Influence of total dissolved gas-supersaturated water on silver carp (Hypophthalmichthys molitrix)

Lu Cao,Rui-feng Liang*,You-cai Tuo,Yong Li,Ke-feng Li

State Key Laboratory of Hydraulics and Mountain River Engineering,Sichuan University,Chengdu 610065,China

Influence of total dissolved gas-supersaturated water on silver carp (Hypophthalmichthys molitrix)

Lu Cao,Rui-feng Liang*,You-cai Tuo,Yong Li,Ke-feng Li

State Key Laboratory of Hydraulics and Mountain River Engineering,Sichuan University,Chengdu 610065,China

Dam discharges over spillways are the main reason for the gas-supersaturated water generated in rivers.Localaquatic organisms,especially fish,readily develop gas bubble disease(GBD)in a river with total dissolved gas supersaturation(TDGS).Sometimes,fish will die after a specifi c exposure period,which negatively affects the biological equilibrium.In this study,the infl uence of TDGS on silver carp was explored. Silver carp were exposed to gas-supersaturated water with TDGS levels of 140%,135%,130%,125%,120%,and 110%to test the impact of TDGS.Some silver carp died when the TDGS levelwas 120%or higher,and all the silver carp exposed to a TDGS level of 140%died within 10 h.In comparison with the Chinese sucker,Prenant's schizothoracin,and rock carp,silver carp seem less sensitive to TDGS.In addition,we tested the catalase(CAT)activity in the muscles and gills of silver carp afterthey were exposed to gas-supersaturated water with a TDGS levelof 140%.The CAT activity changed with time;itincreased first and then decreased until it was lower than the initial CAT activity.The results of this study can provide basic data for developing a standard for environmental evaluation.

Total dissolved gas supersaturation;Silver carp;CAT activity;Gas bubble disease;Median lethal time;Median lethal concentration

1.Introduction

The upper Yangtze River is located in southwestern China, a region filled with water resources,high dams,and habitats for many commercial fish.When water is discharged over the spillway of a dam,the air around the high-speed water willroll into the downstream water,generating gas-supersaturated water,in which the dissolved gas exceeds the normal level. Totaldissolved gas supersaturation(TDGS)was firstidentified in the Columbia and Snake rivers in the 1960s.After dams were constructed along the Columbia River,the TDGS level reached 125%(Ebel,1969).Weitkamp et al.(2003)pointed out that,during the process of release of water from dams onthe Clark Fork River in 1997,the TDGS level observed was close to 150%.Jiang et al.(2008)found that the TDGS level was over 130%when water was discharged from the Zipingpu Dam.Gas-supersaturated water was also generated in the areas downstream of the Three Gorges Dam,Ertan Dam,and other dams in prototype observation(Qu etal.,2011),and the TDGS levels downstream of the Gongzui,Ertan,and Three Gorges dams were 142.5%,140%,and 138%,respectively.

Gas-supersaturated waterleads to gas bubble disease(GBD) in fi sh(Gorham,1901),and the release of dissolved gas is very slow.Therefore,the TDGS infl uence region reacheshundreds of kilometers(Feng et al.,2010).Because of this,the fi sh in the areas downstream of dams during the discharge period suffer from TDGS.Beiningen and Ebel(1970)detected TDGS levels reaching 143%and a large numberofjuvenile and adultsalmon as well as rainbow trout dying in the downstream area.Lutz (1995)showed that fish dying downstream of the Red Rock Damduring the period from1983 to 1994 were associated with a TDGS levelin excess of 110%.Many researchers then pointedoutthatjuvenile fi sh died from GBD during the period of water discharge into the Columbia River(Parametrix,2003)and the Xin'anjiang Reservoir in China(Wu,1994).Backman et al. (2000)and Backman and Evans(2002)collected adult sockeye salmon and steelhead atthe Bonneville Dam from 1995 to 1999 and found that fi sh quickly developed GBD when the TDGS levelwas higherthan 126%butseldom developed GBD when the TDGS level was lower than 125%.To intensively study the infl uence of TDGS on fish in China,laboratory experiments were performed by some researchers at Sichuan University(Huang et al.,2010;Liu et al.,2011;Chen et al., 2012;Wang etal.,2015).

Silvercarp(Hypophthalmichthysmolitrix)are one ofthe four famous commercial fish widely distributed in major river systems in China(Welcomme,1989).They readily suffer from TDGS.Catalase(CAT)is a common enzyme that is widely distributed in nearly all living organisms,including animals, vegetables,fruits,and microorganisms,and is especially concentrated in the livers.CAT can catalyze the decomposition of H2O2into oxygen and water(Chelikani et al.,2004). Therefore,it is a very important enzyme that protects the cell from oxidative damage.Jin et al.(2010)investigated the toxic effects of pollutants on Carassius auratus by measuring the CATactivity,because the CAT activity can reflectthe degree of toxicity of pollutants.

