Mixture toxicity assessment of cadmium and benzo[a]pyrene in the sea worm Hediste diversicolor.

In the present study, Hediste diversicolor biotransformation and anti-oxidant responses to acute exposure to cadmium (Cd) and to the polycyclic aromatic hydrocarbons benzo[a]pyrene (B[a]P) were investigated. Worms were submitted to 0.2, 0.4 and 1 microM of each contaminant and to their mixture during a period of test of 48h. Following biological responses were measured: (1) NADPH cytochrome c reductase (NADPH cyt c) activity, as phase I biotransformation parameter; (2) gluthathione-S-transferase (GST) activity as a phase II conjugation enzyme, (3) catalase activity as anti-oxidant response and (4) malondialdehyde accumulation (MDA) as lipid peroxydation marker. The cholinergic system was evaluated using the acetylcholinesterase activity (AChE). Exposure to the mixture resulted in low dose level additive effects on the investigated biomarkers. However, worms exposed to 1 microM of the single compounds and to their mixture exhibited the highest MDA accumulation and the lowest enzymatic biomarkers activities suggesting severe toxicological effects. These data should be carefully considered in view of the biological effects of mixture pollutants and particularly in marine sediment ecosystems.

Correlations between dioxin-like and indicators PCBs: potential consequences for environmental studies involving fish or sediment.

Among the numerous PCB congeners, most of the dioxin-like PCBs (DL-PCBs) need to be characterized by hyphenated techniques. It has been shown in several instances that these congeners are well related to the total PCB content in fish. We examined datasets collected mainly in France, on freshwater and marine fish and sediments. A statistical model linking DL- and indicator PCBs was developed for a dataset composed of freshwater fishes, and proved to predict well DL-PCBs from indicator PCBs in all other fish sets, including marine ones. Type II error rates remained low in almost all fish sets. A similar correlation was observed in sediments. Non-dioxin-like PCBs elicit various adverse effects and represent 95% of the total PCBs. A European guideline for them is needed; the correlation between DL- and indicator PCBs could help develop this standard in the future.

Acute effects of benzo[a]pyrene on liver phase I and II enzymes, and DNA damage on sea bream Sparus aurata.

In the present study biotransformation and detoxification responses to acute exposure to the polycyclic aromatic hydrocarbons benzo[a]pyrene (B[a]P) were investigated in the liver of Sparus aurata (sea bream). Sexually immature gilthead sea bream were treated by intraperitoneal injection of B[a]P (20 mg kg(-1)) for 6, 12, 24, and 48 h. B[a]P accumulation was quantified in sea bream liver by mean of gas phase chromatography (GPC-MS) after the various exposure periods. The following biological responses were measured: (1) ethoxyresorufin-O-deethylase (EROD) activity, as a phase I biotransformation parameter; (2) liver glutathione S-transferase (GST) activity as a phase II conjugation enzyme. DNA damage was assessed over time using the single-cell gel electrophoresis comet assay. B[a]P bioaccumulation in the liver resulted in a biphasic curve with an increasing uptake up to 5.55 +/- 0.67 microg g(-1) dry weight after only 6 h exposure and 4.67 +/- 0.68 microg g(-1) dry weight after 48 h exposure. EROD activity showed a nonsymmetrical bell-shaped kinetic with a maximum at 24 h and lower but significant activities at 12 and 48 h with respect to control animals. Hepatic GST activities were only significant after 48 h exposure. Comet assay showed an increase in liver cells DNA damage with a maximum after 48 h exposure reaching up to 12.17 % DNA in the tail.

Acute effects of cadmium on liver phase I and phase II enzymes and metallothionein accumulation on sea bream Sparus aurata.

This research was designed to study Sparus aurata (sea bream) biotransformation and detoxification responses to acute exposure to cadmium (Cd). Sexually immature gilthead sea bream were treated by intraperitoneal injection of Cd chloride (200 microg kg(-1)) for 6, 12, 24 and 48 h. Cd accumulation was quantified in sea bream liver by graphite furnace atomic absorption spectroscopy after the various exposure periods. The following biological responses were measured: (1) ethoxyresorufin-O-deethylase (EROD) activity as phase I biotransformation parameter, (2) liver glutathione-S-transferase (GST) activity as a phase II conjugation enzyme and metallothionein (MT) content as specific response to Cd contamination. Cd bioaccumulation in the liver resulted in an increasing uptake up to 10.3 microg g(-1) wet weight after 48 h of exposure. EROD showed a significant activation only after 6 h exposure and a return to control levels after 12 h. GST revealed significant activation starting from 12 h exposure. MT accumulation in liver showed the same behavior as GST activation.

