Different ecological histories of sea urchins acclimated to reduced pH influence offspring response to multiple stressors.

End-of-the-century predictions on carbon dioxide (CO2) driven ocean acidification and the continuous leakage of pesticides from inland to coastal areas are of concern for potential negative effects on marine species' early life stages which are the most vulnerable to environmental changes. Variations in seawater chemistry related to human activities may interfere with the normal development from embryo to juvenile/adult stage. However, transgenerational studies suggest that the parental generation can influence the offspring phenotype, and thus their performances, based on the environment experienced. Here we compared the transgenerational responses to a multiple stressor scenario in sea urchins (Paracentrotus lividus) that experienced different environments since their settlement: i.e., animals from a highly variable environment, such as the Venice lagoon, versus animals from a coastal area with prevailing oligotrophic conditions in the Northern Adriatic Sea. After long-term maintenance (2 and 6 months) of adult sea urchins at natural and -0.4 units reduced pH, the F1 generations were obtained. Embryos were reared under four experimental conditions: natural and -0.4 pH both in the absence and in the presence of an emerging contaminants' mixture (glyphosate and aminomethylphosphonic acid at environmentally relevant concentrations, 100 µg/L). A significant detrimental effect of both the parental and the filial pH was highlighted, affecting embryo development and growth. Nonetheless, sea urchins from both sites were able to cope with ocean acidification. The 6-months F1 response was better than that of the 2-months F1. Conversely, the F1 response of the sea urchins maintained at natural conditions did not change sensibly after more prolonged parental exposure. An additive but mild negative effect of the mixture was observed, mostly in lagoon offspring. Results suggest that long-term exposure to reduced pH leads to transgenerational acclimation but does not affect susceptibility to the tested pollutants.

Toxicological effects and bioaccumulation of fullerene C60 (FC60) in the marine bivalve Ruditapes philippinarum.

Fullerene C60 (FC60), 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 FC60 to aquatic organisms. Nowadays, only few studies have analysed FC60 effects and bioaccumulation in marine organisms following in vivo exposure. To provide new data about FC60 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 FC60 (1 and 10 µg/L) and cellular and biochemical responses were evaluated in clams' gills, digestive gland and haemolymph. The FC60 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 FC60-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. FC60 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 FC60 compared to the other tissues investigated. Indeed, only Single Cell Gel Electrophoresis and Neutral Red uptake assays showed increased values in FC60-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 FC60 toxicity under the exposure conditions tested: the high damage detected to lipids and proteins could contribute to long-term problems for the organism.

Seawater acidification and emerging contaminants: A dangerous marriage for haemocytes of marine bivalves.

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.

Effects of aminomethylphosphonic acid, the main breakdown product of glyphosate, on cellular and biochemical parameters of the mussel Mytilus galloprovincialis.

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.

Imposex levels and butyltin compounds (BTs) in Hexaplex trunculus (Linnaeus, 1758) from the northern Adriatic Sea (Italy): Ecological risk assessment before and after the ban.

The aim of this study was to compare imposex and butyltin compounds (BTs) data, collected before and after the organotin ban in 2008, in order to assess temporal and spatial variation of the phenomenon, the decline of BT contamination, and the effects on Hexaplex trunculus population in the coastal area of the northern Adriatic Sea, close to the Venice Lagoon. Both in marine and in lagoon sites, the results obtained in 2013-2015 showed a significant decline in the incidence of imposex in respect to those from the 2002 survey. In 2002, lagoon samples exhibited Relative Penis Size Index (RPSI) higher than marine samples, whereas no differences were detected in the recent survey, when all RPSI values were below 0.6%. Vas Deference Sequence Index (VDSI) mean values were over 4 before the ban introduction and below this value after that, indicating more critical conditions for gastropod population in 2002 rather than in 2013-15. Percentage of sterile females was up to 69% in 2002, whilst in the more recent survey no sterile female was found. Range of BT concentrations in gastropods decreased from 252 to 579 to 16-31ng∑BT/g d.w. BT body burdens varied according to a gender dependant pattern, with higher concentrations observed in females than in males. A first attempt to propose a classification based on BT impact on H. trunculus, according to the Water Framework Directive, revealed that most sites were in Bad ecological status before the ban and attained a Poor/Moderate status after that.

A first survey on the biochemical composition of egg yolk and lysozyme-like activity of egg envelopment in the cuttlefish Sepia officinalis from the Northern Adriatic Sea (Italy).

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.

A multibiomarker approach in clams (Ruditapes philippinarum) for a toxicological evaluation of dredged sediments.

