehydrogenase activity, haemocyte lysate lysozyme and acid phosphatase activities), as well as in gills and digestive gland (superoxide dismutase, catalase, glutathione S-transferase and acetylcholinesterase activities). AMPA concentrations in seawater samples from the experimental tanks were also measured in order to correlate the biomarker responses of mussels with their exposure to the actual concentrations of AMPA. The MANOVA analysis demonstrated that the experimental variables considered (exposure dose, exposure duration, and their interaction) affected significantly biomarker responses. Nevertheless, the two-way ANOVA analysis revealed significant effects of AMPA on most of the biomarkers measured. The overall results of this study demonstrated that AMPA can affect cellular and biochemical parameters in mussels, similarly to glyphosate., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Biochemical and cellular responses to low concentrations of TiO 2 nanoparticles (nTiO 2, 1 and 10 μg/L) and bulk (bTiO 2 , 10 μg/L) were evaluated in gills, digestive gland and haemolymph of the clam Ruditapes philippinarum after1, 3 and 7 days' exposure. At 7 days, titanium content was determined in gills and digestive gland. nTiO 2 significantly increased antioxidant enzyme activities in both tissues, and lipid peroxidation in digestive gland at 10 μg/L only, and affected glutathione S-transferase activity. Slighter variations were observed in bTiO 2 -treated clams. A significant Ti bioaccumulation was detected in both gills and digestive gland of 10 μg nTiO 2 /L-exposed clams. In haemolymph, nTiO 2 affected total haemocyte count, haemocyte proliferation, haemocyte diameter and volume, and induced DNA damage. Overall, this study demonstrated that TiO 2 alters most of the biomarkers measured in clams, although responses were differently modulated depending on tissues and exposure conditions, and indicated that nTiO 2 can be accumulated by bivalves, suggesting a potential risk for filter-feeding animals., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In this study, we evaluated the capability of amoxicillin (AMX)-one of the most widely used antibiotics worldwide-to induce oxidative stress in both gills and digestive gland from two bivalve species, the clam Ruditapes philippinarum and the mussel Mytilus galloprovincialis. Superoxide dismutase (SOD) and catalase (CAT) activities, as well as the lipid peroxidation levels (LPO) and protein carbonyl content (PCC), were measured in bivalves exposed to 100, 200 and 400 µg AMX/L for 1, 3 and 7 days. The results obtained demonstrated that AMX affected slightly biomarker responses of molluscs.