Immunological and cytotoxicological responses of the Asian clam, Corbicula fluminea (M.), experimentally exposed to cadmium.

Bivalve molluscs, as filter-feeding organisms, are known to accumulate metals that can produce deleterious effects on organisms. The phagocytic activity of haemocytes and lysosomal alterations in the digestive gland cells were measured in the freshwater Asian clam exposed to cadmium, in order to assess the possible use of immunocompetence and lysosomal responses as biomarkers of freshwater quality. Clams were exposed in the laboratory to nominal concentrations of 3, 10, 21.4, 46.5 and 100 microg 1(-1) of cadmium and sampled after 7, 15 and 30 days of exposure. The results show a decrease of phagocytic activity after only 7 days of exposure to 10 microg 1(-1) of cadmium. This response was also observed as the exposure time was increased. Lysosomes in the digestive cells increased in size and number after 7 days of exposure as cadmium concentration increased. After 30 days of exposure, a decrease in size and number indicated a change in the response to the metal from concentrations of 46.5 microg 1(-1) of cadmium. A dose and time response both in phagocytic activity of haemocytes and lysosomal structure demonstrated a possible use of these biomarkers in freshwater biomonitoring.

Preliminary study of responses in mussel (Mytilus edilus) exposed to bisphenol A, diallyl phthalate and tetrabromodiphenyl ether.

Environmental pollutants with hormonal activity including bisphenol, diallyl phtalate and tetrabromodiphenyl ether, have the potential to alter gonadal development and reproduction in aquatic wildlife. Little is known about the biological impact of environmentally relevant concentrations in mussels. To investigate some aspects of their potential estrogenic action, mussels were continuously exposed during 3 weeks. Gonadal development and vitellogenin like protein levels were examined. Bisphenol (50 microg/l) induced the expression of phospho-proteins in females and spawning in both sexes. Diallyl phthalate and tetrabromodiphenyl ether decreased phospho-protein levels in both sexes and induced spawning in males. Moreover, severe damaging effects on ovarian follicles and ovocytes were observed in both bisphenol A- and tetrabromodiphenyl ether-exposed female mussels.

Scale of classification based on biochemical markers in mussels: application to pollution monitoring in Mediterranean coasts and temporal trends.

A battery of biochemical parameters was used to evaluate the response of mussels to a contaminated coastal environment. A multimarker approach was developed, establishing a scale for the classification of the water quality in European coastal sites (BIOMAR European programme). This study allows the evaluation of the temporal trends of this scale when applied to selected sites of European Mediterranean coast (BEEP Biological Effects of Environmental Pollution in Marine Coastal Ecosystems: European programme). Acetylcholinesterase activity (AChE) is highly sensitive to organophosphorus and carbamate insecticides and, to some extent, also to heavy metals. Catalase activity (CAT) and lipid oxidation (evaluated as malonedialdehyde) are markers of oxidative stress, glutathione S-transferase (GST) activity is related to conjugation of organic compounds and benzo(a)pyrene hydroxylase activity (BPH) is a marker of effect of certain planar organic compounds (e.g. polycyclic aromatic hydrocarbons, PAHs). These parameters were measured either in gills (AChE, GST) or digestive gland (BPH, GST, CAT, MDA). For each biomarker, a discriminatory factor was calculated (maximum variation range/confidence interval) and a response index was allocated. For each site, a Multimarker Pollution Index (MPI) was calculated as the sum of the response index of each of the five more discriminating biomarkers. As the result of our calculation method, the quality of the coastal environment at each site can be classified according to a five levels scale. Samples collected for five cruises in May 2001, 2002, 2003, and September 2001 and 2002 showed MPI evolutions. The results show that water quality can be classified from class 1 (clean areas in some sites of France, Italy and Spain) to class 4 (high pollution in main harbours). Results of the use of the biomarker scale in WP3 (Work Package Concernant Biomonitoring Programmes in Mediterranean Sea) during the BEEP programme make a strong contribution to the establishment of standardized strategies and methods for internationally agreed protocols for biomarker-based monitoring programmes. In comparison with scale pollution methodology used in the BIOMAR programme, the main contribution of BEEP was (1) to select from discriminatory analysis the biomarkers to be included in calculation of scale pollution; (2) to improve the use of the biomarker index in order to identify the main contaminants by analysis of individual contributions to the MPI; and (3) to apply methodology for temporal trends at sampled sites.

Monitoring pollution in Tunisian coasts: application of a classification scale based on biochemical markers.

