olive flounder , paralichthys olivaceus has a flattened oval body , mainly occurs at the benthos in water depths of 10200 m , and is widely distributed in korea and east asia ( han et al . , 2007 ) .
it is the most important cultured fish in korea , with approximately 40,922 tons of this species having been produced in 2012 ( park et al . , 2012 ; park et al . , 2015 ) ; correspondingly large nursery and transport processes are associated with the mass culture of olive flounder ( hur et al .
, 2007 ; park et al . , 2012 ; park et al . , 2015 ) .
however , the lack of knowledge of the physiological changes that occur in the olive flounder during transport has resulted in large mortalities .
olive flounder produced at jeju island , korea , are transported predominantly by truck and ship , and the temperature and dissolved oxygen concentration are controlled during the sometimes long transport period by pumping air , and adding liquefied oxygen and ice to improve fish survival . to minimize stress and reduce the chance of fish mass mortality ( ferreira et al . , 1984 ) during handling and long periods of transportation ,
an effective high - density transport method is essential to reduce costs and minimize losses ( ferreira et al .
the development of new equipment and transport procedures is having a positive effect on the transportation of live fish .
for example , the use of non - toxic salt has been shown to reduce stress and result in higher survival rates ( tomasso et al . , 1980 ; carmichael et al . , 1984 ;
carmichael & tomasso , 1988 ) , and the use of low concentrations of calcium chloride is also a cheap and effective improvement in the handling and transportation of live fish ( grizzle et al . , 1985 ;
the use of anesthetics is also an effective procedure for transporting fish ( park et al . , 2009a ) .
anesthetic use in aquaculture reduces fish energy use , enables efficient transportation , increases ease of handling when measurements are taken , reduces pain and trauma for the fish , reduces handling stress ( park et al . , 2009b ) , and reduces bacterial infections , especially during surgery .
water - temperatureinduced anesthesia , which is recognized as stable in fish , can be used without a mandatory period of withdrawal prior to human consumption , as is required for chemical anesthetics ( summerfelt & smith , 1990 ) .
the mode of application as well as any local regulations and legislation dictate the choice of anesthetic .
currently , only tricaine methanesulfonate ( ms-222 ) and metomidate are registered for veterinary use in fish , although researchers have access to many other compounds not available for public use ( park et al . , 2011 ) .
lengthy withdrawal periods are mandatory for chemical anesthesia of food fish prior to harvest , and this has led to an interest in the search for less persistent and more natural anesthetics ; among these is clove oil ( park et al . ,
the active ingredient in clove oil is eugenol ( 4-allyl-2- methoxyphenol ) , which is inexpensive and a safe anesthetic that has been used as a substitute for commonly used compounds including ms-222 ( park et al . , 2011 ) .
compared with ms-222 , clove oil is characterized by more rapid induction of anesthetic effects , prolonged recovery times , and a narrower range of safe doses in at least three teleost species ( sladky et al . , 2001 ) .
clove oil is also safe , inexpensive , and non - toxic for the environment , and does not require a withdrawal period compared with other anesthetic chemicals ( park et al . , 2008 ) .
clove oil has been studied as an anesthetic in a number of species ( park et al . ,
the aim of this study was to determine the optimum concentrations of clove oil for anesthesia in olive flounder over a range of temperature conditions .
stress responses in the fish and water parameters were also analyzed during a simulated transport experiment involving various clove oil concentrations .
in march 2016 , specimens of olive flounder , paralichthys olivaceus ( standard length : 46.6  0.45 cm ; body weight : 500.7  11.67 g ) , were obtained from the institute of marine living modified organisms , pukyung national university , korea .
the fish were transported to the fishery genetics and breeding sciences laboratory of the korea maritime and ocean university , korea , and reared in a recirculating culture system consisting of a circulation pump , an aeration system , and a temperature control system .
the temperature of the water in the tube were controlled so that it was equal to the temperature of the water in the anesthetic and recovery phases of the experiment .
