Methanol, Giemsa stain and H2O2 were obtained from Biopack Co. Acetylcholine, BHT, DNTB and MDA were purchased from Sigma-Aldrich (Darmstadt, Germany). Decabromodiphenyl ether (BDE-209) was provided by Sigma Aldrich.
2.2 Chemical analysis
Quantitative analysis of BDE-209 was carried out by Shimadzu Model 2010 GC-MS equipped with an AOC-20i auto injector (Shimadzu, Japan), using negative chemical ionization (NCI) in the selected ion monitoring (SIM) mode. A CP-Sil 13 CB (12.5 m × 0.25 mm i.d., 0.2 µm film thickness) capillary column was used. Ion fragments m/z 79, 81, 486.7, and 488.7 were monitored for BDE-209, according to the method proposed by Peng et al. (2009). The quantification was performed by external standard method. The limit of quantification was 0.6 ng L− 1.
2.3 Model organism
Fish males of Gambusia affinis were obtained from an unpolluted permanent pond located in El Volcán, San Luis Province, Argentina (33°15’01” S, 66°11’43” W) with the authorization of the Ministry of Environment, Agriculture and Production of the province of San Luis (N° Res. 49-PMA-2019). Mature adults of G. affinis were transported and acclimated to the laboratory under controlled conditions (photoperiod 16:8; temperature 25 ± 1°C; daily renewed dechlorinated tap water: pH 7.12, conductivity 412 µS.cm− 1, hardness 186 mg.L− 1 CaCO3, alkalinity 250 mg.L− 1 CaCO3, nitrate 0.6 mg.L− 1) for two weeks before conducting the experiments, according to the protocols of the Institutional Animal Care and Use Committee (CICUA protocol Q-322/19), from National University of San Luis.
2.4 Ecotoxicity bioassay
Toxicity tests were performed following standardized methods proposed by the USEPA (2002) with minor modifications for local species (Vera-Candioti et al. 2010). All experiments were conducted putting five individuals per replicate in 1 L glass jars, with 1 g L− 1 density of the organisms (n = 15 per treatment). Negative and positive controls as well as increasing gradient concentrations of BDE-209 with test solutions replaced every 24 h in acute exposure (96 h) were used. Fishes were starved for 24 h before initiation of the experiments and were not fed during the bioassays. Bioassays were carried out under controlled conditions such as photoperiod (16:8), daily renewal of the solutions, bioterium temperature (25 ± 1°C). Dechlorinated tap water (pH 7.12; conductivity 412 µS.cm− 1; hardness 186 mg CaCO3 L− 1; alkalinity 250 mg CaCO3 L− 1; chloride 7.1 mg.L− 1; nitrate 0.6 mg.L− 1; sulphate; 17 mg.L− 1; sodium 21.5 mg.L− 1; calcium 54.3 mg.L− 1; potassium 1.9 mg.L− 1 and magnesium 8.1 mg.L− 1) was used for the experiments. The BDE-209 sublethal concentrations for fish exposures were selected based on literature according to previous reports in zebrafish model (Garcia-Reyero et al. 2014; Zhu et al. 2016; Han et al. 2017), and considering the environmental relevant concentrations reported on literature (Mackintosh et al. 2015; McGrath et al. 2017). G. affinis were exposed to environmental concentrations equal to 10, 25, 50 and 100 µg.L− 1 of BDE-209, dissolved in methanol. The positive control group was prepared adding methanol (100 µg.L − 1). The solvent final concentration was the same in all the treatments, always lower than 0.1% (Kaviraj et al. 2004) All treatments (control and BDE-209 exposed groups) were carried out by triplicate. To evaluate biochemical and cellular biomarkers, fishes were euthanized by dissection at the operculum level, then the blood samples were extracted and biochemically analysed. To evaluate these endpoints, all fishes were anesthetized and sacrificed after 48 and 96 h of exposure. The same procedures were followed in all the treatments.