Over the past decade, molecular, biochemical and cellular markers have been extensively used in pollution monitoring of aquatic environments. Biochemical markers have been selected among early molecular events occurring in the toxicological mechanisms of main contaminants. This paper assesses the marine environment quality along the Tunisian coasts using a statistical approach. Clams (Ruditapes decussatus) were collected during the four seasons of 2003 on seven different sites from the Tunisian coasts. Oxidative stress was evaluated in gills using catalase activity (Cat), neutral lipids and malonedialdehyde accumulation. Glutathione S-transferase activity is related to the conjugation of organic compounds and was evaluated in both, gills and digestive glands. Acetylcholinesterase activity was evaluated as the biomarker of exposure to organophosphorous, carbamate pesticides and heavy metals. For each biomarker, a discriminatory factor was calculated and a response index allocated. For each site, a global response index was calculated as the sum of the response index of each biomarker. Discriminant analysis shows significant differences between sites and seasons compared with control sample. Faroua (site 1) and Menzel Jemile (site 2) seem to be the less polluted with respect to the other sites for all seasons. Gargour (site 6) shows the highest Multimarker Pollution Index during the four seasons, indicating higher contamination level.

Biochemical and histological responses in mussel (Mytilus edulis) exposed to North Sea oil and to a mixture of North Sea oil and alkylphenols.

Several environmental chemicals are suspected to be responsible for adverse health effects on the reproductive system in various organisms. During this work, environmentally relevant concentrations of North Sea oil were used alone or in combination with alkylphenols and additional PAH to study the effect on vitellogenin-like protein expression and gonadal development in mussels. North Sea oil (0.5 ppm) induced the expression of phospho-proteins in both sexes indicating that some compounds are oestrogen-mimics. This induction was not seen in samples dosed with the mixture but signs of toxic effects were observed in the gonads. Indeed, numerous degenerating ovarian follicles in females and foci, similar to vertebrate melanomacrophage centres, were observed in testes.

Toxic effects of some major polyaromatic hydrocarbons found in crude oil and aquatic sediments on Scenedesmus subspicatus.

The green alga, Scenedesmus subspicatus was exposed for 7 days to a series of PAHs (polyaromatic hydrocarbons) of increased molecular weight from two to five rings [naphthalene (Nap), anthracene (Ant), phenanthrene (Phe), pyrene (Pyr) and benzo(a)pyrene (BaP)]. The toxicity measured as population growth inhibition by individual PAH to the S. subspicatus followed the order: BaP>Pyr>Ant>Phe>Nap. These results confirmed that the toxicity potential of PAHs seems to be strongly influenced by their physico-chemical properties (aqueous solubility, K(ow), coefficient of volatilization, etc.) and the conditions of algae culture (light, presence of nitrate ions, etc.). Consequently, Nap, Phe and Ant having low k(ow) values and low coefficient of volatilization values were less toxic than BaP with the highest k(ow) value, indicating for example why Nap with the lowest EC(50) value was nearly 2 x 10(5) times lower than that of BaP. Moreover, nitrate ions seemed to act directly on the degree of hydroxylated radical reactivity of PAHs, since BaP always remained the most toxic of the compounds tested. The results were also agreed with the QSAR model for toxicity prediction of PAHs to many aquatic organisms.

Validation of biomarkers for impact evaluation of aqueous industrial waste in mesocosms.

The use of exposure biomarkers in measuring the impact of aqueous waste holds promise because such tools have short response times, are flexible in use and may give an indication about the type of pollution. However, their ecological significance has not yet been demonstrated. It is necessary to validate these responses under controlled conditions before using such biomarkers for biomonitoring. The TotalFinaElf company has developed a pilot scheme incorporating such controlled conditions. This pilot is a dynamic open mesocosm (16 channels 40 m in length supplied with river water). The research programme currently carried out in the "Pilot Rivers" aims at validating biochemical parameters (components of phases I and II (de)toxication metabolism and propionylcholinesterase activity), measured in a fresh water bivalve Corbicula fluminea as a biomarker of water quality. The comparison between biomarker responses and community ones (reference) gives information about the precocity and sensitivity of these biomarker responses. Pure substances (trichloroethylene (TCE), cadmium (CD) and anthracenic oil (AO)) have been injected during one month. Biomarker responses are as sensitive as the most sensitive community response in the presence of CD and AO. With TCE, community responses are more sensitive. Precocity of biomarker response is observed only in the presence of CD.

Effects of dimethoate on snail B-esterase and growth as a function of dose, time and exposure route in a laboratory bioassay.

