Article pubs.acs.org/est
Zebrafish (Danio rerio) Eleutheroembryo-Based Procedure for Assessing Bioaccumulation J. Sanz-Landaluze,*,† M. Pena-Abaurrea,§ R. Muñoz-Olivas,† C. Cámara,† and L. Ramos*,§ †
Department of Analytical Chemistry, Faculty of Chemistry, University Complutense de Madrid, Ciudad Universitaria, 28040 Madrid, Spain § Department of Instrumental Analysis and Environmental Chemistry, IQOG (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain S Supporting Information *
ABSTRACT: 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 chemicalś 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).
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for evaluating BCFs in chemicals.7 The guideline proposes evaluating the chemical content in fish tissue and exposure solution at a given analyte concentration at increasing exposure times up to 42 days under well-defined environmental conditions. Each experiment involves more than one hundred adult fish and demands a huge analytical work making the assay extremely expensive in terms of reagents,8 time, and animal suffering. Although a recent modification of this guideline allows reducing the number of fish under specific circumstances,9 any alternative to this type of laborious in vivo experiment should be welcomed. In fact, REACH regulation also highlights the appropriateness of reducing the number of tests involving animals and encourages the developmentof ecotoxicological evaluations based on animal-free approaches alternative to the existing in vivo testing methods.5 There are two possible: (i) those involving mathematical models that aim to predict a chemical property or biological activity for a test
INTRODUCTION Bioaccumulation is defined as the accumulation of chemicals in an organism through any route, including breathing, ingestion, or direct contact. For aquatic species, this is usually defined by the bioconcentration factor (BCF), which is calculated as the ratio between the analyte concentration in the fish (CB) at the maximum uptake time and that of the surrounding media (CW) at a steady state (SS), BCFSS = CB/CW.1,2 When the SS is not reached, the BCF can be calculated by fitting the uptake and depuration processes to the first-order kinetic model BCFk = k1/k2, where k1 and k2 are the constants of chemical uptake and depuration, respectively.3,4 According to Article 14(1) of the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) regulation, registrants manufacturing or importing substances in amounts above 10 tons per year must conduct a chemical safety assessment, including data concerning persistence, bioaccumulation and toxicity evaluation.5 More than 4600 registered compounds already fulfill these requirements.6 The bioconcentration test 305 has been proposed by the Organization for Economic Co-operation and Development (OECD) and can be considered a widely accepted procedure © XXXX American Chemical Society
Received: September 25, 2014 Revised: December 22, 2014 Accepted: December 24, 2014
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DOI: 10.1021/es504685c Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
Table 1. Comparison of the BCF Calculated Using the Alternative Zebrafish Eleutheroembryos-Based Procedure for the 15 Studied Analytes Against BCFs Reported in Other Data Bases for Fishes with Specification of the Experimental Conditions Used in Each Case EPI suite BCFBAFc
Japan METI-NITE datab
using this methodology d
CAS no.
log Kowa
sodium selenite
10102−18−8
−7.33
10 1
