Miren Pena-Abaurrea

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.

Identification of Potential Novel Bioaccumulative and Persistent Chemicals in Sediments from Ontario (Canada) Using Scripting Approaches with GC×GC-TOF MS Analysis

This work describes a single and fast approach using a filtering script as a means of prioritizing sample processing of data acquired by GC×GC-TOF MS for the identification of potentially novel persistent and bioaccumulative halogenated chemicals. The proposed script is based on the recognition of a generic halogenated isotope cluster pattern that allows for the simultaneous detection of chlorinated, brominated, or mixed halogen-substituted compounds in a single classification. Once developed, the script was applied to the identification of organohalogens in stream sediments collected across the southern region of Ontario (Canada). Classified peaks were first compared with available analytical standards and reference libraries to confirm the known chemicals. Unknown potential persistent organic pollutants (POPs) were evaluated for occurrence within the samples and high resolution mass spectrometry was used in order to identify some of the most prevalent compounds in the samples and resulting in the identification of three decachlorinated dechlorane analogs (C18H14Cl10), two undecachlorinated dechlorane species (C18H13Cl11), and a novel mixed chloro/bromo-carbazole (C12H5NCl2Br2) in a number of sediments analyzed. Relative peak abundances of these unknown halogenated compounds were in the same order of magnitude or slightly higher than levels observed for conventional POPs detected in the samples.

ANTHROPOGENIC AND NATURALLY-PRODUCED ORGANOBROMINATED COMPOUNDS IN BLUEFIN TUNA FROM THE MEDITERRANEAN SEA

Anthropogenic compounds, such as polybrominated diphenyl ethers (PBDEs), together with naturally-produced organobromines, such as methoxylated PBDEs (MeO-PBDEs), polybrominated hexahydroxanthene derivatives (PBHDs), 2,4,6-tribromoanisole (TBA) and a mixed halogenated monoterpene (MHC-1), were measured in muscle from 26 farmed and wild bluefin tuna (Thunnus thynnus) caught in the Mediterranean Sea. This species is ecological attractive because of the changes of geographic habitat throughout its long lifespan which affect its feeding. PBDE concentrations were similar between tuna samples of different groups (17-149 ng g(-1) lipid weight - lw in farmed tuna, 25-219 ng g(-1)lw in longline fished tuna and 26-126 ng g(-1)lw in net-fished tuna). However, higher concentrations of naturally-produced MeO-PBDEs and PBHDs were observed in the two types of wild tuna (longline fished and net-fished) compared to farmed tuna suggesting that wild tunas come easily in contact with sources of these compounds. In all cases PBHDs presented the highest contribution to the sum of organobromines (50% in farmed tuna and >90% in wild tuna). TBA was detected at low concentrations (<6 ng g(-1)lw), while MHC-1 was found at higher concentrations (up to 42 ng g(-1)lw) in farmed tuna. The estimated daily ingestion of PBDEs from tuna was 830 ng PBDEs day(-1), regardless of the origin of the tuna. While this value is approximately 600 times lower than the minimum risk level set by the US Department of Health and Human Services, it is approximately eight times higher than the total intake of PBDEs via diet, suggesting that consumption of tuna can add considerably to the total daily intake of PBDEs.

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.

Comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry for the identification of organobrominated compounds in bluefin tuna.