Recently,mass fish deaths have been reported(Zhu,2010; CCTV,2014),and there are large numbers of high and superhigh dams being constructed in China.Therefore,the threat of TDGS is becoming more and more serious.This study focused on the influence of TDGS on silver carp by exposing them to different levels of gas-supersaturated water to find the time that 50%mortality occurred at a specifi c TDGS level(median lethaltime,LT50)and the TDGS levelatwhich 50%mortality occurred at a specifi c time(median lethal concentration, LC50).CAT activities were also measured to explore the physiological change of silver carp.

2.Materials and methods

2.1.Experimental fish and instruments

All the silver carp in the experiments were six months old and transported from Sichuan Fisheries Research Institute,in China.Before the experiments,they were placed in fully aerated water for four days withoutfeeding.The water temperature was stable at 21°C and the pH value was held at7.1 to 7.8.

The experimentalinstruments included Point Four Trackers (Point Four Systems Inc.,Canada),water thermometers, rulers,electronic scales,and magnifying glasses.

2.2.Acute-lethal experiments

Gas-supersaturated water was generated in a laboratory by pumps,compressors,and autoclaves.Air from the compressor mixed with the water from the pump in the autoclave,generating gas-supersaturated water.Then,through adjustment of the ball valve to change the release of dissolved gas,different levels of gas-supersaturated water were generated(Fig.1).The TDGS level in the fresh water was 100%.

Twenty fi sh were placed into tanks with respective TDGS levels of 140%,135%,130%,125%,120%,110%,and 100% for 72 h.The behavior and the time to death of each fi sh were observed and recorded during the experimentalperiod.LT50of each TDGS level and LC50at 12,24,48,and 72 h were calculated to analyze the influence of gas-supersaturated water on silver carp.

2.3.CAT activity test

The CAT activity can reflect the physiological situation of silver carp and the influence of TDGS on silver carp.Thus,in this study,the CAT activity was chosen as a detectable substance to value the TDGS effects and the method of determination was based on Montavon et al.(2007).H2O2can be absorbed at a 240 nm wavelength.Because of the reaction of CAT and H2O2,the content of H2O2and the absorbance will decrease.Thus,the change of absorbance can reflect the activity of CAT.In this experiment,fish were sampled and killed, and 0.2 g muscles and gills were weighed and homogenized with liquid nitrogen.Then,a 1.6 mL phosphate buffer solution (with a pH value of 7.8)was added to centrifuge the homogenized muscles and gills(at 12000 r/min and 4°C for 25 min).The supernatant was the enzyme extraction.The absorbance of a phosphate buffer solution(with a pH value of 7.0)was adjusted to zero.Then,the change of the absorbance of a test solution,containing 2.75 mL of phosphate buffer solution(with a pH value of 7.0),0.15 mL of H2O2solution (with a concentration of 0.3%),and 0.1 mL of enzyme extraction,over 1 min was measured.The CAT activity can be calculated using Eq.(1)and the CAT activity is expressed in the unit of U/g fresh weight(FW):

where ACATis the CAT activity;⊿240is the change of absorbance over 1 min;Vtis the volume of phosphate buffer solution with a pH value of 7.8,and Vt=1.6 mL;W is the weightof muscles or gills,and W=0.2 g;Vsis the volume of enzyme extraction,and Vs=0.1 mL;and t is the time of testing the absorbance,and t=1 min.

Fig.1.Gas-supersaturated water generation system.

According to the results of the acute-lethal experiment, LT50at the TDGS level of 140%was 5.62 h,so 5.62 h shouldbe the upper limit of the CAT activity test.From the beginning to 5 h later,three fish were taken out every hour to detect the CAT activity in their fresh muscles and gills.Atthe same time, three fish in fresh water were taken out in the same way,but the CAT activity was only tested at the beginning,middle,and end of the experiment,because the CAT activity was almost stable if the environment of the fi sh remained unchanged.

A comparison of the CAT activity of fish in gassupersaturated water and that in fresh water shows physiological changes in the fish exposed to gas-supersaturated water.

2.4.Statistical analysis

LT50and LC50were calculated using the probability unit method and linear regression.To calculate LT50,logarithms of the time to death of each fi sh and the corresponding probability units of mortality were dotted in a coordinate system.A fitting line was generated,from which the logarithms of LT50corresponding to the probability units of 50%mortality could be obtained.LC50was calculated the same way as LT50,but the x-axis and y-axis were changed to the logarithms of the TDGS level and the corresponding probability units of mortality at a specific time,respectively.

