Automated high-throughput Vibrio fischeri assay for (eco)toxicity screening: Application to ionic liquids

REQUIMTE, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
Ecotoxicology and Environmental Safety (Impact Factor: 2.76). 03/2012; 80:97-102. DOI: 10.1016/j.ecoenv.2012.02.013
Source: PubMed


An automated high-throughput Vibrio fischeri assay was developed and further applied to the evaluation of ionic liquids (ILs) (eco)toxicity. The assay was based on the reduction of bacterial bioluminescence in the presence of test compounds and the results were presented as EC(50). The assays were performed with eight commercially available ILs with distinct cationic head groups, alkyl side chains and anions. EC(50) values between 6.5 and 691.9 mmol L(-1) were obtained for the tested ILs, being hmim [Cl] the most toxic and bmim [Cl] the less toxic ones, confirming the influence of the different structural elements. Moreover, all the tested ILs exhibited a (eco)toxicity lower than Cu(II), used as a positive control during the optimization and analysis steps. The automated assay assured the precise control of the contact time between V. fischeri and test compound by means of a simple protocol that guaranteed adequate aspiration and handling of the solutions as well as the precise implementation of a computer controlled stop period. Furthermore, a significant reduction of the assay costs was achieved through automation mainly by a drastic reduction of the volume of bacterial suspension and test compound. The methodology was validated by comparison with a microplate assay; it was stated that the results, obtained after a 3min contact time, changed proportionally relatively to Cu(II) in both assays. This confirmed the applicability of the methodology as an (eco)toxicity screening assay, with reduction of time and increase of robustness and repeatability (n=10; rsd<1.1%). It is expected that due to its simplicity and reduced cost the developed assay can be integrated in the early stage of development of new compounds as a rapid screening test.

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Available from: Paula C.A. G. Pinto, Feb 10, 2015
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    • "This type of structure presents chemical and biological behaviors similar to those of cationic surfactants, a recognized toxic class of compounds (Ivankovic and Hrenovic, 2010). This relationship has also been postulated by other authors, who verified greater toxicity for different aquatic organisms for compounds structurally similar to surfactants (Pinto et al., 2012; Pretti et al., 2009). Additionally, (Hex) 3 (TDec)P [Cl] presents a cation with long lateral chain lengths and, therefore, higher lipophilicity compared with the other tested ILs. "
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    ABSTRACT: Ionic liquids (ILs), also known as liquid electrolytes, are powerful solvents with a wide variety of academic and industrial applications. Bioassays with aquatic organisms constitute an effective tool for the evaluation of ILs' toxicity, as well as for the prediction and identification of possible moieties that act as toxicophores. In this work, the acute toxicity of six ILs and two commonly used organic solvents was evaluated using freshwater organisms: Daphnia magna, Raphidocelis subcapitata and Hydra attenuata. The bioassays were performed by exposing the organisms to increasing concentrations of the ILs and observing D. magna immobilization, R. subcapitata growth inhibition, and the morphological or mortality effects in H. attenuata. The results demonstrate that the tested organisms are not equally susceptible to the ILs, e.g., bmpyr [BF4] was the least toxic compound for R. subcapitata, N1,1 [N1,1,1OOH] for D. magna and emim [Tf2N] for H. attenuata. This highlights the importance of applying a battery of assays in toxicological analysis. Additionally, Hydra proved to be the most tolerant species to the tested ILs. According to their hazard rankings, the tested ILs are considered practically harmless or moderately toxic, except (Hex)3(TDec)P [Cl], which was classified as highly toxic. The ILs were revealed to be more harmful to aquatic systems than the tested organic solvents, reaffirming the need to analyze carefully the (eco)toxicological impact of these compounds. The present study provides additional data in the evaluation of the potential hazard and the impact of ILs in the environment. Copyright © 2015 Elsevier Ltd. All rights reserved.
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    ABSTRACT: A fully automated Vibrio fischeri methodology based on sequential injection analysis (SIA) has been developed. The methodology was based on the aspiration of 75 mu L of bacteria and 50 mu L of inhibitor followed by measurement of the luminescence of bacteria. The assays were conducted for contact times of 5, 15, and 30 min, by means of three mixing chambers that ensured adequate mixing conditions. The optimized methodology provided a precise control of the reaction conditions which is an asset for the analysis of a large number of samples. The developed methodology was applied to the evaluation of the impact of a set of ionic liquids (ILs) on V. fischeri and the results were compared with those provided by a conventional assay kit (Biotox (R)). The collected data evidenced the influence of different cation head groups and anion moieties on the toxicity of ILs. Generally, aromatic cations and fluorine-containing anions displayed higher impact on V. fischeri, evidenced by lower EC50. The proposed methodology was validated through statistical analysis which demonstrated a strong positive correlation (P>0.98) between assays. It is expected that the automated methodology can be tested for more classes of compounds and used as alternative to microplate based V. fischeri assay kits.
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    • "The methodologies applied in this work were adapted from the assays developed before for carboxylesterase [21] and V. fischeri. [30]. The carboxylesterase assay was based on the hydrolysis of 4- methylumbeliferyl acetate (MUA) with formation of a fluorescent product [31]. "
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