Toxicity assessment of ionic liquids with Vibrio fischeri: An alternative fully automated methodology

Accepted Manuscript Title: Toxicity assessment of ionic liquids with Vibrio fischeri: An alternative fully automated methodology Author: Susana P.F. Costa Paula C.A.G. Pinto Rui A.S. Lapa M.L´ucia M.F.S. Saraiva PII: DOI: Reference:

S0304-3894(14)00892-9 http://dx.doi.org/doi:10.1016/j.jhazmat.2014.10.049 HAZMAT 16366

To appear in:

Journal of Hazardous Materials

Received date: Revised date: Accepted date:

8-7-2014 16-10-2014 31-10-2014

Please cite this article as: Susana P.F.Costa, Paula C.A.G.Pinto, Rui A.S.Lapa, M.L´ucia M.F.S.Saraiva, Toxicity assessment of ionic liquids with Vibrio fischeri: An alternative fully automated methodology, Journal of Hazardous Materials http://dx.doi.org/10.1016/j.jhazmat.2014.10.049 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Toxicity assessment of ionic liquids with Vibrio fischeri: An alternative fully automated methodology Susana P.F. Costa a, Paula C.A.G. Pinto a,*[email protected], Rui A.S. Lapa [email protected], M.Lúcia M.F.S. Saraiva a,* a

REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto, Portugal *

Corresponding author. Tel.: +351 220428670; fax: +351 226093483.

Graphical abstract

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fx1 Highlights

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A fully automated Vibrio fischeri methodology was developed. The automated methodology was applied to the evaluation of the toxicity of seven ILs. The use of mixing chamber ensured adequate mixing conditions. A rigorous control of the reaction conditions was obtained through computer control. Toxicity results were discussed considering the structural particularities of ILs

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A fully automated Vibrio fischeri methodology based on sequential injection analysis (SIA) has been developed. The methodology was based on the aspiration of 75 μL of bacteria and 50 μL of inhibitor followed by measurement of the luminescence of bacteria. The assays were conducted for contact times of 5, 15 and 30 minutes, 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.

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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®).

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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. Abbreviations: ILsionic liquids; SIAsequential injection analysis; PAHsPolycyclic aromatic hydrocarbons; emim [Ms]1-ethyl-3-methylimidazolium methanesulfonate; emim [Tf2N]1ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide; emim [Ac]1-ethyl-3methylimidazolium acetate; chol [Ac]choline acetate; bmpy [Cl]1-butyl-4-methylpyridinium chloride; bmpy [BF4]1-butyl-4-methylpyridinium tetrafluoroborate; N4,4,4,4 [Cl]tetrabutylammonium chloride

Keywords: Ionic liquids; Vibrio fischeri; automated SIA system; microplate 1 Introduction In the European Union, one of the most unbending demands during the development of new chemical entities is to assure the standard criteria established in legislation, namely on REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals), guaranteeing not only the quality and efficiency but also the security of novel compounds. Thus, manufactures became responsible for assessing and managing the risks posed by chemicals and for providing trustworthy safety information to their users. In this context, the potential hazard of the compounds to humans and environment must be determined, ideally by a battery of tests, in the initial stage of their development.

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In the context of in vitro toxicological testing, bioassays are the most reliable tool to assess the impact of chemicals once they overcome some of the limitations inherent to chemical analysis, through the analysis of organism's susceptibility to test compounds, along with the evaluation of the involved mechanisms. A wide range of organisms of different trophic levels, including fish, rats, algae, plants, bacteria and cultured cell lines, have been extensively applied to the experimental determination of ecotoxicity. Even though test systems at higher trophic levels enable a more accurate understanding of compound's effects on complex environments, they present evident drawbacks in terms of applicability, namely laborious and extensive techniques as well as considerable reagents' consumption. Distinctively, molecular and cellular tests are generally simpler to apply and present lower costs while providing easily interpreted data. Due to their fast execution, theses assays constitute valuable screening tools [1–3].

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In the aquatic field, one of the most widespread toxicological bioassay is the V. fischeri bioluminescence assay, due to its easy interpretation and good relation speed/cost when compared with others tests. Furthermore, this bioluminescent assay exhibits excellent correlation with bioassays performed in higher organisms such as algae, crustacean or fish and higher sensibility than other bacterial assays [3]. In many countries, it is used as reference method to water samples analysis (ASTM D5660–1995, GB/T 15,441–1995, EN ISO 38,412–1990) [2]. The assay is based on the inhibition of the luminescence emitted by the aquatic bacteria V. fischeri, which is related to its cellular metabolism, allowing the extrapolation between compound toxicity and light intensity reduction [1,3].

