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 BCF's 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 BFC's found, representing an interesting approach to get spatial distribution of metals that reinforces the toxicokinetic information.

Method for quantifying NSAIDs and clofibric acid in aqueous samples, lumpfish (Cyclopterus lumpus) roe, and zebrafish (Danio rerio) eleutheroembryos and evaluation of their bioconcentration in zebrafish eleutheroembryos.

Pharmaceuticals such as nonsteroidal anti-inflammatory drugs (NSAIDs) and lipid regulators are being repeatedly detected at low concentrations (pg · mL-1-ng · mL-1) in the environment. A large fraction of these compounds are ionizable. Ionized compounds show different physico-chemical properties and environmental behavior in comparison to their neutral analogs; as a consequence, the quantification methods currently available, based on the neutral molecules, might not be suitable to detect the corresponding ionized compounds. To overcome this problem, we developed a specific analytical method to quantify NSAIDs and lipid regulators (i.e., ibuprofen, diclofenac, naproxen, and clofibric acid) and their ionized compounds. This method is based on three steps: (1) the extraction of the organic compounds with an organic solvent assisted with an ultrasonic probe, (2) the cleaning of the extracts with a dispersive SPE with C18, and (3) the determination of the chemical compounds by GC-MS (prior derivatization of the analytes). We demonstrated that the proposed method can successfully quantify the pharmaceuticals and their ionized compounds in aqueous samples, lumpfish eggs, and zebrafish eleutheroembryos. Additionally, it allows the extraction and the cleanup of extracts from small samples (0.010 g of wet weight in pools of 20 larvae) and complex matrixes (due to high lipid content) and can be used as a basis for bioaccumulation assays performed with zebrafish eleutheroembryos in alternative to OECD test 305.

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.

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).

Comparison of bioconcentration of ionic silver and silver nanoparticles in zebrafish eleutheroembryos.

The production of silver nanoparticles has reached nowadays high levels. Bioconcentration studies, information on persistence and toxicity are fundamental to assess their global risk and thus necessary to establish legislations regarding their use. Previous studies on silver nanoparticle toxicity have determined a clear correlation between their chemical stability and toxicity. In this work, experimental conditions able to assure silver nanoparticles stability have been optimized. Then, zebrafish (Danio rerio) eleutheroembryos were exposed to ionic silver and to Ag NPs for comparison purposes. A protocol alternative to the OECD 305 technical guideline was used. To determine silver concentration in both the eleutheroembryos and the exposure media, an analytical method consisting in ultrasound assisted extraction, followed by inductively coupled plasma mass spectrometry and graphite furnace atomic absorption spectrometry, was developed. Then, bioconcentration factors were calculated. The results revealed that ionic silver was more accumulative for zebrafish eleutheroembryos than nanoparticles at the levels tested.

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.

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.

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.

Miniaturized extraction methods of triclosan from aqueous and fish roe samples. Bioconcentration studies in zebrafish larvae (Danio rerio).

Triclosan, an antibacterial and antifungal agent, is widely used in household and personal care products, processed foods and food packaging, etc., and thus also released into the environment. Triclosan is acutely and chronically toxic to aquatic organisms and bioaccumulates in fish tissue. Here, we propose a new miniaturized triclosan extraction method for aqueous and fish roe samples, based on the use of a vortex mixer and an ultrasonic probe, respectively, and useful for triclosan determination by gas chromatography coupled to a micro electron capture detector. Different solvents for extraction and sorbents for clean-up purposes were tested. Multivariate optimization of the variables affecting ultrasonic extraction (ultrasound radiation amplitude, sonication time, sample temperature, and the ratio of sample amount and extracting volume) was carried out. Solvent extraction using ethyl acetate and further clean-up with mixed bed cartridges with two layers of Florisil® and Florisil® impregnated with 10% sulfuric acid only for fish roe samples was finally selected. Extraction efficiencies of up to 95% and 90%, and detection limits of 0.165 ng ml(-1) and 2.7 ng g(-1) for aqueous and fish roe samples were obtained, respectively. The optimized method was used in bioconcentration studies with zebrafish larvae (Danio rerio), as an alternative method to the Organization for Economic Cooperation and Development technical guideline 305. Bioconcentration values, BCF = 2,630 and 2,018 at exposure concentrations of 30 and 3 µg L(-1), respectively, were assessed. These results are in agreement with those reported in the literature, showing the feasibility of the method for estimation of toxicokinetic parameters and bioconcentration factors using zebrafish larvae instead of adult fishes, reducing considerable animal testing, as suggested by the European legislation.

