Gut bacterial community structure (Porcellio scaber, Isopoda, Crustacea) as a measure of community level response to long-term and short-term metal pollution

Prokaryotes are of high importance in the assessment of environmental pollution effects. Due to fast responsiveness of bacterial communities to environmental physicochemical factors, it is difficult to compare results of bacterial community investigations on the temporal and spatial scale. To reduce the effects of variable physicochemical environmental conditions on bacterial microbiota when investigating the specific impact of contaminants on bacterial communities, we investigated the bacterial community in the gut of terrestrial isopods (Porcellio scaber, Isopoda, Crustacea) from clean and metal-polluted environments. Animals were collected from a chronically mercury-polluted site, a chronically multiple metal-(Cd, Pb, Zn) polluted site, and two reference sites. In addition, animals from an unpolluted site were laboratory exposed to 5 microg Hg/g food in order to compare the effect of acute and chronic Hg exposure. The bacterial gut microbiota was investigated by temporal temperature gradient gel electrophoresis (TTGE) and clone library construction based on polymerase chain reaction amplified 16S rRNA genes. The major bacterial representatives of the emptied gut microbiota in the animals from the chronically polluted environments seemed not affected when analyzed by TTGE. The detailed bacterial community structure investigated by 16S rRNA clone library construction, however, showed that the community from the Hg-polluted site also was affected severely (242.4 operational taxonomic units [OTU] in the polluted and 650.6 OTU in the unpolluted environment). When animals were acutely exposed to mercury, changes of bacterial community structures already were seen on TTGE profiles and no additional analysis was needed. We suggest the use of P. scaber gut bacterial community structure as a measure of effects caused by both long- and short-term exposure to pollution.     

Lysosomal responses in the digestive gland of the freshwater mussel, Dreissena polymorpha, experimentally exposed to cadmium

In order to examine the possible use of lysosomal response as a biomarker of freshwater quality, structural changes of lysosomes were measured by image analysis in the digestive gland of the zebra mussel, Dreissena polymorpha, exposed in laboratory conditions to cadmium. Mussels were exposed to the metal (10 and 200 microg/L) for 3 weeks and randomly collected after 7 and 21 days. At each treatment day, digestive tissues were excised and beta-glucuronidase activity was revealed in cryotome sections. Four stereological parameters were calculated: lysosomal volume density, lysosomal surface density, lysosomal surface to volume ratio, and lysosomal numerical density. The changes observed in this study reflected a general activation of the lysosomal system, including an increase in both the number and the size of lysosomes in the digestive gland cells of mussels exposed to cadmium. The digestive lysosomal response in zebra mussels was related to exposure time and to metal concentration, demonstrating the potential of this biomarker in freshwater biomonitoring.     

Lysosomal responses as a diagnostic tool for the detection of chronic petroleum pollution at Todos os Santos Bay, Brazil

Coastal marine environments, especially semienclosed systems such as bays, are under unrelenting stress caused by urban and industrial development. Biomonitoring plays a vital role in strategies to identify, assess, and control stressors. However, due to the magnitude of the challenge there is a demand for new and innovative approaches to provide timely and accessible information to environmental managers and policy makers. The present work aimed to assess hydrocarbon levels in sediments from petroleum-related industrial areas at Todos os Santos Bay (Brazil) and associate them to toxicity-induced responses (neutral red retention (NRR) assay) by the burrowing clam Anomalocardia brasiliana. Surface sediments collected during the dry and rainy seasons were analyzed for aliphatic and aromatic hydrocarbons. At the control site, hydrocarbon levels were low and mainly biogenic. The aliphatic hydrocarbon ("total unresolved complex mixture," alkanes, and isoprenoids) concentrations indicated a chronic situation with very little "fresh" oil contamination at the oil-related sites. The polycyclic aromatic hydrocarbons indicated sites moderately contaminated by chronic oil and some pyrolytic input. The effects of those contaminants were assessed by the lysosomal NRR assay applied to A. brasiliana hemocytes. Sediment toxicity at the oil-related sites was evidenced by the lowered capacity of the lysosomes to retain the neutral red dye compared to results from the control site. This research indicates that the NRR assay is a useful and efficient screening technique able to discriminate polluted from clean sites.     