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Initially, the assay was applied to the analysis of water samples, but over time it was extended to the assessment of the hazard of other types of sample such as turbid and colored samples [4] and a wide range of compounds: drugs [5,6], phenols [7,8], pesticides [9] and polycyclic aromatic hydrocarbons (PAHs) [10,11].

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Due to its versatility and wide applicability, the V. fischeri assay was commercialized as a kit in the late 1970 s under the name of Microtox [1,10]. Throughout time other kits have been commercialized, such as BioTox™, LUMIStox® and ToxAlert® with the purpose of evaluating the toxicity of chemical compounds in water, and as Microtox® test, most of them rely on the use of specific equipment to perform the measurement of luminescence and bacteria conditioning. However, the acquisition of a specific apparatus for a single assay is not viable for the majority of the laboratories and consequently some alternatives have emerged.

Microplate approaches have been proposed as adaptations of the methodology to a microscale with the concern of lowering costs and increase applicability. This strategy enabled the increase of sample throughput and the commercial use of V. Fischeri as screening-tool [10]. Nonetheless, these techniques, from a practical perspective, can pose some difficulties to less trained operators in terms of the effective control of the contact time between sample and bacteria, since this depends on the operator ability to synchronize the addition of sample and perform luminescence readings at predefined periods of time.

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In this context, automation emerged in this field aiming the downsizing of the assay conditions as well as the reduction of the dependence of the assay on the operator. Komaitis et al. [12] tried to overcome the difficulties of synchronization of the V. fischeri assay through the development of an automated flow injection analysis (FIA) methodology. Despite the good synchronization of bacteria and sample mixing, the technique involves a considerable waste of reagents and demands the physical reconfiguration of the system to adapt it to different determinations, which is not practical when a large number of samples has to be analyzed. More recently, an automated system resorting to sequential injection analysis (SIA) was developed to overcome some of the limitations described before, which are outweighed through the unique mode of operation of SIA [13]. This technique is based on non-specific apparatus and its operation mode relies on the forward and reversed flow of well-defined zones of sample and reagent solutions through a selection valve, controlled by computer, which enables a precise control of the reaction conditions and lower operator interference. Moreover, it is a versatile, low-cost and robust methodology with large applicability in analytical chemistry presenting the possibility of performing multi determinations with the same manifold coupled to a variety of detectors [14–16]. The abovementioned SIA assay was developed as a screening method with toxicity evaluation after just 3 min. Nevertheless, even though this methodology overcame the issue of synchronization it does not furnish information comparable to that provided by microplate and standard assays since sequential aspiration does not guarantee effective mixing of solutions. Moreover, since it was designed as a screening the tool, the assay time was reduced making it incomparable to conventional procedures.

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Thus, the purpose of this work was the development of a fully automated V. fischeri assay not only with controlled aspiration and mixing conditions but also with the possibility of providing data after distinct contact times. It was also intended to apply the optimized SIA system to the evaluation of the toxicity of a set of ionic liquids (ILs). ILs correspond to a large class of salts, composed by cations and anions with melting points below 100 °C. Although it was originally believed they were “environmentally friendly”, due to distinctive properties, such as non-flammability, low volatility, high thermal and chemical stability, there is a risk of water contamination, due to their water solubility [17]. The inhibition of V. fischeri and other microorganisms by some ILs supports this hypothesis, showing that these compounds might not be so innocuous as previously thought. Furthermore, the V. fischeri assay proved to be useful to clarify the impact of some ILś structural elements, in particular the effect of different alkyl chain lengths, cation cores and anion moieties on their overall toxicity [18]. From an instrumental perspective, the implementation of IL based assays in SIA system has been performed systematically in recent years with distinct objectives confirming the adequacy of this technique to the particularities of these solvents, with advantages in terms of costs, safety and environmental impact through the reduction of effluents [19].

Ultimately, it was intended to develop a fully automated technique based on the V. fischeri bioluminescence assay, as an alternative approach to microplate based assay kit procedures. 2 Materials and methods 2.1 Reagents

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All solutions were prepared using chemicals of analytical reagent grade and high purity water (milli-Q) with specific conductance less than 0.1 μS cm−1. The carrier used in the SIA system was NaCl 2%, with pH adjusted to 7. This solution was also used for the preparation of the diluted solutions of ILs. Lyophilized V. fischeri and corresponding dilution solution were obtained from BioTox™ test kit. All the tested ILs (Fig. 1) were obtained from SigmaAldrich Cooperation and were stored at room temperature in a carefully controlled anhydrous environment. A solution of Cu (II) 5.86 mg L−1 (0.09 mmol L−1) was used as positive control for the daily calibration of the system.

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2.2 Apparatus

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The analytical flow system (Fig. 2) used for the evaluation of ILs toxicity incorporated a 10port multiposition Cheminert™ selection valve and a syringe module Bu1S from Crison Instruments S.A. (Allela, Barcelona, Spain).