all fish were fasted for 24 h prior to the commencement of experiments , and were adapted to 400-l glass tubes , which were maintained at the same temperatures as the experimental water tempeatures ( 15 , 20 , and 25c ) . for the anesthetic experiment ,
20 specimens of each experimental group were randomly removed from the breeding tube using a net , and transferred to water in an aerated 50 l plastic aquarium , to which anesthetic solution ( clove oil containing 85% eugenol ; sigma , usa ) had been added .
when a fish had become anesthetized , it was immediately transferred to a recovery tank . the time to achieve anesthesia ( anesthesia time ) and the recovery time
the anesthetic effects of clove oil were investigated at five concentrations ( 10 , 20 , 30 , 60 , 90 , 120 and 150 ppm ) ; these were prepared using a stock solution of clove oil dissolved in 95% methanol ( sigma , usa ) at a ratio of 1:10 .
the anesthesia time was measured from when the fish was placed in the anesthetic - containing water to when it was perfectly sedated and had minimal opercular movement ( stage a6 , table 1 ) , while the recovery time was measured from the time the fish was placed in the recovery tank to when it again exhibited normal swimming and responsiveness to visual stimulation ( stage r6 , table 1 ) .
table 1 is decision - based framework for categorizing anesthetic effects , derived from park et al .
( 2011 ) , and shows the stages of anesthesia and recovery that were used as endpoints in the present study , including a6 ( minimal opercular movement only ) and r6 ( normal swimming , responsiveness to visual stimuli ) ( summerfelt and smith , 1990 ; park et al . , 2011 ) . during the experiment the fish showed several stages in the development of anesthesia , including slowed swimming and side - to - side rolling ( stage a2 ) through to opercular movement only and no reaction when it was flipped onto its other side ( stage a6 ) . at stage a6 , individual fish
recovery was defined as the point at which erratic swimming began , and included the fish righting its balance and flipping itself back over when turned , ( stage r5 ) and normal swimming , as well as responsiveness to visual stimuli ( stage r6 ) . to investigate the stress response of fish to anesthetic exposure
, blood samples were extracted from five randomly selected fish prior to exposure ( 0 h ) , and at 1 , 6 , 12 , 24 , and 48 h following anesthetic treatment .
the blood was transferred to capillary tubes and analyzed following centrifugation at 200  g for 10 min .
the plasma was collected and stored at 80c ( sw - uf-200 freezer ; samwon freezing engineering , busan , korea ) until analyzed . the cortisol concentration in 50 l samples
was measured using a cortisol radioimmunoassay kit ( coat - a - count tkco cortisol ria kit ; dpc , usa ) .
the sample was added to 100 ml of antiserum and incubated for 45 min at 37c , then 1,000 ml of separation reagent was added .
the mixture was placed in a refrigerator at 4c for 15 min , then centrifuged at 1,200  g for 15 min .
the supernatant was assayed using an automatic gamma radiation counter ( cobra ; packard , usa ) .
the plasma glucose concentration was analyzed according to the method of raabo & terkildsen ( 1960 ) , using a glucose assay kit ( kit 510 , sigma , st louis , mo , usa ) ; the production of h2o2 by glucose oxidase in the presence of o - dianisidine was measured as an absorbance increase at 450 nm .
blood lactic acid concentrations were analyzed using an automatic blood analyzer ( reflotron ; boehringer mannheim , germany ) . at 10 days following analysis of the anesthetic effect and the stress response ,
the respiration of fish was assessed using a respirometer chamber comprising a sealed acrylic resin box having a wall thickness of 8 mm and overall dimensions of 10 cm ( width )  50 cm ( length )  10 cm ( height ) .
the hose providing inflow water to the respirometer was equipped with a temperature - controlled heater to maintain the water at 20  0.3c , cartridge filters ( 10 m and 3 m ) to remove particles from the incoming water , and a flow - through ultraviolet lamp to reduce the likelihood of oxygen consumption by microbes .
the water salinity was maintained optimum anesthetic salinity by brackish water , and optimum anesthetic salinity of each group were analyzed by anesthetic experiment .