Airborne total gaseous mercury and exposure in a Venezuelan mining area

This paper presents a short-term monitoring study of total gaseous mercury (TGM) in air, and exposure to airborne mercury. The evaluation was carried out in polluted mining sites (El Callao, Venezuela), where for decades mercury has been used in diverse stages of gold mining activities. The contamination is mainly due to emission of Hg0 during gold amalgamation and burning, which can cause direct human health risks. Total gaseous mercury (TGM) in air was analysed in mill, jewellery and indoor house sites, and at different heights (height profiles near the surface) at polluted and not polluted sites. Mercury concentration in air was measured with a portable mercury analyser (Lumex Ra-915+). Time weighted average mercury (TWA) was calculated for the evaluation of mercury exposure. TWA values ranged between 0.28 microg m(-3) and >100 microg m(-3). These measurements were done during sunny and dry days. In the case of mills and gold workshops, the values were over the limit recommended by the World Health Organization to exposure (25 microg m(-3)) and NIOSH limit (50 microg m(-3)). Indoors in a house, the air Hg average value was 2.58 microg m(-3) exceeding EPA (0.3 microg m(-3)) and ATSDR (1 microg m(-3)) guidelines. The mercury concentration at different height profiles, varied between 1766 microg m(-3) and 0.014 microg m(-3). Mercury height profiles were described by a power function model of the form c(Hg) = ah(-b), where a parameter describes the magnitude of Hg emission. For polluted sites there was a significant correlation between a and Hg in soil or Hg emission from soil to air, while b is only significantly correlated with air temperature. An air and soil mercury measurement transect was carried out at a mill site up to a distance of 1000 m, and it was observed that the air mercury concentration decreases with increasing distance from the mill site, and inversely to Hg soil content.     

Hemocytic Lysosome Response in the Snail Helix aspersa After Exposure to the Fungicide Copper Oxychloride

This study investigated the use of lysosomal responses of hemocytes of the common garden snail, Helix aspersa, as biomarker of stress due to exposure to the fungicide copper oxychloride. The neutral red retention (NRR) time assay was employed for this purpose. Two groups of snails were exposed to 80 μg g⁻¹ and 240 μg g⁻¹ copper oxychloride in their food, respectively, for a period of 6 weeks. They were compared with a control group to which no copper oxychloride was added. The two groups exposed to the fungicide exhibited significantly higher (p < 0.001) whole body copper concentrations (200.85 ± 53.5 and 272.24 ± 67.15 μg g⁻¹ dry mass, respectively), and significantly shorter (p < 0.001) NRR times (10.22 ± 3.53 and 2.67 ± 2.83 min, respectively), after 6 weeks, compared to the control group (67.85 ± 31.08 μg g⁻¹ dry mass and 24.44 ± 8.35 min). In both exposure groups NRR times became progressively shorter as body copper concentrations increased over time. Thus, both exposure concentration and exposure time of copper oxychloride were shown to be important factors influencing lysosomal responses (and therefore NRR times) of H. aspersa hemocytes. It was concluded that these responses in this species, as measured by the NRR time assay, could be considered a useful cellular biomarker of stress resulting from exposure to copper oxychloride. Received: 8 December 1999/Accepted: 31 May 2000     

Isopod gut microflora parameters as endpoints in toxicity studies

Terrestrial isopods Porcellio scaber (Crustacea) were fed for five weeks on food contaminated by 250, 500, or 1,000 microg of Cd/g or for 10 d on diets with 50 or 250 microg Cd/g food. In both experiments, fecal production rate and colony forming units (CFUs) in the guts were determined. In addition, at the end of 10 d, each distinct colony morphotype obtained in gut samples was purified and characterized. Isolates were separated into 25 groups based on morphological and biochemical characteristics. These bacterial groups were used as units for calculating Shannon equitability indices (J) for each gut. The relative frequencies of the 25 bacterial units were determined in both cadmium groups (50 or 250 microg Cd/g food) and in the control. Cadmium-induced perturbations observed in the gut microbial communities were (1) increased number of morphologically distinct bacterial isolates in the group fed low-cadmium-dosed food (50 microg Cd/g) and reduced number of morphologically distinct bacterial isolates in the group fed high-cadmium-dosed food (250 microg Cd/g) compared with the control, (2) increased or decreased relative frequencies of almost all 25 bacterial units provoked by cadmium-contaminated food, (3) time-dependent increased numbers of gut CFUs in cadmium-fed animals (dose dependence was not observed), and (4) significant changes in community structure described by Shannon equitability indices at lower levels of food contamination (50 microg Cd/g) only. Gut microflora parameters are proposed as additional endpoints in the standardized single-species toxicity test with the terrestrial isopod P. scaber as a means of increasing the ecological relevance of the results.     