The Lagoon of Venice is often dredged for channel maintenance. To avoid harmful consequences to the ecosystem, a proper disposal of bottom sediments requires a preliminary evaluation of its potential toxicity before excavation. Here we evaluated the effects of polluted sediments on clams (Ruditapes philippinarum) using a multibiomarker approach. Bivalves were exposed for 3 and 14 days to five sediment samples collected along a navigation canal between Venice historical centre and the industrial area of Porto Marghera. Immunological, antioxidant, detoxification, and neurotoxicity biomarkers were analysed in haemolymph, gill, and digestive gland. As a control, sediment collected far from pollution sources was used. Two experiments were performed to assess potential seasonal/gametogenic influence in clam sensitivity. A different response of clam biomarkers was observed during the two experiments and among sampling sites. Clams' digestive gland resulted to be the most sensitive tissue analysed showing significant differences among sites in all biomarkers analysed. Greater differences were present due to seasonality rather than exposure. The concentrations of metals and organic pollutants increased from the city centre to the industrial area, highlighting the influence that industrial activities had on the lagoon ecosystem. However, bioaccumulation in clams did not follow the same clear pattern, suggesting low bioavailability of compounds due to relatively high organic matter content. Biomarkers modulation was mainly driven by metals, both present in sediments and bioaccumulated. In comparison, effects of organic pollutants on the biomarkers tested were negligible. Other sources of contamination not investigated (e.g. pesticides) were suggested by neurotoxicity biomarkers alteration.

Can ecological history influence immunomarker responses and antioxidant enzyme activities in bivalves that have been experimentally exposed to contaminants? A new subject for discussion in "eco-immunology" studies.

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.

Immunotoxic effects of triclosan in the clam Ruditapes philippinarum.

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.

New evidences in the complexity of contamination of the lagoon of Venice: polybrominated diphenyl ethers (PBDEs) pollution.

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.

Transgenerational effects and phenotypic plasticity in sperm and larvae of the sea urchin Paracentrotus lividus under ocean acidification.

In marine organisms, differing degree of sensitivity to ocean acidification (OA) is expected for each life stage, and disturbance at one stage can carry over into the following stage or following generation. In this study we investigated phenotypic changes of sperm and larvae of the sea urchin Paracentrotus lividus in response to different pH conditions (8.0, 7.7, 7.4) experienced by the parents during gametogenesis. In sperm from two-months exposed males, sperm motility, velocity, ATP content, ATP consumption and respiration rate were evaluated at three pH values of the activating medium (8.0, 7.7 and 7.4). Moreover, larvae from each parental group were reared at pH 8.0 and 7.7 for 20 days and larval mortality and growth were then assessed. Sperm motility and respiration rate were not affected either by exposure of males to low pH or by the post-activation pH. Sperm velocity did not differ among post-activation pH values in all sperm groups, but it decreased slower in sperm developed under acidified conditions, suggesting the presence of positive carryover effect on sperm longevity. This positive carryover effect of exposure of males to low pH values was highlighted also for the sperm ATP content, which was higher in these groups of sperm. ATP consumption rate was affected by post-activation pH with higher values at pH 8.0 in sperm from males maintained at control condition and pH 7.7 while the energy consumption appeared to be differently modulated at different experimental conditions. A negative carry over effect of OA was observed on survival of larvae from parents acclimated at pH 7.4 and additive negative effects of both parental and larval exposure to low pH can be suggested. In all groups of larvae, decreased somatic growth was observed at low rearing pH, thus larvae from parents maintained at low pH did not show an increased capability to cope with OA.

Zinc oxide, titanium dioxide and C60 fullerene nanoparticles, alone and in mixture, differently affect biomarker responses and proteome in the clam Ruditapes philippinarum.

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 nTiO2 and 1 µg/L FC60 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.

Combined effects of temperature and salinity on functional responses of haemocytes and survival in air of the clam Ruditapes philippinarum.

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.

Effects of a mixture of glyphosate, 17α-ethynylestradiol and amyl salicylate on cellular and biochemical parameters of the mussel Mytilus galloprovincialis.

In this study the effects of a mixture of glyphosate (herbicide), 17a-ethinylestradiol (synthetic estrogen) and amyl salicylate (fragrance) to the mussel Mytilus galloprovincialis were evaluated. Mussels were exposed for 7 days to two realistic concentrations of the mixture (10 and 100 ng/L) and the effects on total haemocyte counts, haemocyte diameter and volume, haemocyte proliferation, haemolymph lactate dehydrogenase activity and haemocyte lysate lysozyme activity were measured. In addition, superoxide dismutase, catalase, acetylcholinesterase, glutathione-S-transferase and glutathione reductase activities were measured in gills and digestive gland. The survival-in-air test was also performed. Results demonstrated that the mixture affected both cellular and biochemical biomarkers, but not tolerance to aerial exposure of M. galloprovincialis. The negative effects recorded in this study suggested that more efforts should be done to assess the ecotoxicological risks posed by contaminant mixture to aquatic invertebrates.