the respirometer chambers were prepared and labeled according to the measurement times and anesthetic concentrations . to determine the most appropriate experimental concentrations of clove oil and lidocaine - hcl
, we performed a pilot experiment for 6 h. to prevent mortality in the experimental sample , the anesthetized fish were maintained at several stages of anesthesia during the pilot study , ranging from normal swimming , opercular movement , and general movement ( stage a1 ) , to slow swimming and side - to - side rolling ( stage a2 ) ( summerfelt and smith , 1990 ; park et al . , 2011 ) .
the anesthetic effects of clove oil were determined at four concentrations : 0 ( control ) , and 1 , 2 , and 3 ppm .
prior to measuring the dissolved oxygen ( do ) concentration , the concentrations of nh4 and co2 were measured using a spectrophotometer ( dr2800 , hach , loveland , colorado , usa ) , and the respiration frequency ( gill cover movement ) of the fish was measured using a counter and a digital timer .
the do concentration was measured with an o2 electrode and a multi - data logger system ( oxyguard , denmark ) .
one- and two - way analysis of variance ( anova ) were performed for the data among treatments , and duncan s test was performed when significant differences were found ( p < 0.05 ; spss statistics package spss 9.0 ; spss inc .
unless otherwise stated , differences were considered to be statistically significant at p < 0.05 .
no samples of olive flounder , paralichthys olivaceus died from stress as a result of exposure to the anesthetic during the experiment .
table 2 shows the results of a twoway anova test for the effect of clove oil dose and water temperature on anesthesia .
the exposure and recovery times were affected by the dose concentration of clove oil , and by water temperature .
table 3 shows the anesthetic effects of clove oil at each concentration and water temperature .
the anesthesia time decreased significantly as both the concentration of clove oil and the water temperature increased .
for each temperature , the anesthesia time decreased as the concentration of clove oil increased .
furthermore , at each concentration of clove oil , the anesthesia time decreased as water temperature increased .
the recovery time in each temperature increased significantly as the concentration of clove oil increased .
in addition , the anesthesia time for each concentration of clove oil decreased as the water temperature increased .
thus , the recovery time at each temperature was related to changes in the concentration of clove oil , and the recovery time increased as water temperature increased . based on an optimal anesthesia time of approximately 1 min , the optimal concentrations of clove oil for olive flounder were 120 ppm at 15c , and 90 ppm at 20c and 25c .
the ratio of the recovery time to the anesthetic exposure time , as a function of the concentration of clove oil and the water temperature , is shown in fig .
the recovery time / exposure time ratio gradually increased as the clove oil concentration increased in each water temerature treatment . at 90 ppm , 120 ppm , and 150 ppm ,
the ratio increased significantly as water temperature increased ; however , at other concentrations the ratio showed no clear relationship with water temperature .
results of clove oil dose and water temperature on anesthesia among olive flounder were shown in table 3 .
values in the same column not sharing common superscripts are significantly different ( p < 0.05 ) .
2 shows the stress response of the fish to clove oil anesthesia over 48 h. the plasma cortisol concentration at each temperature increased significantly up to 12 h. after 1 h of exposure the plasma cortisol concentrations were 8  1.4 , 10  1.0 , and 14  1.3 g / dl at 15c , 20c , and 25c , respectively . after 6 h
the plasma cortisol concentrations were 18  1.0 , 23  1.2 , and 30  1.3 g / dl at 15c , 20c , and 25c , respectively .
thus , the rate of increase in the plasma cortisol concentration increased with increasing water temperature up to 12 h. after this time point , the cortisol concentration at each temperature decreased significantly to 48 h. at 12 h the plasma cortisol concentrations were 35  1.7 , 36  1.8 , and 35  1.4 g / dl at 15c , 20c , and 25c , respectively , while at 24 h were 20  2.4 , 13  3.0 , and 10  1.7 g / dl , and at 48 h were 5  0.6 , 2  0.5 , and 1  0.4 g / dl , respectively .
thus , the rate of decrease in the plasma cortisol concentration increased as the water temperature increased .
actually n=20 for each experiment because the means and se were calculated separately for each group .