Amoxicillin (AMX) is one of the most widely used antibiotics worldwide, and its levels in aquatic ecosystems are expected to be detectable. At present, information concerning the toxic effects of AMX on non-target aquatic organisms, such as bivalves, is scarce. Consequently, in this study, we investigated for the first time the effects of AMX on the haemocyte parameters of two bivalve species, the clam Ruditapes philippinarum and the mussel Mytilus galloprovincialis, which share the same habitat in the Lagoon of Venice, in order to compare the relative sensitivity of the two species. The bivalves were exposed to 100, 200 and 400 μg AMX/L for 1, 3 and 7 days, and the effects on the total haemocyte count (THC), the diameter and volume of the haemocytes, haemocyte proliferation, lactate dehydrogenase (LDH) activity in cell-free haemolymph, the haemolymph pH, and the formation of micronuclei were evaluated. The actual concentrations of AMX in the seawater samples from the experimental tanks were also measured. Overall, the obtained results demonstrated that AMX affected slightly the haemocyte parameters of bivalves. In addition, no clear differences in terms of sensitivity to AMX exposure were recorded between the two bivalve species., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The cuttlefish Sepia officinalis is an important fishery resource in the Northern Adriatic Sea (Italy). During reproduction, fertilised eggs are released by adult females in coastal waters and embryo development can take over two months. During this period, embryos rely on nutrients and other substances, such as immune factors, provided by the female in egg yolk. In cephalopods in general, and specifically in the common cuttlefish, little information is available on yolk biochemical composition and substances included in egg envelopment. In the present study, the main biochemical components of egg yolk and the presence of antimicrobial substances in egg envelopment of S. officinalis were determined for the first time. Statistically significant differences in total egg weight and egg yolk weight were observed among batches from different females. Egg and yolk weights were positively correlated, with yolk representing the 13% (±5%) of the total egg weight. Total proteins were the main biochemical component (46%) of egg yolk, followed by total carbohydrates plus glycogen (39%) and lipids (15%). Statistically significant differences among batches were recorded in egg yolk total protein amounts, lipids, carbohydrates and glycogen, but no correlations were found between egg yolk weight and the biochemical components. The Petri dish and the quantitative spectrophotometric assays revealed the presence of lysozyme-like activity in egg gelatinous envelopment., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The smooth venus clam Callista chione is a commercially exploited bivalve species that lives on the sandy bottom of the Italian coast of the Northern Adriatic Sea. Currently, no information is available in the literature about the haemocytes of this bivalve species. In this study, we performed a morpho-functional characterisation of the haemocytes of C. chione. In freshly collected haemocytes, the total haemocyte count (THC) (measured by a Coulter Counter) varied markedly among individuals, and the mean number of haemocytes was 1.2 (×10(6)) cells mL haemolymph(-1). The mean values for the haemocyte diameter and volume were 4.2 μm and 77.8 fL, respectively. In some cases, higher THC values were related to a smaller haemocyte size, but no correlation was detected between the THC and haemocyte diameter or between THC and cell volume. Conversely, a positive correlation was observed between cell diameter and volume. Two haemocyte types were distinguished by light microscopy: granulocytes (76%), with evident cytoplasmic granules, and hyalinocytes (24%), with a few or no granules. After adhesion to slides and fixation, the cell diameter was approximately 10 μm for granulocytes and 7 μm for hyalinocytes. The granules of the granulocytes were stained in vivo with Neutral Red, indicating that they were lysosomes. The granulocytes and hyalinocytes were further distinguished as basophils and acidophils. Both the granulocytes and the hyalinocytes were able to phagocytise yeast cells. Of 2643 cells that were counted, 2007 (76%) showed phagocytic activity. The granulocytes and hyalinocytes were both positive for some hydrolytic enzymes, whereas they were not positive for peroxidase or phenoloxidase. The two types of haemocytes also produced superoxide anion. Overall, this preliminary study indicates that both the granulocytes and hyalinocytes of C. chione are immune effector cells., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In this study, in vitro effects of ibuprofen (IBU) on the immune parameters of the colonial ascidian Botryllus schlosseri were evaluated. Haemocytes were exposed for 1h to 0 (control), 100 and 1000 μg IBU/L and the effects on haemocyte viability and morphology (shape factor), lysosomal membrane stability (Neutral Red Retention Assay), phagocytic activity, apoptosis (TUNEL reaction), hydrolytic (acid phosphatase) and oxidative (phenoloxidase and peroxidase) enzyme activities were evaluated. The exposure of haemocytes to IBU did not affect significantly their viability, but increased the percentage of cells with round shape. IBU caused a significant reduction in both phagocytic activity and lysosomal membrane stability. The percentage of haemocytes positive to TUNEL reaction (indicative of DNA fragmentation) increased significantly after IBU exposure. Significant decreases in the percentage of haemocytes positive to acid phosphatase were recorded at 1000 μg/L of IBU. Conversely, no significant variations were recorded in the percentage of haemocytes positive to phenoloxidase and peroxidase. Results obtained indicate that exposure of ascidian haemocytes to IBU induces marked alterations in cell functionality. Immunomarkers measured in this study are sensitive, rapid and reproducible. However, their responsiveness and biological relevance will need to be verified for in vivo exposure., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Fluoxetine is an antidepressant used worldwide for the treatment of depression and other psychological disorders. The occurrence of fluoxetine in aquatic environments has been demonstrated. However, there is a lack of information about the effects of fluoxetine on non-target species, such as bivalve molluscs. In the present study, the effects of fluoxetine on the immune parameters of the clam Venerupis philippinarum were evaluated for the first time. Clams were exposed to various sublethal concentrations of fluoxetine (0, 1, 5, 25, 125, 625 μg l⁻¹) for 7 days, and the effects on the total haemocyte count (THC), the diameter and volume of haemocytes, haemocyte proliferation, Neutral Red uptake (NRU), and lysozyme activity in cell-free haemolymph (CFH) were evaluated. In addition, acetylcholinesterase (AChE) activity was measured in clam gills as a biomarker of neurotoxicity. A significant increase in THC values was observed in clams exposed to 25 μg l⁻¹ compared with controls, whereas no significant variations were recorded in either the diameter or the volume of haemocytes. Haemocyte proliferation increased significantly in animals exposed to 25, 125 and 625 μg l⁻¹ compared with controls. NRU decreased significantly in the haemocytes of clams exposed to 1 or 5 μg l⁻¹, whereas NRU returned to control values in clams exposed to the highest fluoxetine concentrations tested (25-625 μg l⁻¹). No significant alterations were observed in CFH lysozyme activity, whereas gill AChE activity decreased significantly in clams exposed to 1 or 5 μg l⁻¹. Overall, the obtained results demonstrated that fluoxetine markedly affected immune parameters and AChE activity in clams., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In this study, the seasonal variations in the morphometric features and in the cellular and biochemical parameters of the haemolymph were investigated in both male and female crabs (Carcinus aestuarii). Crabs were seasonally (November 2010-August 2011) collected from the Lagoon of Venice, and the moult stage, weight, width and length of the carapace, and width and length of the bigger chela were evaluated. In addition, the total haemocyte count (THC), haemocyte diameter and volume, haemolymph glucose and total protein levels, and haemolymph phenoloxidase (PO) and N-acetyl-β-glucosaminidase (NAG) activities were measured. The results demonstrated that the collected crabs were all in the intermoult stage and that the males were bigger than the females. A two-way ANOVA revealed a significant effect of season on the THC and the haemocyte volume and a significant influence of gender on the haemocyte diameter. Season and gender significantly affected the haemolymph glucose concentration, whereas haemolymph protein levels were dependent only on the season. In addition, both season and gender significantly influenced the PO and NAG activities in the haemolymph. Overall, the results demonstrated that crab morphometric features as well as haemolymph cellular and biochemical parameters varied markedly as a function of both season and gender., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Numerous studies have demonstrated that environmental parameters affect bivalve immunomarkers. In the present study, we tested the hypothesis that clams (Venerupis philippinarum) collected in sites with different environmental conditions respond differently to experimental contaminant exposure. Clams were collected at two sites within the Lagoon of Venice that are influenced differently by both anthropogenic impact and natural conditions: Marghera, which is characterised by relatively high contamination levels and restricted clam fishing, and Chioggia, which is inside a licensed clam culture area that is characterised by lower contamination levels. Total haemocyte count, haemocyte diameter and volume, lysozyme activity in both haemocyte lysate and cell-free haemolymph, superoxide dismutase and catalase activities in gills and digestive glands were measured at time 0 (clam sampling time), after 7 days of acclimation in the laboratory and after 1, 3 and 7 days of copper exposure. Interestingly, statistical analyses (three-way ANOVA and Canonical Correlation Analysis) revealed persistent differences in the biological responses of clams from the two sampling sites before and after copper exposure. Conversely, the influence of copper on cellular and biochemical parameters was negligible. Overall, the results obtained indicated that animals with a different ecological history respond differently to experimental contaminant exposure. In addition, this study suggested that immunomarkers and other biomarkers might be used to determine the origin of fishing products., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The effects of ibuprofen (IBU) on the immune parameters of the clam Ruditapes philippinarum were investigated after a 7-day exposure to sublethal IBU concentrations (0, 0 + ethanol, 100, 500, and 1000 μg/L). Total haemocyte count (THC), haemocyte diameter and volume, haemocyte proliferation and uptake of the vital dye Neutral Red (NR) were measured. The cytotoxicity (assessed by the lactate dehydrogenase assay, LDH) and the capability of IBU to induce DNA fragmentation (indicative of apoptosis) were also investigated. The exposure of clams to the highest IBU concentration significantly reduced their THC, whereas no significant changes were observed in either the diameter or volume of haemocytes. Significant increases in haemocyte proliferation were recorded in clams that were exposed to the two highest tested concentrations of IBU. Exposure of clams to 1000 μg IBU/L significantly reduced NR uptake and increased haemolymph LDH activity. Conversely, IBU did not induce DNA fragmentation in haemocytes. Although the IBU concentrations tested in this study were higher than those generally recorded in aquatic environments, results obtained indicate that exposure of clams to IBU induces significant alterations in the immune parameters and suggest potential immunosuppression in treated clams., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