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The syringe module was equipped with a glass syringe of 5 mL total dispense volume (Hamilton Bonaduz AG, Bonaduz, Switzerland), driven by a stepper motor. Solenoid headvalves allow the commutation of the syringe either to the manifold or to the solutions container. The communication between the controller and the modules was done using a RS232C serial protocol. A PC was used as controller.

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The software used for instrumental control was developed using Visual Basic and communication with instruments was accomplished by means of a RS-232C asynchronous protocol, using embedded dynamic libraries. The implementation of the control algorithm was based in the use of a set of interdependent timers allowing a sequential output of the commands as well as the evaluation of the equipment status. The experimental conditions, previously established in a multi column table, comprise the status of valves, speed of the pump, the time to be spent in each step and the loops to be executed, as well as data acquisition and processing.

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The exchange options were classified as in/out, allowing optional disconnection from (or coupling to) the manifold line; both in dispense (upward/ + ) or in pickup (downward/-) piston movements.

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Manifold components, including the holding coil (2 m), were connected by means of 0.8 mm i.d. PTFE tubing. Three mixing chambers with an internal volume of ca. 500 μL, each containing a magnet, were connected to the selection valve to guarantee adequate mixing conditions between the tested compound and bacteria. The chambers provided also the possibility of testing simultaneously different contact times. A FP-2020 Plus model CL detector (Jasco, Easton, MD, USA) incorporating a flow cell, consisting of a spiraled 0.8 mm i.d. PTFE tube, with an internal volume of 100 μL that was

positioned in front of a highly sensitive end-on photomultiplier, was used to perform chemiluminescence measurements. Analytical signals were also recorded on a Kipp & Zonen BD 111 strip chart recorder. V. fischeri was kept under constant stirring during the assays by means of a Crison Micro ST 2039 magnetic stirrer. To control the temperature of the assay, a water bath thermostatized at 15 °C by means of a P Select temperature controller with recirculation was used. V. fischeri suspension, carrier solution and solutions of ILs were immersed on the water bath during the assays. A Synergy HT, Bio-Tek Instruments plate reader was used for the implementation of the microplate assays.

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2.3 Reconstitution of lyophilized V. fischeri

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Lyophilized V. fischeri was reconstituted and stabilized according to the instructions provided in BioTox™ assay kit. Briefly, the dilution solution of V. fischeri was added to the lyophilized bacteria and the bacterial suspension was stabilized 1 h at 4 °C. After, the bacterial suspension was equilibrated at 15 °C for another hour. Throughout the assays, the bacterial suspension was kept at the same temperature and under constant stirring to avoid deposition of the cells in the bottom of the container.

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2.4 Sequential injection procedure

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The analytical cycle optimized for the evaluation of the aquatic toxicity of ILs, in the SIA system, is schematized in Table 1. In each assay (1 concentration of IL), an aliquot of 75 μL of bacteria was aspirated between two aliquots of 25 μL of inhibitor (steps 1–3). Then, the flow was reversed and the reaction zone was propelled to the mixing chamber in inlet 5, during 13 s (step 4). The reaction zone remained in this chamber for 5 minutes. These 4 steps were repeated two more times, by changing only the port of the selection valve, to fill the other two mixing chambers (in inlets 6 and 7) with the reaction zone for the assays of 15 and 30 min. After 5 min (step 6), the reaction zone was aspirated from the mixing chamber in inlet 5 (step 7) and propelled to the detector (step 8). A signal proportional to the V. fischeri bioluminescence inhibition for 5 min was obtained. Steps 9 to 11 corresponded to the cleaning of the mixing chamber with NaCl 2%, pH 7 and propulsion of the washing content to waste. After 15 and 30 min, steps 7 to 12 were repeated for the mixing chambers in inlets 6 and 7, respectively. A complete analytical cycle enabled the evaluation of the effect of one concentration of inhibitor after contact times of 5, 15 and 30 minutes.

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The described procedure was applied to the study of the effect of increasing concentrations of ILs on V. fischeri. Each concentration was evaluated in triplicate. The real concentration of inhibitor in the SIA system was calculated through the application of an experimentally determined dispersion factor correction. The range of concentrations was stipulated specifically for each compound in order to get inhibition profiles amenable to mathematical treatment. For each tested IL, EC50 values were calculated as the effective concentration of inhibitor causing a decrease of 50% in V. fischeri bioluminescence. The calculations were performed

by means of polynomial correlations established, in the inhibition assays, from the inhibition profiles. A blank assay was performed, every 2 hours, in the absence of inhibitor corresponding to the maximum bioluminescence emitted by the bacteria. 2.5 Microplate reader assay

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With comparison purposes, the effect of ILs on V. fischeri was also evaluated by means of a microplate assay according to the instructions provided in BioTox™ test kit. For this, 100 μL of sample were added to 150 μL of bacteria, corresponding to the same proportion of IL/V. fischeri used in the SIA assay. After a predefined contact time (5, 15 and 30 min), bioluminescence inhibition was determined through punctual determinations in the microplate-reader. The microplate temperature was stabilized before the assay and between readings.

