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Comparative ecotoxicity of potential biofuels to water flea (Daphnia magna), zebrafish (Danio rerio) and Chinese hamster (Cricetulus griseus) V79 cells
Science of the Total Environment 631–632 (2018) 216–222
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Comparative ecotoxicity of potential biofuels to water flea (Daphnia magna), zebrafish (Danio rerio) and Chinese hamster (Cricetulus griseus) V79 cells Sebastian Heger a,1, Miaomiao Du a,1, Kevin Bauer a, Andreas Schäffer b,c,d, Henner Hollert a,c,d,e,⁎ a
RWTH Aachen University, Institute for Environmental Research, Department of Ecosystem Analysis, Aachen, Germany RWTH Aachen University, Institute for Environmental Research, Environmental Biology and Chemodynamics, Aachen, Germany c Chongqing University, College of Resources and Environmental Science, Chongqing, China d Nanjing University, State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing, China e Tongji University, College of Environmental Science and Engineering, State Key Laboratory of Pollution Control and Resource Reuse, Shanghai, China b
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Both 1 octanol and 2 butanone caused teratogenic and lethal effects on zebrafish embryos. • Exposure to 1 octanol induced significant effects at concentrations ≥2.0 mg L−1. • 1 Octanol exerts much higher ecotoxicity than 2 butanone to D. magna and zebrafish embryos.
a r t i c l e
i n f o
Article history: Received 20 January 2018 Received in revised form 2 March 2018 Accepted 3 March 2018 Available online xxxx Editor: D. Barcelo Keywords: Ecotoxicity Biofuels Daphnia magna Danio rerio Micronucleus assay
a b s t r a c t The ecotoxicity of two biofuel candidates (1 octanol and 2 butanone) was investigated by an integrative test strategy using three bioassays: the acute immobilisation test with water flea (D. magna), the fish embryo acute toxicity test with zebrafish (Danio rerio) and the in vitro micronucleus assay with Chinese hamster (Cricetulus griseus) V79 cells. The median effective concentration (EC50) values were 14.9 ± 0.66 mg L−1 for 1 octanol, and 2152.1 ± 44.6 mg L−1 for 2 butanone in the D. magna test. Both 1 octanol and 2 butanone caused teratogenic and lethal effects on zebrafish embryos, while exposure to 1 octanol significantly induced these effects at concentrations ≥2.0 mg L−1. These results indicate that 1 octanol exert much higher ecotoxicity than 2 butanone to D. magna and zebrafish embryos. Moreover, both 1 octanol and 2 butanone did not cause significant genotoxic effects, while their metabolites significantly induced micronuclei in V79 cells. The present study proposed an integrative test approach to evaluate the potential ecotoxicity of biofuels using simple, quick and inexpensive bioassays. © 2018 Elsevier B.V. All rights reserved.
⁎ Corresponding author: Department of Ecosystem Analysis, ESA, Worringerweg 1, 52074 Aachen, Germany. E-mail address: [email protected] (H. Hollert). 1 Contributed equally to this work as co-first authors of this paper.
https://doi.org/10.1016/j.scitotenv.2018.03.028 0048-9697/© 2018 Elsevier B.V. All rights reserved.
1. Introduction Increasing energy demand, greenhouse gas emissions and air pollution from fossil fuels make switching to renewable, low-carbon and
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environment-friendly fuels a high priority (Fargione et al., 2008; Sendzikiene et al., 2007). Biofuels are considered to be potential alternatives for fossil fuels due to their promising benefits. The application of biofuels could maintain a better energy balance, reduce environmental pollution and slow down global climate change (Bluhm et al., 2012). With the support of European Union and the United States, biofuels technology and industry have accelerated during the last decades (Heger et al., 2012). However, there are various economic and environmental issues with biofuels production and use, including the “food vs fuel” debate, environmental concerns and impact on water resources (Srinivasan, 2009; Stephanopoulos, 2007). Recently, concerns have increased regarding their potentially adverse impacts on the environment and human health (Bluhm et al., 2012; Heger et al., 2016). Scientists believe that prior to manufacturing and distribution, risk assessment of novel tailor-made biofuels should be conducted in parallel to the biofuels development (Heger et al., 2012). The Cluster of Excellence “Tailor-Made Fuels from Biomass” (TMFB) at RWTH Aachen University takes an interdisciplinary approach to research on new synthetic fuels obtained from biomass. Within this cluster, the research area called “fuel design process” makes it possible to derive novel, optimized biofuels that are cost-effective in production and that facilitate an efficiency-optimized combustion. In parallel, a test strategy for a comprehensive hazard assessment of biofuels is developed to investigate the ecotoxicological impact of the biofuels. This strategy provides a framework for integrating ecotoxicology principles and tools into designing safer biofuels, thereby minimizing potential environmental effects as early as possible (Maertens et al., 2014). The biofuels 1 octanol and 2 butanone are two promising candidates currently investigated within TMFB, because both of them showed superior combustion and emission performance than diesel and gasoline (Heuser et al., 2015; Hoppe et al., 2016). Moreover, 1 octanol and 2 butanone can be also used widely in industrial processes and chemical technology, e.g., 1 octanol as a precursor to perfumes (Falbe et al., 2000) and 2 butanone as an industrial solvent (Turner and McCreery, 1981). However, data about their potential effects on the environment and human health is scarce. The objective of the present study was to evaluate the potential ecotoxicity of 1 octanol and 2 butanone. The acute immobilisation test (Daphnia magna), fish embryo acute toxicity test (Danio rerio) and in vitro micronucleus assay for genotoxicity (Chinese hamster V79 cells) were conducted to investigate their ecotoxicological effects on different species from different trophic levels. Furthermore, our study also aims to develop a simple and reliable methodology for screening and evaluating toxicological potential of various biofuel candidates, as well as to adapt ‘green toxicology’ in the sustainable development of biofuels. Green toxicology refers to the application of toxicology in the sustainable development and production of new less harmful materials and chemicals in terms of environmental and human health impacts (Crawford et al., 2017). 2. Materials and methods 2.1. Chemicals All chemicals used in this study were purchased from Sigma-Aldrich (Munich, Germany): 1 octanol (purity: ≥99%), 2 butanone (purity: ≥99.7%), 3,4 dichloroaniline (DCA), calcium chloride dihydrate (CaCl2·2H2O), magnesium sulfate heptahydrate (MgSO4·7H2O), sodium hydrogen carbonate (NaHCO3), potassium chloride (KCl), ethylmethane sulfonate (EMS), cyclophosphamide (CPP), methanol, glacial acetic acid and acridine orange. 2.2. Daphnia magna acute immobilisation test Individuals of D. magna STRAUS (clone 5) were kept in ~80 mL of Elendt M4 medium (OECD 202:2004) at 20 ± 1 °C and a 16:8 h light: dark cycle. D. magna were fed three times a week with Desmodesmus