This study presents the first evaluation of the current pollution by polybrominated diphenyl ethers (PBDEs) of surface sediments from the Lagoon of Venice. We focused the research on tri-to hepta-BDEs, the main components of penta- and octa-mixtures, which are considered to be the most toxic for the biocoenosis. The results pointed out a quite homogeneous contamination of this keystone European transitional environment, with ∑ (13)PBDEs values ranging from 0.39 to 6.78 ng/g dry weight; these values reflect low to moderate pollution levels, which is in conformity to other coastal European ecosystems. The average PBDE profile of the lagoon sediments follows this decreasing trend of congeners: BDE-47>BDE-99> >BDE-190>BDE-28>BDE-153>BDE-154>BDE-138, BDE-183, and BDE-17, which is similar to the worldwide distribution pattern. BDE-47 and BDE-99 revealed a recent use of a penta-BDE mixture, while the presence of hepta-BDEs (BDE-183 and BDE-190) in all of the sites can indicate the actual use of a deca-BDE formulation, because these congeners are considered to be debrominated byproducts of BDE-209 degradation.

The effects of 7 days' exposure to differing Triclosan (TCS) concentrations (300, 600, and 900 ng/L) were investigated in the clam Ruditapes philippinarum. Vitellogenin (Vg)-like protein levels in haemolymph and digestive gland from males and females, gill acetylcholinesterase (AChE) activity, superoxide dismutase (SOD) and catalase (CAT) activities in gills and digestive gland, and gill lipid peroxidation (LPO) were measured. The highest TCS concentrations decreased significantly Vg levels in male haemolymph and digestive gland, whereas no significant variations were found in females. The highest TCS concentrations increased significantly SOD activity in gills, but decreased it in digestive gland. No changes in CAT activity were observed. In gills, TCS reduced significantly AChE activity, but it did not induce significant variations in LPO. Our study demonstrates that TCS alters biochemical parameters in R. philippinarum, even at environmentally realistic concentrations, and suggests differing modes of action of the contaminant, in clams at least., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The effects of Triclosan (TCS) on the immune parameters of the clam Ruditapes philippinarum were investigated after a 7-day exposure to sublethal TCS concentrations (300, 600, and 900 ng l(-1)). Hemocytes from controls and exposed clams were collected, and the effects of TCS on total hemocyte count (THC), hemocyte diameter and volume, uptake of the vital dye Neutral Red (an indicator of pinocytosis), lysozyme activity, and proliferation were evaluated. The cytotoxicity, which was evaluated using the lactate dehydrogenase assay, and the capability of TCS to induce DNA fragmentation, which indicated apoptosis, were also investigated. The exposure of clams to all tested TCS concentrations significantly decreased THC, whereas the highest tested TCS concentration significantly reduced the diameter and volume of hemocytes. TCS negatively affected pinocytosis and the proliferation of hemocytes, but it did not alter hemocyte lysozyme activity. TCS exerted cytotoxic effects and induced DNA fragmentation in hemocytes of R. philippinarum. Overall, these results suggest a relationship between TCS exposure and changes in the measured immune parameters and indicate immunosuppression in TCS-treated clams.