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3 Results and discussion

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In the following sections, the optimization of the SIA methodology and its application to the evaluation of the effect of ILs in V. fischeri bioluminescence will be presented. The results obtained in the automated methodology will also be compared with those provided by the microplate assay.

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3.1 Optimization of the SIA system

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In this work, the screening SIA system developed by Pinto et al. [13] was adapted to enable the implementation of inhibition studies with contact times of 5, 15 and 30 min, as usually performed in conventional procedures. Thus, the reaction coil of the former system was replaced by 3 mixing chambers that guaranteed complete mixing of the aspirated solutions (bacteria and inhibitor), without operator intervention. This kind of device has been extensively used in flow analysis and in particular in SIA systems to improve the mixture of solutions aspirated sequentially, leading usually to an enhancement of the sensitivity and repeatability of the methodologies [20]. In this case, the incorporation of three mixing chambers enabled the simultaneous implementation of assays with contact times of 5, 15 and 30 minutes. This modification required the optimization of the parameters directly affected by the new assay conditions. The optimization studies comprised the selection of flow rate and dispensed volume to the mixing chambers, optimization of bacteria and sample volumes and studies of the influence of temperature on bacteria stability, inside the mixing chamber. A solution of Cu(II) 0.09 mmol L−1was used as inhibitor in the optimization assays. The propulsion flow rate to the mixing chambers is a determinant factor studied during the optimization of automated flow assays incorporating these devices. The optimization of this parameter must be performed in parallel with the optimization of the time necessary to propel the reaction zone to the chambers. Only the coordination between these two parameters ensures that only the reaction zone enters into the mixing chamber, preventing the entry of carrier and consequently the dilution of the reaction zone. The best compromise between sensitivity and repeatability was achieved through the propulsion of the reaction zone to the mixing chamber during 13 seconds, at a flow rate of 1 mL min−1. Regarding V. fischeri, its volume was tested between 50 and 100 μL and as a compromise between cost and sensitivity the studies proceeded with 75 μL of bacterial suspension.

The volume of inhibitor was fixed at 50 μL since as can be seen in Fig. 3 for higher volumes (75 and 100 μL) a dilution of the reaction zone was registered. 3.2 Evaluation of ILs (eco) toxicity through automated and batch assays The developed methodology was applied to the evaluation of ILs aquatic toxicity. Particular emphasis was given to the impact of different structural elements in the overall toxicity, namely specific cations and anions,. The respective inhibition profiles are shown in Fig. 4 and the calculated EC50 values for 5, 15 and 30 min of contact time are represented in Tables 2 and 3 for the automated and batch assays, respectively.

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In terms of head groups, the effect of imidazolium, choline, pyridinium and ammonium in the toxicity of was evaluated. The impact of anions to V. fischeri was assessed through the analysis of five groups commonly used in ILs: methanesulfonate, bistriflimide, acetate, chloride and tetrafluoroborate.

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Regarding to cation effect, comparing V. fischeri inhibition in the presence of emim [Ac] (EC50 = 16.4 mmol L−1) and chol [Ac] (EC50 = 22.4 mmol L−1), a higher EC50 was obtained with the last one, i.e. the choline element presented lower toxicity than the imidazolium one. These results are in good agreement with literature data, since the quaternary ammonium cation incorporating polar hydroxyl group is expected to have relatively low toxicity [21,22]. For instance, Hou et al. [21] demonstrated that the cholinium core is less harmful to acetylcholinesterase than the imidazolium one. It was also evidenced that ILs with nonaromatic groups present generally lower toxicity than the ones incorporating aromatic rings as can be seen from the results of N4,4,4,4 [Cl] (EC50= 3.65 mmol L−1) and bmpy [Cl] (EC50= 2.23 mmol L−1). This tendency was already observed by some authors, for different aquatic organisms [23–26]. Although it is not possible to establish a direct comparison between imidazolium and pyridinium cores, emim [Ms] (EC50 = 127 mmol L−1) and emim [Tf2N] (EC50 = 2.69 mmol L−1) exhibited higher EC50 than bmpy [Cl] (EC50 = 2.23 mmol L−1) and bmpy [BF4] (EC50 = 0.313 mmol L−1). The apparent higher toxicity of the pyridinium core to V. fischeri observed in the assays is in accordance with the described effect of an extra element in the ring [27].