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subspicatus and once a week with yeast (1 mg L−1). Medium was renewed once a week. The 48-h acute immobilisation tests using D. magna were conducted according to the OECD Test No. 202 (OECD). To avoid the potential effect from plastic, Hamilton syringes were used to prepare exposure solutions by adding test substances in M4 medium, and 20 mL glass vials sealed with PTFE caps were used for exposure. D. magna neonates (b24 h) were exposed to 1 octanol and 2 butanone, respectively. The exposure concentrations for 1 octanol were 5.0, 7.5, 10.0, 15.0 and 20.0 mg L−1 and those for 2 butanone were 500, 1000, 2000, 3000, 4000 and 5000 mg L−1. All exposure concentrations were chosen based on our range finding experiments. Four glass vials were used for each exposure concentration (20 mL exposure solution and 5 D. magna neonates per vial). M4 medium without test substances was used as negative control (NC). All D. magna were kept at 20 ± 1 °C and a 16:8 h light:dark cycle. There was no feeding during the test. D. magna unable to swim for 15 s after gentle stirring were considered immobile. The number of immobilised D. magna was recorded after 24 h and 48 h of exposure. The criterion for our tests to be valid was that no more than 10% of D. magna in the control group showed immobilisation or other signs of disease or stress. Each test was carried out in three replicates. 2.3. Fish embryo acute toxicity test Zebrafish embryos were obtained from Fraunhofer Institute for Molecular Biology and Applied Ecology in Aachen. Adult zebrafish were maintained in a 250 L glass tank with water at 26 ± 1 °C and a simulated 14 h daylight period. Fish were fed twice one day with TetraMin flakes (Tetra GmbH, Melle, Germany), and Nauplius larvae of Artemia spp. For spawning, glass dishes were placed into the glass tank to collect eggs, and then fertilized eggs between the 2-cell and 8-cell state were selected for exposure. The fish embryo acute test (FET) was conducted for 96 h according to the OECD Test No. 236 (OECD) with minor modifications (Bluhm et al., 2016). To avoid the potential effect from plastic, Hamilton syringes were used to prepare exposure solutions, and 10 mL glass vials were used for exposure. Newly fertilized zebrafish eggs were examined under a microscope, and those developed normally and reached at least 16-cell stage were chosen for the exposure. Then those eggs were randomly transferred into glass vials with artificial water (294 mg L−1 CaCl2·2H2O, 123.3 mg L−1 MgSO4·7H2O, 63 mg L−1 NaHCO3, 5.5 mg L−1 KCl). The exposure concentrations for 1 octanol were 2.0, 5.0, 10.0, 15.0, 20.0 and 30.0 mg L−1, and those for 2 butanone were 500, 1000, 1500, 2000, 3000, 4000 and 5000 mg L−1. Each exposure concentration consisted of four glass vials (10 mL exposure solution and 5 embryos per vial). Artificial water was used as negative control (NC), and 3.7 mg L−1 3,4 dichloroaniline in medium was performed as positive control (PC). All the eggs were kept at 26 ± 1 °C and subjected to a 14:10 h light:dark cycle. Each egg was examined under the microscope every 24 h, and lethality, abnormal development as well as hatching were recorded. All experiments were conducted in three replicates. 2.4. Micronucleus assay with V79 cells Chinese hamster lung fibroblast cell line V79 was cultivated in Dulbecco's Modified Eagle Medium (DMEM) with phenol red and glutamine (Invitrogen, Carlsbad, CA, USA) supplemented with 9% fetal calf serum (FCS, Biowest, Nuaillé, France) and 1% penicillin/streptomycin solution (Sigma-Aldrich). Cells were maintained at 37 °C, 5% carbon dioxide and 90% humidity. Cells are passaged three times per week. The micronucleus assay was performed according to the international guideline (ISO/DIS 21427-2) and as detailed in Reifferscheid et al. (2008) with slight modifications. V79 cells were seeded onto microscopic glass cover slips (18 × 18 mm) in crystallizing dishes covered
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Fig. 1. Acute toxicity induced by 48-h exposure to 1 octanol (A) and 2 butanone (B) on D. magna. The data are presented as the mean ± SD (n = 3). NC: negative control.
with aluminium foil and glass covers, and were incubated for 6 h. Subsequently, the medium was replaced by supplemented DMEM with 9% FCS (treatment without S9 mix) or supplemented FCS-free DMEM with S9 mix (treatment with S9 mix). Then different concentrations of 1 octanol (3.125, 6.25, 12.5 and 25.0 mg L−1) or 2 butanone (3220, 4025, 4830, 5635 and 6440 mg L−1) were added into the test culture. DMEM was used as negative control (NC); 350 mg L−1 EMS (without S9) and 2.5 mg L−1 CPP (with S9) in DMEM were performed as positive controls. The cultures without S9 were incubated for 24 h at 37 °C, 5% carbon dioxide and 90% humidity. The cultures with S9 were incubated for 4 h at 37 °C, 5% carbon dioxide and 90% humidity. After 4 h, the medium was removed and then fresh supplemented DMEM with 9% FCS was added and incubated for further 20 h. At the end of incubation, the cells were treated for 5 mins in a 1:1 mixture of PBS and methanol/glacial acetic acid (4:1 v/v) solution, and were fixed for 5 min with methanol/glacial acetic acid (4:1 v/v) solution. After air-drying, the glass cover slips were mounted onto glass slides and the cells were stained with 0.004% acridine orange diluted in PBS. 1000 cells per slip were randomly selected and analysed for micronucleus under a fluorescence microscope (Nikon GmbH, Düsseldorf, Germany) at 200× magnification coupled to an automated slide-loader system (PL 200 Slide Loader, Prior Scientific Instruments GmbH, Jena, Germany) using the software NIS-elements v4.13.03 (Nikon GmbH).