The effects of nonylphenol (NP) on functional responses of haemocytes of the colonial ascidian Botryllus schlosseri were evaluated. Haemocytes were exposed to 0.1, 1 and 10 μM NP and the effects on haemocyte viability, adhesion, morphology, lysosomal membrane stability, phagocytic activity and early apoptosis were evaluated. Haemocyte viability and adhesion were not affected by NP. Phagocytic activity and the amoebocytic index decreased significantly at all the concentrations tested, while exposure of haemocytes to 1 and 10 μM NP caused a significant increase in the diffusion of Neutral Red into the cytosol. The percentage of cells positive to Annexin-V (indicative of early apoptosis) increased significantly at 1 and 10 μM NP. Results obtained suggested a relationship between NP and alterations in functional responses of haemocytes in B. schlosseri. Biomarkers measured resulted sensitive, rapid and reproducible, even if their responsiveness will be evaluated after in vivo exposure of animals to NP., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The effects of temperature on cellular and biochemical parameters of the crab Carcinus aestuarii were evaluated. Crabs were kept for 7 days at 4, 17 (reference value) and 30 °C (salinity of 35 psu), and total haemocyte count (THC), haemocyte volume, haemocyte proliferation, phenoloxidase (PO) activity in both haemocyte lysate (HL) and cell-free haemolymph (CFH), CFH total protein and glucose levels, superoxide dismutase (SOD) and catalase (CAT) activities in both gills and digestive gland were evaluated. The lowest and the highest temperature significantly decreased THC, whereas haemocyte volume and haemolymph glucose concentration did not differ significantly among experimental conditions. Haemolymph protein concentration significantly reduced in crabs maintained at 30 °C, when compared with that of animals kept at 4 and 17 °C. Haemocyte proliferation increased significantly in crabs kept at 4 and 30 °C, when compared with that of crabs held at 17 °C. Likewise, a significantly higher PO activity was recorded in CFH from crabs kept at 4 and 30 °C, than in control crabs. Conversely, PO activity did not vary significantly in HL. With regard to antioxidant enzyme activities, a significant decrease in CAT activity was observed in gills from crabs kept at 4 °C, when compared to that of crabs kept at 17 °C and 30 °C. Results obtained demonstrated that the highest and lowest temperature tested influenced crab biological responses, and indicated that C. aestuarii modulated its cellular and biochemical parameters (mainly haemocyte proliferation, CFH protein concentrations and CFH PO activity) in order to cope with temperature., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Crustacea experience periods of starvation during moulting or when limited food availability occurs. The effects of starvation on Crustacea physiological responses have been demonstrated, whereas the effects of starvation on Crustacea immune parameters remain to be more fully studied. In the present study the effects of starvation on immune parameters and antioxidant enzyme activities of the crab Carcinus aestuarii were evaluated for the first time. Treated crabs were starved for 7 days, whereas control crabs were fed daily with mussels. Total haemocyte count (THC), haemocyte diameter and volume, haemocyte proliferation, cell-free haemolymph (CFH) glucose and total protein levels, and phenoloxidase (PO) activity in both haemocyte lysate (HL) and CFH were measured in crabs. In addition, superoxide dismutase (SOD) and catalase (CAT) activities were evaluated in both gills and digestive gland from crabs, in order to evaluate whether starvation induced oxidative stress in C. aestuarii. THC increased significantly in starved crabs, with respect to controls, whereas no significant variations were observed in haemocyte diameter, volume and proliferation. In CFH of starved animals glucose concentration significantly increased, whereas total protein concentration significantly reduced. A significantly higher PO activity was recorded in HL from starved crabs, than in control crabs. Conversely, PO activity did not vary significantly in CFH. Starvation did not cause significant alterations in antioxidant enzyme activities in both gills and digestive gland. Results obtained demonstrated that starvation influenced crab immune parameters, but did not induce oxidative stress. Results also indicated that C. aestuarii can modulate its cellular and biochemical parameters in order to cope with starvation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The combined effects of temperature and salinity on both immune responses and survival in air of the clam, Ruditapes philippinarum, were evaluated for the first time. The animals were kept for 7 days at three differing temperature (5 °C, 15 °C, 30 °C) and salinity values (18 psu, 28 psu, 38 psu), and effects of the resulting 9 experimental conditions on total haemocyte count (THC), Neutral Red uptake (NRU), haemolymph protein concentration, and lysozyme activity in both haemocyte lysate (HL) and cell-free haemolymph (CFH) were evaluated. The survival-in-air test was also performed. Two-way ANOVA analysis revealed that temperature influenced significantly THC and NRU, whereas salinity and temperature/salinity interaction affected NRU only. Temperature and salinity did not influence significantly HL and CFH lysozyme activity, as well as haemolymph total protein content. Survival-in-air test is widely used to evaluate general stress conditions in clams. In the present study, temperature and salinity were shown to influence the resistance to air exposure of R. philippinarum. The highest LT₅₀ (air exposure time resulting in 50% mortality) value was recorded in clams kept at 18 psu and 15 °C, whereas the lowest value was observed in clams kept at 28 psu and 30 °C. Overall, results obtained demonstrated that temperature and salinity can affect some functional responses of haemocytes from R. philippinarum, and suggested a better physiological condition for animals kept at 15 °C temperature and 18 psu salinity., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

