World Aquaculture December 2019

WWW.WA S.ORG • WORLD AQUACULTURE • DECEMBER 2019 51 • Preparation ofwater-soluble fraction Crude oil and tap water were mixed at a 1:10 volume ratio and the mixture placed on a magnetic stirrer. After continuous high-speed stirring for 24 h, the solution was allowed to sit undisturbed for 3 h to allow separation of the aqueous phase from the solid phase. The water-soluble fraction was stored at 4-6C. Ultraviolet spectrophotom- etry was used to determine crude oil concentration. The characteristic absorption of oil and its products in the ultraviolet area can be par- titioned into aromatic compounds with benzene (250-260 nm), crude oil (225 and 254 nm) and com- pounds with conjugated double bonds (215-230 nm). • Preparation of dispersantwater-soluble fraction A dispersant (BH-X) and the water-soluble fraction were mixed at a 1:20 volume ratio and then stirred at high-speed for 1 h. After 1 h standing, the milky mixture was the dispersant - water soluble fraction (“experimental liquor”) that included dispersed oil droplets and dispersant (Huang 2010). The diluent experimental liquor was mixed with tap water and aerated for more than 1 d. During the experiment, water temperature was 20 ± 2C, aerated most of the time, and the pHwas 8.0 ± 0.2. • Experimentalmethod Goldfish juveniles were randomly divided into eight groups: two control groups, two low concentration groups (0.82 mg/L), two average concentration groups (2.10 mg/L) and two high concentration groups (5.20 mg/L). There were ten goldfish juveniles in each group. Fish were sampled on days 1, 3, 5, 7 and 9 after the beginning of the experiment. Gills were carefully removed and placed into a small bag after washing with distilled water. Two gills were selected for each experimental group and each sampling time. Samples were stored at -20 C until determination. DNAwas extracted and purified using standard methods. DNA damage was assessed with the alkaline unwinding assay (Shugart 1999). Under certain alkaline unwinding conditions, the quantity of double-stranded DNA changing into single-stranded DNA is directly proportional to the number of fractures on the double- stranded DNA polynucleotide chain. Fluorescent Hoechst dye 33258 c (diphenyl imidazole) and double-stranded DNA form stable fluorescent products. If combined with single-stranded DNA, its fluorescence intensity is ½ that of double-stranded DNA. Thus, we can distinguish double-stranded from single-stranded DNA. This unwinding analysis process in this experiment was as follows. The extracted DNA samples were separated into three aliquots for testing double, single and alkali unwinding fluorescence value. Samples were shaken and kept in the dark for 15 min before measuring fluorescence at an excitation wavelength of 360 nm and an emission wavelength of 450 nm. DNA integrity is usually expressed as an F value, which was calculated according to the formula: F = (X auDNA -X ssDNA ) / (X dsDNA - X ssDNA ). In the formula, X is the fluorescence value, dsDNA is double- stranded DNA, ssDNA is single-strand DNA and auDNA is DNA after alkali unwinding (Apraiz et al . 2009). Results and Discussion The DNA integrity of goldfish gills in the control group did not change significantly during the experiment and held constant at about 85 percent (Table 1, Fig. 2). For the low, middle, and high concentration groups, the trend in DNA integrity was similar. DNA integrity decreased rapidly over the first three days and thereafter decreased less rapidly. By day 9, the three groups exposed to oil, irrespective of concentration, reached similar DNA integrity values of about 20 percent. In general, there was an inverse relationship between the concentration of experimental liquor and the DNA FIGURE 2. DNA integrity changes of goldfish gills under different concentrations of experimental liquor. TABLE 1. The DNA integrity of juvenile Carassius auratus gills at different exposure time and concentrations. Time (d) Control group Low concentration Middle concentration High concentration group group group 1 0.85±0.08 0.64±0.14 0.57±0.08 0.36±0.16 3 0.79±0.09 0.36±0.08* 0.25±0.13* 0.21±0.10* 5 0.83±0.12 0.25±0.10* 0.25±0.09* 0.22±0.09* 7 0.85±0.06 0.26±0.09* 0.26±0.07* 0.20±0.07* 9 0.83±0.09 0.21±0.04* 0.19±0.06* 0.21±0.06* Note: The data in the table were mean ± standard error, which indicated the degree of DNA damage; * noted that the results showed a significant difference (0.01 <P <0.05) between the data and the corresponding control group data by the t-test (Double sample equal variance hypothesis). ( C O N T I N U E D O N P A G E 5 2 )