Lethal and estrogenic effects of 4-nonylphenol in the cockle Cerastoderma glaucum

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.     

Immunotoxicity of the xenoestrogen 4-nonylphenol to the cockle Cerastoderma glaucum

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.     

Lethal and estrogenic effects of 4-nonylphenol in the cockle Cerastoderma glaucum

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 LC₅₀ value was 0.3 mg 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.     

First evidence of altered vitellogenin-like protein levels in clam Tapes philippinarum and in cockle Cerastoderma glaucum from the Lagoon of Venice

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.     

First evidence of altered vitellogenin-like protein levels in clam Tapes philippinarum and in cockle Cerastoderma glaucum from the Lagoon of Venice

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.     

Immunotoxicity of the xenoestrogen 4-nonylphenol to the cockle Cerastoderma glaucum

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 () levels, acid phosphatase and lysozyme-like activities in both haemocyte lysate (HL) and cell-free haemolymph (CFH) were evaluated. Exposure of cockles to 0.1 mg/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 μm 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 levels was observed. Conversely, significant increases (p < 0.05) in acid phosphatase activity were observed in CFH from 0.05 and 0.1 mg/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/l NP. 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.     

Impact of digenean parasite infection on metallothionein synthesis by the cockle (Cerastoderma edule): a multivariate field monitoring

Metallothioneins (MT) are proteins that play an important role in metabolism of essential metals and detoxification of trace metals from living organisms. Their synthesis is induced by metal pollution but can also be exacerbated by other factors such as reproduction processes. In this context, we monitored MT concentrations in a cockle Cerastoderma edule (marine bivalve) population and highlighted the effect of a castrating digenean parasite, Labratrema minimus. In spent cockles, MT levels were low (ca. 5 nmol sites g(-1), fresh weight) but slightly higher in parasitized individuals. During gametogenesis, MT synthesis increased in all cockles, but concentrations were lower in parasitized individuals (18 against 27 nmol sites g(-1), fw in unparasitized cockles) in relation with gonad damage by parasites. Therefore, it is suggested that parasite infection in cockles can modulate MT synthesis that could consequently interfere with the response of these protective proteins in case of metal contamination.     

Lethal and sublethal effects of dredging on reef corals

Effects of dredging on a coral reef are described. Under water light values at a depth of 12–13 m were reduced from about 30% to less than 1% surface illumination. Colonies of coral species which are inefficient sediment rejectors (Porites astreoides) lost their zooxanthellae and died. Calcification rates in Madracis mirabilis and Agaricia agaricites were observed to decrease by 33%. The period of suppressed calcification exceeds that of environmental disturbance.     

Exposure of the clam Tapes philippinarum to 4-nonylphenol: changes in anti-oxidant enzyme activities and re-burrowing capability

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.     

Estrogen equivalent concentration of individual isomer-specific 4-nonylphenol in Ariake sea water, Japan

Concentrations of 4-nonylphenol (NP) were determined by isomer-specific quantification of individual NP isomers based on relative response factor (RRF) quantification with GC–MS in combination with steam distillation extraction. Concentrations of NP in the Ariake Sea decreased with distance from the river mouth (St.A; 49 ng NP/l) to offshore areas (St.C; 11 ng NP/l). Even the least concentration in water from St.C in Ariake Sea was sufficient to have adverse effects on barnacles. The isomers, NP1–NP14 were separated by GC–PFC and identified structurally with NMR. The isomers varied in estrogenic activity with NP7 exhibiting the greatest estrogenic activity with a potency that was approximately 1.9 × 10⁻³ that of 17β-estradiol (E2) in recombinant yeast screen system. The coefficient of variation (CV) of NP isomer’s concentrations among three samples at St.A, B and C were 4–75%. This suggests that NP isomers might be independently degraded in aquatic environmental samples. The predicted estrogenic activity of measured concentrations of NP in Ariake Sea was 2.7–3.0-fold greater than the measured estrogen agonist activity.     

Effects of 4-nonylphenol exposure in mussels (Mytilus galloprovincialis) and crabs (Carcinus aestuarii) with particular emphasis on vitellogenin induction

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 LC₅₀ 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 μg g⁻¹ d.w. in mussels, and from 3.6 to 37 μg 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.     

Phenolic endocrine disrupting chemicals (EDCs) in Anzali Wetland, Iran: elevated concentrations of 4-nonylphenol, octhylphenol and bisphenol A

We have studied the distribution and value of phenolic endocrine disrupting chemicals (EDCs) in surface sediment samples taken from Anzali Wetland, Iran. These samples were collected from 22 stations during the time span of June-May 2010. In each of the sampling stations, we detected 4-nonylphenol (4-NP), octylphenol (OP), and bisphenol A (BPA) with maximal concentrations of 29, 4.3, and 7 μg g(-1) dry weight (dw), respectively. High levels of alkylphenols (APs) and BPA were also found near urban areas. Furthermore there were no significant differences between those stations in terms of the detected levels. One of the important factors in controlling the fate of these compounds in the aquatic environment appeared to be Total Organic Carbon (TOC). Hierarchical cluster analysis showed differences in the biomarker characteristics of EDCs and TOC between the stations. Our findings indicate that EDCs are ubiquitous in sediments from northeast Wetlands of Iran, contaminating the aquatic habitats in this area.     