2.5. Statistical analysis For the tests using D. magna and zebrafish embryos, the median effective concentrations (EC50) and median lethal concentrations (LC50) were calculated using the software ToxRat Professional (ToxRat Solutions GmbH, Alsdorf, Germany). For the micronucleus assay, analysis of variance (one way ANOVA with Dunnett's post-hoc test) was performed to determine the statistical differences between the treatments and control groups using the software GraphPad Prism 6.02 (GraphPad Inc., San Diego, CA). The criterion for statistical significance was p b 0.05. All data were expressed as the mean ± standard deviation (SD) (n = 3).
3. Results and discussion 3.1. D. magna acute immobilisation test After 48 h exposure, the EC50 values were 14.9 ± 0.66 mg L−1 for 1 octanol, and 2152.1 ± 44.6 mg L−1 for 2 butanone (Fig. 1 and Table 1). The results indicated that 1 octanol showed toxicity values that was 144-fold of 2 butanone. Based on classification criterion by Commission of the European Communities (96/67/EEC), 1 octanol could be categorized as harmful to aquatic organisms (EC50 10– 100 mg L−1), while 2 butanone could be classified as nontoxic to aquatic organisms (EC50 ˃ 100 mg L−1). Kühn et al. (1989) reported that the EC50 values of 1 octanol on D. magna were 26 mg L−1 for 24-h acute test. The shorter exposure time (24 h) and test conducted in open vessel maybe the reason why the EC50 in their report was higher than our results. The 48-h EC50 values of 2 butanone reported in other studies were ˃520 mg L−1 (Leblanc, 1980) and 5100 mg L−1 (Pedersen and Petersen, 1996) by acute toxicity test on D. magna.
3.2. Fish embryo acute toxicity test Our results revealed concentration-dependent effects of 1 octanol and 2 butanone on zebrafish embryos (Figs. 2, 3). Their EC50 and LC50 values (Table 1) indicated that 1 octanol was much more toxic to zebrafish embryos than 2 butanone. Exposure to 1 octanol induced a significant increase in embryo mortality and morphological defects even at our lowest concentration (2.0 mg L−1), while 2 butanone showed significant effects at 500 mg L−1 (p b 0.05). As shown in Fig. 4, both 1 octanol and 2 butanone induced different morphological alterations on zebrafish embryos and larvae, i.e., pericardial edema, yolk sac edema, lack of pigmentation, eye defects. However, the severe spinal curvature was only found in 1 octanol exposure groups (Fig. 4E). Previous studies reported that the 96-h LC50 of 1 octanol by acute toxicity test to 30-d-old fathead minnows was 13.5 mg L−1 (Veith et
Table 1 Acute toxicity results of 1 octanol and 2 butanone.
1–Octanol 2 Butanone
D. magna
Zebrafish embryos
EC50 48 h
EC50 48 h
LC50 48 h
EC50 96 h
LC50 96 h
14.9 ± 0.66 2152.1 ± 44.6
10.9 ± 1.2 1897.8 ± 234.3
15.3 ± 1.7 4307.6 ± 178.5
7.7 ± 3.3 822.1 ± 183.1
10.0 ± 3.9 2627.8 ± 1090.4
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Fig. 2. Zebrafish embryo toxicity induced by exposure to 1 octanol. (A): Effect rate at 48 hpf; (B): mortality rate at 48 hpf; (C): effect rate at 96 hpf; (D): mortality rate at 96 hpf. The data are presented as the mean ± SD (n = 3). NC: negative control; PC: positive control.
al., 1983), and the 48-h LC50 for 1 octanol from static acute toxicity test with 24-d-old medaka was 21 mg L−1 (Carlson et al., 1998). These values for LC50 were higher than the results in present study, which might be due to that fish embryos are more sensitive than juvenile fish (Mohammed, 2013). 1 octanol has been identified as nonpolar narcotic on fathead minnows (Veith et al., 1983). After exposure to 1 octanol, a syndrome of hypoactivity was identified in juvenile fathead minnows, but no unusual change in their body morphology
(Drummond and Russom, 1990). Moreover, exposure to 1 octanol caused neurological effects on startle response and predation on medaka (Carlson et al., 1998). The 96-h LC50 for 2 butanone on zebrafish was 3200 mg L−1 (Pedersen and Petersen, 1996), similar to the results in present study. Maes et al. (2012) found that exposure to 2 butanone resulted in pericardial edema, jaw defect, microcephaly, impaired circulation and developmental delay on zebrafish embryos and larvae. Moreover,
Fig. 3. Zebrafish embryo toxicity induced by exposure to 2 butanone. (A): Effect rate at 48 hpf; (B): mortality rate at 48 hpf; (C): effect rate at 96 hpf; (D): mortality rate at 96 hpf. The data are presented as the mean ± SD (n = 3). NC: negative control; PC: positive control.
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Fig. 4. Morphological defects of zebrafish embryos and larvae after exposure to 1 octanol and 2 butanone. (A): Negative control at 48 hpf; (B): 10 mg L−1 1 octanol at 48 hpf; (C): 3000 mg L−1 2 butanone at 48 hpf; (D): negative control at 96 hpf; (E): 10 mg L−1 1 octanol at 96 hpf; (F): 2000 mg L−1 2 butaone at 96 hpf.
2 butanone also exhibited developmental toxicity in Swiss Mice. After pregnant mice were exposed to 2 butanone vapors, their offspring showed several malformation, cleft palate, fused ribs, missing vertebrae and syndactyly (Schwetz et al., 1991).
3.3. Micronucleus assay with V79 cells Representative photos of cells with micronuclei are shown in Fig. 5. Without metabolic activation (S9 mix), both 1 octanol and 2 butanone did not cause significant induction of micronuclei in Chinese hamster V79 Cells (Fig. 6). However, in the presence of exogenous metabolic activation, both biofuel candidates increased micronucleus frequencies in a concentration-dependent manner. For 1 octanol, significant increase in micronuclei was identified at 12.5 and 25.0 mg L−1 with metabolic activation, while 2 butanone significantly induced micronuclei at high concentrations (4830, 5635 and 6440 mg L−1) with metabolic activation (Fig. 6). Our results show that metabolic activation with an exogenous S9 supplementation could increase the mutagenic potential of 1 octanol and 2 butanone, which suggests that both 1 octanol and 2 butanone might be metabolised by cytochrome P450 depending enzymes to their genotoxic metabolites. Previous study showed that 1 octanol (≥13 mg L−1) could induce spindle disturbances in V79 cells, such as c-mitosis and aneuploidy, which is considered to be an unspecific effect based on the distribution of the lipophilic compound into hydrophobic cell compartments.
