in the method of the present invention , aquatic animals are anesthetized by bubbling a gas or gases containing n 2 o and co 2 into water containing the animals through one or more aeration pipes such as those having a sintered glass tip or an air stone tip to absorb them into water . regarding the effects of n 2 o and co 2 on aquatic animals , it is considered that n 2 o exerts sedative activity and co 2 exerts anesthetic activity . it is impossible to anesthetize animals without vigorous swimming motions only by either of the above activities . when the both activities are combined , aquatic animals can be anesthetized for the first time without vigorous swimming motions as the result of their synergism . this has not been found heretofore in the prior art and is one of important advantages of the present invention . although it is well known that n 2 o is an anesthetic gas and is often used in an operation of the human body , there is no case that n 2 o is applied to an aquatic animal . that is , according to the method of the present invention , n 2 o is firstly applied to aquatic animals in combination with co 2 . the aquatic animals to which the method of the present invention is applied is so - called primary aquatic animals which are cold blooded animals living in water and respiring dissolved oxygen . particularly , the method of the present invention is preferably applied to very valuable high grade marketable animals from the economical point of view . examples of such animals include those belonging to the class pisces such as salmon , trout , char , ayu , carp , crucian carp , goldfish , roach , whitebait , eel , conger eel , sardine , flying fish , sea bass , sea bream , parrot bass , snapper , mackerel , horse mackerel , tuna , bonito , yellowtail , rockfish , fluke , sole , flounder , blowfish , filefish , etc . ; those belonging to the class cephalopoda such as squid , cuttlefish , octopus , etc . ; those belonging to the class pelecypoda such as clam , scallop , ark shell , oyster , etc . ; those belonging to the class gastropoda such as turban shell , abalone , etc . ; and those belonging to the class crustacea such as lobster , prawn , shrimp , crab , squilla , etc . by appropriately selecting conditions such as a flow rate of gas to be bubbled , a ratio of n 2 o to co 2 , an aeration time and the like according to kinds and size of animals to be applied , desired sedative activity of n 2 o and anesthetic activity of co 2 can be exerted to obtain a desired effect of the present invention . although such conditions are varied according to size of a water tank , one standard for conditions in the anesthetizing method of the present invention is as follows . when anesthetizing sea bream ( chrysophrys major ) having 1 kg of body weight in a 650 liter water tank , n 2 o and co 2 are bubbled into sea water at the flow rates of 10 liter / min . and 1 liter / min ., respectively , for 20 minutes or more to bring ph to 6 . 0 or lower . thereby , sea bream is anesthetized calmly . the method of the present invention can be carried out , for example , by putting aquatic animals such as fish in a water tank and bubbling n 2 o and co 2 into water from different nozzles simultaneously under the above conditions , or by preparing a mixed gas of n 2 o and co 2 and bubbling the mixed gas into water from one nozzle . alternatively , it is possible to put aquatic animals in a water tank into which n 2 o and co 2 have been previously bubbled . further , it is possible to firstly bubble n 2 o and then co 2 into water , or to put aquatic animals in a water tank into which n 2 o has been previously bubbled for a certain period of time and then transfer the animals to another water tank into which co 2 is bubbled . these are appropriately selected according to a particular use of the method of the present invention . that is , in the method of the present invention , it is necessary that sufficient sedative activity by n 2 o should be exerted on aquatic animals before exertion of anesthetic activity by co 2 due to lowering of ph of water . in other words , the aeration