different letters on error bars are significantly different among elapsed time in anesthesia groups ( p<0.05 ) .
the do concentrations , respiratory frequencies , ph values , and nh4 and co2 concentrations in the clove oil treatments are shown in tables 4 and 5 , and figs . 3 , 4 , and 5 , respectively .
the do concentration , ph value , and the respiratory frequency decreased in all experimental groups up to 6 h ( table 4 and figs . 3 and 4 ) , whereas the nh4 and co2 concentrations increased in all experimental groups ( table 5 and fig .
measurement times the do concentration in all clove oil treatments was higher than in the control group ( table 4 ) .
at 6 h the ph values were 6.1  0.064 , 6.35  0.068 , 6.57  0.061 , and 6.82  0.066 in the control group and the 1 , 2 and 3 ppm clove oil treatments , respectively , and ph values in all clove oil treatments were significantly higher than that of control group ( fig .
the co2 and nh4 concentrations and the respiratory frequencies decreased with increasing clove oil concentration ( p<0.05 ) . at 6
the respiratory frequencies were markedly less than in the control group ( 2 3  1.9 , 23  1.5 , 23  1.4 , and 42  3.4 , respectively ; fig .
the co2 concentration in the 1 , 2 , and 3 ppm clove oil treatments were lower than that in the control group ( table 5 ) .
with respect to nh4 , at all measurement times the concentrations in the 1 , 2 , and 3 ppm clove oil treatments were lower than that in the control group ( 0.07  0.006 , 0.06  0.009 , 0.05  0.005 , and 0.09  0.007 mg
values in the same column not sharing common superscripts are significantly different ( p<0.05 ) .
values in the same column not sharing common superscripts are significantly different ( p<0.05 ) .
actually n=20 for each experiment because the means and se were calculated separately for each group .
different letters on error bars are significantly different between no anesthesia and clove oil anesthesia groups ( p<0.05 ) .
actually n=20 for each experiment because the means and se were calculated separately for each group .
different letters on error bars are significantly different between no anesthesia and clove oil anesthesia groups ( p<0.05 ) .
actually n=20 for each experiment because the means and se were calculated separately for each group .
different letters on error bars are signifiantly different between no anesthesia and clove oil anesthesia groups ( p<0.05 ) .
determining the safety and reliability of anesthetics , and the associated recovery time , is a prerequisite for choosing the most appropriate anesthetic for a given species .
the anesthesia time is the time required for an animal to reach the anesthetized condition for the species , and the recovery time is the time required for the animal to completely recover ( summerfelt & smith , 1990 ) . the results of this study indicated that clove oil is an effective anesthetic for p. olivaceus . based on the criteria that anesthesia should be achieved within 3 min , that recovery should occur in < 10 min , and that no mortality occur ( park et al . , 2011 ) , we assessed a range of concentrations of clove oil ( 10120 ppm ) and exposure water temperatures of 20c and 25c .
our results clearly indicated that shorter anesthesia times were achieved with higher concentrations of clove oil .
the observed anesthesia times for olive flounder , paralichthys olivaceus exposed to clove oil were similar to previous reports of its effect on other bony fishes including sockeye salmon , oncorhynchus nerka , rock bream , oplegnthus fasciatus , and kelp grouper , epinephelus bruneus ( woody et al . , 2002 ; park et al . , 2008 and 2009b ) .
the dose response curves for olive flounder exposed to clove oil were negatively exponential , with higher doses resulting in reduced times to anesthesia stage a6 ( see table 1 ) .
optimum anesthetic concentrations are usually expected to induce anesthesia within 3 min , with recovery occurring within 10 min ( park et al . , 2011 ) .
we set the optimum anesthesia concentration at approximately 1 min , and at this level found that the optimal clove oil concentrations for olive flounder were 120 ppm at 15c , and 90 ppm at 20c or 25c .
for other species the optimal clove oil concentration for achieving an anesthesia time of approximately 1 min include 80 ppm at 9.4c for sockeye salmon , 150 ppm at 24c and 100125 ppm at 26c for rock bream , and 50 100 ppm at 22c for kelp grouper ( woody et al . , 2002 ; park et al . , 2008 and 2009b ) .
