Zebrafish (Danio rerio) eleutheroembryo-based procedure for assessing bioaccumulation.

This paper reports on the development and preliminary evaluation of a new bioaccumulation test based on the use of zebrafish (Danio renio) eleutheroembryos (72 h after hatching, corresponding to 144 h post fertilization, hpf) as an alternative to adult fish-based procedures for regulatory purposes regarding REACH application. The proposed test accomplished the OECD 305 guideline and consists of a 48 h uptake period followed by a 24 h depuration step. Bioaccumulation experiments were performed for a selected of hyper hydrophobic chemicals (log Kow> 7.6), that is, PCB 136 and PBDE 154 at two concentration levels corresponding roughly to 1% and 0.1% the chemicals LC50(nominal concentrations of 4.0 and 12.0 µg/L for PCB 136, and 1.0 and 5.0 µg/L PBDE 154, respectively). Toxicokinetic models were used to calculate the bioconcentration factors (BCFs) based on of the chemical concentrations found in the contaminated eleutheroembryos and their surrounding media. The experimentally determined accumulation profiles show bioaccumulation by zebrafish eleutheroembryos of both chemicals, and that the process is more complex than simple water-lipid partition. Calculated log BCFs using a first-order accumulation model(3.97 and 3.73 for PCB 136, and 3.95 and 4.29 for PBDE 154) were in the range of those previously reported in the literature. The suitability of this new nonprotected life stage bioaccumulation protocol for BCF estimation was evaluated by application to widely divergent micropollutants with different accumulation mechanisms. The results were compared with those in the MITE-NITE database for adult rice fish (Oryzias latipes).

Non-target analysis of organohalogenated contaminants in deep-sea fishes from the Mediterranean Sea by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry.

The enhanced separation power and identification capabilities make comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC × GC - ToF MS) a valuable instrumental alternative for non-target analysis (NTA). In the present study, GC × GC - ToF MS has been used for the NTA of chlorine- and bromine-containing compounds in composite livers of two scarcely investigated Mediterranean deep-sea fish species, hollowsnout grenadier (Coelorinchus caelorhincus) and roughsnout grenadier (Trachyrhinchus trachyrhinchus). Attention focused on the identification of organohalogenated compounds with structural characteristics similar to those of persistent organic pollutants (POPs). In total, 116 Cl-, Br- and mixed Cl/Br-compounds were either positively or tentatively identified in the analyzed liver samples. Up to 88 of these compounds were legacy POPs, being polychlorinated biphenyls (PCBs) and organochlorinated pesticides (OCPs) the most abundant and frequently detected families. The other 28 identified POP-like compounds were analytes not considered by current regulation and environmental monitoring programs, including, among others, degradation products of specific OCPs, naturally produced organohalogen compounds and several perchlorinated diethyldiphenylmethane-derivatives whose presence in the investigated species is reported here for the first time. The presence of other naturally occurring brominated and mixed halogenated compounds in these fish species is also described for the first time. Our results also showed differences in the accumulation profile of the identified compounds in both species. Thereby, anthropogenic POPs showed higher relative abundances in the livers of roughsnout grenadiers than those in hollowsnout grenadiers, while for naturally occurring compounds the opposite trend or similar levels were found in both species.

Comprehensive ethoxymer characterization of complex alcohol ethoxy sulphate products by mixed-mode high-performance liquid chromatography coupled to charged aerosol detection.

