2.1 Chemicals
Tylosin Tartrate (CAS no.1405-54-5; HPLC ≥ 98.0%) was purchased from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai CHN). Erythromycin-13C, d3 (CAS no. 959119-26-7; HPLC ≥ 99%) and atrazine D5 (CAS no. 1912-24-9; HPLC ≥ 97.0%) were purchased from J&K Scientific (Beijing, CHN). Methanol and acetonitrile with HPLC ≥ 99.9% were obtained from TEDIA (Ohio, USA). Formic acid (HPLC ≥ 88%) was purchased from Kemiou Chemical Reagent Co., Ltd (Tianjin, CHN). Biomarker kits for the determination of SOD, CAT, GP and the content of MDA, GSH, GST were purchased from the Nanjing Jiancheng Co., Ltd. (Nanjing, CHN). Other chemicals used in this study were at least reagent grade.
2.2 Algal cultures
Algal toxicity tests were conducted using organisms, C. vulgaris (FACHB-8) and R. subcapitata (FACHB-271), obtained from Freshwater Algae Culture Collection at the Institute of Hydrobiology (Wuhan, China). Algae culture was performed following the existed Guideline. In brief, both of algae were grown in Blue-Green Medium (BG11), pH 7.1 (OECD, 2011). C. vulgaris and R. subcapitata were cultured in 250 mL Erlenmeyer flasks containing 150 mL BG11 with a controlled temperature (22 ± 2 ℃) and constant illumination (5500 Lux). The initial algal concentrations for C. vulgaris and R. subcapitata were set at 2 × 104 cells mL-1 and 1 × 104 cells mL-1, respectively. By the way, the Erlenmeyer flasks gently shaken manually three times per day. All glassware, stoppers and inoculum used in this study were autoclaved at 121 ℃ for 30 min and all the operations were performed on a clean bench. According to the guidelines (OECD 1984; 2011), the algal suspension, at its exponential phase, was used for further experiments. The cell density was estimated by counting the cell number with a hemacytometer under a microscope, on a daily basis, to plot the growth curve (cell density versus day). The algal stocks were subcultured each week.
2.3 Procedures for the growth inhibition test
The growth inhibition tests for TYN was conducted on the basis of OECD Guidelines.30 In order to determine the concentration of TYN during the experiment, we initially measured EC50s for C. vulgaris and R. subcapitata by plotting the concentration-response curves. Specifically, C. vulgaris and R. subcapitata were exposed to different concentrations of TYN (C. vulgaris: 0, 0.5, 1, 1.5, 3, and 5 mg L-1; R. subcapitata: 0, 0.2, 0.45, 0.8, 1.6, and 3.2 mg L-1) and cultured for 4 days. On day 4, the 4-day EC50s of TYN against both species of algae was calculated by the cell densities of C. vulgaris and R. subcapitata. In this study, the 4-day EC50 of TYN for R. subcapitata was 0.75 mg L-1 (Figure S1), however, TYN could not significantly inhibit the growth of C. vulgaris, the 4-day EC50 of TYN for C. vulgaris was not calculated. According to EC50s value in this study and the value reported in previous study (Table S3), we determined the concentration of TYN in the experiment. The toxicological effects of same concentrations of TYN (0, 0.003, 0.8, 1.2, 1.6 and 3 mg L-1) on R. subcapitata and C. vulgaris were investigated by cultivating the same initial algal biomass as above in 250 mL Erlenmeyer flasks containing 150 mL of sterilized BG11 for 7 days. Each concentration was conducted in triplicates. The cell numbers were measured at regular time intervals (2, 4, and 7 days).
2.4 Measurement of oxidative stress biomarkers
MDA, SOD, GP, GST, CAT and GSH were measured according to the manufacturer's protocol (Nanjing Jiancheng Bioengineering Institute, China) after 7-days exposure. As for the high inhibition at the TYN concentration of 1.6 and 3 mg L-1 on R. subcapitata, only the oxidative stress biomarkers of 0, 0.003, 0.8 and 1.2 mg L-1 were measured. In this study, 5 mL R. subcapitata culture at the concentration of 0 and 0.003 mg L-1 and 50 mL for 0.8 and 1.2 mg L-1 respectively were harvested at day 7 by centrifugation at 4000 rpm min-1 for 10 min in the centrifuge tubes. Meanwhile 5 mL C. vulgaris culture was harvested by the same method. And the centrifugation was weighted again after the supernatant was discarded which was used to calculated the algae wet weight. Then the precipitates was re-suspended by 1mL stroke-physiological saline solution (SPSS) into the centrifuge tubes and centrifuged at 1000 rpm min-1 for 10 min. This process repeated three times. The precipitates was re-suspended by 2mL SPSS at the last time the supernatant was discarded. Then the suspension homogenated in ice water bath for six times (one time per minutes, 30 seconds interval) with a tissue tearor. Then the suspension was used to measure the oxidative stress biomarkers following the protocol. The activity or contents of these biomarkers were measured with a Tecan Infinite® 200 Pro multi-function microplate reader (Tecan Austria Gmbh, Männedorf, Switzerland).
2.5 Measurement of total chlorophyll and carotenoid content
The chlorophyll a , b and carotenoid content were measured following the previous study.37 After 7-days exposure in the growth studies, 5 mL microalgal suspensions from each sample were filtered using a 0.22-μm fiber filter. Then each filter membrane put into a 5 mL centrifuge tube containing 3 mL methanol to extract pigments in a spark free fridge (-80 ℃) for 24 h. All samples were centrifuged at 10,000 rpm min-1 for 10 min. Chlorophyll a and b were estimated using the Wellburn coefficient equation (Equation1; Equation2) (Wellburn 1994) and the carotenoid were estimated using the Lichtenthaler equation (Equation3). Absorbance values (A470, A653, and A666) were measured by Tecan Infinite®200 Pro Mutil Reader.
2.6 Antibiotic analysis
At day 0, The concentrations of TYN in all samples were determined using liquid chromatography tandem-mass spectrometry (LC-MS/MS, Agilent 1290) coupled with solid phase extraction (SPE). Details on the system settings and method validation can be found in Supporting Information. Since the variation between the nominal and measured concentrations was less than 20%, the nominal concentration was used for further investigation (OECD, 2011).
2.7 Statistical methods
The experimental data was analyzed by using The GraphPad Prism 8.0 software (California, USA). The normality test of MDA content, GSH content, antioxidant enzyme activities, pH and pigment contents were determined using the Normality and Lognormality Tests. Further more significant differences between in treatments and controls were calculated using the one way ANOVA Dunnett test. A p value less than 0.05 was considered statistically significant.