J. Sanz-Landaluze

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.

Rapid determination of polycyclic aromatic hydrocarbons (PAHs) in zebrafish eleutheroembryos as a model for the evaluation of PAH bioconcentration.

A simple and fast approach for a novel bioaccumulation test by exposing zebrafish eleutheroembryos to fluorene and anthracene (PAHs) at two concentration levels below 1% of their LC50 is presented. This alternative protocol sets an uptake period of 48 h at 26 °C for bioconcentration of the compounds tested and an additional 24h for depuration. Two different methods to estimate bioconcentration factors (BCFs) of PAHs were used. The first consists in the ratio between concentrations of the tested compounds in the zebrafish eleutheroembryos and the exposure media when a steady state during exposure is reached (BCFss). The second employs the concentration-time profile with the use of a least-square fit to a non-linear model (BCFk). A steady state (ss) was reached after 12h and 22 h for fluorene and anthracene exposure, respectively. Uptake and depuration rate constants obtained were similar for the two exposure levels tested, hence toxicokinetic rate constants appeared to be independent of the exposure level. Bioconcentration factors of 1164 and 817 for fluorene exposure at 0.99 ng mL(-1) and 7.9 ng mL(-1) and of 2089 and 2344 for anthracene at 0.63 ng mL(-1) and 6.1 ng mL(-1) were assessed. The good agreement of the obtained results with those reported in the literature proves the feasibility of the proposed method for estimation of both toxicokinetic parameters and bioconcentration factors. Furthermore, this protocol, has potential to be an alternative to the Organisation for Economic Cooperation and Development (OECD) 305 method, considerably reducing time and associated costs of the test suggested by European legislation.

Analytical performance of two miniaturised extraction methods for triclosan and methyltriclosan, in fish roe and surimi samples

A new and reliable miniaturised QuEChERS-based extraction method combined with a dispersive SPE cleanup procedure for extracting triclosan and methyltriclosan from fish roe and surimi samples was proposed. The effectiveness of different extraction/partition conditions for QuEChERS method was systematically investigated, and the use of acetonitrile extraction solvent and MgSO4, PSA, C18 and Florisil as cleanup reagents was recommended in the final method. Other method based on ultrasonic extraction with ethylacetate and clean-up with SPE was also evaluated for these samples. Different polymeric and silica sorbents for clean up were tested and the combination of Florisil and PSA was finally selected. The performance of these miniaturised sample preparation methods combined with GC-MS with quadrupole detection were compared. Extraction efficiency as well as cleaning effectiveness, laboriousness and speed were taken as criteria for method evaluation. Satisfactory validation parameters, such as linearity, recovery, precision and LODs and LOQs for both developed analytical methods were obtained from fish roe and surimi samples. Finally, both methods were applied to real samples. The sensitivity of the proposed methods was good enough to ensure reliable determination of target analytes at concentration levels commonly found in this kind of samples.

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).

Bioconcentration of ionic cadmium and cadmium selenide quantum dots in zebrafish larvae.

The concern related to the use of nanomaterials is growing nowadays, especially the risk associated with their emission or exposure. One type of nanomaterials that has attracted much attention is quantum dots (QDs). QDs incorporation in consumer goods increases the probability of their entering in the environment and then into living organisms and human. In order to evaluate their potential to be bioconcentrated, zebrafish larvae have been exposed to SeCd/ZnS QDs, after performing an exhaustive characterization of these nanoparticles under the assay conditions. These data were compared with those obtained when zebrafish larvae were exposed to ionic cadmium. Finally, distribution of ionic Cd and QDs in exposed zebrafish larvae have been evaluated by Laser Ablation ICP-MS.

Bioaccumulation and transformation of methylmercury and selenite using zebrafish (Danio Rerio) larvae as a model.