ratio of n 2 o to co 2 is of importance in the case that aeration is carried out simultaneously , or the mixing ratio of n 2 o and co 2 is of importance in the case that a mixed gas is used . although the optimum ratio of n 2 o to co 2 is varied according to kinds of aquatic animals , difference between individuals , water temperature and the like , in general , the ratio of co 2 : n 2 o is in the range of , preferably , 1 : 1 to 1 : 100 , more preferably , 1 : 8 to 1 : 30 . even if n 2 o is increased more than this range , no more sedative effect before exertion of anesthetic activity by co 2 can be expected and therefore it is disadvantage from the economical point of view . on the other hand , when co 2 is increased , ph is dropped to the prescribed point before exertion of sedative effect by n 2 o , which results in the &# 34 ; exaltation phase at initial stage under anesthesia &# 34 ; of the animals to cause vigorous swimming motions . this is undesirable . particularly , in the case of anesthetization of sea bream , blowfish , flounder , lobster and the like , aeration is carried out at the co 2 : n 2 o ratio of 1 : 8 to 1 : 10 until ph becomes 6 . 2 to 5 . 8 . in the case of anesthetization of yellowtail , filefish , squid and the like , aeration is carried out at the co 2 : n 2 o ratio of 1 : 10 to 1 : 30 until ph becomes 6 . 8 to 6 . 3 . thereby , it is possible to exert desired sedative and anesthetic activities smoothly . by the way , dissolved oxygen may be decreased by bubbling of these gases into water to cause an oxygen deficit state , which results in weakening of the animals . therefore , it is desired to also bubble oxygen into water in such a manner that dissolved oxygen in water is kept in a concentration of not less than 5 ppm , for example , by monitoring the concentration with a dissolved oxygen ( do ) meter . the method of the present invention can be carried out at ambient temperature and cooling of water is not required . in order to awaken the animals from anesthesia , for example , fish under anesthesia are transferred to another water tank into which n 2 o and co 2 are not bubbled . preferably , at this stage , oxygen is bubbled into water to increase dissolved oxygen . thereby , fish can be readily awakened from anesthesia within a short period of time , for example , several minutes to several ten minutes . the following examples further illustrate the present invention in detail but are not to be construed to limit the scope thereof . in this example , anesthetization is carried out by using an apparatus as shown in fig1 . in the apparatus of fig1 n 2 o gas and co 2 gas are bubbled into a sea water tank ( 1 ) containing aquatic animals from a n 2 o bomb ( 2 ) and co 2 bomb ( 3 ) each of which has a control valve and a flowmeter through an aeration pipe ( 4 ). o 2 gas is also bubbled from a o 2 bomb ( 5 ) through an aeration pipe ( 6 ). the tank ( 1 ) is equipped with a do meter ( 7 ) and a ph meter ( 8 ) to monitor ph and do conditions . about 600 liter of sea water was filled in the 650 liter tank ( 1 ), and two yellowtails ( seriola quinqueradiata , about 5 kg of body weight ), two sea breams ( chrysophrys major , about 1 kg of body weight ) and two flounders ( paralichtys olivaceus , about 700 g of body weight ) were put in the tank . the tank was aerated with 10 liter / min . of n 2 o and 1 liter / min . of co 2 as well as o 2 with monitoring do meter ( 7 ) so as to keep dissolved oxygen at a concentration of at least 7 to 8 ppm . behavior of fish were observed according to change of ph . the initial ph of sea water was 7 . 8 to 7 . 9 and the initial water temperature was 20 ° c . the relation between aeration time , ph and do is shown in table 1 . table 1______________________________________time ( min .) 0 5 6 8 10 15 19 22 25ph 7 . 8 7 . 0 6 . 5 6 . 4 6 . 3 6 . 1 6 . 0 6 . 0 6 . 0do ( ppm ) 5 . 1 6 . 5 6 . 9 7 . 0 7 . 3 7 . 9 8 . 2 8 . 3 8 . 6______________________________________ from immediatley after commencement of aeration , among the fish , yellowtails began to show &# 34 ; surfacing &# 34 ;. after 6 minutes , one of yellowtails was put under anesthesia at ph about 6 . 