Effects of total dissolved gas supersaturated water on lethality and catalase activity of Chinese sucker (Myxocyprinus asiaticus Bleeker)

Effects of total dissolved gas supersaturated water on lethality and catalase activity of Chinese sucker ( Myxocyprinus asiaticus Bleeker) 1 first-author 2 1 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:kefengli@scu.edu.cndujun9100@yahoo.com.cn †E-mail:kefengli@scu.edu.cndujun9100@yahoo.com.cn kefengli@scu.edu.cn 3 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:kefengli@scu.edu.cndujun9100@yahoo.com.cn †E-mail:kefengli@scu.edu.cndujun9100@yahoo.com.cn kefengli@scu.edu.cn 3 3 State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610065, China State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610065, China School of Energy and Environment, Xihua University, Chengdu 610039, China School of Energy and Environment, Xihua University, Chengdu 610039, China Fisheries Institute, Sichuan Academy of Agricultural Sciences, Chengdu 611731, China Fisheries Institute, Sichuan Academy of Agricultural Sciences, Chengdu 611731, China Total dissolved gas (TDG) supersaturation caused by dam sluicing can result in gas bubble trauma (GBT) in fish and threaten their survival. In the present study, Chinese suckers ( Myxocyprinus asiaticus Bleeker) were exposed to TDG supersaturated water at levels ranging from 120% to 145% for 48 h. The median lethal concentration (LC 50 ) and the median lethal time (LT 50 ) were determined to evaluate acute lethal effects on Chinese suckers. The results showed that the LC 50 values of 4, 6, 8, and 10 h were 142%, 137%, 135%, and 130%, respectively. The LT 50 values were 3.2, 4.7, 7.8, 9.2, and 43.4 h, respectively, when TDG supersaturated levels were 145%, 140%, 135%, 130%, and 125%. Furthermore, the biological responses in Chinese suckers were studied by assaying the catalase (CAT) activities in gills and muscles at the supersaturation level of 140% within LT 50 . The CAT activities in the gills and muscle tissues exhibited a regularity of a decrease after an increase. CAT activities in the muscles were increased significantly at 3/5LT 50 ( P < 0.05) and then came back to the normal level. However, there were no significant differences between the treatment group (TDG level of 140%) and the control group (TDG level of 100%) on CAT activities in the gills before 3/5LT 50 ( P > 0.05), but the activities were significantly lower than the normal level at 4/5LT 50 and LT 50 ( P < 0.05). Total dissolved gas supersaturation Median lethal time (LT 50 ) Median lethal concentration (LC 50 ) Chinese sucker Catalase Introduction 1 The flood sluicing from a dam will carry atmospheric air and dissolve it when hitting the plunge-pool, and can become supersaturated with gas under high hydrostatic pressure (Gale et al., 2004 2004 ). Since the 20th century, total dissolved gas (TDG) supersaturated water has been considered a threat to aquatic resources, and is considered a water quality issue. Many studies have been carried out to explore the effects of TDG supersaturation on fish. Huang et al. ( 2010 2010 ) found that juvenile rock carp could survive at supersaturated levels below 120% and showed a strong avoidance response to the supersaturated water with gas saturation above 135%. Salmon and rainbow trout could always avoid 145% and 125% saturation, but were dull to 115% saturation (Stevens et al., 1980 1980 ). Gray et al. ( 1985 1985 ) reported that the 96 h LC 50 values of sea bass and striped mullet were 127.2% and 129.4% respectively at 20 °C. No significant changes of growth rate were found by Liu et al. ( 2011 2011 ) after rock carp were exposed to gas saturation ranging from 100% to 116% for 42 d, but biochemical responses assessed by catalase (CAT) and superoxide dismutase (SOD) activities were obviously found. As existing findings are applicable to specific species or rivers only, research on TDG supersaturation thus far has not been sufficient to set up a limit level for water quality assessment in China. With high or superhigh dams being built upstream of the Yangtze River in recent years, the issue of TDG supersaturation is becoming more and more serious. TDG levels above 138% were observed downstream from the Three Gorges dam and the Ertan dam by field observation during dam sluicing (Li et al., 2009 2009 ; Qu et al., 2011 2011 ). Fish were found dead from gas bubble disease