The present work describes a simultaneous mixed-mode high performance liquid chromatography (HPLC) method combined with a universal and non-selective-response detector for the complete ethoxymer profiling of alcohol ethoxy sulphate mixtures. The optimized HPLC methodology combines the dual hydrophilic (HILIC) and reversed-phase selectivity of a surfactant-type column in order to render a comprehensive and simultaneous separation of more than 50 endogenous ethoxymers in a single analysis. Furthermore, an accurate quantitation of every single analyte was achieved using a final universal charged aerosol detector (CAD) including specific mathematical processing tools. Results obtained helped describing a complete alkyl chain and ethoxymer distribution of the investigated AES samples. Method validation evidences provided reliability of the individual ethoxymer contributions determined with the proposed HPLC-CAD methodology. Regarding accuracy including independent nuclear magnetic resonance (NMR) experiments, an excellent correlation was found between the structural information provided by a COSY NMR spectrum and the CAD results regarding the mono/polyethoxylated and the non-ethoxylated/ethoxylated distribution. Additional calculations including the average molecular weight and the degree of ethoxylation for the reference AES sample showed minimum differences (relative error < 1 %) between the two considered techniques. An outstanding precision and linearity along the working concentration range (r2>0.999) was also observed. The individual limit of detection for the target sulphate ethoxymers was determined to be in the low ppm range. Further validated distribution profiles for a large number of AES samples demonstrated the applicability of the optimized HPLC-CAD methodology to routine surfactant screenings. Therefore, the hereby developed methodology provided extensive information regarding the detailed individual ethoxymer profile of AES formulations, which can be extremely useful for the surfactant industry in order to gain information on specific synthesis routes and/or detergency properties.

Ultrasound-assisted extraction followed by disposable pipette purification for the determination of polychlorinated biphenyls in small-size biological tissue samples.

The use of solid-phase extraction pipette tip (also called disposable pipette extraction, DPX) has been evaluated for the purification of environmentally relevant polychlorinated biphenyls (PCBs) in fatty extracts obtained by ultrasound-assisted extraction with a sonication probe from small-size biological tissues. Complete sample treatment involved only 50 mg of sample and was completed in ca. 15 min with minimal sample manipulation and reagents consumption (i.e., 1.5 mL of n-hexane and 0.8 g of acidic silica). The performance of the proposed methodology for the intended determination was firstly evaluated by determination of the endogenous PCB levels in a naturally contaminated internal reference material. The determined concentrations showed a good agreement with those obtained using a more conventional sample preparation procedure previously validated in our laboratory (recoveries, as compared to levels determined using the latter method, were in the 85-123% range for a large majority of the studied congeners, and the relative standard deviations were in general lower than 14%). Results obtained for the analysis of reference food samples and certified reference materials NIST 1945 and 1947 demonstrated that, when combined with gas chromatography coupled to ion trap mass spectrometry working in the tandem mode, GC-ITD(MS/MS), the proposed methodology allowed accurate determination of most of the investigated PCBs and that 50 mg of sample sufficed for the screening of less abundant toxic congeners.

Miniaturized selective pressurized liquid extraction of polychlorinated biphenyls and polybrominated diphenyl ethers from feedstuffs.

A new miniaturized pressurized liquid extraction (PLE) with in-cell purification method has been developed for the simultaneous extraction of endogenous prioritary and toxic polychlorinated biphenyls (PCBs) and environmentally relevant tri- to deca-brominated diphenyl ether (PBDE) congeners from different feed matrices. Parameters affecting the efficiency of the selective PLE process, such as sorbent:matrix ratio, volume and nature of the extraction solvent, PLE working mode, extraction time and temperature, and amount of co-sorbents, were optimized. n-Hexane and n-hexane:dichloromethane (1:1, v/v) were used as extraction solvents. 8-mL of organic solvents and 3.5 g of sorbents sufficed for complete sample treatment. Only 0.25 g of feed sample were required for accurate determination of the endogenous PCBs studied using gas chromatography with a micro-electron capture detector (GC-µECD) during method development, and for PBDE analysis using either GC-µECD or gas chromatography with negative chemical ionization-quadrupole mass spectrometry (GC-NCI-qMS). Gas chromatography coupled to ion trap detection working in tandem mode, GC-ITD (MS/MS), was used for final PCB confirmation. Additional purification of the sample extracts was not required. The performance of the complete PLE-based method was evaluated at two spiking levels, 0.4 and 4 ng/g wet weight. Recoveries in the range 60-120% were obtained for PCBs, while those of PBDEs ranged from 86% to 114% for most of the target analytes. The relative standard deviations were in general lower than 20%. The optimized procedure was applied to the determination of the investigated PCBs and PBDEs in a variety of feed samples.

