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Biomonitoring is a scientific method for assessing the health status of the environment to natural and synthetic chemicals, based on sampling and analysis of organism's tissues and fluids. This technique relies on the knowledge that chemicals induce markers reflecting this exposure: the marker may be the chemical itself. Different models have been used to study the bioavailability and the bioaccumulation of contaminants: the Biota-Sediment Accumulation Factor (BSAF) predicts that in the organism's tissue the chemical residue can be estimated from the partitioning of the compound between the lipid fraction and the organic carbon in the sediment. In this work the BSAFs of NP and PAH in Tapes philippinarum from the Venice lagoon were calculated, with the aim to verify if the routine biomonitoring studies are reliable in contaminated sites. A field campaign from October 2003 to June 2004 in three sites of the Venice lagoon was realised: Marghera characterised by high contamination levels of industrial origin, Campalto located close to a sewage treatment plant and Poveglia benchmark site, located near one of the lagoon mouth. Results showed that Marghera and Campalto sediments were more contaminated by NPs and PAHs than Poveglia. In each sampling date, the highest NP concentrations (64-247 ng/g d.w.) were measured in Marghera sediments while the lowest one (28-99 ng/g d.w.) in Poveglia. The higher mean concentrations of PAHs were found in Marghera sediments in April and June (516 and 364 ng/g d.w. respectively), while in October and January the higher values were detected in Campalto, with a mean PAH concentration of 821 and 668 ng/g d.w., respectively. Different trends were observed in the contamination of clams: the Poveglia site, in April, showed the highest NP and PAH contamination with values, respectively, of 181 and 1160 ng/g f.w.. From these values, BSAFs were calculated, which resulted always highest at Poveglia for both classes of contaminants. The BSAF trend appears to be inversely related to the contamination level of the sites. Environmental stressors can have an impact on the health status of the clams, altering their normal feeding behavior and, as a probable consequence, reducing the bioaccumulation rates. This could mean that the concentration measured in biota are not necessarily representative of the biota exposure with the risk of losing the direct correlation between sediment and biota concentrations in contaminated sites.

Nonylphenol (NP) is reported to exert estrogenic effects in marine organisms, being able to mimic the action of endogenous estrogens. As a consequence, NP can induce vitellogenin (Vg) synthesis, the major precursor of the egg-yolk proteins. In our previous study [Matozzo and Marin, 2005. Environ. Res., 97, 43-49], we found that exposure for 7 days of the clams Tapes philippinarum to NP caused a dose-related induction of Vg in both haemolymph and digestive gland of males. In the present study, Vg induction was evaluated in sexually undifferentiated clams exposed to NP: for 7 days to 0, 0 + acetone, 0.025, 0.05, 0.1, and 0.2 mg NP/L and for 14 days to 0, 0 + acetone, 0.025, 0.05, and 0.1 mg NP/L. Vg was determined in both haemolymph and digestive gland by the alkali-labile phosphate (ALP) assay. In the haemolymph Ca2+ levels were also measured. Exposure for 7 days to 0.2 mg NP/L resulted in significant increases (p < 0.05) in ALP in both aemolymph and digestive gland with respect to controls, whereas no difference was observed in Ca2+ levels. After 14 days exposure, ALP significantly (p < 0.05) increased in digestive gland from 0.05 and 0.1 mg NP/Lexposed clams. Increases (p < 0.05) in Ca2+ levels were recorded in haemolymph from clams exposed to 0.025 and 0.05 mg NP/L. Results obtained demonstrates that NP induces Vg synthesis in T. philippinarum also far from the reproductive period. As endocrine disruption may cause fertility reduction and alteration in sex ratio, a condition of potential risk for clam populations in estuarine areas is highlighted. This study was supported by grants from Co.Ri.La (Second Research Programme, 2004-2006).

The impact of experimental dredging was assessed on the venus clam Chamelea gallina from seabeds of the Lagoon of Venice. Commercial-sized clams were collected using high water pressure and mechanical sorting (HP), as in professional fishing, and low water pressure without sorting (LP). To highlight the effects of acute stress due to the different approaches in harvesting clams, various biological responses were evaluated at both organism and cell level. Filtration and respiration rates showed opposite behaviour,the former significantly decreasing, the latter increasing, when mechanical stress was higher. Consequently, lower values of scope for growth in HP clams were observed. The survival in air test exhibited significantly lower median survival time values (LT50) in more severely stressed animals. The haematocrit value and phagocytic index significantly decreased in HP samples, revealing a detrimental effect on mechanisms of immunological defence. Acid phosphatase and b-glucuronidase activity was also affected by increasing mechanical stress, even though an opposite trend was shown in the two investigated enzymes. Lastly,higher damage levels were detected in shells of HP clams.

In the last few years, the Manila clam (Tapes philippinarum) has been subjected to intense fishing effort in the Lagoon of Venice owing to their commercial importance. Because of the lack of data concerning fishing-induced immunomodulation in bivalves, the consequences of fishing impact on functional responses of clam haemocytes were investigated. Clams were seasonally collected from September 2000 to July 2001 from three sites: S. Angelo, a freefishing area, and Chioggia, inside a licensed area for clam culture, in two zones characterised by fishing and nonfishing, respectively. Haematocrit, phagocytosis, Neutral Red retention time, and lysozyme and superoxide dismutase activities were evaluated. No clear fishing effect was recorded, whereas a seasonal pattern of the cell parameters analysed was revealed. Alterations in functional responses of haemocytes seem to be more closely dependent on seasonal variations in both environmental parameters and physiological status of clams than on stress caused by fishing, and they may also be related to adaptation strategies of bivalves.

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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.

Environmental stresses are responsible of HSPs induction, that guarantee tolerance of otherwise lethal conditions. This study was performed to evaluate HSP70 expression in Chamelea gallina (Bivalvia) digestive gland in response to oxygen, temperature, salinity changes and B[a]P exposure. The results obtained confirm general induction of HSP70 in all the stress conditions tested.

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.

SOD activity and expression were evaluated in haemocytes of Chamelea gallina exposed for seven days to three different temperatures. The values recorded for the acitivity of Total SOD, Mn-SOD and CuZn-SOD were significantly lower in specimens kept at 30° C. Even the results of immumological analysis (Western Blotting) revealed a minor expression at the end of the exposure at the same temperature for Mn-SOD, CuZn-SOD and EC-SOD. The high mortality recorded at 30° C is probably related to a lower phagocytosis activity and to a lower production of ROS, a useful defence against foreign elements.

I ROS rivestono un importante ruolo microbicida e allo stesso tempo sono responsabili di molteplici danni a diverse macromolecole biologiche (stress ossidativi). Per contrastare lo stress ossidativo gli organismi aerobi dispongono di sistemi di difesa. In particolare la superossido dismutasi (SOD) è il principale enzima coinvolto nella dismutazione dello ione superossido (O2-•). Dal momento che le variazioni della tensione di ossigeno e della temperatura sono responsabili di potenziali rischi immunologici, con la presente ricerca ci proponiamo di valutare gli effetti della anossia/riossigenazione e della temperatura sull'attività ed espressione della SOD negli emociti del bivalve Chamelea gallina.