Bioaccumulation and possible transformation of methylmercury and selenite has been checked on a 72 h-cycle of bioaccumulation and depuration using larvae from zebrafish. The larvae were exposed to methylmercury and selenite at concentrations of 1% and 0.1% of their LC(50) values. Quantitative extraction of methylmercury and selenite from exposed larvae was achieved by using ultrasonic probe-assisted extraction (USP), thus reducing extraction time and solvent consumption. Extracted species collected at different exposure times were characterized and quantified by liquid chromatography coupled to ICP-MS. Bioconcentration factors (BCFs) were estimated by two procedures: (i) as the ratio of the contaminant concentration in larvae and exposure media (BCF(48 h)) and (ii) fitting contaminant concentration in larvae to bioaccumulation models that describe uptake and depuration processes (BCF(k)). The BCFs obtained for methylmercury were 5000 and 2333 for larvae exposed to 1 μg L(-1) and 10 μg L(-1), respectively; while for selenite the BCF was 74 for larvae exposed to 10 μg L(-1). The good correlation between the BCFs found and those previously reported in the literature shows the proposed method as a good and promising alternative to the OECD Bioconcentration Test 305. Actually, the use of zebrafish larvae reduces the bioaccumulation test time from forty two (OECD Bioconcentration Test 305) to three days. In addition, potential biotransformation of both methylmercury and selenite was evaluated by LC-ICP-MS. For this purpose, a method for species extraction in small size samples by using ultrasonic probe sonication was developed.

Evaluation of arsenic biotransformation by Iberian green frog during metamorphosis

This study examined the effects of arsenic exposure and the biotransformation capacity of developing Iberian green frogs (Rana perezi) with the main aim of finding valid biomarkers to monitor pollution and toxicological problems. Total arsenic concentration and arsenic species distribution were determined using liquid chromatography coupled with inductively coupled plasma-mass spectrometry (LC-ICP-MS). The level of accumulation of arsenic and its species distribution differed depending on the metamorphosis stage and level of exposure. Tetramethylarsonium cation was the major compound detected and continuously increased in concentration during frog development. Results suggest that rather low As accumulation is obtained and that Rana perezi metabolised arsenic during embryonic and larval development.

In vivo bioconcentration of a metal mixture by Danio rerio eleutheroembryos

Exposure to heavy metals has represented one of the most serious health risks of environmental pollution over the last 50 years. Most of the bioconcentration studies that have been carried out to date explored only individual contaminants, unlike the real situations that occur in the environment. In this work, zebrafish eleutheroembryos were exposed to a mixture of CH3Hg(II), iAs(III), Ag(I) and Cd(II), and new BCFs were calculated and compared with those calculated from single metal exposures. In both cases, experimental conditions meet the OECD Test 305 conditions established for aquatic systems. In addition, spatial imaging obtained by laser ablation coupled to inductively plasma mass spectrometry (LA-ICP/MS), has been directly performed in these samples providing complementary information. The new BCFs have revealed some differences compared to single metal exposures when eleutheroembryos were exposed to the metal mixture, especially for iAs(III) and Cd(II). LA-ICP/MS images are in good agreement with the BFCs found, representing an interesting approach to get spatial distribution of metals that reinforces the toxicokinetic information.

Evaluation of chromatographic columns packed with semi- and fully porous particles for benzimidazoles separation.

The behavior of 15 benzimidazoles, including their main metabolites, using several C18 columns with standard or narrow-bore diameters and different particle size and type were evaluated. These commercial columns were selected because their differences could affect separation of benzimidazoles, and so they can be used as alternative columns. A simple screening method for the analysis of benzimidazole residues and their main metabolites was developed. First, the separation of benzimidazoles was optimized using a Kinetex C18 column; later, analytical performances of other columns using the above optimized conditions were compared and then individually re-optimized. Critical pairs resolution, analysis run time, column type and characteristics, and selectivity were considered for chromatographic columns comparison. Kinetex XB was selected because it provides the shortest analysis time and the best resolution of critical pairs. Using this column, the separation conditions were re-optimized using a factorial design. Separations obtained with the different columns tested can be applied to the analysis of specific benzimidazoles residues or other applications.