Fig. 5. Photomicrographs of micronucleus in V79 cells after treatment with 1 octanol (A) and 2 butanone (B). Micrographs were captured at 1000× magnification.
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Fig. 6. Induction of micronuclei in V 79 cells treated with 1 octanol (A) and 2 butanone (B) at different concentrations. (*) p b 0.05. The data are presented as the mean ± SD (n = 3). NC: negative control.
However, the concentrations given in this study might be higher than the true concentrations due to experimental errors (Önfelt, 1987). Odonoghue et al. (1988) found that 2 butanone was not genotoxic by a battery of in vitro assays including Salmonella (Ames) assay, L5178/TK+/− mouse lymphoma assay, BALB/3T3 cell transformation assay, unscheduled DNA synthesis and micronucleus assay. The only evidence of genotoxicity for 2 butanone was mitotic chromosome loss at a very high concentration in the yeast Saccharomyces cerevisiae (Zimmermann et al., 1985). The results in the present study indicated that 1 octanol showed much higher ecotoxicity than 2 butanone to D. magna, zebrafish embryos and Chinese hamster V79 cells. Previous studies reported that a large variety of chemicals were more toxic in acute tests to D. magna than to D. rerio, e.g., metals, pesticides and solvents (Martins et al., 2007); while our results about acute toxicity of 1 octanol and 2 butanone showed that there were no significant differences between D. magna and D. rerio.
4. Conclusion The International Energy Agency has a goal for biofuels to provide 27% of total transport fuels by 2050 (Tanaka, 2011). Considering this large potential market for biofuels in the future and their inevitable release into the environment, identification of potential ecotoxicity for biofuels during their early development can help protect the entire environment and saving research resource from biofuels harmful to the environment and human health. The present study combined three widely used test systems to assess different effects (acute toxic effects, developmental effects, teratogenic effects and genotoxic effects) of two biofuel candidates, which proposed an integrative test approach to evaluate the potential ecotoxicity of biofuels using simple, quick and inexpensive bioassays. Our results can provide information for the whole cluster to establish innovative and sustainable processes for tailor-made biofuels with high efficiency and low pollutant emissions. More research resources in the cluster will be given priority to environment-friendly biofuel candidates at their early developmental stage to avoid harmful effect on the environment and human health.
Author contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. # Sebastian Heger and Miaomiao Du contributed equally to this work as co-first authors of this paper.
Acknowledgements This work was performed as part of the Cluster of Excellence “Tailormade fuels from biomass”, which is funded by the German federal and state governments to promote science and research at German universities. The authors would like to kindly thank Nikon Germany and Prior Scientific for their contribution to this study as a sponsor of the Student's Lab Fascinating Environment. References Bluhm, K., Heger, S., Seiler, T.-B., Hallare, A.V., Schäffer, A., Hollert, H., 2012. Toxicological and ecotoxicological potencies of biofuels used for the transport sector—a literature review. Energy Environ. Sci. 5, 7381–7392. Bluhm, K., Seiler, T.-B., Anders, N., Klankermayer, J., Schaeffer, A., Hollert, H., 2016. Acute embryo toxicity and teratogenicity of three potential biofuels also used as flavor or solvent. Sci. Total Environ. 566, 786–795. Carlson, R.W., Bradbury, S.P., Drummond, R.A., Hammermeister, D.E., 1998. Neurological effects on startle response and escape from predation by medaka exposed to organic chemicals. Aquat. Toxicol. 43, 51–68. Commission of the European Communities, 1996. Technical Guidance Document in Support of Commission Directive 93/67/EEC on Risk Assessment for New Notified Substances and Commission Regulation (EC) N. 1488/94 on Risk Assessment for Existing Substances. Office for Official Publications of the European Communities. Crawford, S.E., Hartung, T., Hollert, H., Mathes, B., van Ravenzwaay, B., Steger-Hartmann, T., et al., 2017. Green toxicology: a strategy for sustainable chemical and material development. Environ. Sci. Eur. 29, 16. Drummond, R.A., Russom, C.L., 1990. Behavioral toxicity syndromes - a promising tool for assessing toxicity mechanisms in juvenile fathead minnows. Environ. Toxicol. Chem. 9, 37–46. Falbe, J., Bahrmann, H., Lipps, W., Mayer, D., 2000. Alcohols, aliphatic. Ullmann's Encyclopedia of Industrial Chemistry. Fargione, J., Hill, J., Tilman, D., Polasky, S., Hawthorne, P., 2008. Land clearing and the biofuel carbon debt. Science 319, 1235–1238. Heger, S., Bluhm, K., Agler, M.T., Maletz, S., Schaffer, A., Seiler, T.B., et al., 2012. Biotests for hazard assessment of biofuel fermentation. Energy Environ. Sci. 5, 9778–9788. Heger, S., Bluhm, K., Brendt, J., Mayer, P., Anders, N., Schäffer, A., et al., 2016. Microscale in vitro assays for the investigation of neutral red retention and ethoxyresorufin O deethylase of biofuels and fossil fuels. PLoS One 11, e0163862. Heuser, B., Mauermann, P., Wankhade, R., Kremer, F., Pischinger, S., 2015. Combustion and emission behavior of linear C 8 oxygenates. Int. J. Engine Res. 16, 627–638. Hoppe, F., Burke, U., Thewes, M., Heufer, A., Kremer, F., Pischinger, S., 2016. Tailor-made fuels from biomass: potentials of 2 butanone and 2 methylfuran in direct injection spark ignition engines. Fuel 167, 106–117. ISO/DIS 21427-2. Water Quality - Evaluation of Genotoxicity by Measurement of the Induction of Micronuclei - Part 2: ‘Mixed Population’ Method Using the Cell Line V79. Kühn, R., Pattard, M., Pernak, K.-D., Winter, A., 1989. Results of the harmful effects of water pollutants to Daphnia magna in the 21 day reproduction test. Water Res. 23, 501–510. Leblanc, G.A., 1980. Acute toxicity of priority pollutants to water flea (Daphnia magna). Bull. Environ. Contam. Toxicol. 24, 684–691. Maertens, A., Anastas, N., Spencer, P.J., Stephens, M., Goldberg, A., Hartung, T., 2014. Food for thought … green toxicology. ALTEX 31, 243–249. Maes, J., Verlooy, L., Buenafe, O.E., De Witte, P.A., Esguerra, C.V., Crawford, A.D., 2012. Evaluation of 14 organic solvents and carriers for screening applications in zebrafish embryos and larvae. PLoS One 7, e43850.