Reduction and methylation of mercury in the terrestrial isopod Porcellio scaber (Crustacea) and its environment

Reduction and methylation of inorganic mercury in Porcellio scaber (Isopoda, Crustacea) and its environment were studied, using a purpose-built experimental setup where Hg cycling was followed using 203Hg2+ tracer in experiments without and with isopods. In experiment without isopods, daily reduction of 203Hg2+ to 203Hg0 under sterile and nonsterile conditions was measured for three weeks to assess the contribution of bacteria to this process. In experiments with isopods, daily release of 203Hg0 was measured for two weeks. Total mercury (T203Hg) and monomethylmercury (Me203Hg) in whole animals, gut, digestive glands (hepatopancreas), food (hazelnut leaves), and feces were measured to obtain the assimilation and distribution of mercury in the animals, to investigate the origin and fate of Me203Hg, and, finally, to assess the mass balance of mercury in the experimental system. Experiment without isopods showed the important role of bacteria in reduction of 203Hg2+ to 203Hg0, especially in the first day of the experiment. Experiments with isopods showed that formation of 203Hg0 depended on the 203Hg2+ concentration in the food. The contribution of the isopod's digestive flora in reduction of 203Hg2+ to 203Hg0 was negligible. Approximately 3% of T203Hg and 2% of Me203Hg consumed was assimilated by the animals. Methylation of 203Hg2+ occurred already in the leaves before they were consumed by the isopods. Assimilation of Me203Hg from the food surprisingly was low. Also, a loss of Me203Hg was noticed when comparing assimilated and excreted Me203Hg versus consumed Me203Hg. This may be explained by the assumption that demethylation of MeHg prevailed over methylation of Hg2+ in the animal's digestive system, leading to excretion of ingested mercury as Hg2+.     

Effect of Environmental Pollutant Mixtures on Acid DNase Activity in Mussel <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis>: Ex Situ and In Situ Study

Acid DNase activity in marine organisms displays a sensitive response to pollutants. In the present study, ex situ effect of toxic seawater organic extracts (Rijeka, Vranjic, Plo?e) and in situ impact of environmental factors at referent (St. Andrew) and a polluted site (ACI Rovinj) on acid DNase in the digestive gland and gills of mussels was investigated. In the digestive gland, enzyme activity was related to potential toxicity of seawater organic extracts. Seawater organic extracts showed an increase of the enzyme activity in digestive gland following exposure. In in situ study, polluted environment affected mussel’s acid DNase activity in digestive gland, when compared to gills. Acid DNase activity in the digestive gland expressed significant difference between the referent and polluted site, particularly from early spring through summer. Both ex situ and in situ studies displayed more sensitive acid DNase response to the presence of toxic mixtures in the digestive gland.     

Relationship between lysosomal membrane destabilization and chemical body burden in eastern oysters (Crassostrea virginica) from Galveston Bay,Texas, USA

Lysosomal destabilization was measured by using hemocytes of eastern oysters (Crassostrea virginica) collected along a chemical concentration gradient in Galveston Bay, Texas, USA. Results of the lysosomal response were compared to concentrations of organic compounds and trace elements in oyster tissue. Concentrations (on a dry-wt basis) ranged from 288 to 2,390 ng/g for polycyclic aromatic hydrocarbons (PAHs), 38 to 877 ng Sn/g for tri-n-butyltin (TBT), 60 to 562 ng/g for polyclorinated biphenyls (PCBs), and 7 to 71 ng/g for total DDT. Trace element concentrations (on a dry-wt basis) ranged from 1.1 to 4.0 microg/g for Cd, 105 to 229 microg/g for Cu, 212 to 868 microg/g for Al, and 1,200 to 8,180 microg/g for Zn. The percentage of destabilized lysosomes ranged from 34 to 81%. A significant positive correlation (p < 0.05) was observed between lysosomal destabilization and body burden of organic compounds (PAHs, PCBs, TBT, and chlorinated pesticides). No significant correlation was found between metal concentrations and lysosomal destabilization. Based on lysosomal destabilization, the study sites in Galveston Bay can be placed in one of three groups: healthy (Hanna Reef and Confederate Bay), moderately damaged (Offats Bayou and Todd's Dump), and highly damaged (Yacht Club and Ship Channel). Lysosomal destabilization that is consistent with toxic chemical body burdens supports previous observations that lysosomal membranes are damaged by toxic chemicals and indicates that this method can serve as an early screening tool to assess overall ecosystem health by using oysters.     