5 and the other one was also put under anesthesia at ph 6 . 3 after 10 minutes . they rose to the surface with lying on their backs . regarding sea breams , after 15 minutes , they showed &# 34 ; surfacing &# 34 ; at ph about 6 . 1 and , after 19 minutes , two sea breams were put under anesthesia at ph about 6 . 0 . they lay on their sides and rose to the surface . regarding flounders , since they lay on the bottom of the tank without significant motions from the beginning of aeration , it was difficult to confirm when they were put under anesthesia . however , it was considered that they were put under anesthesia at ph about 6 . 0 because , when they were pecked with a stick and turned over at this ph , they did not return to the original positions . about 350 liter of sea water was filled in the 400 liter tank as shown in fig1 and two filefishes ( stephanolepis cirrhifer , about 20 cm of body length ), two squid ( dorytenthis kensaki , about 20 cm of body length ) and two lobsters ( panulirus japonicus , about 20 cm of body length ) were put in the tank . the tank was aerated with 8 liter / min . of n 2 o as well as co 2 with adjusting the flow rate in the range of 0 . 35 to 1 . 5 liter / min . according to ph drop . o 2 was also continuously bubbled into sea water with monitoring by do meter ( 7 ) so as to keep dissolved oxygen at a concentration of at least 7 to 8 ppm . the initial ph of sea water was 8 . 0 and the initial water temperature was 19 . 5 ° c . the changes of the flow rate , ph and do are shown in table 2 . table 2______________________________________time ( min .) 0 4 7 9 14 25 34 50 56n . sub . 2 o ( liter / min ) 8 . 0 8 . 0 8 . 0 8 . 0 8 . 0 8 . 0 8 . 0 8 . 0 -- co . sub . 2 ( liter / min ) 1 . 0 0 . 35 0 . 35 0 . 35 0 . 35 1 . 0 1 . 5 1 . 5 -- ph 8 . 0 7 . 1 7 . 0 6 . 8 6 . 7 6 . 7 6 . 4 6 . 0 5 . 9do ( ppm ) 9 . 7 10 . 9 10 . 6 10 . 3 8 . 6 14 . 1 8 . 6 7 . 5 12 . 0______________________________________ after 4 minutes from commencement of aeration , filefishes began to show &# 34 ; surfacing &# 34 ; at ph of about 7 . 1 . the body color became reddish and , after 9 minutes , they rose to the surface at ph about 6 . 8 . after 14 minutes , they were completely put under anesthesia without vigorous swimming motions . regarding squids , after 7 minutes , one of them rose to the surface at ph 7 . 0 and put under anesthesia with closing eyes . the other one was also put under anesthesia calmly after 14 minutes without vigorous swimming motion or ejection of ink . regarding lobsters , after 14 minutes , they began to show such behavior that they moved backward with enfolding their tails under abdomens , which is characteristic of anesthetic effect , at about 6 . 4 . after 56 minutes , it was confirmed that they were put under anesthesia calmly at ph of about 5 . 9 . the same tank as used in example 1 was used and two sea breams ( 1 kg of body weight ) and two flounders ( 700 g of body weight ) were put in the tank in which n 2 o and co 2 had been previously dissolved . the ph of sea water was 6 . 0 and dissolved oxygen was 8 . 8 to 9 . 0 ppm . as the results , it was confirmed that sea breams were put under anesthesia after 2 to 3 minutes and flounders were put under anesthesia after 3 to 5 minutes . all fish did not show vigorous swimming motions . when the aquatic animals put under anesthesia in the above examples 1 to 3 were transferred into ordinary sea water adjusted to do of at least 7 to 8 ppm only by aeration with o 2 , all of them completely awakened from anesthesia within 3 to 5 minutes , although there were individual differences . no animal died in these anesthetization experiments under the proper ph and do conditions . in this example , anesthetization is carried out by using an apparatus as shown in fig2 . in the apparatus of fig2 a mixed gas of n 2 o and co 2 are bubbled into a sea water tank ( 1 ) containing aquatic animals from a mixed gas bomb ( 2 ) having a control valve and a flowmeter through an aeration pipe ( 3 ). o 2 gas is also bubbled from a o 2 bomb ( 4 ) through an aeration pipe ( 5 ). the tank ( 1 ) is equipped with a do meter ( 6 ) and a ph meter ( 7 ) to monitor ph and do conditions . sea water was filled in the tank ( 1 ) having the inner volume of about 1 . 