the relationship between water temperature and anesthesia time followed a negative exponential curve , with increasing water temperature resulting in decreased anesthesia time .
a similar inverse relationship has also been reported in studies of anesthesia by clove oil in other fish species , including european sea bass , dicentrarchus labrax and gilthead sea bream , sparus aurata ( constantinos et al . , 2005 ) .
for these species , lower temperatures resulted in significantly longer induction of anesthesia and recovery times ( constantinos et al . , 2005 ) .
in addition , kelp grouper anesthetized with clove oil , and greenling , hexagrammos decagrammus anesthetized with lidocaine - hcl exhibited similar trends ( park et al .
, 2003 ; constantinos et al . , 2005 ; park et al . , 2008 ) .
1988 ) reported an anesthesia time of 93.0  7.5 s for olive flounder at 100 ppm lidocaine - hcl , and that the optimum concentration of lidocaine - hcl was 200 ppm .
comparison of the anesthetic potency of clove oil and lidocaine - hcl showed that olive flounder were more sensitive to clove oil than to lidocaine - hcl ; clove oil more effectively immobilized fish at lower doses than did lidocaine - hcl in the same anesthesia time .
if the ratio of the recovery time to the anesthesia time is greater than 1 , the recovery time is longer than the anesthesia time . at each water temperature in our study , the ratio of the recovery time to the anesthesia time increased as the anesthesia concentration increased .
that is , as the anesthetic concentration increased , the increase in the recovery time was greater than the decrease in the anesthesia time .
in contrast , as the anesthesia concentration was reduced , the rate of decrease in the recovery time was greater than the rate of increase in the anesthesia time .
similar results demonstrating proportional changes in the clove oil concentration and the ratio of the recovery time to the anesthesia time have been reported for rock bream ( park et al . , 2009b ) .
cortisol is an indicator of a stress response in fish subject to various stressors , and is involved in metabolism and ecologic balance ( mommsen et al . , 1999 ) .
in addition , cortisol affects the body balance , carbohydrates , proteins , and lipids ( mommsen et al . , 1999 ) .
cortisol levels in fish suggest that anesthesia , salinity , do , ph , nh4 , and acidic conditions can affect fish metabolism and hormone levels ( mommsen et al . , 1999 ) .
( 1999 ) , it means they made the annually changeable temperature , fresh water to sea water , salinity , waste water and do .
plasma cortisol is recognized as a useful indicator of stress in fish ( schreck , 1982 ; park et al . , 2008 ) .
elevated plasma cortisol levels were reported in red drum ( sciaenops ocellatus ) simultaneously exposed to the anesthetics ms- 222 and quinaldine ( massee et al . , 1995 ) .
barton & iwama ( 1991 ) stated that  usually , phenomenon that plasma cortisol concentration of fishes rises by stress is first order reaction , phenomenon that plasma glucose concentration rises is result of second - order first order reaction by hormone rise reaction by stress . 
the plasma cortisol concentration in anesthetized olive flounder returned to approximately the concentration in control fish 48 h post - administration of clove oil .
if the metabolic rate is high , the anesthetic is absorbed more rapidly through the gills ( summerfelt & smith , 1990 ) .
the metabolism of olive flounder at 25c was expected to be higher than at the other temperatures tested .
the results showed that the anesthesia and recovery times at 25c were faster than at the other temperatures tested . to confirm and interpret these observations
the patterns of decline in the do concentration in water in the control , clove oil , and lidocaine - hcl treatments were consistent with the trends reported in studies of the amur minnow , rhynchocypris steindachneri and winter flounder , pleuronectes americanus ( park et al .
these results suggest that high levels of stress induced by handling or netting cause high levels of oxygen consumption during the early stages of transportation .