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Concerning to the anion effect in ILs toxicity, three different moieties incorporating 1-ethyl3-methylimidazolium as head group (emim [Ms], emim [Tf2N] and emim [Ac]) were tested. The results revealed a significant contribution of bistriflimide to ILs toxicity. It is known that perfluoronated ions are potential toxic elements, due to their hydrolysis to hydrofluoric acid. In particular, bistriflimide, with six fluorine atoms, is potentially dangerous for the environment [28], as confirmed by the obtained results. The effect of other fluorinecontaining anion was determined and the fluorine toxicity persisted in comparison with the respectively halide (bmpy [BF4] = 0.313 mmol L−1 and bmpy [Cl] = 2.23 mmol L−1, respectively). All these considerations are also valid for the microplate assay. Additionally, as can be seen in Table 2 and 3, the EC50 values obtained in both SIA and microplate assays are globally similar for all the tested compounds. This similarity is particularly relevant when compared with the discrepant results present in literature, in terms of EC50, for the same compound and microorganism [29–32]. This is probably associated to the difficulty in controlling the reaction conditions that was obviated in the approach herein presented.

To confirm if the apparent parallelism observed between the obtained results, both in terms of EC50 and hazard ranking, was statistically acceptable, a statistical analysis of the results was performed for all compounds and contact times. A Pearson's analysis was applied and revealed a strong positive correlation (P  > 0.98) between automated and batch assays. The nonparametric Wilcoxon test allowed equally to verify if the analysis of ILs toxicity through SIA system and microplate approaches presents statistical differences in terms of EC50 values. Statistical results showed that, for a 2-sided significance level of 0.05 (p = 0.211), no significant changes were observed. Similarly to standards methods (EN ISO 11,348), the sensitivity of the developed automated methodology is in the range of mg L−1. The repeated analysis of variable concentrations of distinct ILs (n = 5) did not conduct to relative standard deviations higher than 10% which is acceptable considering the particularities of the studied compounds.

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4 Conclusions

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In this work, the toxicity of seven ILs to the aquatic bacteria V. fischeri was evaluated in a fully automated SIA system. The obtained results proved that the structural elements that constitute the studied ILs, namely the cation head group and anion moiety, determine their toxicity. It was observed that the aromatic rings incorporated in the cation core affect more the toxicity of ILs than the non-aromatic groups, since ILs containing choline and tetrabutylammonium as cations presented higher EC50 than the ones containing imidazolium and pyridinium. In terms of anion, as expect according to literature [28], the compounds containing fluorine in the anion composition (bistriflimide and tetrafluoroborate) presented higher toxic effect to bacteria than other anion moieties.

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The developed automated SIA system for the evaluation of ILs' aquatic toxicity through the analysis of their effect on V. fischeri showed to be simple and robust while providing reliable data by means of a non-specific manifold. It is important to highlight that the assembled apparatus can have utility in the majority of laboratories to perform a large variety of analytical measurements by means of small instrumental rearrangements and adequate instructions. It is also an economic and environmentally friendly approach, in which there is a significant reduction of both V. fischeri volume and effluent production when compared with the BioTox™ assay kit that was used for comparison purposes. Additionally, SIA provided a precise control of the reaction conditions (time, mixture and temperature) enabling a good reproducibility of the conditions between the assays. Furthermore, the fully automated mode of operation of the system demands reduced intervention of the operator resulting in a reduction of errors and in higher protection of the operator particularly important during the analysis of toxic chemicals.

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Acknowledgements This work received financial support from the European Union (FEDER funds through COMPETE) and National Funds (FCT, Fundação para a Ciência e Tecnologia) through project Pest-C/EQB/LA0006/2013. The work also received financial support from the European Union (FEDER funds) under the framework of QREN through Project NORTE-070124-FEDER-000067. Susana P.F. Costa received PhD grant (SFRH/BD/86381/2012) from FCT. To all financing sources the authors are greatly indebted.

References [1] X.Y.Y. Ma X.C.C. Wang H.H. Ngo W.S. Guo M.N.N. Wu N. Wang Bioassay based luminescent bacteria: Interferences, improvements, and applications Sci. Total Environ. 468 2014 1–11 [2] S. Girotti E.N. Ferri M.G. Fumo E. Maiolini Monitoring of environmental pollutants by bioluminescent bacteria Anal. Chim. Acta 608 2008 2–29 [3] S. Parvez C. Venkataraman S. Mukherji A review on advantages of implementing luminescence inhibition test (Vibrio fischeri) for acute toxicity prediction of chemicals Environ. Int. 32 2006 265–268

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[4] J. Lappalainen R. Juvonen K. Vaajasaari M. Karp A new flash method for measuring the toxicity of solid and colored samples Chemosphere 38 1999 1069–1083