downstream from the Three Gorges dam (Tan, 2006 2006 ). Chinese suckers dwelling in the Yangtze River were threatened by TDG supersaturation. However, existing research mainly focuses on the current resource situation, reproduction and artificial culture, molecular biology and fodder (Chen, 2008 2008 ). The effect of TDG supersaturation on Chinese suckers has not been reported so far. In this study, the median lethal concentration (LC 50 ), the median lethal time (LT 50 ), and the CAT activity of tissues in the Chinese sucker were measured to evaluate the effects of acute lethality and the biological response in fish exposed to TDG supersaturated water. It can be expected that the research results will provide scientific data for operational schemes of hydropower plants and the formulation of water quality standards in China. Materials and methods 1 Experimental fish and instrument 2 The present study was carried out by using half-year-old Chinese suckers ( Myxocyprinus asiaticus Bleeker) which were transported from Sichuan Fisheries Research Institute, China, in November 2011. Before the experiment, they were placed in tanks of the State Key Laboratory of Hydraulics and Mountain River Engineering (Sichuan University, China) for 8 d to adapt to living conditions with stable water temperature at 21 °C and pH 7.1–7.8. The fish were fed on Limnodrilus hoffmeisteri every day, but were not fed during the trial. Water temperature, TDG level, pH value, and dissolved oxygen (DO) value were monitored daily using a mercury thermometer, point four tracker (Point Four Systems Inc., Canada), digital pH meter (JENCO Model 6010, China), and dissolved oxygen meters (Oxi 3210 SET 3 Inc., Germany), respectively. The experimental conditions were as follows: water temperature 21 °C, pH 7.1–7.8, and DO 7.5–8.0 mg/L. The weight of the fish was (39.54±7.63) g (mean±standard deviation (SD)), while the length was (11.54±0.91) cm (mean±SD). Acute lethality experiment 2 The experiment system was based on Huang et al. ( 2010 2010 ) to generate the TDG supersaturated water. Twenty fish were held in each tank with TDG levels of 145%, 140%, 135%, 130%, 125%, 120%, and 100% for 48 h, respectively. The behaviour of fish was observed, and the death time of each fish was recorded during the trial. The LT 50 was calculated in probit methods, while the LC 50 was analyzed through linear interpolation (Zhou and Zhang, 1989 1989 ). Measurement of CAT activity 2 The group with the supersaturated level of 140% was selected to conduct the biological response experiment according to the LT 50 calculated in Section 2.2. Forty fish were used in the treatment group (TDG level of 140%) and the control group (TDG level of 100%), respectively. The repeated test was carried out at the same time. Three fish from both the treatment group and control group were sampled every other 1/5LT 50 . They were narcotized with MS-222 immediately and weight and length were measured. Gills and muscles with a weight of 1.5 g were excised from each fish and ground under liquid nitrogen, and then they were extracted with the method described by Wang et al. ( 2010 2010 ). The 1.5 ml reaction mixture liquid contained 0.05 mol/L sodium phosphate buffer (pH 7.0) and 0.1 mmol/L ethylene diamine tetraacetic acid (EDTA). The enzyme extraction was gained by centrifugation at 4 °C. The CAT activity was determined by assaying the supernatant with an ultraviolet spectrophotometer according to Montavon et al. ( 2007 2007 ). The enzyme-activity unit of CAT was defined as the amount of the enzyme which caused a decrease of 0.01 on the absorbance of 0.6% (v/v) hydrogen peroxide (H 2 O 2 ) per min under the wave length of 240 nm. Statistical analysis 2 The software SPSS was used to analyze the differences of CAT activities between the treatment group and the control group. The level of significant differences was set at P < 0.05. The figures of LC 50 , LT 50 , and the changes of the CAT activities were determined using Excel 2003. Results 1 Acute lethality 2 At the beginning of the trial, fish in each group swam everywhere in the tanks. After a few minutes, they gathered together at the corner of the tanks. Fish in the tank with the TDG level of 145% began to swim alone after 1 h. Some fish started to swim quickly and suddenly, but were slow in reacting about 1 h later. The phenomenon of sidestroke was observed. Sometimes they leaped in