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.

Zebrafish larvae as a model for the evaluation of inorganic arsenic and tributyltin bioconcentration.

The European REACH legislation establishes the need to study the toxicity, persistence and bioaccumulation of those chemicals with an exceeding production of 100tons and/or chemicals considered PBTs substances (Persistence, Bioaccumulation and Toxicity). Currently, the OECD technical guideline 305 is the most used protocol to determine bioconcentration factors of contaminants in aquatic environments. However, this procedure implies high cost and amount of adult fishes. Zebrafish (Danio Rerio) has been selected since this animal model has several advantageous features over other vertebrates, mainly fast embryonic development and easy growth. The analytical methodology here developed has been applied to calculate the bioconcentration factors (BCFs) of two contaminants: inorganic arsenic and tributyltin (measured as arsenic and tin). The method is based on the use of an ultrasonic probe assisted extraction for accelerating the sample treatment followed by detection using graphite furnace atomic absorption spectrometry with Zeeman correction (ZGFAAS). Results obtained for the BCFs values are in good agreement with previously reported data on freshwater aquatic organisms. In the case of arsenic, after exposing larvae to concentrations of 5 and 50µgL(-1), very low BCFs were observed (between 2.2 and 9.5); while for tributyltin, the BCFs observed were within the range 840-1280 after exposure to concentrations of 0.2 and 2.0µgL(-1), respectively. This study shows the use of zebrafish larvae together with the proposed analytical approach as a promising alternative to the OECD 305 test to evaluate the BCFs of classical and emergent contaminants.

Miniaturisated method for the analysis of polycyclic aromatic hydrocarbons in leaf samples.

A new methodology is proposed for monitoring 16 priority polycyclic aromatic hydrocarbons, using tree leaves as passive samplers, by means of a mini-ultrasonic probe coupled with reversed-phase liquid chromatography (RP-LC) and fluorescence (FL) detection. Separation and detection of the 16 PAHs were completed in 19 min, using a 3 microm (particle size) C(18) column RP-LC with acetonitrile-water gradient elution. The ultrasonic probe device used was equipped with a 2 mm titanium tip, and sample and solvent amounts used were just 50 mg and 1 mL, respectively. Multivariate optimisation of the variables affecting extraction was conducted by means of full factorial analysis to determine which of the variables were significant. A central composite design was applied to define surface responses and to calculate optimal values for the variables. The accuracy of the method was determined by both analysis of a Certified Reference Material with a similar matrix (IAEA-140 OC, seaweed) and by comparison of the results obtained with those from a previously developed method. The proposed analytical method avoids some of the main problems encountered in the determination of PAH in complex matrices; no clean-up step is necessary, consequently sample preparation time and costs can be significantly reduced. The developed method was applied to determine PAH in leaf samples from medlar and red and white mangrove trees, situated near PAH pollution sources.

Accelerated extraction for determination of polycyclic aromatic hydrocarbons in marine biota.

A rapid and simple method is proposed for determination of polycyclic aromatic hydrocarbons (PAH) in complex matrices such as marine biota. The method uses sonication, by means of an ultrasonic probe, as a new tool for assisted extraction, coupled with reversed-phase liquid chromatography (RP-LC) with fluorescence detection (FL) for determination of 16 US EPA priority PAH. Separation and detection of the 16 PAH were complete in 45 min by RP-LC with a C18 column and acetonitrile-water gradient elution. Multivariate optimisation of the variables affecting extraction (ultrasound radiation amplitude, sonication time, and temperature of the water-bath in which the extraction cell was placed) was conducted. The accuracy of the method was determined by analysis of a certified reference material and comparison of the results obtained with those from another method (microwave-assisted extraction and GC-MS). The new technique avoids the main problems encountered in the determination of PAH in complex matrices such as marine biota, and no clean-up step is necessary. The method was applied to determination of PAH in estuarine biota samples from the Urdaibai estuary (Biscay, Spain).