Mercury, selenium, and cadmium in human autopsy samples from Idrija residents and mercury mine workers

Total Hg and Se concentrations were determined in autopsy samples of retired Idrija mercury mine workers, Idrija residents living in a Hg-contaminated environment, and a control group with no known Hg exposure from the environment. In selected samples we also checked the presence of MeHg. The highest Hg concentrations were found in endocrine glands and kidney cortex, regardless of the group. MeHg contributed only to a negligible degree to the total mercury concentrations in all analyzed samples. In the Hg-exposed groups the coaccumulation and retention of mercury and selenium was confirmed. Selenium coaccumulation with a Hg/Se molar ratio near 1 or higher was notable only in those tissue samples (thyroid, pituitary, kidney cortex, nucleus dentatus) where the mercury concentrations were >1 microg/g. After tissue separation of such samples the majority of these elements were found in the cell pellet. Because the general population is continuously exposed to Cd and possibly also to Pb from water, food, and/or air, in some samples the levels of these elements were also followed. In all examined control tissue samples the average values of Cd (kidney cortex, thyroid, hippocampus, cortex cerebellum, nucleus dentatus) and Pb (thyroid, hippocampus) exceeded the average values of Hg. Cd concentrations were the highest, particularly in kidney cortex and thyroids (microg/g), but no relationship between Cd and Se concentration was evident at the tissue level. Regarding the results in the control group, it is debatable which element is the more hazardous for the general population as concerns neurotoxicity.     

Atmospheric distribution and deposition of mercury in the Idrija Hg mine region, Slovenia

The atmospheric distribution and deposition of Hg in the area of the former Idrija Hg-mine, Slovenia, were investigated. Mapping of air Hg⁰ concentrations was performed to assess the spatial distribution and major sources of mercury to the atmosphere in the area. In addition, analyses of mercury speciation in the air over Idrija were performed during a 4-day sampling campaign in September 2006 to better understand the fate and transformation of Hg in the atmosphere of this specific mercury polluted site. The speciation results were then compared to the results of mercury speciation in the wet and throughfall deposition sampled on a precipitation event basis from October 2006 to September 2007. The Hg⁰ concentration in air was mostly below 10 ng m⁻³, with the highest concentration in the area of the former smelter complex exceeding 5000 ng m⁻³. Mercury-bearing airborne particles (TPM) seem to dominate the atmospheric Hg deposition, which revealed noticeable variations between precipitation events (11–76 ng m⁻² day⁻¹), mostly as a function of the amount of precipitation. Hg in precipitation was largely (∼50%) associated with the particulate phase (THg_P). No correlation was found between the THg_P and the dissolved phases (THg_D), suggesting that particulate phase Hg is mostly the result of dry deposition. In the throughfall, significantly higher (2–10 fold) Hg concentrations than in associated event precipitation were observed, mostly due to Hg in the particulate phase (∼70% THg). As shown by SEM/EDXS microscopy, an important amount of mercury in the precipitation and throughfall samples is due to the presence of cinnabar particles as a result of the aeolian erosion of cinnabar-containing surfaces in the area.     

Pyrene biotransformation products as biomarkers of polycyclic aromatic hydrocarbon exposure in terrestrial Isopoda: concentration-response relationship,and field study in a contaminated forest