5 m 3 and three sea breams ( about 1 . 5 kg of body weight ) and three blowfishes ( fugu rubripes , about 500 g of body weight ) were put in the tank . the tank was aerated with 30 liter / min . of a mixed gas of co 2 ( 10 %) and n 2 o ( 90 %). o 2 gas was also bubbled into sea water with monitoring do meter ( 6 ) so as to keep dissolved oxygen at a concentration of at least 10 ppm . after 30 minutes , one sea bream rose to the surface lying on its back at ph about 6 . 2 and it was confirmed that it was put under anesthesia . thereafter , the fish were put under anesthesia one after another calmly and , after 40 minutes , all fish were put under anesthesia at ph of about 6 . 0 without vigorous swimming motions . then , aeration with the mixed gas of n 2 o and co 2 was discontinued . while a small amount of o 2 was bubbled so as to keep the concentration of do of at least 7 to 8 ppm , the entire water tank was transported about 150 km distance over 4 hours by a motor truck . after transport , the state of each fish was scarcely different from that just after putting under anesthesia , although 4 . 5 hours already elapsed . when the fish were transferred into ordinary sea water aerated with only o 2 , three blowfishes and one sea bream completely awakened from anesthesia within several minutes . the reamining two sea breams completely awakened from anesthesia after several ten minutes , although they showed &# 34 ; surfacing &# 34 ; like behavior . various kinds of aquatic animals were put in each water tank as shown in fig1 ( a : 1 . 5 m 3 , b : 650 liter , c : 400 liters , d : 100 liter ) and experiments were carried out according to the same manner as described in example 1 . the results are shown in table 3 . as seen from table 3 , the method of the present invention is useful for anesthetizing various aquatic animals . table 3__________________________________________________________________________ ph at time initial required size anesthe - for anesthe - length weight ratio of tization tizationanimals ( cm ) ( kg ) co . sub . 2 : n . sub . 2 o stage ( min .) tank__________________________________________________________________________piscessea bream ( chrysophrys 30 1 . 0 - 1 : 8 - 1 : 10 5 . 8 - 6 . 2 10 - 20 a , bmajor ) 1 . 5flounder ( paralichtys 30 0 . 7 1 : 8 - 1 : 10 5 . 8 - 6 . 2 10 - 20 b , colivaceus ) yellowtail ( seriola 50 - 3 - 1 : 10 - 1 : 30 6 . 3 - 6 . 8 15 - 25 a , bquinqueradiata ) 60 5filefish ( stephanolepis 15 0 . 2 1 : 10 - 1 : 30 6 . 3 - 6 . 8 5 - 10 ccirrhifer ) mackerel ( scomber 25 0 . 5 1 : 8 - 1 : 10 6 . 0 - 6 . 5 20 - 30 djaponicus ) horse mackerel ( trachurus 20 0 . 2 1 : 8 - 1 : 10 6 . 0 - 6 . 5 10 - 20 djaponicus ) scorpion fish ( sebastiscus 20 0 . 3 1 : 8 - 1 : 10 6 . 3 - 6 . 8 10 - 20 dmarmoratus ) sardine ( sardinops 15 0 . 1 1 : 8 - 1 : 10 6 . 3 - 6 . 8 10 - 20 d melanosticta ) blowfish ( fugu 20 0 . 5 1 : 8 - 1 : 10 5 . 8 - 6 . 2 10 - 20 arubripes ) carp ( cyprinus 20 0 . 7 1 : 8 - 1 : 10 4 . 3 - 4 . 5 30 - 60 dcarpio ) crucian carp ( carassius 15 0 . 5 1 : 8 - 1 : 10 3 . 5 - 4 . 5 30 - 60 dauratus ) ayu ( plecoglossus 15 0 . 2 1 : 8 - 1 : 10 4 . 5 - 5 . 0 30 - 40 daltivelis ) goldfish 5 -- 1 : 8 - 1 : 10 3 . 5 - 4 . 5 30 - 60 dcephalopodasquid ( dorytenthis 20 0 . 3 1 : 10 - 1 : 30 6 . 3 - 6 . 8 10 - 20 ckensaki ) octopus ( octopus 20 0 . 3 1 : 10 - 1 : 30 6 . 3 - 6 . 8 10 - 20 cvulgaris ) crustacealobster ( panulirus 20 0 . 3 1 : 8 - 1 : 10 5 . 6 - 6 . 0 20 - 30 cjaponicus ) prawn ( penaeus 15 -- 1 : 8 - 1 : 10 5 . 8 - 6 . 2 20 - 30 cjaponicus ) swimming crab ( neptunus 15 -- 1 : 8 - 1 : 10 5 . 8 - 6 . 2 20 - 30 ctrituberculatus ) river crab ( potamon 5 -- 1 : 8 - 1 : 30 5 . 8 - 6 . 