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Comparative ecotoxicity of potential biofuels to water flea (Daphnia magna), zebrafish (Danio rerio) and Chinese hamster (Cricetulus griseus) V79 cells Sebastian Heger a,1, Miaomiao Du a,1, Kevin Bauer a, Andreas Schäffer b,c,d, Henner Hollert a,c,d,e,⁎ a
RWTH Aachen University, Institute for Environmental Research, Department of Ecosystem Analysis, Aachen, Germany RWTH Aachen University, Institute for Environmental Research, Environmental Biology and Chemodynamics, Aachen, Germany c Chongqing University, College of Resources and Environmental Science, Chongqing, China d Nanjing University, State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing, China e Tongji University, College of Environmental Science and Engineering, State Key Laboratory of Pollution Control and Resource Reuse, Shanghai, China b
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Both 1 octanol and 2 butanone caused teratogenic and lethal effects on zebrafish embryos. • Exposure to 1 octanol induced significant effects at concentrations ≥2.0 mg L−1. • 1 Octanol exerts much higher ecotoxicity than 2 butanone to D. magna and zebrafish embryos.
a r t i c l e
i n f o
Article history: Received 20 January 2018 Received in revised form 2 March 2018 Accepted 3 March 2018 Available online xxxx Editor: D. Barcelo Keywords: Ecotoxicity Biofuels Daphnia magna Danio rerio Micronucleus assay
a b s t r a c t The ecotoxicity of two biofuel candidates (1 octanol and 2 butanone) was investigated by an integrative test strategy using three bioassays: the acute immobilisation test with water flea (D. magna), the fish embryo acute toxicity test with zebrafish (Danio rerio) and the in vitro micronucleus assay with Chinese hamster (Cricetulus griseus) V79 cells. The median effective concentration (EC50) values were 14.9 ± 0.66 mg L−1 for 1 octanol, and 2152.1 ± 44.6 mg L−1 for 2 butanone in the D. magna test. Both 1 octanol and 2 butanone caused teratogenic and lethal effects on zebrafish embryos, while exposure to 1 octanol significantly induced these effects at concentrations ≥2.0 mg L−1. These results indicate that 1 octanol exert much higher ecotoxicity than 2 butanone to D. magna and zebrafish embryos. Moreover, both 1 octanol and 2 butanone did not cause significant genotoxic effects, while their metabolites significantly induced micronuclei in V79 cells. The present study proposed an integrative test approach to evaluate the potential ecotoxicity of biofuels using simple, quick and inexpensive bioassays. © 2018 Elsevier B.V. All rights reserved.
⁎ Corresponding author: Department of Ecosystem Analysis, ESA, Worringerweg 1, 52074 Aachen, Germany. E-mail address: [email protected] (H. Hollert). 1 Contributed equally to this work as co-first authors of this paper.
https://doi.org/10.1016/j.scitotenv.2018.03.028 0048-9697/© 2018 Elsevier B.V. All rights reserved.
1. Introduction Increasing energy demand, greenhouse gas emissions and air pollution from fossil fuels make switching to renewable, low-carbon and
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environment-friendly fuels a high priority (Fargione et al., 2008; Sendzikiene et al., 2007). Biofuels are considered to be potential alternatives for fossil fuels due to their promising benefits. The application of biofuels could maintain a better energy balance, reduce environmental pollution and slow down global climate change (Bluhm et al., 2012). With the support of European Union and the United States, biofuels technology and industry have accelerated during the last decades (Heger et al., 2012). However, there are various economic and environmental issues with biofuels production and use, including the “food vs fuel” debate, environmental concerns and impact on water resources (Srinivasan, 2009; Stephanopoulos, 2007). Recently, concerns have increased regarding their potentially adverse impacts on the environment and human health (Bluhm et al., 2012; Heger et al., 2016). Scientists believe that prior to manufacturing and distribution, risk assessment of novel tailor-made biofuels should be conducted in parallel to the biofuels development (Heger et al., 2012). The Cluster of Excellence “Tailor-Made Fuels from Biomass” (TMFB) at RWTH Aachen University takes an interdisciplinary approach to research on new synthetic fuels obtained from biomass. Within this cluster, the research area called “fuel design process” makes it possible to derive novel, optimized biofuels that are cost-effective in production and that facilitate an efficiency-optimized combustion. In parallel, a test strategy for a comprehensive hazard assessment of biofuels is developed to investigate the ecotoxicological impact of the biofuels. This strategy provides a framework for integrating ecotoxicology principles and tools into designing safer biofuels, thereby minimizing potential environmental effects as early as possible (Maertens et al., 2014). The biofuels 1 octanol and 2 butanone are two promising candidates currently investigated within TMFB, because both of them showed superior combustion and emission performance than diesel and gasoline (Heuser et al., 2015; Hoppe et al., 2016). Moreover, 1 octanol and 2 butanone can be also used widely in industrial processes and chemical technology, e.g., 1 octanol as a precursor to perfumes (Falbe et al., 2000) and 2 butanone as an industrial solvent (Turner and McCreery, 1981). However, data about their potential effects on the environment and human health is scarce. The objective of the present study was to evaluate the potential ecotoxicity of 1 octanol and 2 butanone. The acute immobilisation test (Daphnia magna), fish embryo acute toxicity test (Danio rerio) and in vitro micronucleus assay for genotoxicity (Chinese hamster V79 cells) were conducted to investigate their ecotoxicological effects on different species from different trophic levels. Furthermore, our study also aims to develop a simple and reliable methodology for screening and evaluating toxicological potential of various biofuel candidates, as well as to adapt ‘green toxicology’ in the sustainable development of biofuels. Green toxicology refers to the application of toxicology in the sustainable development and production of new less harmful materials and chemicals in terms of environmental and human health impacts (Crawford et al., 2017). 2. Materials and methods 2.1. Chemicals All chemicals used in this study were purchased from Sigma-Aldrich (Munich, Germany): 1 octanol (purity: ≥99%), 2 butanone (purity: ≥99.7%), 3,4 dichloroaniline (DCA), calcium chloride dihydrate (CaCl2·2H2O), magnesium sulfate heptahydrate (MgSO4·7H2O), sodium hydrogen carbonate (NaHCO3), potassium chloride (KCl), ethylmethane sulfonate (EMS), cyclophosphamide (CPP), methanol, glacial acetic acid and acridine orange. 2.2. Daphnia magna acute immobilisation test Individuals of D. magna STRAUS (clone 5) were kept in ~80 mL of Elendt M4 medium (OECD 202:2004) at 20 ± 1 °C and a 16:8 h light: dark cycle. D. magna were fed three times a week with Desmodesmus