In this study, biotransformation products of pyrene were measured in the hepatopancreas of terrestrial isopods as biomarkers of polycyclic aromatic hydrocarbon (PAH) exposure. These products--pyrene-1-glucoside, pyrene-1-sulfate, an unknown pyrene conjugate, and 1-hydroxypyrene--were quantitated using high-performance liquid chromatography (HPLC) with fluorescence detection. In a controlled exposure experiment, a linear relationship was established between pyrene exposure and pyrene metabolite concentrations in the hepatopancreas of Porcellio scaber Latr. To this end, isopods of the species P. scaber were exposed to a range of pyrene concentrations spiked to their food. A linear response was found for all pyrene conjugates in the range 0.67 to 67 microg/g of pyrene (dry wt). Hepatopancreatic pyrene metabolite concentrations were also measured in isopods (P. scaber and Oniscus asellus L.) from PAH-contaminated field sites. The sites and the inhabiting isopods were located in a gradient of atmospheric PAH deposition caused by a nearby blast furnace plant. The highest levels of conjugated 1-hydroxypyrene in the hepatopancreas were 3.8 pmol/g fresh weight (pyrene-1-glucoside) and 2.8 pmol/g fresh weight (pyrene-1-sulfate) (expressed on whole-body basis). The levels of the pyrene metabolites correlate with reported pyrene concentrations in spite of these sites. As pyrene is one of the most predominant PAHs, analysis of its metabolites provides a good tool for environmental risk assessment of ecosystems with regard to PAH exposure, bioavailability, and biotransformation.     

Inhibition of acetylcholinesterase activity in the terrestrial isopod Porcellio scaber as a biomarker of organophosphorus compounds in food

This paper describes the toxicity of organophosphorus pesticide diazinon in juvenile and adult terrestrial isopods Porcellio scaber (Isopoda, Crustacea). The woodlice were exposed to different concentrations of diazinon added to food (5, 10, 50, and 100 or 150 micrograms/g dry food). Weight change and food assimilation efficiency were determined two and four weeks after the exposure. The activity of acetylcholinesterase (AChE) in surviving animals was measured at the end of the experiment. The results show that woodlice exposed to diazinon do not significantly differ from controls in growth and feeding rate. The reduction of AChE activity was observed at the lowest diazinon exposure (5 and 10 micrograms/g dry food). These results suggest that AChE activity might prove a useful biomarker, indicating low levels of organophosphates in food.     

How terrestrial snails can be used in risk assessment of soils

Among soil invertebrates, terrestrial snails are herbivorous and detritivorous organisms exposed to polluted soils by both digestive and cutaneous routes. Using laboratory-reared snails (Helix aspersa aspersa), we describe how the effects of contaminants on survival and growth of snails can be evaluated in laboratory bioassays. A national ring test was performed to assess the effect of Cd added to the soil or to the food. The ecotoxicity of sewage sludge also was evaluated. The present results demonstrate that toxicity depends on both the pollutants and the exposure route. Cadmium was sixfold more toxic for snails exposed via food contamination (median effective concentration [EC50], 68-139 microg/g) than via soil contamination (EC50, 534-877 microg/g), whereas the opposite occurred with the sewage sludge (EC50, 55% of sludge in the food and 10% of waste in the soil). A logistic relationship linked growth inhibition and internal Cd concentrations, which can reach 2,000 microg/g in the viscera of snails exposed to 626 microg/g in the food. No clear trend was found between Cu, Zn, Pb, Cr, and Ni concentrations in the sludge and in snail tissues. These data enabled the development of an international standard, which should enhance the use of terrestrial gastropods for both fundamental research and routine risk assessment in the terrestrial environment.     

Assessment of seasonal variability of cytochemical responses to contaminant exposure in the blue mussel Mytilus edulis (complex)

A selected suite of cytochemical parameters in Mytilus edulis are altered in response to field and laboratory exposure to chemical contaminants. These biomarkers include lysosomal stability, nicotinamide adenine dinucleotide phosphate (NADPH)-ferrihemoprotein reductase activity, liposfuscin deposition, and accumulation of lysosomal and cytoplasmic unsaturated neutral lipid. Normal variations in physiological processes (influenced by exogenous seasonal changes in temperature, salinity, food availability, etc.) may alter the sensitivity of these biomarkers to contaminant exposure. To address this issue, M. edulis (complex) were sampled monthly from a reference nonurban site (Coupeville, Penn Cove) and a polluted urban site (Seacrest, Elliott Bay) in Puget Sound, WA, for a period of 15 months. Physiological measurements including total length, total weight, somatic and mantle weights (an indication of gonadal development and reproductive status), condition index, and the presence or absence of hemic neoplasia (HN, or leukemia) were recorded. Significant differences in lysosomal stability, lysosomal and cytoplasmic unsaturated neutral lipids, lipofuscin deposition, and NADPH-ferrihemoprotein reductase activity in cells of the digestive gland or digestive tubules were generally found in mussels taken throughout the year from Seacrest compared to mussels sampled from Coupeville, consistent with exposure to chemical contaminants. No seasonally influenced suppression of the entire suite of parameters as measures of contaminant exposure was evident. Therefore these biomarkers can be used to evaluate contaminant exposure in mussels throughout the entire year.     