the air and then fell at the bottom of the tank without swimming ability. After a while, these phenomena repeated constantly until they died on the water surface with mouths open and lots of bubbles in the fins. Fish in the groups of 140%, 135%, 130%, and 125% showed the same phenomena as those in the group of 145%. On the other hand, no dead fish were found in the group of 120% or 100%. The LT 50 and LC 50 are shown in Figs. 1 1 and 2 2 . The Chinese suckers died a few hours after the trial started with levels greater than 125%. However, the LT 50 dramatically increased when the gas saturation level was 125%. The LT 50 was not measured in the group with the gas saturation level of 120%, for no dead fish were found in it. Since all fish in the groups of levels above 125% died within a few hours, the LC 50 was calculated at 4, 6, 8, and 10 h, respectively. CAT activity 2 As shown in Fig. 3 3 , CAT activities varied with different TDG levels, exposure time, and tissues. As shown in Fig. 3a 3a , the CAT activities in muscles of Chinese suckers exposed to gas saturation level of 140% first increased and then dropped. They were significantly ( P < 0.05) increased to the maximum at 3/5LT 50 in the treatment group compared with the control group. And then they dropped back to the normal level ( P > 0.05). Values were expressed as mean±SD. * P < 0.05 Fig. 3b 3b showed the same tendency in gills as that in Fig. 3a 3a , but no significant differences were found between the treatment group and the control group during the increasing stage ( P > 0.05). They began to drop after 3/5LT 50 , and were significantly lower in the treatment group than in the control group at 4/5LT 50 and LT 50 ( P < 0.05). Discussion 1 Influence factors on the lethality by TDG supersaturation 2 Many studies have shown that different tolerances for TDG supersaturation occurred in different fish. Beeman et al. ( 2003 2003 ) described the LT 50 of five different fish in the Columbia River. Table 1 1 shows that the sensibility to TDG supersaturation varies in different fish under the same condition. Compared with the fish in Table 1 1 , Chinese sucker showed a moderate-level tolerance to TDG supersaturation. Date are from Beeman et al. ( 2003 2003 ) 2 Species 2 LT 50 (h) 125% 130% Largescale sucker 17.0 9.5 Longnose sucker 56.0 30.0 Northern pikeminnow 15.3 10.5 Redside shiner 116.0 31.0 Walleye 169.0 62.0 Results indicated that the tolerance to TDG supersaturation varied with age. Arntzen et al. ( 2009 2009 ) found that the chum salmon egg had a strong tolerance to the supersaturation levels ranging from 110% to 115%. Bohl ( 1997 1997 ) pointed out that steelhead trout fry could survive when the supersaturation level was 102%, but serious traumas were found in gills, skeletons, vessels, and the nervous system. However, the initial level causing the death of adult salmons was 116%. Some factors played an important role in lethality of fish exposed to TDG supersaturation, such as water temperature and supersaturation levels. Nebeker et al. ( 1978 1978 ) found that mortality of the rainbow trout and the Chinook salmon increased with rising temperatures. However, the silver salmon and the red salmon were not affected. Alderdice and Jensen ( 1985 1985 ) and Jensen et al. ( 1986 1986 ) reported that fish in the groups with high TDG levels died earlier than those with lower TDG levels. Huang et al. ( 2010 2010 ) indicated mortality increased with the rising TDG level. Thus, keeping the experimental conditions steady was of great significance, such as stable temperature at 21 °C. Furthermore, there are different tolerances in different fish exposed to the TDG supersaturated water owing to the individual differences of fish. In this study, the fact that three fish were sampled from each group every time and the repeated test was carried out at the same time could decrease individual differences. This study showed the similar regularity as that in Huang et al. ( 2010 2010 ). The LT 50 in the group of 130% dramatically increased compared with the group of 125%. Moreover, no dead fish were found in the group of 120%. Under the experimental conditions, the threshold of the acute TDG level in Chinese sucker could be determined at 125%. However, TDG supersaturation of 138% has been measured downstream from the Three Gorges dam with height of 185 m. And at the upper reaches of the Chinese sucker protection