The aim was to study the effects of dimethoate on enzymatic targets and on the growth of Helix aspersa for different times and modes of exposure under laboratory conditions. Young snails were exposed to increasing dimethoate concentrations in the food (D.exp) or in an artificial substrate (S.exp) for 1, 2, 7 and 14 days. Both acetylcholinesterase (AChE) and carboxylesterase (CaE) activities were measured in the foot of the snails for each concentration and exposure time tested. Growth was evaluated after 7 days of exposure. AChE inhibition, dose-dependent for all lengths of exposure, was stronger in S.exp. AChE was more sensitive than CaE for both modes of exposure. IC50(-7) days was 38.3 micrograms g-1 in D.exp and 11.7 micrograms g-1 in S.exp for AChE and was higher than 150 micrograms g-1 in two exposure modes for CaE. AChE activity decreased from the first day to reach maximum inhibition after 7 days of exposure. As noted for B-esterase activities, growth inhibition was stronger in S.exp and was only significant for AChE inhibition of > 90%. The present results show that AChE activity could be used to give early warning of toxic effects of dimethoate in terrestrial gastropods.

Hazard identification by methods of animal-based toxicology.

This paper is one of several prepared under the project "Food Safety In Europe: Risk Assessment of Chemicals in Food and Diet" (FOSIE), a European Commission Concerted Action Programme, organised by the International Life Sciences Institute, Europe (ILSI). The aim of the FOSIE project is to review the current state of the science of risk assessment of chemicals in food and diet, by consideration of the four stages of risk assessment, that is, hazard identification, hazard characterisation, exposure assessment and risk characterisation. The contribution of animal-based methods in toxicology to hazard identification of chemicals in food and diet is discussed. The importance of first applying existing technical and chemical knowledge to the design of safety testing programs for food chemicals is emphasised. There is consideration of the presently available and commonly used toxicity testing approaches and methodologies, including acute and repeated dose toxicity, reproductive and developmental toxicity, neurotoxicity, genotoxicity, carcinogenicity, immunotoxicity and food allergy. They are considered from the perspective of whether they are appropriate for assessing food chemicals and whether they are adequate to detect currently known or anticipated hazards from food. Gaps in knowledge and future research needs are identified; research on these could lead to improvements in the methods of hazard identification for food chemicals. The potential impact of some emerging techniques and toxicological issues on hazard identification for food chemicals, such as new measurement techniques, the use of transgenic animals, assessment of hormone balance and the possibilities for conducting studies in which common human diseases have been modelled, is also considered.

Interest of a multibiomarker approach in the assessment of freshwater ecosystem quality: laboratory and field studies.

The aims of this study were to validate several biochemical parameters as biomarkers of pollution in the freshwater bivalve Corbicula fluminea and to underline the interest of a multibiomarker approach in environmental biomonitoring. The study was divided into a laboratory exposure to 4 doses of trichloroethylene, toluene, cadmium chloride or a coal tar fraction for 5 days and a field exposure for one week in 5 sites surrounding an industrial effluent outlet. Whatever the product was, parameters that exhibited significant responses were mainly parameters related to oxidative stress and components of phase I metabolism. As a result of discriminant analysis, doses were clearly discriminated from the control and from each other. Likewise, products were discriminated from each other, based on results of the whole parameter responses obtained for the first dose. Concerning the field experiment, all biochemical parameters assayed exhibited significant responses for sites located downstream of the effluent outlet, compared to the upstream reference site. Through a discriminant analysis, sites could be distinguished from each other in terms of pollution intensity. In order to characterise pollution at a qualitative level, further laboratory and field studies are needed to obtain typical profiles for the main pollutants present in freshwater ecosystems.

Absence of adverse effects of sodium metabisulphite in manufactured biscuits: results of subacute (28-days) and subchronic (85-days) feeding studies in rats.

Sulphites are extensively used in the food and drinks industry. Their toxicity has been previously evaluated by addition to the diet or drinking water of laboratory animals. Because interactions between sulphites and food constituents occur, the present work was conducted to determine the subacute and subchronic toxicity of sulphite-bound compounds in a finished product: manufactured biscuits. The studies were performed on Sprague Dawley, rats for 28 and 85 days of dietary exposure. Diets were prepared from sulphited or untreated (controls) biscuits with the addition of sugar, protein, vitamins and minerals according to the nutritional requirements of the animals. Groups of 10 male and 10 female rats were administered diets containing sulphited biscuits at levels of 0, 10, 35 and 75%, corresponding to 10-15, 35-45, 150-170 and 310-340 mg SO2/kg diet. In both studies, no death or clinical abnormalities were reported. Growth rate, food consumption and food conversion efficiency were not affected by treatment. No dose-related changes were observed for haematology, clinical chemistry, ocular examination, renal-function, urinalysis, organ weights or gross and microscopic examinations. The liver concentrations of vitamins A, B1, C and E were not significantly changed except for an increase in vitamin E in high-dose males after 28 days' exposure. Based on these data, the no-observed-adverse-effect level (NOAEL) of sulphites in baked biscuits was judged to be 310 mg SO2/kg diet or 25 mg/kg body weight/day.