SC

RI

[5] L. Bolelli Z. Bobrovová E. Ferri F. Fini S. Scandurra G. Fedrizzi S. Girotti Bioluminescent bacteria assay of veterinary drugs in excreta of food-producing animals J. Pharm. Biomed. Anal. 42 2006 88–93

U

[6] T. Backhaus M. Scholze L.H. Grimme The single substance and mixture toxicity of quinolones to the bioluminescent bacterium Vibrio fischeri Aquat. Toxicol. 49 2000 49–61

A

N

[7] M.T. Cronin T.W. Schultz Validation of Vibrio fisheri acute toxicity data: mechanism of action-based QSARs for non-polar narcotics and polar narcotic phenols Sci. Total Environ. 204 1997 75–88

D

M

[8] G. Repetto A. Jos M.J. Hazen M.L. Molero A. del Peso M. Salguero P.D. Castillo M.C. Rodriguez-Vicente M. Repetto A test battery for the ecotoxicological evaluation of pentachlorophenol Toxicol. in Vitro 15 2001 503–509

TE

[9] M.J. Ruiz L. Lopez-Jaramillo M.J. Redondo G. Font Toxicity assessment of pesticides using the microtox test: application to environmental samples Bull. Environ. Contam. Toxicol. 59 1997 619–625

CC

EP

[10] D. Hirmann A.P. Loibner R. Braun O.H. Szolar Applicability of the bioluminescence inhibition test in the 96-well microplate format for PAH-solutions and elutriates of PAHcontaminated soils Chemosphere 67 2007 1236–1242

A

[11] A.P. Loibner O.H. Szolar R. Braun D. Hirmann Toxicity testing of 16 priority polycyclic aromatic hydrocarbons using Lumistox Environ. Toxicol. Chem. 23 2004 557–564 [12] E. Komaitis E. Vasiliou G. Kremmydas D.G. Georgakopoulos C. Georgiou Development of a fully automated flow injection analyzer implementing bioluminescent biosensors for water toxicity assessment Sensors 10 2010 7089–7098 [13] P.C.A.G. Pinto S.P.F. Costa J.L.F.C. Lima M.L.M.F.S. Saraiva Automated highthroughput Vibrio fischeri assay for (eco) toxicity screening: Application to ionic liquids Ecotox. Environ. Safe. 80 2012 97–102

[14] C.I.C. Silvestre P.C.A.G. Pinto M.A. Segundo M.L.M.F.S. Saraiva J.L.F.C. Lima Enzyme based assays in a sequential injection format: A review Anal. Chim. Acta 689 2011 160–177 [15] R.B.R. Mesquita A.O.S.S. Rangel A review on sequential injection methods for water analysis Anal. Chim. Acta 648 2009 7–22 [16] P.C.A.G. Pinto M.L.M.F.S. Saraiva J.L.F.C. Lima Sequential injection analysis as a tool for implemention of enzymatic assays in ionic liquids Talanta 77 2008 479–483 [17] P. Wasserscheid , T. Welton , Ionic liquids in Synthesis Second Ed. Wiley-VCH Verlag GmbH & Co. KGaA Weinheim 2008;

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[18] S.P.M. Ventura C.S. Marques A.A. Rosatella C.A.M. Afonso F. Goncalves J.A.P. Coutinho Toxicity assessment of various ionic liquid families towards Vibrio fischeri marine bacteria Ecotox. Environ. Safe. 76 2012 162–168

SC

RI

[19] E. Cunha P.C.A.G. Pinto J.P.S. Carvalho M.L.M.F.S. Saraiva Automated carboxylesterase assay for the evaluation of ionic liquids' human toxicity J. Hazard. Mater. 244 2013 563–569

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[20] E.A.G. Zagatto J.M.T. Carneiro S. Vicente P.R. Fortes Mixing chambers in flow analysis: a review J. Anal. Chem. 64 2009 524–532

A

N

[21] X.D. Hou Q.P. Liu T.J. Smith N. Li M.H. Zong Evaluation of Toxicity and Biodegradability of Cholinium Amino Acids Ionic Liquids Plos One 8 2013

D

M

[22] S.P. Ventura F.A. Silva A.M. Goncalves J.L. Pereira F. Goncalves J.A. Coutinho Ecotoxicity analysis of cholinium-based ionic liquids to Vibrio fischeri marine bacteria Ecotox. Environ. Safe. 102 2014 48–54

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[23] D.J. Couling R.J. Bernot K.M. Docherty J.K. Dixon E.J. Maginn Assessing the factors responsible for ionic liquid toxicity to aquatic organisms via quantitative structure - property relationship modeling Green Chem. 8 2006 82–90

CC

EP

[24] C. Pretti C. Chiappe I. Baldetti S. Brunini G. Monni L. Intorre Acute toxicity of ionic liquids for three freshwater organisms: Pseudokirchneriella subcapitata, Daphnia magna and Danio rerio Ecotox. Environ. Safe. 72 2009 1170–1176