zone, dams more than 200 m high, like Xiluodu dam and Baihetan dam, are being built or prepared. TDG levels in the protection zone may be getting much higher than 125%. Not only the survival is under threat, but also the reproduction may be seriously affected, which needs further studying. CAT activity 2 As is known, the reactive oxygen species (ROS) including oxygen radical and H 2 O 2 are being produced during the oxygen metabolism. H 2 O 2 is being transformed to hydroxyl radicals easily, which does harm to cells. However, it can be cleared away by CAT efficiently. Research has shown that environmental stresses can lead to oxidative stress in fish, usually shown as behavioral disorders, increasing amount of ROS, changes of enzyme activities, and organismal death. Liu ( 2006 2006 ) reported that there was a positive correlation between the amount of ROS in the blood and the stocking densities of Chinese sturgeon. Qu et al. ( 2006 2006 ) found the CAT activities in gills and muscles of tilapia exposed to phenol increased at first and then dropped in the groups with lower concentrations, while the CAT activities were controlled in the higher groups. Since the TDG supersaturation was treated as a water quality issue, biological responses to TDG supersaturation in fish have been found. Newcomb ( 1974 1974 ) mentioned that serum potassium and phosphate increased while serum albumin, calcium, and cholesterol decreased when the juvenile steelhead trout was exposed to nitrogen supersaturation. Liu et al. ( 2011 2011 ) showed a trend that the CAT and SOD activities in gills and muscles of rock carp exposed to TDG levels ranging from 100% to 116% for 42 d increased first and then dropped. The present study indicated that CAT activities in gills were higher than those in muscles, which agrees well with Liu et al. ( 2011 2011 ). In this study the CAT activities in gills did not increase significantly in the treatment group. The reason may be that the CAT activities were induced briefly or the gills had already been damaged before they were increased significantly. In addition, there is a phenomenon that CAT activities in muscles were increased significantly. It indicated that the muscles were already affected by the TDG supersaturation. From then on, CAT activities began to drop. A possible explanation is that damage of the gills slowed down the respiration. The transport of TDG supersaturation to the inside of the body was blocked, and as a result the body was dying. These findings can provide a means to evaluate how seriously the fish are damaged by the TDG supersaturated water downstream from dams. Suggestions 2 Since more and more high dams are being built or prepared in China, problems in reducing the TDG supersaturation need to be solved urgently. Qu et al., ( 2011 2011 ) reported that the gas saturation downstream from a dam was associated with energy dissipation structures, spill rate, and operation patterns; the greater the ratio of spill rate vs. power flow was, the higher TDG supersaturation levels were generated. Orlins and Gulliver ( 2000 2000 ) reported that TDG supersaturation could be substantially reduced through changing the spillway configurations equipped at the Wanapum dam. In order to reduce the gas saturation to a safe level, the water level can be lowered in advance to extend the flood discharge time and decrease the spill rate through the reservoir regulation, or choose surface or under-flow dissipation instead of ski-jump energy dissipation. The results of this study can facilitate future scheduling schemes for hydropower plants along the Yangtze River and help with the establishment of the water environment standards in China. References DF Alderdice , JOT Jensen . 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Method of Toxicity Tests on Aquatic Creatures The authors are grateful to Mr. Wen-min YI, Mr. Xun-chi PU, Ms. Bo LI, Mr. Song WU, Mr. Zhi-guang ZHANG, Ms. Juan WEI, Mr. Zhong-luan YAN, Ms. Lu CAO, etc. (Sichuan University, China) for their help during the experiments. 10.1631/jzus.B1200022 Project supported by the National Natural Science Foundation of China (No. 50979063) and the Scientific Research Foundation for Young Teachers of Xihua University (No. Z1120412), China 2012-01-16 2012-09-10 2012-05-30 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2012 2012 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 10 13