For the first time, a functional study of haemocytes from the crab Carcinus aestuarii was performed in order to evaluate their involvement in immune responses. Total haemocyte count (THC), phagocytosis, haemolymph opsonisation properties, hydrolytic and oxidative enzyme activities, and production of intracellular superoxide anion were evaluated. A great variability in THC was recorded among individuals, and haemocyte mean number was 6.4 (x10(6)) cells/ml haemolymph. Although only hyalinocytes were able to phagocytose yeast cells or Zymosan, phagocytic index was low (3%) and did not increase significantly (4%) after pre-incubation of yeast and Zymosan in cell-free haemolymph, suggesting that haemolymph did not have opsonising properties. All haemocyte types produced superoxide anion, whereas only granulocytes were positive to the hydrolytic enzymes assayed. In addition, only granulocytes were positive to phenoloxidase activity. Both Petri dish and spectrophotometric assays revealed a very low lysozyme-like activity in cell-free haemolymph (CFH) and haemocyte lysate (HL), although enzyme activity was higher in CFH than in HL. Interestingly, normalisation of data as to total protein content in CFH and HL resulted in an opposite situation, lysozyme-like activity being higher in HL than in CFH. This demonstrated that haemolymph of C. aestuarii has a high quantity of total proteins, functional properties of which need to be better investigated in future studies. Overall, the results obtained in the present study indicated that C. aestuarii haemocytes are not very active phagocytic cells, but they are more active in terms of both hydrolytic and oxidative enzyme activities and superoxide anion production., (Copyright 2009 Elsevier Ltd. All rights reserved.)

An integrated biological-chemical approach is necessary to evaluate correctly the environmental status of bodies of water, as suggested by the EU Water Framework Directive. The shore crab Carcinus aestuarii, sampled in the Lagoon of Venice (NE Italy), was used as a biomonitor species, and the chemical concentrations of 42 organic pollutants (HCHs, PAHs, PCBs, DDTs, PBDEs), biological responses related to neurotoxicity (AChE inhibition), detoxification mechanisms (CYP450 induction) and endocrine alterations (vitellogenin-like protein induction) were measured at the same time. The responsiveness of biomarkers as predictors (or descriptors) of chemical contamination was evaluated by multivariate regression analysis, revealing good predictor potential for the selected biomarkers. Biomonitoring in the Lagoon of Venice revealed a predominance of DDT and PCB compounds, especially near industrial sites or large cities. Endocrine alterations, not always correlated with the presence of measured compounds, were also detected in many areas, suggesting exposure to compounds able to interfere with the crab endocrine system., (Copyright 2009 Elsevier Ltd. All rights reserved.)

In the present study, three biomarkers, acetylcholinesterase (AChE) activity, vitellogenin (Vg)-like proteins, and ethoxyresorufin O-deethylase (EROD) activity, were evaluated in male Carcinus aestuarii crabs collected from four sites in the Lagoon of Venice, two in the northern lagoon area and two in the southern one. Results revealed differences among sites in relation to each specific biomarker. Hence, a suite of biomarkers can be used to discriminate sampling sites according to types of pollution, reflecting differing conditions of anthropogenic impact. Comparisons between the northern and southern lagoon areas suggest that the latter are in better condition for Vg-like protein level and AChE activity. The lower EROD activity recorded in crabs from the northern sites suggests a detrimental inhibitory effect of contaminants, rather than a lack of induction due to lower pollution levels.

Effects of benzo(a)pyrene [B(a)P] (at a nominal concentration of 0.5 mg/L) on immune responses of the clam Chamelea gallina were investigated after 1, 7, and 12 days exposure. Total hemocyte count (THC), hemocyte volume, phagocytic activity, lysozyme-like activity in both hemocyte lysate (HL) and cell-free hemolymph (CFH) were measured. As unexpected alterations in hemocyte adhesion capability were observed in short-term hemocyte cultures for phagocytosis assays after a 1-day exposure, an adhesion test (not included in the original experimental setup) was performed after 7 and 12 days of exposure only. The survival-in-air test was carried out to evaluate general stress conditions in B(a)P-exposed clams. No alterations in THC was observed, whereas exposure for 7 and 12 days to B(a)P significantly decreased phagocytic activity and adhesion capability when compared with controls. Significant decreases in lysozyme activity were observed in CFH and HL, with respect to controls. B(a)P was also shown to alter the resistance to air exposure of clams. The LT(50) values fell from 9 days in control clams to 7 days in 1-day-exposed animals, and from 6 days in control clams to 5 days in 7-day-exposed bivalves. No significant variations in LT(50) values were recorded after 12 days of exposure. Results highlight a relationship between B(a)P exposure and alterations in hemocyte functionality and suggest that the contaminant induced irreversible immunosuppression in C. gallina, by altering phagocytic activity, adhesion capability, and enzymatic activity. Conversely, reduction in resistance to air exposure was reversible, suggesting that impairment of important physiological functions of clams occurred in the first phases of exposure only.

Estrogenic potency of 17-β estradiol (E2) was evaluated for the first time in both sexually undifferentiated (during resting phase, at the very early stage of gametogenesis) and differentiated (in pre-spawning phase) clams (Tapes philippinarum), following waterborne exposure for 7 and 14 days to various E2 concentrations (0, 0+acetone, 5, 25, 50, 100 and 1000ng/L). Vitellogenin (Vg)-like protein levels were evaluated in both haemolymph and digestive glands by the alkali-labile phosphate (ALP) assay. In the resting phase of clams, exposure for 7 days to all E2 concentrations tested resulted in significant increases in ALP in haemolymph with respect to controls. Conversely, no statistically significant differences were observed in digestive gland from E2-exposed bivalves, compared with controls. After 14-days exposure, ALP significantly increased in haemolymph from 1000ng E2/L-exposed clams and in digestive gland from 5ng E2/L-exposed animals. Interestingly, in the pre-spawning phase of clams, significant decreases in ALP levels were recorded in haemolymph from females exposed for 7 days, whereas no differences were observed in males. No significant variations were observed in ALP content in digestive gland from both females and males. ALP levels in haemolymph were shown to increase significantly in females exposed for 14 days to 50ng E2/L, and in males exposed to 100ng E2/L. Exposure for 14 days to all the E2 concentrations tested resulted in significant increases of ALP levels in the digestive gland of females. Conversely, variations in ALP content of male digestive glands were not significant. Results obtained appeared controversial and somewhat difficult to explain. Indeed, no univocal response by clams was observed after 7 and 14 days exposure to E2. Further studies need to elucidate the role of E2 in promoting Vg induction in clams.