Lysosomal response of earthworm (Eisenia fetida) coelomocytes to the fungicide copper oxychloride and relation to life-cycle parameters

Juvenile specimens of the earthworm species Eisenia fetida Savigny, 1826, were exposed in the laboratory to a range of concentrations of the fungicide copper oxychloride. After an eight-week exposure period, the neutral-red retention (NRR) times were measured in the coelomocytes of all the worms that had matured. Life-cycle traits (survival, biomass change, cocoon production, cocoon mass, hatching of cocoons, number of hatchlings) were also monitored. Dose-related effects on NRR times, maturation, growth, and reproduction parameters were determined (one-way analysis of variance, p < 0.05). The lowest effect concentration for substrate copper (Cu) measured for the NRR time was 8.9 mg/kg (dry wt). Growth was severely affected at the highest exposure concentration (330 mg/kg substrate dry wt), at which no worms matured and no NRR times could be measured. Negative linear correlations (p < 0.05) were apparent between the NRR times and substrate and body Cu concentrations. Positive linear relationships were found between the NRR times and some life-cycle parameters (p < 0.05). This study showed that the NRR assay can indicate toxic stress due to Cu exposure at an early stage, and that NRR times can be linked to effects on certain life-cycle traits.     

Effect of Pollution History on Immunological Responses and Organ Histology in the Marine Mussel Mytilus edulis Exposed to Cadmium

The effect of previous toxicant exposure (i.e., exposure history) on an organism’s response to re-exposure to the toxicant is of considerable interest. The marine mussel Mytilus edulis was collected from reference and polluted sites in southwest England, and groups of mussels from each site were exposed to 20 μg/L CdCl₂ for 0, 1, 4, and 8 days and compared with unexposed controls. End points evaluated were tissue metal and electrolyte concentrations, haemolymph chemistry, haemocyte characteristics [counts, neutral red uptake (NRU), and phagocytosis], histology, and expression of metallothionein gene (mt10) expression in digestive glands. Field-collected animals differed by collection site for some end points at time zero, at which time tissue Fe and Pb concentrations were greater and NRU and condition index lower in mussels from the polluted site. Subsequent exposure to cadmium (Cd) in the laboratory caused Cd accumulation mainly in digestive gland, but there were no site-specific effects on tissue trace-metal concentrations. NRU, phagocytosis, and haemolymph Na⁺ and K⁺ concentrations differed among sites and Cd treatment, but there were no clear trends. Exposure to Cd resulted in lower Ca²⁺ concentrations in gill, digestive gland, and haemolymph in animals from the polluted site compared with controls (Kruskal–Wallis, p ≤ 0.05). Lesions, including necrosis, inflammation, and neoplasia, were observed in animals from the polluted site, but the frequency of these lesions appeared to decrease unexpectedly after Cd exposure. Expression of mt10 increased 3-fold in Cd-exposed animals from the polluted site compared with all other groups (Kruskal–Wallis, p = 0.01). We conclude that Cd exposure affected some immune responses in M. edulis, but pre-exposure history influenced toxicological outcomes of Cd exposure in the laboratory.     

DNA adducts, benzo(a)pyrene monooxygenase activity, and lysosomal membrane stability in Mytilus galloprovincialis from different areas in Taranto coastal waters (Italy)