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subspicatus and once a week with yeast (1 mg L−1). Medium was renewed once a week. The 48-h acute immobilisation tests using D. magna were conducted according to the OECD Test No. 202 (OECD). To avoid the potential effect from plastic, Hamilton syringes were used to prepare exposure solutions by adding test substances in M4 medium, and 20 mL glass vials sealed with PTFE caps were used for exposure. D. magna neonates (b24 h) were exposed to 1 octanol and 2 butanone, respectively. The exposure concentrations for 1 octanol were 5.0, 7.5, 10.0, 15.0 and 20.0 mg L−1 and those for 2 butanone were 500, 1000, 2000, 3000, 4000 and 5000 mg L−1. All exposure concentrations were chosen based on our range finding experiments. Four glass vials were used for each exposure concentration (20 mL exposure solution and 5 D. magna neonates per vial). M4 medium without test substances was used as negative control (NC). All D. magna were kept at 20 ± 1 °C and a 16:8 h light:dark cycle. There was no feeding during the test. D. magna unable to swim for 15 s after gentle stirring were considered immobile. The number of immobilised D. magna was recorded after 24 h and 48 h of exposure. The criterion for our tests to be valid was that no more than 10% of D. magna in the control group showed immobilisation or other signs of disease or stress. Each test was carried out in three replicates. 2.3. Fish embryo acute toxicity test Zebrafish embryos were obtained from Fraunhofer Institute for Molecular Biology and Applied Ecology in Aachen. Adult zebrafish were maintained in a 250 L glass tank with water at 26 ± 1 °C and a simulated 14 h daylight period. Fish were fed twice one day with TetraMin flakes (Tetra GmbH, Melle, Germany), and Nauplius larvae of Artemia spp. For spawning, glass dishes were placed into the glass tank to collect eggs, and then fertilized eggs between the 2-cell and 8-cell state were selected for exposure. The fish embryo acute test (FET) was conducted for 96 h according to the OECD Test No. 236 (OECD) with minor modifications (Bluhm et al., 2016). To avoid the potential effect from plastic, Hamilton syringes were used to prepare exposure solutions, and 10 mL glass vials were used for exposure. Newly fertilized zebrafish eggs were examined under a microscope, and those developed normally and reached at least 16-cell stage were chosen for the exposure. Then those eggs were randomly transferred into glass vials with artificial water (294 mg L−1 CaCl2·2H2O, 123.3 mg L−1 MgSO4·7H2O, 63 mg L−1 NaHCO3, 5.5 mg L−1 KCl). The exposure concentrations for 1 octanol were 2.0, 5.0, 10.0, 15.0, 20.0 and 30.0 mg L−1, and those for 2 butanone were 500, 1000, 1500, 2000, 3000, 4000 and 5000 mg L−1. Each exposure concentration consisted of four glass vials (10 mL exposure solution and 5 embryos per vial). Artificial water was used as negative control (NC), and 3.7 mg L−1 3,4 dichloroaniline in medium was performed as positive control (PC). All the eggs were kept at 26 ± 1 °C and subjected to a 14:10 h light:dark cycle. Each egg was examined under the microscope every 24 h, and lethality, abnormal development as well as hatching were recorded. All experiments were conducted in three replicates. 2.4. Micronucleus assay with V79 cells Chinese hamster lung fibroblast cell line V79 was cultivated in Dulbecco's Modified Eagle Medium (DMEM) with phenol red and glutamine (Invitrogen, Carlsbad, CA, USA) supplemented with 9% fetal calf serum (FCS, Biowest, Nuaillé, France) and 1% penicillin/streptomycin solution (Sigma-Aldrich). Cells were maintained at 37 °C, 5% carbon dioxide and 90% humidity. Cells are passaged three times per week. The micronucleus assay was performed according to the international guideline (ISO/DIS 21427-2) and as detailed in Reifferscheid et al. (2008) with slight modifications. V79 cells were seeded onto microscopic glass cover slips (18 × 18 mm) in crystallizing dishes covered
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Fig. 1. Acute toxicity induced by 48-h exposure to 1 octanol (A) and 2 butanone (B) on D. magna. The data are presented as the mean ± SD (n = 3). NC: negative control.
with aluminium foil and glass covers, and were incubated for 6 h. Subsequently, the medium was replaced by supplemented DMEM with 9% FCS (treatment without S9 mix) or supplemented FCS-free DMEM with S9 mix (treatment with S9 mix). Then different concentrations of 1 octanol (3.125, 6.25, 12.5 and 25.0 mg L−1) or 2 butanone (3220, 4025, 4830, 5635 and 6440 mg L−1) were added into the test culture. DMEM was used as negative control (NC); 350 mg L−1 EMS (without S9) and 2.5 mg L−1 CPP (with S9) in DMEM were performed as positive controls. The cultures without S9 were incubated for 24 h at 37 °C, 5% carbon dioxide and 90% humidity. The cultures with S9 were incubated for 4 h at 37 °C, 5% carbon dioxide and 90% humidity. After 4 h, the medium was removed and then fresh supplemented DMEM with 9% FCS was added and incubated for further 20 h. At the end of incubation, the cells were treated for 5 mins in a 1:1 mixture of PBS and methanol/glacial acetic acid (4:1 v/v) solution, and were fixed for 5 min with methanol/glacial acetic acid (4:1 v/v) solution. After air-drying, the glass cover slips were mounted onto glass slides and the cells were stained with 0.004% acridine orange diluted in PBS. 1000 cells per slip were randomly selected and analysed for micronucleus under a fluorescence microscope (Nikon GmbH, Düsseldorf, Germany) at 200× magnification coupled to an automated slide-loader system (PL 200 Slide Loader, Prior Scientific Instruments GmbH, Jena, Germany) using the software NIS-elements v4.13.03 (Nikon GmbH).
2.5. Statistical analysis For the tests using D. magna and zebrafish embryos, the median effective concentrations (EC50) and median lethal concentrations (LC50) were calculated using the software ToxRat Professional (ToxRat Solutions GmbH, Alsdorf, Germany). For the micronucleus assay, analysis of variance (one way ANOVA with Dunnett's post-hoc test) was performed to determine the statistical differences between the treatments and control groups using the software GraphPad Prism 6.02 (GraphPad Inc., San Diego, CA). The criterion for statistical significance was p b 0.05. All data were expressed as the mean ± standard deviation (SD) (n = 3).