2 15 - 25 ddehaani ) __________________________________________________________________________ in this example , anesthetization is carried out by using an apparatus as shown in fig3 . in the apparatus of fig3 o 2 gas and n 2 o gas are bubbled into a 1st water tank ( 1 ) containing fry from an o 2 bomb ( 2 ) and a n 2 o bomb ( 3 ), each of which has a control valve and a flowmeter , through aeration pipes ( 5 ) and ( 4 ), respectively . likewise , o 2 gas and co 2 gas are bubbled into a 2nd water tank ( 6 ) from the above o 2 bomb ( 2 ) and a co 2 bomb ( 7 ) through aeration pipes ( 5 ) and ( 8 ), respectively . the 1st tank ( 1 ) is equipped with a do meter ( 9 ) and the 2nd tank ( 6 ) is equipped with a do meter ( 9 ) and a ph meter ( 10 ) to monitor ph and do conditions . ten sea bream fry ( about 10 cm of body length ) were put in the 1st tank filled with 30 liter of sea water at a temperature of 23 ° c . at ph 8 . 0 . o 2 gas was intermittently bubbled into the tank so as to keep 5 ppm of dissolved oxygen by monitoring with the do meter . then , n 2 o gas was bubbled into the tank at the flow rate of 1 . 5 liter / min until sedative effect on the fry was exerted . as the result of aeration with n 2 o , after 15 minutes , initial effect of n 2 o was exerted and relatively active swimming motions was observed in comparison with a control group of fry in an ordinary water tank . after 20 minutes , the fry began to rise to the surface and thereby sedative effect of n 2 o was confirmed . that is , the fry put their heads down and their tail fins up and , following the ascending current due to bubbling from the aeration pipes , they rose to the surface . then , they intended to sink toward the bottom by themselves . they repeated these motions several times . thereafter , some of them laid on their sides . however , they were not put under anesthesia only by aeration with n 2 o gas . after bubbling n 2 o gas at the flow rate of 1 . 5 liter / min . for 35 minutes , the fry were transferred to the 2nd tank . in the 2nd tank , co 2 was bubbled at the flow rate of 1 . 0 liter / min . for about 3 minutes to lower ph of sea water from 8 . 0 to 5 . 9 . as the result , all the fry were calmly put under anesthesia without vigorous swimming motions as observed in the control group which was not treated with n 2 o . by using the same apparatus used in example 6 , n 2 o gas was bubbled into the 1st tank at the flow rate of 1 . 5 liter / min . for 35 minutes . then , ten sea bream fry were put therein and allowed to stand for 20 minutes . thereafter , the fry were transferred into the 2nd tank into which co 2 gas had been bubbled at the flow rate of 1 . 0 liter / min . for about 3 minutes to lower ph to 5 . 9 . as the result , after 1 . 5 minutes , one fry showed vigorous swimming motion . however , after 3 minutes , all fry were put under anesthesia . about 350 liter of sea water was filled in the 400 liter tank ( 1 ) as shown in fig1 and , according to the same manner as described in example 1 , two yellowtails ( about 5 kg of body weight ), two sea breams ( about 1 kg of body weight ) and two flounders ( about 700 g of body weight ) were put in the tank . only co 2 was bubbled into the tank at the flow rate of 1 . 5 liter / min . o 2 was also bubbled into the tank small poritons by monitoring with do meter . behavior of fish were observed according to change of ph by comparing with the results in example 1 . the relation of aeration time , ph and do is shown in table 4 . table 4______________________________________time ( min .) 0 2 3 5 7 10ph 6 . 7 6 . 1 6 . 0 5 . 8 5 . 6 -- do ( ppm ) 6 . 0 6 . 3 6 . 8 6 . 9 6 . 6 -- ______________________________________ from immediatey after commencement of aeration , all fish began to show &# 34 ; surfacing &# 34 ; and vigorous swimming motions . particularly , yellowtails jumped up from the tank and were not put under anesthesia , even ph dropped to the prescribed value . at about ph 5 . 8 , all fish were put under anesthesia . however , yellowtails were damaged such as bleeding from the gills and , when they were transferred to ordinary sea water at the end of the experiment , one of yellowtails did not awaken from anesthesia and died .
an improved method for anesthetizing aquatic animals which comprises anesthetizing the animals in water with nitrous oxide and carbon dioxide .