This study evaluates comprehensive two-dimensional gas chromatography (GC×GC) coupled to time-of-flight mass spectrometry (GC×GC-ToF MS) for the simultaneous analysis of several classes of organobromines (OBs), including polybrominated diphenyl ethers (PBDEs), polybrominated biphenyls (PBBs), methoxylated PBDEs (MeO-PBDEs), several halogenated naturally produced compounds (HNPs) and eight novel brominated flame retardants (NBFRs), polybrominated hexahydroxanthene derivates (PBHDs), 2,4,6-tribromoanisole and a mixed halogenated compound (MHC-1), in bluefin tuna muscles. The proposed methodology maximised separation of both within and among OB families, and among these and other halogenated micropollutants detected in these samples and co-extracted matrix components. Special attention has been paid to solve co-elution problems observed during the analysis of OBs with one-dimensional GC-based techniques. Satisfactory separation among several relevant PBDEs and MeO-PBDEs has been obtained allowing their unambiguous determination in a single run. Additional studies were conducted to identify selected NBFRs and HNPs. 2,4-Dibromoanisole, a dibromophenol isomer and hexabromobenzene were identified in the investigated samples. Several new tri- and tetra-BHD derivates were also identified, indicating that these compounds could apparently exist as structured families in nature. In addition, a tetrabrominated diMeO-biphenyl and two tetrabrominated diMeO-BDEs were also tentatively identified.

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.

Halogenated indigo dyes: A likely source of 1,3,6,8-tetrabromocarbazole and some other halogenated carbazoles in the environment

In recent years, a number of halogenated carbazoles have been detected in environmental samples. These emerging contaminants have been shown to be persistent and possess dioxin-like toxicological potential. The goal of this research was to examine the literature to determine likely anthropogenic origin(s) of halogenated carbazoles in the environment. The scientific literature indicated a number of pathways by which 1,3,6,8-tetrabromocarbazole could form in the manufacture of 5,5,7,7-tetrabromoindigo. The U.S. production history of 5,5,7,7-tetrabromoindigo correlates well with the concentration rise, decline, and disappearance of 1,3,6,8-tetrabromocarbazole in dated Lake Michigan sediments. Additionally, other halogenated carbazoles that have been found in environmental sediments can be explained by the production of other halogenated indigo dyes. 1,8-dibromo-3,6-dichlorocarbazole can be accounted for by the manufacture of 7,7-dibromo-5,5-dichloroindigo, while 1,3,6,8-tetrachlorocarbazole was found at relatively high concentration near the outfall of a U.S. manufacturer of 5,5,7,7-tetrachloroindigo. Carbazoles containing an iodo-substituent can be explained by the use of iodine as a catalyst in the manufacture of halogenated indigo dyes. 3,6-Dichlorocarbazole measured in soils and dibromocarbazoles measured in more recently deposited sediments are not easily rationalized on the basis of an indigo related source and may be related to other anthropogenic sources or natural origins.

Characterization and Biological Potency of Mono- to Tetra-Halogenated Carbazoles

This paper deals with the characterization and aryl hydrocarbon receptor (AhR) agonist activities of a series of chlorinated, brominated, and mixed bromo/chlorocarbazoles, some of which have been identified in various environmental samples. Attention is directed here to the possibility that halogenated carbazoles may currently be emitted into the environment as a result of the production of carbazole-containing polymers present in a wide variety of electronic devices. We have found that any carbazole that is not substituted in the 1,3,6,8 positions may be lost during cleanup of environmental extracts if a multilayer column is utilized, as is common practice for polychlorinated dibenzo-p-dioxin (dioxin) and related compounds. In the present study, (1)H NMR spectral shift data for 11 relevant halogenated carbazoles are reported, along with their gas chromatographic separation and analysis by mass spectrometry. These characterization data allow for confident structural assignments and the derivation of possible correlations between structure and toxicity based on the halogenation patterns of the isomers investigated. Some halogenated carbazoles exhibit characteristics of persistent organic pollutants and their potential dioxin-like activity was further investigated. The structure-dependent induction of CYP1A1 and CYP1B1 gene expression in Ah-responsive MDA-MB-468 breast cancer cells by these carbazoles was similar to that observed for other dioxin-like compounds, and the magnitude of the fold induction responses for the most active halogenated carbazoles was similar to that observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). 2,3,6,7-Tetrachlorocarbazole was one of the most active halogenated carbazoles and, like TCDD, contains 4 lateral substituents; however, the estimated relative effect potency for this compound (compared to TCDD) was 0.0001 and 0.0032, based on induction of CYP1A1 and CYP1B1 mRNA, respectively.

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 chemicaĺs 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).

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.