Since it is often difficult to estimate possible adverse effects due to contamination in selected ecosystems, multi-species biomonitoring may provide more information, taking into account different routes of exposure, ecological roles and metabolic capabilities of animals. In this context, we exposed for 7 days the mussel Mytilus galloprovincialis and the crab Carcinus aestuarii to 4-nonylphenol (NP), a well-known xenoestrogen. In mussels (0-0.2 mg NP l(-1)), we measured NP bioaccumulation in soft tissues and vitellogenin (Vg)-like protein levels in digestive glands from both males and females by the alkali-labile phosphate assay (ALP). As no reference data were available for crab exposure, the NP 96-h LC(50) value was previously determined. Then, in sublethally exposed (0-1.0 mg NP l(-1)) male crabs, NP bioaccumulation and Vg levels were measured in hemolymph, gonads and digestive gland. Bioaccumulation of NP increased from 43 to 371 microg g(-1) d.w. in mussels, and from 3.6 to 37 microg g(-1) d.w. in crabs, depending on the NP concentration in water. Dose-dependent Vg-like protein induction was observed in both species, appearing to be related to NP bioaccumulation, although a partial decrease was recorded at the highest concentration tested. A similar trend was observed in both digestive gland and gonad of exposed crabs; Vg increased to a lesser extent, although significantly, in hemolymph. Results demonstrated that NP induces Vg synthesis both in male and female mussels, as well as in male crabs. On the basis of the responsiveness of both species investigated, a multi-species approach is indicated in biomonitoring programmes.

The lethal and sublethal effects of the xenoestrogen 4-nonylphenol (NP) were evaluated in the cockle Cerastoderma glaucum. In a 96-h lethality test, bivalves were exposed to 0, 0+ acetone, 0.19, 0.38, 0.75, 1.5 and 3.0 mg NP/l. The 96-h LC(50) value was 0.3mg NP/l. No mortality was observed at 0.1 mg NP/l. The potential estrogenicity of NP was studied in both sexually undifferentiated (resting phase) and differentiated (pre-spawning phase) cockles, exposed for 7 and 14 days to 0, 0+ acetone, 0.0125, 0.025, 0.05, and 0.1 mg NP/l. Vitellogenin (Vg)-like protein levels were determined in both haemolymph and digestive gland by the alkali-labile phosphate (ALP) assay. In the resting phase, exposure for 7 days to 0.1 mg NP/l resulted in significant increases in ALP in both haemolymph and digestive gland, compared with controls. A significant increase was also observed in digestive gland of animals exposed to 0.0125 mg NP/l-exposed animals. After 14 days of exposure, haemolymph ALP levels were significantly increased in exposed animals at all NP concentrations tested, whereas no difference was recorded in digestive gland. In the pre-spawning phase, exposure for 7 days to NP significantly increased ALP levels in haemolymph from males exposed at all NP concentrations tested, whereas no significant variations were found in haemolymph from females. NP (0.05 and 0.1 mg/l) was also shown to increase ALP concentrations significantly in digestive gland of males, but not in those of females. Likewise, after 14 days' exposure, ALP levels significantly increased in haemolymph from males only at 0.1 mg NP/l. Conversely, NP caused significant increases in ALP levels in digestive gland from both males (at all NP concentrations tested) and females (at 0.025 and 0.1 mg NP/l). These results demonstrate that NP induces Vg synthesis in C. glaucum. Interestingly, males were more responsive to NP than females.

The in vivo effects of 4-nonylphenol (NP) on functional responses of haemocytes from the cockle Cerastoderma glaucum were investigated after 7 days exposure to sublethal NP concentrations (0, 0+acetone, 0.0125, 0.025, 0.05 and 0.1 mg/l NP). Haemocytes from both controls and exposed cockles were collected, and the effects of NP on total haemocyte count (THC) and volume of circulating cells, intracellular superoxide anion (O(2)(-)) levels, acid phosphatase and lysozyme-like activities in both haemocyte lysate (HL) and cell-free haemolymph (CFH) were evaluated. Exposure of cockles to 0.1mg/l NP significantly increased THC (p<0.05) with respect to controls. Analysis of haemocyte size frequency distribution showed that the haemocyte fraction of about 7-8 microm in diameter and 250 femtolitres in volume increased markedly in cockles exposed to the highest NP concentration tested. Apoptosis resulting in cell volume reduction in NP-exposed animals cannot be excluded. No statistically significant variation in intracellular O(2)(-) levels was observed. Conversely, significant increases (p<0.05) in acid phosphatase activity were observed in CFH from 0.05 and 0.1mg/l NP-exposed animals; no significant differences in enzyme activity were recorded in HL. Lysozyme-like activity also increased significantly in CFH from cockles exposed to 0.05 mg/l NP (p<0.05) and 0.1 mg/l NP (p<0.001). Instead, lysozyme-like activity decreased significantly (p<0.05) in the HL of animals exposed to 0.05 mg/lNP. Our results suggest that NP induces variations in the functional responses of haemocytes of C. glaucum, mainly by reducing cell membrane stability and promoting cell degranulation.