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[25] S. Stolte M. Matzke J. Arning A. Boschen W.R. Pitner U. Welz-Biermann B. Jastorff J. Ranke Effects of different head groups and functionalised side chains on the aquatic toxicity of ionic liquids Green Chem. 9 2007 1170–1179 [26] P. Luis I. Ortiz R. Aldaco A. Irabien A novel group contribution method in the development of a QSAR for predicting the toxicity (Vibrio fischeri EC50) of ionic liquids Ecotox. Environ. Safe. 67 2007 423–429 [27] S.P.M. Ventura A.M.M. Gonçalves T. Sintra J.L. Pereira F. Gonçalves J.A.P. Coutinho Designing ionic liquids: the chemical structure role in the toxicity Ecotoxicology 22 2013 1– 12

[28] H. Wang S.V. Malhotra A.J. Francis Toxicity of various anions associated with methoxyethyl methyl imidazolium-based ionic liquids on Clostridium sp. Chemosphere 82 2011 1597–1603 [29] R.F.M. Frade C.A.M. Afonso Impact of ionic liquids in environment and humans: An overview Hum. Exp. Toxicol. 29 2010 1038–1054 [30] K.M. Docherty C.F. Kulpa Toxicity and antimicrobial activity of imidazolium and pyridinium ionic liquids Green Chem. 7 2005 185–189

PT

[31] J. Ranke K. Molter F. Stock U. Bottin-Weber J. Poczobutt J. Hoffmann B. Ondruschka J. Filser B. Jastorff Biological effects of imidazolium ionic liquids with varying chain lengths in acute Vibrio fischeri and WST-1 cell viability assays Ecotox. Environ. Safe. 58 2004 396– 404

RI

[32] M.T. Garcia N. Gathergood P.J. Scammells Biodegradable ionic liquids Part II. Effect of the anion and toxicology Green Chem. 7 2005 9–14

N

U

SC

Fig. 1 Chemical structures of the studied ILs: (1) 1-ethyl-3-methylimidazolium methanesulfonate (emim [Ms]); (2) 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl) imide (emim [Tf2N]); (3) 1-ethyl-3-methylimidazolium acetate (emim [Ac]); (4) choline acetate (chol [Ac]); (5) 1-butyl-4-methylpyridinium chloride (bmpy [Cl]); (6) 1-butyl-4-methylpyridinium tetrafluoroborate (bmpy [BF4]); (7) tetrabutylammonium chloride (N4,4,4,4 [Cl]).

M

A

Fig. 2 Schematic representation of the SIA manifold. C: carrier (NaCl 2%, pH 7); S: syringe; HC: holding coil; SV: selection valve; D: detector; IL: ionic liquid; MC: mixing chambers (containing a magnet each).

D

Fig. 3 Effect of different inhibitor volumes (10 to100 μL) in V. fischeri bioluminescence.

TE

Fig. 4 Experimental toxicity data of ILs tested in SIA system, for 5 min of contact time (average of 3 replicates).

EP

Table 1 Analytical cycle for the evaluation of V. fischeri bioluminescence in the presence of the tested ILs. Flow rate

Valve position

Time (s)

1 2 3 4 5 6

1 2 3 5/6/7 10 5/6/7

3 4.5 3 13 20 277/417/1090

(mL min ) 0.5 1 0.5 1 -----

7

5/6/7

13

1

212.5

8

9

40

2

---

A

CC

Step

-1

Volume (µL) 25 75 25 217 2775 ----

Event Aspiration of inhibitor Aspiration of V. fischeri Aspiration of inhibitor Propulsion to mixing chamber Filling of the syringe with carrier Stop period Aspiration of reaction zone from the mixing chamber Propulsion to detector

Flow rate

Valve position

Time (s)

9

5/6/7

12

(mL min ) ---

10

5/6/7

12

---

---

11 12

8 10

20 20

-----

--3058

Step

Volume (µL)

-1

---

Event Propulsion of carrier to the mixing chamber Aspiration of carrier from the mixing chamber Propulsion of cleaning solution to waste Filling of the syringe with carrier

Table 2 Results of the inhibition of V. fischeri by ILs expressed as EC50 for 5, 15 and 30 min of contact time in the SIA system. EC50 (mmol EC50(mmol L-1) L-1)

EC50 EC (mg L(mmol L-1) 1 50 )

16.4 ± 1.3 13.0 ± 0.7 22.2 ± 1.4 17.8 ± 0.8 2.23 ± 0.11 1.29 ± 0.01

9.62 ±2.38 11.3 ± 0.9 0.885 ± 0.064 bmpy [BF4] 0.12 - 0.41 0.313 ± 0.238 ± 0.038 0.191 ± 0.017 0.007 N4,4,4,4 [Cl] 0.29 – 12 3.65 ± 0.93 1.98 ± 0.06 1.44 ± 0.08

