K. Le Menach

Optimisation of the microwave-assisted extraction in open cell of polycyclic aromatic hydrocarbons from soils and sediments : Study of moisture effect

Sample preparation remains a long and critical step for organic contaminant analyses in the environment. Classical extraction methods, like Soxhlet extraction, are time and solvent consuming. Some new techniques have been developed in the last few years (supercritical fluid extraction, microwave-assisted extraction, pressurized liquid extraction). Among them, microwave-assisted extraction at atmospheric pressure has appeared an interesting alternative method to Soxhlet extraction. In this present study the extraction of polycyclic aromatic hydrocarbons (PAHs) from solid environmental matrices (soils and sediments) by a microwave-assisted method has been studied. In a first part the experimental conditions have been optimised and different parameters adjusted using the SRM 1941a as a test matrix: time and power of irradiation, nature of solvent, percentage of water. Among all the parameters, the quantity of water is of primary importance to maximize the recoveries. With the optimised conditions applied to the SRM 1941a, the results obtained by the microwave-assisted extraction with 30% of water are in a good agreement with those obtained by Soxhlet. When compared to the certified values, the global recovery obtained by microwave-assisted extraction with 30% of water is 90%. The relative standard deviations obtained for the microwave-assisted extraction with 30% of water are in the same range as those given by the Soxhlet extraction and in good agreement with the certified values (relative standard deviations below 15%). The optimal conditions (30% of water, 30 ml of dichloromethane, 30 W, 10 min) have then been applied to the extraction of PAHs from various matrices (certified and natural ones; soils, sediments, sewage sludge) and the concentrations of PAHs obtained by microwave-assisted extraction are compared to those obtained by Soxhlet extraction. For all the samples tested in this study the recoveries for the microwave-assisted extraction using 30% of water are very good (more than 85%) in comparison to the Soxhlet extractions. The reproducibility of such an optimised procedure is also very correct (under 15% for ΣPAHs for all the matrices). The method developed has appeared to be not matrix-dependent.

Relationship between PAH biotransformation as measured by biliary metabolites and EROD activity, and genotoxicity in juveniles of sole (Solea solea)

Polycylic aromatic hydrocarbons (PAHs) are ubiquitous contaminants in the marine environment. Their toxicity is mainly linked to the ability of marine species to biotransform them into reactive metabolites. PAHs are thus often detected at trace levels in animal tissues. For biomonitoring purposes, this findings have two main consequences, (i) the determination of the PAH tissue concentration is not suitable for the evaluation of individual exposure to PAHs (ii) it can explain sometimes the lack of correlations obtained with relevant markers of toxicity such as genotoxicity biomarkers. The aim of the present study was to better investigate the link between PAH exposure and genotoxicity in marine flatfish. During a laboratory experiment, juvenile soles were exposed for four weeks to a mixture of three PAHs, namely benzo[a]pyrene, fluoranthene and pyrene, followed by one week of depuration. Fish were exposed via the trophic route to a daily PAH concentration of 120 μg/g food. Fish were sampled at different time points. The bioavailability and the biotransformation of PAHs were assessed by the measurement of biliary metabolites using a sensitive UPLC MS/MS method. The 7-ethoxyresorufine-O-deethylase was also measured in liver subcellular fractions as a biomarker of phase I biotransformation activities. Genotoxicity was assessed in parallel by the measurement of DNA strand breaks in fish erythrocytes by the alkaline comet assay. During this study, the high amount of PAH metabolites produced in sole demonstrated the bioavailability of PAHs and their biotransformation by fish enzymes. A positive correlation was observed between the level of hydroxylated PAH metabolites and genotoxicity as measured by the alkaline comet assay.

Fast and efficient extraction methods for the analysis of polychlorinated biphenyls and polybrominated diphenyl ethers in biological matrices

This paper describes fast and simple extraction methods for the determination of polychlorinated biphenyls and polybrominated diphenyl ethers in biological matrices. Four extraction protocols were tested. The first protocol used microwave-assisted extraction combined with two purification steps. The second one was similar, except that microwave-assisted extraction was replaced by accelerated solvent extraction. The third one combined extraction/purification by accelerated solvent extraction with final purification on a silica gel column. The last one combined microwave-assisted extraction with purification on an acidic silica gel column. The protocols were tested on various matrices: a spiked matrix, two certified matrices (SRM 2977, WMF 01), and natural matrices (mysids and fish). All of the protocols produced good performance in terms of recovery and reproducibility. The two last protocols showed promising results in terms of applicability to natural matrices, as they required a minimum of sample handling and minimal amounts of solvent and time. These methods allowed at least 24 samples to be handled per day, and could easily be used for routine analysis.

Detection of early effects of a single herbicide (diuron) and a mix of herbicides and pharmaceuticals (diuron, isoproturon, ibuprofen) on immunological parameters of Pacific oyster (Crassostrea gigas) spat

In the context of massive summer mortality events of the Pacific oyster Crassostrea gigas, the aim of this study was to investigate the early effects on genes, enzymes and haemocyte parameters implicated in immune defence mechanisms in C. gigas oysters exposed to a potentially hostile environment, i.e. to an herbicide alone or within a mixture. Following 2 h of exposure to the herbicide diuron at 1 μg L(-1), the repression of different genes implicated in immune defence mechanisms in the haemocytes and the inhibition of enzyme activities, such as laccase-type phenoloxidase (PO) in the plasma, were observed. The inhibition of superoxide dismutase (SOD) activity in the plasma was also observed after 6 and 24 h of exposure. In the mixture with the herbicides diuron and isoproturon, and the pharmaceutical ibuprofen, catecholase-type PO activity in the plasma and the percentage of phagocytosis in the haemocytes were reduced after 6 h of exposure. Our results showed that early effects on molecular, biochemical and cellular parameters can be detected in the presence of diuron alone or within a mixture, giving an insight of its potential effect in situations that can be found in natural environments, i.e. relatively high concentrations for short periods of time.