Potential biomarkers of trichloroethylene and toluene exposure in Corbicula fluminea.

Freshwater clams Corbicula fluminea were exposed in aquariums to four doses of trichloroethylene-TCE-(1.56 up to 100 mg/1) or toluene-TOL-(7.5 up to 60 mg/1) for 5 days. At the end of exposure, components of (de)toxification metabolism of phases I and II, parameters related to oxidative stress and propionylcholinesterase activity were assayed. Determination of TCE and TOL concentrations in water revealed an important evaporative loss during the experiment, characteristic of acute and occasional contaminations by such products occurring in the environment. Appropriate statistical methods such as ANOVA, Tukey test and discriminant analysis underlined the relevance of cytochromes P450 and P418, NADH-cytochrome c reductase, catalase, peroxided and peroxidizable lipids and net peroxidation as biomarkers of exposure to these solvents in C. fluminea. This experiment emphasised the importance of a multi-biomarker approach in environmental surveys and will be completed further by mesocosm studies.

Safety considerations of DNA in food.

Recombinant DNA techniques are capable of introducing genetic changes into food organisms that are more predictable than those introduced through conventional breeding techniques. This review discusses whether the consumption of DNA in approved novel foods and novel food ingredients derived from genetically modified organisms (GMOs) can be regarded as being as safe as the consumption of DNA in existing foods. It concludes that DNA from GMOs is equivalent to DNA from existing food organisms that has always been consumed with human diets. Any risks associated with the consumption of DNA will remain, irrespective of its origin, because the body handles all DNA in the same way. The breakdown of DNA during food processing and passage through the gastrointestinal tract reduces the likelihood that intact genes capable of encoding foreign proteins will be transferred to gut microflora. The review does not specifically address food safety issues arising from the consumption of viable genetically modified microorganisms but it shows that the likelihood of transfer and functional integration of DNA from ingested food by gut microflora and/or human cells is minimal. Information reviewed does not indicate any safety concerns associated with the ingestion of DNA per se from GMOs resulting from the use of currently available recombinant DNA techniques in the food chain.

Combined effects of okadaic acid and cadmium on lipid peroxidation and DNA bases modifications (m5dC and 8-(OH)-dG) in Caco-2 cells.

Okadaic acid (OA) is a marine toxin, a tumour promoter and an inducer of apoptosis. It mainly inhibits protein-phosphatases, protein synthesis and enhances lipid peroxidation. Cadmium (Cd) is known to be carcinogenic in animals and humans (group 1 according to the International Agency for Research on Cancer (IARC) classification). Cd also induces oxidative stress in living organisms. Since they are sometimes found simultaneously in mussels, we have evaluated in the present investigation, the lipid peroxidation, as malondialdehyde (MDA) production, in the variation of the ratios of 8-(OH)-dG/10(5)dG and m5dC/(dC + m5dC) induced by OA and/or Cd in Caco-2 cells. When cells were treated exclusively by OA (15 ng/ml) or Cd (0.625 and 5 microg/ml) for 24 h, protein synthesis was inhibited (by 42 +/- 5%, 18 +/- 13%, and 90 +/- 4% respectively) while MDA production was 2,235 +/- 129, 1710 +/- 20, and 11,496 +/-1,624 pmol/mg protein respectively. In addition, each toxicant induced modified bases in DNA; increases in oxidised bases and methylated dC. The combination of OA and cadmium was more cytotoxic and caused more DNA base modifications; the ratio m(5)dC/(m(5)dC + dC) was increased from 3 +/- 0.15 to 9 +/- 0.15 and the ratio 8-(OH)-dG/10(5) dG also (from 36 +/- 2 to 76 +/- 6). The combination of OA and Cd also increased the level of MDA (1,6874 +/- 2,189 pmole/mg protein). The present results strongly suggest that DNA damage resulting from the oxidative stress induced by these two toxicants may significantly contribute to increasing their carcinogenicity via epigenetic processes.