N

4.4 - 35 4.7 - 88 0.23 - 8.2

45.3 400

Aquatic toxicity[18]b

Harmless Practically harmless Harmless Harmless Practically harmless Moderately toxic Practically harmless

TE

a

1637 1843 164

D

M

A

emim [Ac] chol [Ac] bmpy [Cl]

U

SC

RI

PT

Concentration Compound range(mmol Vibrio Vibrio Vibrio Vibrio L-1) fischeri for fischeri for 5 fischerifor 15 fischerifor 30 min ± min± SDa min ± SDa 30 min SDa emim [Ms] 10.2 - 352 127 ± 9 63.1 ± 1.5 60.1 ±7.4 14083 emim [Tf2N] 0.59 - 4.7 2.69 ± 0.30 2.40 ± 0.08 2.14 ± 1.02 837

Mean values and standard deviation of three replicates.

b

CC

EP

A substance with an EC50 between 100 and 1000 mg L is considered practically harmless, from 10 to 100 mg L−1 moderately toxic, from 1 to 10 mg L−1 slightly toxic and from 0.1 to 1 mg L−1 highly toxic [18].

A

Table 3 Results of the inhibition of V. fischeri by ILs expressed as EC50 for 5, 15 and 30 min of contact time in the microplate approach. EC50 Concentrati Compou (mmol L−1)Vib on range nd rio fischeri for (mmol L−1) 5 min ± SDa emim 2.5 – 350 111 ± 10 [Ms] emim 0.59 – 4.7 3.34 ± 0.01 [Tf2N]

EC50 (mmol L−1)Vib rio fischeri for 15 min ± SDa

EC50 (mmol L−1)Vib rio fischeri for 30 min ± SDa

EC50 Aquatic (mg L−1)Vibr toxicity io fischeri [18]b for 30 min

81.8 ± 7.6

44.7 ± 16.9

10474

Harmless

2.60 ± 0.17

2.17 ± 0.02

849

Practicall y

EC50 Concentrati Compou (mmol L−1)Vib on range nd rio fischeri for (mmol L−1) 5 min ± SDa emim 4.4 – 35 [Ac] chol [Ac] 4.7 – 88

EC50 (mmol L−1)Vib rio fischeri for 15 min ± SDa

EC50 (mmol L−1)Vib rio fischeri for 30 min ± SDa

EC50 Aquatic (mg L−1)Vibr toxicity io fischeri [18]b for 30 min harmless

18.5 ± 1.3

14.7 ± 4.3

6.25 ± 2.89

1064

Harmless

21.4 ± 1.2

21.7 ± 3.9

20.5 ± 2.2

3343

2.81 ± 0.50

1.09 ± 0.04

0.921 ± 0.038 171

Harmless Practicall y harmless Moderate ly toxic Practicall y harmless

bmpy [Cl]

0.23 – 8.2

bmpy [BF4]

0.12 – 0.41 0.265 ± 0.007 0.240 ± 0.001 0.208 ± 0.008 49.3

N4,4,4,4 [Cl]

0.29–12

1.89 ± 0.04

Mean values and standard deviation of three replicates.

b

525

PT

2.45 ± 0.02

RI

a

4.12 ± 0.09

U

SC

A substance with an EC50 between 100 and 1000 mg L-1 is considered practically harmless, from 10 to 100 mg L-1 moderately toxic, from 1 to 10 mg L-1 slightly toxic and from 0.1 to 1 mg L-1 highly toxic [18]. H3 C

1

N

N+

A

N

H3 C

N

CC

H3 C

A

5

7

O

N+ –O

4

EP

3

TE

D

M

H3 C

CH3

6

–N

O

F

S

F

O

F

O

F

S O

F F

2

Fig. 1 V. fischeri NaCl 2%, pH 7 (15 ºC) (15 ºC) HC SV C (15 ºC)

2

1

10

3

D

9 4 5 6

7

8

S

PT

WASTE

RI

IL (15 ºC)

SC

MC (15 ºC)

U

Fig. 2

N

70

A

50 40

M

% inhibition

60

30 20

D

10

A

CC

Fig. 3

20

40 60 80 inhibitor volume (µL)

EP

0

TE

0

100

120

100 80

Chol [Ac]

% inhibition

emim [Ac] 60

emim [Tf2N] bmpy [Cl]

40

N4,4,4,4 [Cl] emim [Ms]

20

bmpy [BF4] 0 -2

-1

Cu 0

1

2

3

PT

log C (mmol L -1 )

A

CC

EP

TE

D

M

A

N

U

SC

RI

Fig. 4