3.Results

3.1.Acute-lethal experiments

In the first 1.25 h,the fi sh at each TDGS level swam in the usual way and did not show any abnormal action.Fish at the TDGS level of 140%began behaving abnormally after 1.25 h. First,they became irritable,swam faster,and moved up and down.This situation continued for about 2 h.Then,their action began to slow down,and their bodies lost balance and gradually grew sluggish.With the passage of time,their bodies began rotating and spiraling in the water,and then suddenly accelerated when exposed to a stimulus.In the fi nal10-20 min, the fish lost their ability to swim,died,and stayed at the bottom or surface of the water.All the fish that died experienced this process before their death.However,the fish in other groups showed abnormal activity later on in the experiment and the activity lasted longer.In the 72-h experiment, only the fish at TDGS levels of 110%and 100%did not die. The phenomena in the dead fi sh were almost the same: exophthalmia,gills and fi n bleeding,bubbles formed in fins, and so on.

The time to death of fish at different TDGS levels was recorded and drawn in Fig.2.All the fish at the TDGS levels of 140%,135%,and 130%died within 10,20,and 54 h, respectively.However,only 55%and 25%of the fish died at the TDGS levels of 125%and 120%,respectively.

According to the regulation of death,LT50of the fish in gas-supersaturated water was calculated,except at the TDGS level of 120%,at which less than 50%of the fish died within 72 h.The LT50values at TDGS levels of 140%,135%,130%, and 125%were 5.62,11.22,28.84,and 67.71 h,respectively.

Fig.2.Mortality of silver carp at different TDGS levels.

In this study,the LC50values for exposure time in gassupersaturated water at 12,24,48,and 72 h was calculated. The LC50values decreased from 137.47%at 12 h to 130.91% at 24 h,126.11%at 48 h,and 122.96%at 72 h.

3.2.CAT activity test

The CAT activity in muscles and gills of the fi sh exposed to fresh water and gas-supersaturated water with a TDGS levelof 140%is shown in Fig.3 from the beginning of the experiment to 5 h later.In fresh water,the CAT activity was stable and only changed slightly,and the CAT activity was about 190 U/g in the muscles and 380 U/g in the gills.When silver carp were exposed to gas-supersaturated water,the CAT activity decreased at fi rst and then increased signifi cantly within 2 h. As time continued,the CAT activity decreased slowly and it was fi nally lower than that in fresh water.The same process occurred in the muscles and gills of silver carp,but the CAT activity in muscles decreased faster than in gills.

4.Discussion

4.1.Acute-lethal experiments

The occurrence of mortality in each TDGS group was the same as in Bouck(1980),in which the death of silver carp was slow ateach TDGS levelatthe beginning,there was a massive spike in deaths in the middle of the time period,and towards the end there were few dead fi sh as those remaining had a strong tolerance.The mortality in this study was mainly associated with two factors:the TDGS leveland exposed time.When exposed to a lowerTDGS level(110%),no fi sh died.However,silvercarp at TDGS levels of125%and 120%began to die after48 h and 60 h of exposure,respectively.Untilthe end of this experiment,only 25%ofthe silvercarp died ata TDGS levelof120%and 55%of the silvercarp died ata TDGSlevelof125%.Similarresultswere found in other studies.Gu¨ltepe etal.(2011)found no mortality occurring in their experiment,in which rainbow trout were placed in gas-supersaturated water with TDGS levels of 104% and 115%.The fi sh at a TDGS level of 115%even showed an increase in swimming activity and panic episodes.

Fig.3.CAT activity in muscles and gills of silver carp in gassupersaturated water with TDGS level of 140%and in fresh water.

Matthew et al.(2000)conducted laboratory experiments to evaluate the relationship between GBD and mortality in Chinook salmon and steelhead.They pointed outthatthere was a substantial amount of inter-individual variation in terms of susceptibility to GBD.Beeman et al.(2003)also compared the LT50values of different species of fish exposed to gassupersaturated water in the Columbia River,and showed a variation with regard to both TDGS levels and differentspecies. For differentsizes of fi sh of the same species,the tolerances to different TDGS levels also differed(Gunnarslietal.,2009).For silvercarp,LT50presented an increase by multiple times with the decrease ofthe TDGS level.Forexample,LT50was increased by 5.6 h when the TDGS level diminished from 140%to 135%. However,LT50was increased by 38.87 h when the TDGS level diminished from 130%to 125%.The results of silver carp were compared with those of Chinese sucker,Prenant's schizothoracin,and rock carp through experiments performed atthe State Key Laboratory of Hydraulics and Mountain River Engineering of Sichuan University,in China,with the same experimental equipment and methods(Chen et al.,2012;Liu et al.,2011; Huang et al.,2010;Wang et al.,2015).Fig.4 shows the LT50values of these three species and silver carp at TDGS levels of 140%,135%,and 130%.Itindicates differenttolerances to gassupersaturated water.Itisobviousthatthe tolerance ofsilvercarp is greaterthan thatofotherfi sh.However,athigher TDGS levels, the LT50values of these four species show similarresults within 5 h.Thus,when the TDGS levelis high enough,the fish in this gas-supersaturated water will die within a very short time. Furthermore,when fish are exposed to low gas-supersaturated water,different fish show different tolerances.Some fi sh,like Chinese sucker,rock carp,and Prenant's schizothoracin,die within shortertime compared with silvercarp(Chen etal.,2012; Liu etal.,2011;Wang etal.,2015).