For the first time, morpho-functional characterisation of haemocytes from the cockle Cerastoderma glaucum was performed to identify circulating cell types and to study their involvement in immune responses. Haemocyte mean number was 5.5 (x 10(5)) cells/mL haemolymph. Two main haemocyte types were found in haemolymph: granulocytes (85%), about 10 microm in diameter and with evident cytoplasmic granules, and hyalinocytes (15%), 8 to 14 microm in diameter, with a few or no granules. Most of the cytoplasmic granules stained in vivo with Neutral Red, indicating that they were lysosomes. On the basis of haemocyte staining properties, granulocytes and hyalinocytes were further classified as basophils and acidophils. Acidophil hyalinocytes were the largest haemocyte type (about 14 microm in diameter) and had an eccentric nucleus and a large cytoplasmic vacuole. Both granulocytes and hyalinocytes (except acidophils) were able to phagocytise yeast cells, although the basal phagocytic index was very low (about 2%). It increased significantly (up to 26%) after pre-incubation of yeast in cell-free haemolymph, suggesting that haemolymph has opsonising properties. Haemocytes also produced superoxide anion. Moreover, both granulocytes and hyalinocytes (except acidophils) were positive for some important hydrolytic and oxidative enzymes. Lysozyme-like activity was recorded in both cell-free haemolymph and haemocyte lysate, although enzyme activity in cell lysate was significantly higher. Results indicate that haemocytes from C. glaucum are effective cells in immune responses.

The effects of high temperatures on the clam, Chamelea gallina, generally recognised as a low tolerant bivalve species, were studied by evaluating some functional responses of the haemocytes. The animals were kept for 7days at 20, 25 and 30 degrees C and total haemocyte count (THC), phagocytosis, lysozyme activity (in both haemocyte lysate and cell-free haemolymph), activity and expression of the antioxidant enzyme superoxide dismutase (SOD) (in both haemocyte lysate and cell-free haemolymph) were chosen as biomarkers of exposure to high temperatures. The survival-in-air test was also performed. During the experiment, the clams showed differing burrowing behaviour: the animals kept at 20 and 25 degrees C burrowed completely, whereas at 30 degrees C the clams progressively emerged from the sediment and then remained on the surface. The highest temperature significantly increased THC, whereas it decreased the phagocytic activity of haemocytes. The haemocyte size frequency distribution in clams kept at 30 degrees C showed that the cell population of about 8-10microm was markedly reduced compared to clams kept at 20 and 25 degrees C. In clams maintained at 25 degrees C, lysozyme activity was significantly increased in haemocyte lysate, whereas it was markedly decreased in cell-free haemolymph. Total SOD activity significantly decreased in haemocytes from clams held at 30 degrees C whereas it increased in cell-free haemolymph from clams held at 25 degrees C and 30 degrees C. A significant decrease in haemocyte Mn-SOD and Cu/Zn-SOD activities was found with increasing temperature. In cell-free haemolymph, the highest Mn-SOD activity was recorded at 30 degrees C, whereas the Cu/Zn-SOD activity showed no significant changes in clams maintained at different temperatures. SOD isoform expression exhibited different patterns in haemocyte lysate and cell-free haemolymph. The resistance to air exposure of clams kept at 30 degrees C was shown to decrease significantly, LT(50) values fell from 6days in clams kept at 20 degrees C and 25 degrees C to 4days in those kept at 30 degrees C.

Possible xenoestrogenic effects were investigated, for the first time, in two bivalve species from the Lagoon of Venice (Italy): the clam Tapes philippinarum and the cockle Cerastoderma glaucum. Bivalves were collected far from their reproductive phase at the very early stage of gametogenesis (January), and in the pre-spawning period (June) in six sites. Vitellogenin (Vg)-like proteins (a biomarker of exposure to estrogenic compounds) were measured by the alkali-labile phosphate method (ALP), in both haemolymph and digestive gland from males and females (when it was possible to distinguish sex by microscopic observation of gonadal tissue). Haemolymph calcium concentrations (a parameter considered closely related to the presence of Vg-like proteins) and the bivalve condition index (CI) were also measured. In both seasons, bivalves collected at Campalto (near a sewage treatment plant) and Marghera (a highly contaminated area) had higher Vg-like protein levels, particularly in haemolymph, than animals from the other sampling sites. Interestingly, CI had high values in these polluted sites. In June only, Vg-like proteins and Ca(2+) levels in haemolymph exhibited similar trends in both bivalve species at most sampling sites. The responsiveness of bivalves to environmental xenoestrogens was higher in June, allowing better discrimination among sites. The present study demonstrates that animals from highly polluted areas have increased Vg-like protein levels. As endocrine disruption due to exposure to estrogenic compounds may cause fertility reduction, alterations in the sex ratio, and a decrease in reproductive rate, a condition of potential risk for bivalve populations in estuarine areas is highlighted.