Evaluation of comprehensive two-dimensional gas chromatography-time-of-flight-mass spectrometry for the analysis of polycyclic aromatic hydrocarbons in sediments.

This study evaluates the feasibility of comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC × GC-ToF MS) for the determination of the 15+1 EU PAHs in sediments. Experimental variables affecting the injection, chromatographic separation and analytical detection of the analytes have systematically been optimised. Under finally proposed conditions, a satisfactory resolution among critical pairs/groups of PAHs, including benz[a]anthracene, cyclopenta[cd]pyrene and chrysene, the three benzofluoranthene isomers, indeno[1,2,3-cd]pyrene from dibenz[a,h]anthracene (DahA), and DahA from dibenz[a,c]anthracene, has been achieved using DB-5 × BPX-50 as column combination with a run time of 1 h. The feasibility of the method for the analysis of real-life samples has been demonstrated by accurate determination of relevant target PAHs in the certified harbour sediment BCR-535 (deviations among certified concentration values and those calculated using the proposed method lower than 3%); and by successful application to sediments sampled from a relevant protected area located in the South of Spain. The low methodological limits of detection (LODs) obtained for most of the targeted PAHs (in the 5.7-60 µg/kg range, as calculated for real samples) guarantied accurate quantification of the target compounds at the low levels expected in these types of pristine matrices. The strong retention experienced by the heaviest dibenzopyrene isomers included in the study resulted in relatively high LODs for these analytes; nevertheless, these compounds were detected at concentration levels above the corresponding LOD in some of the analysed sediments. In addition, the enhanced identification power provided by GC × GC-ToF MS for the identification of non-target analytes allowed the tentative identification of a group of polynuclear aromatic thiophenes in some of the test samples. Finally, the potential of the use of normalised bubble plots for the fast screening of the potential PAH sources has been demonstrated.

Bioconcentration of pesticides in zebrafish eleutheroembryos (Danio rerio).

The feasibility of a bioaccumulation test based on the use of zebrafish eleutheroembryos as an alternative to adult-individual-based approaches for REACH application has been evaluated for three test compounds, chlorpyrifos, dicofol and atrazine. Following the OECD 305 guidelines, zebrafish eleutheroembryos (72 h after hatching, hpf) were separately exposed to the investigated pesticides at two nominal concentrations below 1% of its corresponding LC(50). The uptake experiments lasted for 48 h. Then, the exposure medium was replaced by a non-contaminated medium for depuration experiments (up to 72 h). Zebrafish eleutheroembryos (larvae 144 hpf, i.e. at the end of the depuration step) and their corresponding exposure media was sampled at ten different times during each experiment and the concentration of the investigated pesticide determined in both the organisms and in the exposure medium. The experimentally determined pesticide accumulation profiles in the eleutheroembryos demonstrated that atrazine has a very fast accumulation kinetic, reaching steady sate (SS) within 24h. Chlorpyrifos and dicofol did not reach the SS within the 48-h uptake experiments although they exhibit higher accumulations than the former pesticide. Two toxicokinetic models were used to calculate the bioconcentration factor (BCF) of the studied pesticide in zebrafish eleutheroembryos. In the former, the BCF was calculated under SS conditions (BCF(SS)). The second was used when the compounds did not reach the SS during the uptake experiment (BCF(k)). Log BCF values of 3.55 and 3.84 for chlorpyrifos; 0.6 and 1.17 for atrazine, and 3.90 for dicofol were experimentally calculated at selected exposure concentrations. These values have been compared with those reported in related bioaccumulation studies and official databases.