3. Results and discussion 3.1. D. magna acute immobilisation test After 48 h exposure, the EC50 values were 14.9 ± 0.66 mg L−1 for 1 octanol, and 2152.1 ± 44.6 mg L−1 for 2 butanone (Fig. 1 and Table 1). The results indicated that 1 octanol showed toxicity values that was 144-fold of 2 butanone. Based on classification criterion by Commission of the European Communities (96/67/EEC), 1 octanol could be categorized as harmful to aquatic organisms (EC50 10– 100 mg L−1), while 2 butanone could be classified as nontoxic to aquatic organisms (EC50 ˃ 100 mg L−1). Kühn et al. (1989) reported that the EC50 values of 1 octanol on D. magna were 26 mg L−1 for 24-h acute test. The shorter exposure time (24 h) and test conducted in open vessel maybe the reason why the EC50 in their report was higher than our results. The 48-h EC50 values of 2 butanone reported in other studies were ˃520 mg L−1 (Leblanc, 1980) and 5100 mg L−1 (Pedersen and Petersen, 1996) by acute toxicity test on D. magna.
3.2. Fish embryo acute toxicity test Our results revealed concentration-dependent effects of 1 octanol and 2 butanone on zebrafish embryos (Figs. 2, 3). Their EC50 and LC50 values (Table 1) indicated that 1 octanol was much more toxic to zebrafish embryos than 2 butanone. Exposure to 1 octanol induced a significant increase in embryo mortality and morphological defects even at our lowest concentration (2.0 mg L−1), while 2 butanone showed significant effects at 500 mg L−1 (p b 0.05). As shown in Fig. 4, both 1 octanol and 2 butanone induced different morphological alterations on zebrafish embryos and larvae, i.e., pericardial edema, yolk sac edema, lack of pigmentation, eye defects. However, the severe spinal curvature was only found in 1 octanol exposure groups (Fig. 4E). Previous studies reported that the 96-h LC50 of 1 octanol by acute toxicity test to 30-d-old fathead minnows was 13.5 mg L−1 (Veith et
Table 1 Acute toxicity results of 1 octanol and 2 butanone.
1–Octanol 2 Butanone
D. magna
Zebrafish embryos
EC50 48 h
EC50 48 h
LC50 48 h
EC50 96 h
LC50 96 h
14.9 ± 0.66 2152.1 ± 44.6
10.9 ± 1.2 1897.8 ± 234.3
15.3 ± 1.7 4307.6 ± 178.5
7.7 ± 3.3 822.1 ± 183.1
10.0 ± 3.9 2627.8 ± 1090.4
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Fig. 2. Zebrafish embryo toxicity induced by exposure to 1 octanol. (A): Effect rate at 48 hpf; (B): mortality rate at 48 hpf; (C): effect rate at 96 hpf; (D): mortality rate at 96 hpf. The data are presented as the mean ± SD (n = 3). NC: negative control; PC: positive control.
al., 1983), and the 48-h LC50 for 1 octanol from static acute toxicity test with 24-d-old medaka was 21 mg L−1 (Carlson et al., 1998). These values for LC50 were higher than the results in present study, which might be due to that fish embryos are more sensitive than juvenile fish (Mohammed, 2013). 1 octanol has been identified as nonpolar narcotic on fathead minnows (Veith et al., 1983). After exposure to 1 octanol, a syndrome of hypoactivity was identified in juvenile fathead minnows, but no unusual change in their body morphology
(Drummond and Russom, 1990). Moreover, exposure to 1 octanol caused neurological effects on startle response and predation on medaka (Carlson et al., 1998). The 96-h LC50 for 2 butanone on zebrafish was 3200 mg L−1 (Pedersen and Petersen, 1996), similar to the results in present study. Maes et al. (2012) found that exposure to 2 butanone resulted in pericardial edema, jaw defect, microcephaly, impaired circulation and developmental delay on zebrafish embryos and larvae. Moreover,
Fig. 3. Zebrafish embryo toxicity induced by exposure to 2 butanone. (A): Effect rate at 48 hpf; (B): mortality rate at 48 hpf; (C): effect rate at 96 hpf; (D): mortality rate at 96 hpf. The data are presented as the mean ± SD (n = 3). NC: negative control; PC: positive control.
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Fig. 4. Morphological defects of zebrafish embryos and larvae after exposure to 1 octanol and 2 butanone. (A): Negative control at 48 hpf; (B): 10 mg L−1 1 octanol at 48 hpf; (C): 3000 mg L−1 2 butanone at 48 hpf; (D): negative control at 96 hpf; (E): 10 mg L−1 1 octanol at 96 hpf; (F): 2000 mg L−1 2 butaone at 96 hpf.
2 butanone also exhibited developmental toxicity in Swiss Mice. After pregnant mice were exposed to 2 butanone vapors, their offspring showed several malformation, cleft palate, fused ribs, missing vertebrae and syndactyly (Schwetz et al., 1991).
3.3. Micronucleus assay with V79 cells Representative photos of cells with micronuclei are shown in Fig. 5. Without metabolic activation (S9 mix), both 1 octanol and 2 butanone did not cause significant induction of micronuclei in Chinese hamster V79 Cells (Fig. 6). However, in the presence of exogenous metabolic activation, both biofuel candidates increased micronucleus frequencies in a concentration-dependent manner. For 1 octanol, significant increase in micronuclei was identified at 12.5 and 25.0 mg L−1 with metabolic activation, while 2 butanone significantly induced micronuclei at high concentrations (4830, 5635 and 6440 mg L−1) with metabolic activation (Fig. 6). Our results show that metabolic activation with an exogenous S9 supplementation could increase the mutagenic potential of 1 octanol and 2 butanone, which suggests that both 1 octanol and 2 butanone might be metabolised by cytochrome P450 depending enzymes to their genotoxic metabolites. Previous study showed that 1 octanol (≥13 mg L−1) could induce spindle disturbances in V79 cells, such as c-mitosis and aneuploidy, which is considered to be an unspecific effect based on the distribution of the lipophilic compound into hydrophobic cell compartments.
Fig. 5. Photomicrographs of micronucleus in V79 cells after treatment with 1 octanol (A) and 2 butanone (B). Micrographs were captured at 1000× magnification.