In addition,the LC50value for silver carp was calculated. The results show that when the TDGS level is 137.47%,50% of fi sh will die within 12 h,and if the TDGS level diminishes to 122.96%,50%mortality occurs from 12 h to 72 h.In comparison,at a TDGS level of 130%,50%mortality occurs in Chinese sucker within 10 h(Chen et al.,2012).Silver carp have a stronger tolerance to TDGS but the impact of TDGS still exists.

4.2.CAT activity

CAT is the main enzyme whose activity changes when the environment changes.In this experiment,CAT activities were measured to value the influence of gas-supersaturated water on silver carp.Gu¨ltepe et al.(2011)chose carbonic anhydrase to study the effects of gas-supersaturated water on rainbow trout, as carbonic anhydrase is also an important enzyme in fish. They found thatwhen fi sh were exposed to gas-supersaturated water,the activity of carbonic anhydrase was reduced.Liu (2006)pointed out the relationship between the stocking densities of Chinese sturgeon and CAT activity in blood.Qu et al.(2006)found that the CAT activity in gills and muscles of tilapia increased fi rst and decreased under the normal level when exposed to phenol.

When fi sh were exposed to gas-supersaturated water,the CATactivitiesofrock carp and Chinese suckerwere recorded by Liu et al.(2011)and Chen etal.(2012),respectively.Liu etal. (2011)exposed rock carp to gas-supersaturated waterwith a low TDGS level for 42 days,and found that the CAT activity increased first and then decreased.However,there is no such pattern in Chinese sucker.In thisstudy,the CATactivity ofsilver carp also increased fi rstand then decreased,similarly to thatin rock carp.However,silver carp were exposed to highly gassupersaturated water and rock carp were exposed to low-level gas-supersaturated water.When silver carp were exposed to highly gas-supersaturated water,the CATactivity in the gills and muscles suddenly increased.Then,the CAT activity began to decrease when silvercarp adapted to the environment.The CAT activity continued to decrease until silver carp died due to the severe poisoning by TDGS.

Fig.4.LT50of Chinese sucker,rock carp,Prenant's schizothoracin, and silver carp at TDGS levels of 130%,135%,and 140%.

The influence of TDGS on silver carp is a continuous process.Reducing the TDGS level or changing the exposure process may be an effective way to diminish the harm to silver carp or other fish,but this requires more research.

5.Conclusions

(1)Death of silver carp occurred when they were exposed to gas-supersaturated water for three days and the TDGS level reached 120%.In contrast,silvercarp survived forthree days at a TDGS level of 110%.Thus,when silver carp stayed in lowlevel gas-supersaturated water,they could survive,but the duration for which they can survive requires more study.

(2)In comparison with Chinese sucker,Prenant's schizothoracin,and rock carp,silver carp survived longer in lowlevel gas-supersaturated water,but they were all unable to survive for a long time in highly gas-supersaturated water.

(3)When exposed to highly gas-supersaturated water,silver carp showed some changes in their bodies.The CAT activity suddenly increased,but,after a period of time,the CAT activity recovered.However,the toxicity of TDGS caused the reduction of CAT activity until the fish died.

Acknowledgements

The authors are grateful to Mr.Shi-chao Chen,Ms.Bo Li, Mr.Wen Jiang,Mr.Song Wu,and Mr.Zhi-guang Zhang at Sichuan University in China,for their help during the experiments.They also appreciate the advice of Ms.Xiao-jia He for modifying this paper.

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Received 24 March 2016;accepted 26 July 2016

Available online 6 January 2017

This work was supported by the National Natural Science Foundation of China(Grant No.51379136).

*Corresponding author.

E-mail address:ruifengliangscu@126.com(Rui-feng Liang). Peer review under responsibility of Hohai University.

?2016 Hohai University.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license(http:// creativecommons.org/licenses/by-nc-nd/4.0/).