Measurement of acetylcholinesterase (AChE) is widely used as a biomarker of exposure to neurotoxic compounds in aquatic environments. In the present study, AChE activity was monitored in the clam Tapes philippinarum in the Lagoon of Venice, with the aim of evaluating the possible exposure of animals to neurotoxic compounds. Two sampling strategies were followed. In the first case, temporal and spatial variations of AChE activity were measured in clams collected seasonally (from October 2003 to June 2004) in 3 sites of the Lagoon: Campalto (site 1), near a sewage treatment plant, Marghera (site 2), a highly polluted area, and Poveglia (site 3), a reference site. The condition index (CI) of clams was also calculated. In the second case, AChE activity was measured in clams collected at the end of seasonal samplings in 12 sites located throughout the Lagoon: areas licensed for clam farming (sites 5 to 11), nearby canals characterised by waste waters from agricultural areas (sites 4, 12, 13, 14), and a reference site outside the Lagoon (site 15). Results revealed a significant reduction in AChE activity in clams from Marghera in October, January and April, indicating their probable exposure to neurotoxic compounds originating in the industrial zone of Porto Marghera. A seasonal trend in AChE activity was also observed, enzyme activity being higher in January and lower in June. Clams from Campalto generally had higher CI values than those from Marghera and Poveglia. However, no particular correlation between AChE activity and CI was found. Lastly, marked differences in AChE activity were recorded when comparing enzyme activity of clams from various sites in the Lagoon of Venice with those of clams collected at site 15. Significant reductions in AChE activity were observed in animals collected in both nearby canals and licensed areas, indicating the homogeneous spatial distribution of potentially neurotoxic compounds throughout the Lagoon. It is probable that sediment mobilisation caused by clam harvesting and the passage of shipping plays an important role in resuspension of persistent contaminants having neurotoxic activity.

Vitellogenins (Vg) are precursors of egg-yolk proteins (vitellins) constituting energy reserves for embryos. In oviparous females, Vg are produced in the liver (or equivalent organs) under the control of endogenous estrogens, released into the bloodstream (plasma or hemolymph), and stored in developing oocytes. Although Vg levels are generally undetectable in male plasma, they may significantly increase in (xeno-)estrogen-exposed males. The aim of the present study was to evaluate estrogenic effects of 4-nonylphenol (NP) in sexually differentiated males and females of the Manila clam Tapes philippinarum. Clams were exposed for 7 days to various sublethal NP concentrations (0, 0+acetone, 0.025, 0.05, 0.1, and 0.2 mg NP/L) and Vg-like protein levels were evaluated in both hemolymph and digestive glands of mollusks by the alkali-labile phosphate assay. This method, based on the determination of phosphates released by Vg, has previously been used to detect Vg concentrations in both fish plasma and bivalve hemolymph. Although Vg levels were generally higher in both hemolymph and digestive gland of T. philippinarum females, males appeared to be more sensitive than females to the estrogenic effects of NP. In males exposed to 0.1 and 0.2 mg NP/L, Vg levels increased significantly in both hemolymph (P < 0.05 and P < 0.01, respectively) and digestive gland (p < 0.05), whereas no changes were observed in those of females. Although the mechanisms by which NP exerts its in vivo endocrine-modulating effects in bivalve mollusks remain unclear, the results of this study demonstrate that NP may induce Vg-like proteins in T. philippinarum, particularly males.

The effects of 4-nonylphenol on anti-oxidant enzyme (superoxide dismutase and catalase) activity in both gills and digestive gland and re-burrowing capability of the clam Tapes philippinarum were investigated after 7 days' exposure to various sublethal concentrations of nonylphenol (NP). NP caused a significant decrease in superoxide dismutase activity in both tissues from the lowest concentration tested, whereas no significant alteration with respect to controls was observed in catalase activity. NP effects on re-burrowing of clams were evaluated in two experiments. In the first, clams exposed to the highest NP concentrations showed a marked delay in re-burrowing 48 h after the end of exposure. Modifications in normal burrowing behaviour were also observed, suggesting that NP acts via narcosis and/or neurotoxic mechanisms. In the second experiment, the elapsed time for 50% of the exposed clams to re-burrow into the sediment (RT(50)) was also calculated in a 24-h re-burrowing response test. At the highest NP concentrations, it was not possible to estimate RT(50) values, as the percentage of clams showing complete burial was less than 50%. The present study demonstrates that NP causes oxidative stress by inhibiting superoxide dismutase activity and greatly affects the re-burrowing capability of T. philippinarum, even at environmentally realistic concentrations.

Lethal and sublethal effects of 4-nonylphenol (NP) were investigated in the clam Tapes philippinarum from the Lagoon of Venice. In a 96-h lethality test, bivalves were exposed to the following NP concentrations: 0, 0+acetone, 0.19, 0.38, 0.75, 1.5, and 3.0mg NP/L. The 96-h LC(50) value was 1.12mg NP/L. No mortality was observed at 0.19mg NP/L, whereas at 1.5 and 3.0mg NP/L both siphons and foot of clams were often cut-off, the animals being unable to withdraw them before shell closure, suggesting the possible narcotic effect of NP. Lower concentrations (0, 0+acetone, 0.025, 0.05, 0.1, and 0.2mg NP/L) were then used to evaluate sublethal NP effects on clearance rate (CR), respiration rate (RR), scope for growth (SFG), and survival in air. Following a 7-day exposure, decreased RRs were found at all NP concentrations tested. Significant decreases in the CR and SFG were only observed at the two highest NP concentrations (0.1 and 0.2mg/L), with SFG reductions reaching 54% and 71%, respectively. Last, the highest concentrations tested significantly decreased the resistance of clams to exposure to air and enhanced the mortality rate. As the lowest effective NP concentrations are similar to environmentally realistic levels, a condition of potential risk for the well-being of clam populations in estuarine areas is highlighted.