2.5.1 Cytogenetical endpoints: MNs assays
Peripheral blood of each fish were smeared onto pre-cleaned slides according to Vera Candioti et al. (2010). Then, slides with blood samples were air dried, fixed with methanol (4°C), and stained with Giemsa solution (5%). Finally, the coded slides were quantified. The same researcher carried out all the experiments at microscopy 1000× magnification. The MN frequency was determined by analysing a total of 1000 mature erythrocytes, expressed as the total number of MN per 1000 cells (Fenech 2007)
2.5.2 Biochemical endpoints: CAT, TBARS and AChE activity
Supernatants from homogenates of whole fish were obtained applying the methodology proposed by Brodeur et al. (2017), with minor modifications. Briefly, post-mitochondrial supernatant was prepared (in ice bath cooling) from a 1 mL homogenate of fish tissues with a buffer Tris 50 mM (pH 7.4) containing 1 mM EDTA and sucrose 0.25 M. The homogenate was centrifuged at 1 104 ×g for 10 min at 4°C. The supernatant was used to measure the following biochemical biomarkers: i) the CAT activity was determined by measuring the decomposition of hydrogen peroxide at 240 nm (37°C, 2 min), using a molar extinction coefficient of 43.6/M cm. The reaction mixture consisted of 20 µL of pure sample, 40 µL of H2O2 (10 %, v/v) and 1900 µL of PBS (pH 7, 100 mM); ii) the lipid peroxidation was determined by the reaction of thiobarbituric acid-reactive substances (TBARS) according to the method of Buege and Aust (1978), with minor modifications to aquatic vertebrates. The lipid peroxidation in whole fish was determined by measuring the formation of the colour produced during the TBARS reaction. To this end, fish homogenate (20 µL) and 380 µL of the reaction mixture (trichloroacetic/ thiobarbituric acid) were incubated at 90.0 ± 0.5°C for 15 min; then the coloured product was cooled and centrifuged at 7500× g for 8 min. Finally, the absorbance was measured at 530 nm. Lipid peroxidation or TBARS levels were expressed as mmol MDA mg− 1 protein (Buege et al. 1978); and iii) AChE activity was determined by the method of Ellman (1961). The reaction mixture consisted of 150 µL of PBS (100 mM, pH 8), 50 µL of acetylthiocholine iodide (1 mM), 150 µL of 5,5'-dithiobis-(2-nitrobenzoic acid) (0.5 mM) and 10 µL of pure sample. The change in absorbance was recorded at 412 nm (37°C, 1 min). The enzymatic activity was calculated using a molar extinction coefficient of 14.150 M− 1 cm− 1. Protein concentration was determined according to the Bradford method (Bradford 1976). All biochemical enzyme reactions and protein determinations were measured using a spectrophotometer (Rayleigh - Model UV2601 UV/VIS - Double Beam Spectrophotometer, China).
2.5.3 Individual endpoints
Mortality was considered as the lethal endpoint; so, fishes were examined daily, and the mortality criterion was the lack of sudden swimming in response to gentle touching. Dead individuals were fixed in formaldehyde (10%, v/v). The lethal concentration (LC-50), No Observed Effect Concentration (NOEC), and the Lowest Observed Effect Concentration (LOEC) values were determined in fishes at each exposure time.
The altered swimming activity in fishes was evaluated by direct observation according to parameters proposed by Little and Finger (1990), characterized by changes in water column position (surfacing, resting on bottom), swimming posture (head-up swimming), body movements (increased or decreased waveform of body movement) or swimming patterns (frequent turns or spiralling), since they often occur during toxicant exposure. Extreme cases such as a loss of coordination, convulsive movements or loss of equilibrium were also considered. The all-or-none occurrence of activity level has been proposed as sublethal biomarkers, and successfully used to describe swimming alterations after the exposure to several environmental stressors (Shuman-Goodier and Propper 2016).
2.5.4 Integrative response of biomarkers
To integrate the different results, two methods were reliably performed: i) the Integrated Biomarker Response Index (IBR) was calculated, according to Baudou et al. (2019), considering the following biomarkers: CAT, TBARS, AChE, swimming activity and MNs. The IBR provides a numeric value that integrates all these responses. Higher IBR values indicate higher stress levels (Baudou et al. 2019); ii) the Principal Component Analysis (PCA) was used to determinate the implications of each biomarker at each concentration taken into account in this study. The significance of correlations was examined by simple linear regression and correlation analyses obtained with R software v.2.11.1. The level of significance was set at α = 0.05 for all tests, unless otherwise indicated.
2.6 Statistical analysis
The LC-50 values, concentration response curves and ecotoxicological parameters such as slope and correlation coefficient at different sampling times (24 to 96 h) with 95% of confidence limits were estimated, using the U.S. EPA Probit Analysis (Finney 1952) with the package “ecotoxicology” for R software v.2.11.1 (R Core Team 2010, October 14, 2015). All significance test for regression and correlation were performed according to Zar (2010).
The proportion of fishes affected by test chamber was calculated for mortality, swimming activity, CAT, TBARS, AChE and MNs. These parameters were subsequently angular-transformed. ANOVA one-way with Dunnett’s test was performed to compare the different test concentrations to the control group, and to obtain NOEC and LOEC values. Homogeneity of variances and normality, for ANOVA assumptions, were corroborated with Barlett’s test and x2 test, respectively. When these assumptions could not be met, a non-parametric test was performed (Zar 2010).