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Fig. 6. Induction of micronuclei in V 79 cells treated with 1 octanol (A) and 2 butanone (B) at different concentrations. (*) p b 0.05. The data are presented as the mean ± SD (n = 3). NC: negative control.
However, the concentrations given in this study might be higher than the true concentrations due to experimental errors (Önfelt, 1987). Odonoghue et al. (1988) found that 2 butanone was not genotoxic by a battery of in vitro assays including Salmonella (Ames) assay, L5178/TK+/− mouse lymphoma assay, BALB/3T3 cell transformation assay, unscheduled DNA synthesis and micronucleus assay. The only evidence of genotoxicity for 2 butanone was mitotic chromosome loss at a very high concentration in the yeast Saccharomyces cerevisiae (Zimmermann et al., 1985). The results in the present study indicated that 1 octanol showed much higher ecotoxicity than 2 butanone to D. magna, zebrafish embryos and Chinese hamster V79 cells. Previous studies reported that a large variety of chemicals were more toxic in acute tests to D. magna than to D. rerio, e.g., metals, pesticides and solvents (Martins et al., 2007); while our results about acute toxicity of 1 octanol and 2 butanone showed that there were no significant differences between D. magna and D. rerio.
4. Conclusion The International Energy Agency has a goal for biofuels to provide 27% of total transport fuels by 2050 (Tanaka, 2011). Considering this large potential market for biofuels in the future and their inevitable release into the environment, identification of potential ecotoxicity for biofuels during their early development can help protect the entire environment and saving research resource from biofuels harmful to the environment and human health. The present study combined three widely used test systems to assess different effects (acute toxic effects, developmental effects, teratogenic effects and genotoxic effects) of two biofuel candidates, which proposed an integrative test approach to evaluate the potential ecotoxicity of biofuels using simple, quick and inexpensive bioassays. Our results can provide information for the whole cluster to establish innovative and sustainable processes for tailor-made biofuels with high efficiency and low pollutant emissions. More research resources in the cluster will be given priority to environment-friendly biofuel candidates at their early developmental stage to avoid harmful effect on the environment and human health.
Author contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. # Sebastian Heger and Miaomiao Du contributed equally to this work as co-first authors of this paper.
Acknowledgements This work was performed as part of the Cluster of Excellence “Tailormade fuels from biomass”, which is funded by the German federal and state governments to promote science and research at German universities. The authors would like to kindly thank Nikon Germany and Prior Scientific for their contribution to this study as a sponsor of the Student's Lab Fascinating Environment. References Bluhm, K., Heger, S., Seiler, T.-B., Hallare, A.V., Schäffer, A., Hollert, H., 2012. Toxicological and ecotoxicological potencies of biofuels used for the transport sector—a literature review. Energy Environ. Sci. 5, 7381–7392. Bluhm, K., Seiler, T.-B., Anders, N., Klankermayer, J., Schaeffer, A., Hollert, H., 2016. Acute embryo toxicity and teratogenicity of three potential biofuels also used as flavor or solvent. Sci. Total Environ. 566, 786–795. Carlson, R.W., Bradbury, S.P., Drummond, R.A., Hammermeister, D.E., 1998. Neurological effects on startle response and escape from predation by medaka exposed to organic chemicals. Aquat. Toxicol. 43, 51–68. Commission of the European Communities, 1996. Technical Guidance Document in Support of Commission Directive 93/67/EEC on Risk Assessment for New Notified Substances and Commission Regulation (EC) N. 1488/94 on Risk Assessment for Existing Substances. Office for Official Publications of the European Communities. Crawford, S.E., Hartung, T., Hollert, H., Mathes, B., van Ravenzwaay, B., Steger-Hartmann, T., et al., 2017. Green toxicology: a strategy for sustainable chemical and material development. Environ. Sci. Eur. 29, 16. Drummond, R.A., Russom, C.L., 1990. Behavioral toxicity syndromes - a promising tool for assessing toxicity mechanisms in juvenile fathead minnows. Environ. Toxicol. Chem. 9, 37–46. Falbe, J., Bahrmann, H., Lipps, W., Mayer, D., 2000. Alcohols, aliphatic. Ullmann's Encyclopedia of Industrial Chemistry. Fargione, J., Hill, J., Tilman, D., Polasky, S., Hawthorne, P., 2008. Land clearing and the biofuel carbon debt. Science 319, 1235–1238. Heger, S., Bluhm, K., Agler, M.T., Maletz, S., Schaffer, A., Seiler, T.B., et al., 2012. Biotests for hazard assessment of biofuel fermentation. Energy Environ. Sci. 5, 9778–9788. Heger, S., Bluhm, K., Brendt, J., Mayer, P., Anders, N., Schäffer, A., et al., 2016. Microscale in vitro assays for the investigation of neutral red retention and ethoxyresorufin O deethylase of biofuels and fossil fuels. PLoS One 11, e0163862. Heuser, B., Mauermann, P., Wankhade, R., Kremer, F., Pischinger, S., 2015. Combustion and emission behavior of linear C 8 oxygenates. Int. J. Engine Res. 16, 627–638. Hoppe, F., Burke, U., Thewes, M., Heufer, A., Kremer, F., Pischinger, S., 2016. Tailor-made fuels from biomass: potentials of 2 butanone and 2 methylfuran in direct injection spark ignition engines. Fuel 167, 106–117. ISO/DIS 21427-2. Water Quality - Evaluation of Genotoxicity by Measurement of the Induction of Micronuclei - Part 2: ‘Mixed Population’ Method Using the Cell Line V79. Kühn, R., Pattard, M., Pernak, K.-D., Winter, A., 1989. Results of the harmful effects of water pollutants to Daphnia magna in the 21 day reproduction test. Water Res. 23, 501–510. Leblanc, G.A., 1980. Acute toxicity of priority pollutants to water flea (Daphnia magna). Bull. Environ. Contam. Toxicol. 24, 684–691. Maertens, A., Anastas, N., Spencer, P.J., Stephens, M., Goldberg, A., Hartung, T., 2014. Food for thought … green toxicology. ALTEX 31, 243–249. Maes, J., Verlooy, L., Buenafe, O.E., De Witte, P.A., Esguerra, C.V., Crawford, A.D., 2012. Evaluation of 14 organic solvents and carriers for screening applications in zebrafish embryos and larvae. PLoS One 7, e43850.
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