The aim of this study was to investigate the impact of environmental pollution at different stations along the Taranto coastline (Ionian Sea, Puglia, Italy) using several biomarkers of exposure and the effect on mussels, Mytilus galloprovincialis, collected in October 2001 and October 2002. Five sampling sites were compared with a "cleaner" reference site in the Aeronautics Area. In this study we also investigated the differences between adduct levels in gills and digestive gland. This Taranto area is the most significant industrial settlement on the Ionian Sea known to be contaminated by polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls, heavy metals, etc. Exposure to PAHs was evaluated by measuring DNA adduct levels and benzo(a)pyrene monooxygenase activity (B(a)PMO); DNA adducts were analyzed by 32P-postlabeling with nuclease P1 enhancement in both gills and digestive glands to evaluate differences between DNA adduct levels in the two tissues. B(a)PMO was assayed in the microsomal fraction of the digestive glands as a result of the high expression of P450-metabolizing enzymes in this tissue. Lysosomal membrane stability, a potential biomarker of anthropogenic stress, was also evaluated in the digestive glands of mussels, by measuring the latent activity of beta-N-acetylhexosaminidase. Induction of DNA adducts was evident in both tissues, although the results revealed large tissue differences in DNA adduct formation. In fact, gills showed higher DNA adduct levels than did digestive gland. No significant differences were found in DNA adduct levels over time, with both tissues providing similar results in both years. DNA adduct levels were correlated with B(a)PMO activity in digestive gland in both years (r = 0.60 in 2001; r = 0.73 in 2002). Increases were observed in B(a)PMO activity and DNA adduct levels at different stations; no statistical difference was observed in B(a)PMO activity over the two monitoring campaigns. The membrane labilization period in mussels from some stations was decreased in both years. No statistical differences were established in the membrane labilization times from 2001 to 2002. Our results suggest the existence of different sources and amounts of environmental contaminants at the stations investigated. The formation of DNA adducts confirms the existence of activation pathways in mussels and shows the importance of DNA adduct analysis in the gill tissue in addition to the more commonly used digestive gland; these results confirm the utility of lysosomal membrane stability as a biomarker of general stress. Overall, the integrated use of biomarkers of exposure and the effects of environmental contaminants on living marine organisms may help to better interpret the impact of pollutants in a marine coastal environment.     

Assessment of Seasonal Variability of Cytochemical Responses to Contaminant Exposure in the Blue Mussel <Emphasis Type="Italic">Mytilus edulis</Emphasis> (Complex)

A selected suite of cytochemical parameters in Mytilus edulis are altered in response to field and laboratory exposure to chemical contaminants. These biomarkers include lysosomal stability, nicotinamide adenine dinucleotide phosphate (NADPH)–ferrihemoprotein reductase activity, liposfuscin deposition, and accumulation of lysosomal and cytoplasmic unsaturated neutral lipid. Normal variations in physiological processes (influenced by exogenous seasonal changes in temperature, salinity, food availability, etc.) may alter the sensitivity of these biomarkers to contaminant exposure. To address this issue, M. edulis (complex) were sampled monthly from a reference nonurban site (Coupeville, Penn Cove) and a polluted urban site (Seacrest, Elliott Bay) in Puget Sound, WA, for a period of 15 months. Physiological measurements including total length, total weight, somatic and mantle weights (an indication of gonadal development and reproductive status), condition index, and the presence or absence of hemic neoplasia (HN, or leukemia) were recorded. Significant differences in lysosomal stability, lysosomal and cytoplasmic unsaturated neutral lipids, lipofuscin deposition, and NADPH–ferrihemoprotein reductase activity in cells of the digestive gland or digestive tubules were generally found in mussels taken throughout the year from Seacrest compared to mussels sampled from Coupeville, consistent with exposure to chemical contaminants. No seasonally influenced suppression of the entire suite of parameters as measures of contaminant exposure was evident. Therefore these biomarkers can be used to evaluate contaminant exposure in mussels throughout the entire year. Received: 16 December 2001/Accepted: 30 May 2002     

Cell membrane integrity and internalization of ingested TiO(2) nanoparticles by digestive gland cells of a terrestrial isopod

The present study was motivated by the paucity of reports on cellular internalization of ingested titanium dioxide (TiO(2)) nanoparticles (nano-TiO(2)). The model invertebrate (Porcellio scaber, Isopoda, Crustacea) was exposed to food dosed with nano-TiO(2) containing 100, 1,000, 3,000, or 5,000?μg nano-TiO(2) per gram of food. After 14?d of exposure, the amount of Ti in the entire body was analyzed by inductively coupled plasma-mass spectrometry, and elemental analyses of tissue cross sections were performed by particle induced X-ray emission. In addition, a series of toxicological markers including feeding parameters, weight change, and survival, as well as cytotoxic effects such as digestive gland cell membrane stability, were monitored. Internalization of ingested nano-TiO(2) by the isopod's digestive gland epithelial cells was shown to depend on cell membrane integrity. Cell membranes were found to be destabilized by TiO(2) particles, and at higher extracellular concentrations of nano-TiO(2), the nanoparticles were internalized.