When site matters: Metabolic and behavioural responses of adult sea urchins from different environments during long-term exposure to seawater acidification.

CO2-driven ocean acidification (OA) affects many aspects of sea urchin biology. However, even in the same species, OA effects are often not univocal due to non-uniform exposure setups or different ecological history of the experimental specimens. In the present work, two groups of adult sea urchins Paracentrotus lividus from different environments (the Lagoon of Venice and a coastal area in the Northern Adriatic Sea) were exposed to OA in a long-term exposure. Animals were maintained for six months in both natural seawater (pHT 8.04) and end-of-the-century predicted condition (-0.4 units pH). Monthly, physiological (respiration rate, ammonia excretion, O:N ratio) and behavioural (righting, sheltering) endpoints were investigated. Both pH and time of exposure significantly influenced sea urchin responses, but differences between sites were highlighted, particularly in the first months. Under reduced pH, ammonia excretion increased and O:N decreased in coastal specimens. Righting and sheltering were impaired in coastal animals, whereas only righting decreased in lagoon ones. These findings suggested a higher adaptation ability in sea urchins from a more variable environment. Interestingly, as the exposure continued, animals from both sites were able to acclimate. Results revealed plasticity in the physiological and behavioural responses of sea urchins under future predicted OA conditions.

First Evidence of In Vitro Effects of C6O4-A Substitute of PFOA-On Haemocytes of the Clam Ruditapes philippinarum.

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 ß-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.

Use of a simple empirical model for the accurate conversion of the seawater pH value measured with NIST calibration into seawater pH scales.

The seawater pH measurement is usually quite complicated because that matrix is characterized by a high ionic strength leading to calibration errors if NIST standards are used. For this matrix, different pH scales like the "total hydrogen ion concentration scale" (TOT) and the "seawater scale" (SWS), are defined, and suitable synthetic seawater solutions must be prepared according to standard procedures to calibrate the glass electrode. This work provides a new approach to make seawater pH measurements by using the glass electrode calibrated with the NIST standards (pHNIST) converting the pHNIST into the right TOT or SWS scales by using empirical equations derived from theoretical thermodynamic data: pHTOT=pHNIST+0.10383+4.33⋅10-5TS+3.633⋅10-5T2-4.921⋅10-5S2, and pHSWS=pHNIST+0.097733+4.1059⋅10-5TS+3.5437⋅10-5T2-4.941⋅10-5S2, for the TOT and SWS scales, respectively. These equations are functions of two simple experimental parameters, namely, T = temperature (°C) and S = salinity (PSU, (g/L), Practical Salinity Units). These equations were experimentally validated and the uncertainty of pHTOT and pHSWS was demonstrated to have no statistical difference with the corresponding values obtained following the standard operative procedure (SOP) using commercially unavailable seawater-like buffers. The proposed method has therefore the same performances and it is largely preferable as it avoids long and tedious procedures of the synthetic seawater preparations.

New compounds, old problems. The case of C6O4 - a substitute of PFOA - and its effects to the clam Ruditapes philippinarum.

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 substitute of perfluorooctanoic acid (PFOA). Recently, C6O4 has been detected in aquatic environments, but, at present, no information concerning the effects of C6O4 on aquatic species, such as bivalves, are available in the literature. Therefore, in this study we evaluated for the first time the effects of C6O4 (0.1 and 1 µg/L) and PFOA (1 µg/L) to the clam Ruditapes philippinarum. Short-term (7 days) and long-term (21 days) exposures of clams to the two compounds were carried out and numerous biomarkers were measured in haemocytes/haemolymph, as well as in gills and digestive gland. The MANOVA analysis demonstrated statistically significant effects of the independent variables "treatment", "time" and "treatment-time interaction" on the whole dataset of biomarker responses. The two-way ANOVA analysis performed for each biomarker response indicated that the two compounds affected most of the cellular and tissue parameters measured. Despite preliminary, the results obtained suggested that C6O4 - similarly to PFOA - can affect both cellular and biochemical parameters of clams.

The new PFAS C6O4 and its effects on marine invertebrates: First evidence of transcriptional and microbiota changes in the Manila clam Ruditapes philippinarum.

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.

The role of haemocytes from the crab Carcinus aestuarii (Crustacea, Decapoda) in immune responses: A first survey.

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.