( 1984 ) , who used benzocaine - hcl as an anesthetic in the transport of java tilapia , oreochromis mossambicus . in general , both studies showed that following application of the anesthetic there was a reduction in fish metabolism , which is an indication of declining oxygen consumption . in this study there appeared to be a positive relationship between the concentration of clove oil and the do concentration , insofar as the group treated with the highest concentration of clove oil exhibited the smallest decline in do ( ferreira et al . , 1984 ) . in a similarly designed experiment by park et al .
( 1998 ) , involving amur minnow as the test organism , the same trends in do concentration and ventilation rates was observed over 2 h as were observed over 6 h in our study .
park et al . ( 1998 ) used lower lidocaine - hcl concentrations ( 5 , 10 , and 20 ppm ) and a lower temperature ( 18c , compared with 26c in this experiment ) , but nearly identical trends were found .
this is an important comparison because it indicates the wide - ranging effects of clove oil over a broad spectrum of temperatures . 

( 1995 ) performed a transportation experiment that involved the playfish , xiphophorus maculatus ( gnther ) being treated with three anesthetics : 2-phenoxyethanol ( 200 ppm ) , quinaldine sulfate ( 10 ppm ) , and ms-222 ( 30 ppm ) . at 16
h following anesthetic administration the nh4 concentration in the water in the 2-phenoxyethanol treatment was only 65% that in the control , 20% that in the quinaldine sulfate treatment , and relatively lower than in the ms-222 treatment .
the trends in nh4 concentrations exhibited by the five experimental groups were consistent with the trends reported by park et al .
( 1998 and 2009a ) , insofar as the overall reduction in nh4 excretion was directly related to the anesthetic - induced decline in metabolism .
this study showed that clove oil is effective for anesthetizing olive flounder , and that the anesthetic effect was influenced by water temperature .
park et al . ( 2011 ) reported that the anesthetic effects of clove oil or lidocaine - hcl were significantly affected by water temperature and the anesthetic concentration , decreasing proportionately as these parameters increased .
( 2011 ) suggest that the anesthetic effects of clove oil are similar to those of lidocaine - hcl .
the results of our experiment suggest that clove oil reduced the metabolic activity of olive flounder , thus reducing excretion of nh4 and oxygen consumption by the fish .
clove oil is a cost - effective and efficient anesthetic , and is safe for use and non - toxic to the fish or users ( park et al . , 2003 ) .
in conclusion , clove oil is an effective anesthetic for use in improving the transport of olive flounder and minimizing the stress for olive flounder .
the results of this study will improve the safe laboratory handling and transport of olive flounder , which are commonly required for use in research studies and experiments .
the results of our study should also be useful for aquaculturalists and fish transporters wanting to minimize the stress imposed on fish during transport .
 the optimum concentrations of clove oil as an anesthetic for olive flounder ( paralichthys olivaceus ) and the stress response of the fish to clove oil anesthesia were determined over a range of water temperatures , and investigated in a simulated transport experiment using analysis of various water and physiological parameters . while the time for induction of anesthesia decreased significantly as both the concentration of clove oil and water temperature increased , the recovery time increased significantly ( p<0.05 ) . 
 the plasma cortisol concentration in fish at each temperature increased significantly up to 12 h following exposure ( p<0.05 ) , then decreased to 48 h ( p<0.05 ) . 
 the do dissolved oxygen concentrations , ph values , and the fish respiratory frequencies decreased over 6 h following exposure to clove oil in all experimental groups ( p<0.05 ) , whereas the nh4 
 + and co2concentrations in all experimental groups increased up to 6 h ( p<0.05 ) . 
 the ph values and do concentrations increased with increasing clove oil concentration ( p<0.05 ) in the 6 h following exposure , and the co2 and nh4 
 + concentrations and the respiratory frequencies decreased with increasing clove oil concentration ( p<0.05 ) . 
 the results of this experiment suggest that clove oil reduced the metabolic activity of olive flounder , thus reducing nh4 
 + excretion and o2 consumption . in conclusion 
 , clove oil appears to be a cost - effective and efficient anesthetic that is safe for use and non - toxic to the fish and users . 
 its use provides the potential for improved transportation of olive flounder . 
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION