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In the present study, potential exposure to xenoestrogens was evaluated in the clam Ruditapes philippinarum and the crab Carcinus aestuarii, both from the Lagoon of Venice. Animals were collected in 4 sites (Ca' Roman, Valle di Brenta, Marghera, and Palude del Monte), differently influenced by anthropogenic impact and natural conditions. Vitellogenin (Vg)-like protein levels were measured in digestive gland and haemolymph. The survival-in-air test was also performed on clams, since it is useful in detecting general stress conditions in bivalves. Clams from Palude del Monte had higher Vg levels in haemolymph than those from Valle di Brenta and Ca' Roman. In digestive gland, statistically significant differences in Vg levels were detected in clams from Marghera with respect to those from Valle di Brenta. In crabs, statistically significant higher Vg levels were observed in haemolymph of animals from Palude del Monte, with respect to crabs from Ca' Roman. No statistically significant differences were recorded in crab digestive gland. Significantly increased resistance to air exposure was observed in clams from Palude del Monte, compared with that of clams from the other sites. LT50 values fell from 11 days in clams from Palude del Monte to 5 days in ones from Ca' Roman. The present study demonstrates that measurement of Vg levels is a valuable bio- marker in biomonitoring estuarine environments, highlighting a similar pattern of variation in both species. Although the animals used have different habits, the two-species approach identified a peculiar condition for Palude del Monte, a licensed area for clam culture. [ABSTRACT FROM AUTHOR]

Sea-Nine 211 ™ (4,5-dichloro-2-n-octyl-4-isothiazolin-3-one) has recently been introduced as a primary biocide in the formulations of new antifouling paints for the boat hulls in place of the organotin compounds. In this study, immunotoxic effects of various sublethal concentrations - 0.1 to 10 μM - of Sea-Nine 211 ™ on the edible Manila clam Ruditapes philippinarum were evaluated in vitro. By means of assays with short-term haemocyte cultures, the influence of the xenobiotic on both cell morphology and functionality was evaluated. Although no effect on cell adhesion was observed, severe and irreversible morpho-functional effects occurred similarly to those reported for organotin compounds. At 1 μM (282.2 μg/1), Sea-Nine 211™ exerted significant (p <0.001) inhibitory effects on cell spreading, yeast phagocytosis, hydrolytic enzyme (β-glucuronidase) activity, induced apoptosis and caused oxidative stress, the latter demonstrated through a decrease in cytoplasmic glutathione content. Data suggest that the biocide Sea-Nine 211™ is immunotoxic and represents a potential risk for benthic filter-feeding invertebrates living in the water-sediment interface and, as a consequence of bioaccumulation, for human health. However, further studies need to validate functional responses of clam haemocytes as immunomarkers in field conditions, like those measured in the present study in Sea-Nine 211™-exposed cells. [ABSTRACT FROM AUTHOR]

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 Vglike 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. [ABSTRACT FROM AUTHOR]

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 LT50 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 LT50 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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Thiacloprid affects mussel biochemical responses. • Thiacloprid influences antioxidant enzymes and haemolymph parameters. • Thiacloprid causes histological damage to gills and digestive gland. [ABSTRACT FROM AUTHOR]

It is well known that a wide variety of endocrine-disrupting chemicals (EDCs) (e.g. organotin compounds, heavy metals, herbicides, xenoestrogen compounds) can induce endocrine alterations in aquatic invertebrates, often causing reproductive disorders. Although these phenomena are of great concern, mostly in coastal and estuarine environments, few data are available about the mechanism of action of EDCs, mainly due to lack of knowledge about the invertebrate endocrine system. To understand the possible mechanism of action of EDCs better, the presence and patterns of variation of sexual steroids, progesterone, testosterone and 17β-estradiol were measured in the whole body of the clam Tapes philippinarum by specific and opportunely validated microtitre radioimmunoassays (RIAs). Female steroid concentrations showed similar trends, with higher values during spawning and lower ones in post-spawning and gametogenesis periods. Conversely, in males, sexual steroids varied over the year: progesterone remained unchanged, with values similar to those of females in gametogenesis, whereas testosterone and 17β-estradiol showed a significant decrease during gametogenesis only. Seasonal variations in clam steroid levels may reflect their role in modulating reproduction. All hormones were unexpectedly high in the resting/early developing stage, during which gonadal tissue is scarce, indicating that digestive gland may have a biosynthetic steroid capacity or accumulation. Although these results are preliminary, evaluation of sexual steroids in T. philippinaruni during the gametogenic cycle represent the first step for future research aimed at understanding the physiological role of these hormones and their potential interaction with EDCs. [ABSTRACT FROM AUTHOR]

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 3.0mg NP/l. The 96-h LC50 value was 0.3mg NP/l. No mortality was observed at 0.1mg 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 14days to 0, 0+ acetone, 0.0125, 0.025, 0.05, and 0.1mg 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 7days to 0.1mg 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 0.0125mg NP/l-exposed animals. After 14days 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 7days 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 0.1mg/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 0.1mg 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 0.1mg NP/l). These results demonstrate that NP induces Vg synthesis in C. glaucum. Interestingly, males were more responsive to NP than females. [Copyright &y& Elsevier]

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 0.1mg/lNP). 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 () 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/lNP significantly increased THC (p <0.05) with respect to controls. Analysis of haemocyte size frequency distribution showed that the haemocyte fraction of about 7–8μm in diameter and 250femtolitres 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 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 0.05mg/l NP (p <0.05) and 0.1mg/l NP (p <0.001). Instead, lysozyme-like activity decreased significantly (p <0.05) in the HL of animals exposed to 0.05mg/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. [Copyright &y& Elsevier]

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 (0–0.2mg 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 LC50 value was previously determined. Then, in sublethally exposed (0–1.0mg 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 371μgg−1 d.w. in mussels, and from 3.6 to 37μg 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

A recurrent blastogenetic cycle characterizes colonies of the ascidian Botryllus schlosseri. This cycle starts when a new zooid generation opens its siphons and ends with take-over, when adult zooids cease filtering and are progressively resorbed and replaced by a new generation of buds, reaching functional maturity. During the generation change, massive apoptosis occurs in the colony, mainly in the tissues of old zooids. In the present study, we have investigated the behaviour of haemocytes during the colonial blastogenetic cycle, in terms of the occurrence of cell death and the expression of molecules involved in the induction of apoptosis. Our results indicate that, during take-over, caspase-3 activity in haemocyte lysates increases. In addition, about 20%–30% of haemocytes express phosphatidylserine on the outer leaflet of their plasma membrane, show DNA fragmentation and are immunopositive for caspase-3. Senescent cells are quickly ingested by circulating phagocytes that frequently, having once engulfed effete cells, in turn enter apoptosis. Dying cells and corpses are replaced by a new generation of cells that appear in the circulation during the generation change. [ABSTRACT FROM AUTHOR]

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 Ca2+ 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. [Copyright &y& Elsevier]

Abstract: 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 (×105) cells/mL haemolymph. Two main haemocyte types were found in haemolymph: granulocytes (85%), about 10μm in diameter and with evident cytoplasmic granules, and hyalinocytes (15%), 8 to 14μm 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 14μm 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. [Copyright &y& Elsevier]

In the present study, the effects of differing salinities on some important functional responses of haemocytes from the clam, Chamelea gallina, were investigated. The animals were kept for 7 days at 28‰ (hyposalinity), 34‰ (control) and 40‰ salinity (hypersalinity), and total haemocyte count (THC), haemocyte volume, phagocytosis, lysozyme-like activity (in both haemocyte lysate and cell-free haemolymph) were measured. The survival-in-air test was also performed. Clams kept at 28‰ showed significantly increased THC with respect to animals kept at 34 and 40‰. The analysis of haemocyte size frequency distribution highlighted that in clams kept at 28‰ the haemocyte fraction of about 5 μm in diameter and 50–100 femtolitre in volume increased markedly. Conversely, in animals kept at 40‰ an increase was observed in the haemocyte fraction having about 8–10 μm diameter and 400–500 femtolitre volume. Higher phagocytic activity was recorded in haemocytes from control clams, with respect to that of clams kept at 28 and 34‰. Lysozyme-like activity in haemocyte lysate was shown to increase significantly in animals kept at 28‰ with respect to that of clams kept at 40‰, whereas enzyme activity in cell-free haemolymph from clams kept at 34‰ was significantly higher with respect to that of clams maintained at 40‰. A relationship between phagocytosis and lysozyme secretion is suggested. The resistance to air exposure of clams kept at 28 and 40‰ was shown to decrease significantly; LT50 values fell from 7 days in clams kept at 34‰ to 4 and 5 days in those kept at 28 and 40‰, respectively. Results demonstrated that salinity values far from 34‰ affects the functional responses of haemocytes and reduce the resistance of clams to exposure to air. [ABSTRACT FROM AUTHOR]

The effects of different salinity levels (28, 34 and 40‰) on functional responses of Chamelea gallina haemocytes were evaluated in a two part study dealing with modulations of immune parameters. This part (Part II) of the study was focused on the superoxide dismutase (SODs) activity and expression in haemocyte lysate and cell-free haemolymph. Results of this study established that the exposure of C. gallina specimens at 40‰ salinity provoked a decrease in Mn-SOD and Cu/Zn-SOD activities in haemocyte lysate suggesting a declining superoxide anion generation at the highest salinity tested. Expression of MnSOD was coherent with activity values, while Cu/ZnSOD showed two immunoreactive bands. The former corresponds to the cytosolic Cu/Zn-SOD (16 kDa) was not coherent with the enzyme activity and the second (28–30 kDa) probably attributed to EC-SOD. In cell-free haemolymph, Mn-SOD activity decrease and Cu/Zn-SOD activity increase at 40‰ were observed, likely due to EC-SOD contribution, strongly induced at the same salinity. After EC-SOD detection with two different antibody, we postulate that the EC-SOD like-protein band (29 kDa) may be constitute partly by EC-SOD and probably by Cu/Zn-SOD dimeric form not completely dissociated under reducing condition or a Cu/Zn-SOD degenerated but still recognized by antibody. The cell-free haemolymph increase of EC-SOD at high salinity values plays an important role in immune defence of C. gallina. According to the conclusion of Part I of this study, our data confirmed the destabilizing effect of 40‰ salinity on haemocyte functionality, while to 28‰ exposure, data don’t confirm its stressful action as instead stated by results of Part I. Further studies are necessary to clear up this discrepancy. [ABSTRACT FROM AUTHOR]

Abstract: 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°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°C burrowed completely, whereas at 30°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°C showed that the cell population of about 8–10μm was markedly reduced compared to clams kept at 20 and 25°C. In clams maintained at 25°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°C whereas it increased in cell-free haemolymph from clams held at 25°C and 30°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°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°C was shown to decrease significantly, LT50 values fell from 6days in clams kept at 20°C and 25°C to 4days in those kept at 30°C. [Copyright &y& Elsevier]

To confirm the hypothesis that 4-nonyiphenol (NP), an estrogenic compound, has neurotoxic effects on the clam Tapes philippinarum, in the present study clams were exposed to NP (for 7 days to 0, 0+acetone, 0.0125, 0.025, 0.05, 0.1, and 0.2 mg NP/L, and for 14 days to 0, 0+acetone, 0.0125, 0.025, 0.05, and 0.1 mg NP/L), and the activity of acetylcholinesterase (AChE) was evaluated in gills. The effects on AChE activity were also studied after exposing clams for 96 h to chlorpyrifos (CP) (12 and 24 μg/L), an organophosphorus pesticide known to inhibit AChE in aquatic organisms. Exposure to NP for 7 days significantly increased AChE activity in 0.025, 0.1 and 0.2 mg NP/L-treated clams, whereas no significant alteration in enzyme activity was recorded after 14 days' exposure. This allowed us to exclude AChE inhibition by NP. Interestingly, CP not only did not affect AChE activity, but also significant increases in enzyme activity were recorded in 12-μg CP/L-exposed animals with respect to controls. On the basis of these results, further research will be performed on T. philippinarum by increasing exposure levels, in terms of both time and concentration, to better define the possible application of AChE activity in clams as a biomarker of exposure to organophosphorus pesticides in biomonitoring. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

In our previous study, we demonstrated that the microalgae Phaeodactylum tricornutum can bioaccumulate bisphenol A analogues. Since this microalgae species is part of the diet of marine filter-feeding organisms, such as bivalves, in this study we tested the hypothesis that a diet based on exposed microalgae can exert negative effects on the clam Ruditapes philippinarum. Microalgae were exposed for 7 days to 300 ng/L of bisphenol AF (BPAF), bisphenol F (BPF), and bisphenol S (BPS), alone or as a mixture (MIX), to allow bioaccumulation. Microalgae were then supplied as food to bivalves. After 7 and 14 days of diet, the effects of exposed microalgae were evaluated on a battery of biomarkers measured in haemolymph/haemocytes, gills and digestive glands of clams. In addition, bioaccumulation of the three bisphenols was investigated in clams by UHPLC-HRMS. The results obtained demonstrated that total haemocyte count (THC) increased in clams following ingestion for 7 days of BPAF- and BPF-exposed microalgae, while BPS-exposed microalgae significantly reduced THC after 14 days of diet. MIX- and BPS-exposed microalgae increased haemocyte proliferation. The diet of exposed microalgae affected acid and alkaline phosphatase activity in clams, with an opposite response between haemolymph and haemocytes. Regarding antioxidants, an increase in catalase activity was observed in clams after ingestion of BPA analogue-exposed microalgae. The results also demonstrated marked oxidative stress in gills, the first tissue playing an important role in the feeding process. Oxidative damage was recorded in both the gills and digestive glands of clams fed BPA analogue-exposed microalgae. Alterations in epigenetic-involved enzyme activity were also found, demonstrating for the first time that BPA analogue-exposed food can alter epigenetic mechanisms in marine invertebrates. No bioaccumulation of BPA analogues was detected in clam soft tissues. Overall, this study demonstrated that a diet of BPA analogue-exposed microalgae can induce significant alterations of some important biological responses of R. philippinarum. To our knowledge, this is the first study demonstrating the effects of ingestion of BPA analogue-exposed microalgae in the clam R. philippinarum, suggesting a potential ecotoxicological risk for the marine food chain, at least at the first levels. [ABSTRACT FROM AUTHOR]

There is growing concern for the wide use of perfluorooctanoic 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 of PFOA. 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. [ABSTRACT FROM AUTHOR]