Persistent Organic Pollutants in a marine bivalve on the Marennes-Oléron Bay and the Gironde Estuary (French Atlantic coast) -Part 1: Bioaccumulation

The aim of this study was to determine 1) the relevance of using the Pacific oyster Crassostrea gigas as a sentinel organism, at a juvenile stage, for polycyclic aromatic hydrocarbon (PAH) and persistent organic pollutant (polychlorobiphenyl, PCB, polybromodimethylether, PBDE, and organochlorine pesticide, OCP) contamination, 2) the potential levels of chemical organic contamination in the Marennes-Oléron Bay, and their potential sources and 3) the potential influence of physiological or environmental factors on contaminant body burdens in oysters. To this end, juvenile oysters purchased from an oyster hatchery were transplanted to a reference site, in Bouin, and to different transplantation sites in the Marennes-Oléron Bay, the first oyster production area in France, and in the Gironde Estuary, the biggest estuary in Occidental Europe. Transplantations were done during summer and winter. Whole oyster soft tissues from each site were analysed for PAHs, PCBs, PBDEs and OCPs. Results obtained with a transplantation period of 3months suggest that C. gigas, at the juvenile stage, is a relevant sentinel organism for short-term assessment of contamination for these contaminants. In addition, no significant effects of physiological factors on contaminant body burdens were observed. Principal component analysis revealed two distinct groups of contaminants (PAHs and OPCs, and PCBs and PBDEs) and three groups of sites: 1) the reference site, 2) Les Palles (LP) and Boyard (BOY) in winter and 3) all the other sites. The group of LP and BOY was clearly defined by the levels of PAHs and OCPs, suggesting higher levels of contamination of these chemical compounds on these sites, potentially due to local contamination sources. In addition, no relevant effects of physiological or environmental factors on contaminant body burdens were observed. Results suggest also a predominance of contaminants related to agricultural activities along the Marennes-Oléron Bay, and therefore, further studies on the presence of pesticides in this region should be considered.

Atmospheric Heterogeneous Reactions of Benzo(a)pyrene

Abstract This experimental study deals with heterogeneous reactions of benzo(a)pyrene (BaP) with ozone, nitrogen dioxide and hydroxyl radicals. BaP was adsorbed on silica particles chosen here as a model of mineral atmospheric particles. Compound extractions were assisted by focused microwave and analyses were performed by gas chromatography coupled with mass spectroscopy in single ion monitoring mode. Pseudo-first order rate constants were obtained from the fit of experimental decays of particulate-BaP concentration versus reaction time. Second order rate constants were determined considering the different oxidant gaseous concentrations except for the case of hydroxyl radicals where only a pseudo-first order rate constant was proposed. Values obtained at room temperature are (2.1±0.5)×10−15 cm3 molecule−1 s−1 for (BaP + ozone), (5.8±1.4)×10−16 cm3 molecule−1 s−1 for (BaP + nitrogen dioxide) and (3.4±0.8)×10−2 s−1 for (BaP + OH) reactions. Products have only been investigated for the NO2 and the OH (in the presence of NOx) reactions. 1-, 3- and 6-nitrobenzo(a)pyrenes were detected as degradation products and quantified. Reaction rate constants for product formation are (3.7±0.9)×10−16 cm3 molecule−1 s−1 for 6-NBaP, (2.2±0.6)×10−17 cm3 molecule−1 s−1 for 1-NBaP and (5.3±1.3)×10−17 cm3 molecule−1 s−1 for 3-NBaP. 1-, 3- and 6-nitroBaP account respectively for approximately 5%, 12% and 83% of total nitrated species. If in the presence of only nitrogen dioxide, BaP was totally degraded within few minutes, only 20 to 25 % of the initial BaP led to nitrated compounds when reacting with OH (in the presence of NOx).

Assessment of the effects of discontinuous sources of contamination through biomarker analyses on caged mussels

The present study analysed potential adverse effects of discontinuous sources of contamination, namely the discharge of a combined sewer overflow (CSO) and of runoff in an urban area, the Bay of Santander (North Iberian Peninsula). Water samples and caged mussels were used to analyse concentrations of contaminants and biological responses. Mussels (Mytilus galloprovincialis) were transplanted to a marina receiving runoff from a petrol station and to a CSO discharge site. Samples were collected in synchrony with heavy rains along 62days. Lysosomal membrane stability (LMS) and acyl-CoA oxidase (AOX) activity were measured as core biomarkers and were analysed at all sampling times. Histopathology of digestive gland and gonads, transcription levels of vitellogenin gene, volume density of black silver deposits and micronuclei formation were measured at initial and final stages of the transplant. Chemical analyses of metals, polycyclic aromatic hydrocarbons (PAHs) and endocrine disruptors were performed in water samples and mussel flesh. Mussels accumulated low concentrations of contaminants, which is in accordance with results obtained from exposure biomarkers. AOX activity decreased in all transplanted mussels after the first heavy rain, but this change seems to be related to the seasonal pattern of the enzyme activity. Mussels located close to the CSO discharge site showed a reduction in LMS after the first rain event, when compared to mussels before the transplant and to mussels from the reference location. However, this was attributable to natural environmental changes rather than to pollution. Values of the rest of analysed biomarkers were below threshold values reported for the study area.