Effects of 4-nonylphenol exposure in mussels (Mytilus galloprovincialis) and crabs (Carcinus aestuarii) with particular emphasis on vitellogenin induction

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.     

Effects of 4-nonylphenol exposure in mussels (Mytilus galloprovincialis) and crabs (Carcinus aestuarii) with particular emphasis on vitellogenin induction

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 LC₅₀ 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 μg g⁻¹ d.w. in mussels, and from 3.6 to 37 μg 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.     

Levels of estrogenic compounds in Xiamen Bay sediment, China

Concentrations of seven estrogenic compounds, i.e., estrone (E1), 17β-estradiol (E2), 17α-ethynylestradiol(EE2), diethylstilbestrol (DES), nonylphenol (NP), octylphenol (OP) and bisphenol A (BPA), were determined in sediments and pore water from Xiamen Bay in China, and their distributions and potential risks in the Bay were assessed. Total estrogenic compounds concentrations varied from 49.20 to 1230.69 ng/g dw in sediments and from 102.33 to 4376.60 ng/L in pore water. The highest levels of these compounds were found at Yundang Lagoon. The results showed that estrogenic compounds in Xiamen Bay originated mainly from municipal wastewaters. Compared with other areas, Xiamen Bay was contaminated with high levels of estrogen hormones. This contamination poses a potential threat to benthic organisms. Although a good relationship (r = 0.94) was observed between the estrogenic compounds concentrations and total organic carbon (TOC) contents in sediments, which did not indicate that the sediment organic matter favors the accumulation of the detected estrogenic compounds.     

Occurrence of ten metals in Mytilus edulis L. and Cardium glaucum L. from the Gdańsk Bay

The determination of Fe, Mn, Ni, Pb, Cu, Co, Zn, Cd, Ca and Mg was carried out in the soft tissues and shells of Mytilus edulis and Cardium glaucum from Gdańsk Bay. This paper reports on the influence of size (age) on the metal concentration in molluscs. Both species-dependent variability and regional variations of metal concentrations in molluscs were observed. The correlation coefficients between the concentrations of metal in the soft tissues and shells were determined. The correlation coefficients between hard and soft parts metals were calculated for Mytilus edulis.     

Lethal Effects of Zinc and Nickel on Freshwater Teleosts

Static bioassays were conducted at 20.5£C and the hardness of 260 ppm as CaCO₃ with zinc sulphate and nickel sulphate using Puntius sophore, Rasbora daniconius neilgeriensis, Channa punctatus and Lebistes reticulates. The 96 h LC50 values for all four fish species ranged from 29.88 to 54.95 £ of zinc and 13.57 to 48.83 £ of nickel. Channa punctatus were consistently more sensitive to zinc, while Puntius sophore were to nickel. The behavioural changes, such as loss of equilibrium and shoaling nature, swimming with their bellies upwards and irregular opercular movement were noted.     

Loading effects in Metsähovi from the atmosphere and the Baltic Sea

Loading by atmosphere and by the Baltic Sea cause gravity change at Metsähovi, located 15 km from the open sea. Gravity is changed by both the Newtonian attraction of the loading mass and by the crustal deformation. We have performed loading calculations using appropriate Green's function for both gravity and deformation, for both atmospheric and Baltic loading. The loading by atmosphere has been computed using a detailed surface pressure field from high resolution limited area model (HIRLAM) for north Europe up to 10° distances. Baltic Sea level is modelled using tide gauge records. Calculations show that 1 m of uniform layer of water corresponds to 31 nm s⁻² in gravity and −11 mm in height. Modelled loading is compared with observations of the superconducting gravimeter T020 for years 1994–2002. The combination of HIRLAM and a tide gauge record decreases RMS of gravity residuals by 14% compared to single admittance in air pressure corrections without sea level data. Regression of gravity residuals on the tide gauge record at Helsinki (at 30 km distance) gives a gravity effect of 26 nm s⁻² m⁻¹ for Baltic loading.The gravity station is co-located with a permanent GPS station. We have also associated the loading effects of the atmosphere and of the Baltic Sea with temporal height variations. The range of modelled vertical motion due to air pressure was 46 mm and that due to sea level 18 mm. The total range was 38 mm. The effects of the Baltic Sea and of the atmosphere partly cancel each other, since at longer periods the inverse barometer assumption is valid. Regression of the modelled height on local air pressure gives −0.37 mm hPa⁻¹, corresponding approximately to width 6° for pressure system.We have tested the models using one year of daily GPS data. Multilinear regression on local air pressure and sea level in Helsinki gives the coefficient −0.34 mm hPa⁻¹ for pressure, and −11 mm m⁻¹ for sea level. These match model values. Loading by air pressure and Baltic Sea explains nearly 40% of the variance of daily GPS height solutions.     

First evidence of altered vitellogenin-like protein levels in clam Tapes philippinarum and in cockle Cerastoderma glaucum from the Lagoon of Venice

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.