Integrated use of biomarkers (acetylcholinesterase and antioxidant enzymes activities) in Mytilus galloprovincialis and Mullus barbatus in an Italian coastal marine area

The use of biomarkers to evaluate the biological effects of chemical pollutants in marine organisms represents a recent tool in the monitoring field responding to the need to detect and assess the effects of chemical contaminants on the biota. The aim of the present work was the field application of the integrated use of acetylcholinesterase (AChE) and antioxidant enzymes (catalase--CAT, glutathione peroxidase--GSH-Px), for detecting the possible exposure/effect induced by chemical pollutants in native marine organisms from a coastal marine area, represented by Salento Peninsula (Italy), that shows a coastline of high environmental value, but under constant urban pressure, including agriculture activities, widely diffused in the whole hinterland. Eight sampling stations were chosen: four not urbanized areas considered "uncontaminated" controls and four clearly exposed to anthropogenic impact. The bioindicator species studied were a sessile invertebrate, Mytilus galloprovincialis, and a benthic teleost fish, Mullus barbatus.AChE activity in M. galloprovincialis revealed significant differences among places; the minimum values observed (3.9+/-1.8 nmolmin(-1)mg(-1)) was about 50% reduced with respect to the maximum found (11.4+/-0.9 nmolmin(-1)mg(-1)). The reduction in AChE activity observed in two control stations could be explained by the leaching of pesticides into the sea from the agricultural lands. Moreover, the inhibition of AChE activity by heavy metals besides pesticides, can also explain the reduction of the enzymatic activity observed in an industrialized and harbour area. In M. galloprovincialis AChE activity showed a significant inverse correlation with catalase activity but not with glutathione peroxidase that did not significantly change in animals sampled from the eight stations. Also in M. barbatus AChE activity showed significant differences among places; it was inversely correlated with liver GSH-Px activity, but not with catalase activity, that did not show any significantly variation in animals sampled in the different stations.In conclusion, the integrated use of AChE and antioxidant enzymes (catalase or glutathione peroxidase) in M. galloprovincialis and M. barbatus, two species living in different compartment of marine coastal ecosystem, can find a useful application within the framework of marine coastal environment monitoring programs for detecting the possible exposure/effect induced by chemical pollutants, including pesticides, on living marine organisms.     

Accumulation of polycyclic aromatic hydrocarbons by Neocalanus copepods in Port Valdez, Alaska

Sampling zooplankton is a useful strategy for observing trace hydrocarbon concentrations in water because samples represent an integrated average over a considerable effective sampling volume and are more representative of the sampled environment than discretely collected water samples. We demonstrate this method in Port Valdez, Alaska, an approximately 100 km(2) basin that receives about 0.5-2.4 kg of polynuclear aromatic hydrocarbons (PAH) per day. Total PAH (TPAH) concentrations (0.61-1.31 microg/g dry weight), composition, and spatial distributions in a lipid-rich copepod, Neocalanus were consistent with the discharge as the source of contamination. Although Neocalanus acquire PAH from water or suspended particulate matter, total PAH concentrations in these compartments were at or below method detection limits, demonstrating plankton can amplify trace concentrations to detectable levels useful for study.     

Potential for photoenhanced toxicity of spilled oil in Prince William Sound and Gulf of Alaska waters

Photoenhanced toxicity is the increase in the toxicity of a chemical in the presence of ultraviolet light (UV) compared to a standard laboratory test conducted with fluorescent lighting (minimal UV). Oil products, weathered oil, and specific polycyclic aromatic compounds present in oil are 2 to greater than 1000 times more toxic in the presence of UV. The photoenhanced toxicity of oil to fish and aquatic invertebrates appears to occur through a process of photosensitization, rather than photomodification of the aqueous phase oil. In photosensitization, the bioaccumulated chemical transfers light energy to other molecules causing toxicity through tissue damage rather than a narcosis mechanism. The available evidence indicates that phototoxic components of oil are specific 3-5 ring polycyclic aromatic hydrocarbons (PAHs) and heterocycles. Determinants of photoenhanced toxicity include the extent of oil bioaccumulation in aquatic organisms and the spectra and intensity of UV exposure. No studies have specifically investigated the photoenhanced toxicity of spilled oil in Alaska waters. Although there are substantial uncertainties, the results of this evaluation indicate there is potential for photoenhanced toxicity of spilled oil in Prince William Sound and the Gulf of Alaska. The potential hazard of photoenhanced toxicity may be greatest for embryo and larval stages of aquatic organisms that are relatively translucent to UV and inhabit the photic zone of the water column and intertidal areas. Photoenhanced toxicity should be considered in oil spill response because the spatial and temporal extent of injury to aquatic organisms may be underestimated if based on standard laboratory bioassays and existing toxicity databases. Additionally, the choice of counter measures and oil removal operations may influence the degree of photoenhanced toxicity.     

Consequences of linseed oil spills in salt marsh sediments

In a simulated spill in a salt marsh, linseed oil penetrated rapidly into the sediments at a rate of 10(-7) cm2 s(-1). The oil concentration remained unchanged for the first month after the spill, but 60% of the oil disappeared from the top 30 cm after a further month. The oil adsorbed to and accumulated in the muddy sediments (top 15 cm) leading to decreased sediment permeability, pH, Eh, abundance of plant roots and infauna and to the establishment of anoxic conditions. These changes accompanied transformations in the original fatty acid composition of the linseed oil, mainly associated with a decrease in 18 : 3omega3, an increase in the other fatty acids and the presence of 'new' fatty acids. A rapid increase in the abundance of heterotrophic aerobic and anaerobic bacteria and aerobic oil degrading bacteria, suggested that these micro-organisms degraded the oil. The role of the bacteria in oil degradation was confirmed in laboratory experiments where the fatty acids composition of the linseed oil underwent identical transformations to those obtained in the field. The degradation of linseed oil appears to be a sequential process initiated by aerobic and/or anaerobic bacteria and continued by sulphate reducing bacteria, which were unable to degrade the raw oil.     

Changes in trace metals in Thalassia testudinum after hurricane impacts

Major hurricanes Emily and Wilma hit the Mexican Caribbean in 2005. Changes in trace metals in the seagrass Thalassiatestudinum prior to (May 2004, 2005) and following passage of these hurricanes (May, June 2006) were determined at four locations along a ∼130 km long stretch of coast. Before the hurricanes, essential metals were likely limiting and concentrations of potentially toxic Pb were high in a contaminated lagoon (27.5 μg g⁻¹) and near submarine springs (6.10 μg g⁻¹); the likely sources were inland sewage disposal or excessive boat traffic. After the hurricanes, Pb decreased to 2.0 μg g⁻¹ in the contaminated lagoon probably through flushing. At the northern sites, essential Fe increased >2-fold (from 26.8 to 68.3 μg g⁻¹ on average), possibly from remobilization of anoxic sediments or upwelling of deep seawater during Wilma. Thus, hurricanes can be beneficial to seagrass beds in flushing toxic metals and replenishing essential elements.     

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.     

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 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.