CA2178722C

CA2178722C - Methods for sedating and anaesthetising aquatic organisms

Info

Publication number: CA2178722C
Application number: CA002178722A
Authority: CA
Inventors: Alistair Renfrew Jerrett
Original assignee: AQUI-S NEW ZEALAND Ltd
Current assignee: AQUI-S NEW ZEALAND Ltd
Priority date: 1993-12-23
Filing date: 1994-12-21
Publication date: 2003-06-17
Anticipated expiration: 2014-12-21
Also published as: GB9612687D0; GB2299757A; AU1328695A; NZ277896A; CA2178722A1; GB2299757B; WO1995017176A1; AU1838599A

Abstract

This invention relates to methods of sedating and/or anaesthetising aquatic organisms through contacting the organisms with a sedative or anaesthetic solution. This solution includes as active agent eugenol, isoeugenol or mixtures thereof, with the concentration of the active agent being up to 12.5 mg l-1. Active compositions for use in the methods are also provided.

Description

WO 95/17176 ~ ~ ~ ~ ~ '? PCT/NZ94100148 Methods for sedating and anaesthetising aquatic organisms FIELD OF THE INVENTION
This invention relates to improved methods for sedating and/or anaesthetising aquatic organisms and to compositions for use in such methods.
BACKGROUND OF THE INVENTION
The practice of catching fish or other aquatic organisms usually involves the organisms undergoing some stress. The organisms commonly associate capture with predation and therefore struggle to escape immobilization. This struggle can have a major impact on the post-mortem quality of the tissue of the organism depending upon its duration and at the pre-mortem physical condition of the organism (Lowe, T.E.; Ryder, J.M.;
Carragher, J.F.; Wells, R.M.G. 1993: Flesh quality in snapper, Pagrus auratus, affected by capture stress. Journal of Food Science 58: 770-773).
In aquaculture, the cultured organisms are usually individually handled during their life cycle. With excitable fish species such as Chinook Salmon (Oncorhynchus tshawytscha), great care must be taken to ensure that the animals are not bruised, scaled or in any way disfigured or damaged during handling. A natural, undamaged appearance is often a critical factor in determining the final sale price of the fish.
To achieve optimum product quality during harvesting, the organisms must be maintained in a calm state. One approach which has been investigated is the use of anaesthetics during harvesting. Commonly used anaesthetics such as MS-222, 2-phenoxyethanol, benzocaine and more recently, the sedatives etomidate and metomidate (Kreiberg, H'.
1992: Metomidate Sedation Minimises Handling Stress in Chinook Salmon.
Bulletin of the Aquaccdtural Association of Canada 92-3: 52-54) have been used to minimise damage during handling but their potential residual toxicity to (or misuse by) humans prevents their use during harvesting.
s~~s~,-ru-r~ st~~' Non-toxic non-chemical anaesthesia has also been investigated. Commonly used non-toxic alternatives such as cold anaesthesia (Mittal, A.K. and Whitear, M.
1978: A note on cold anaesthesia of poikilotherms. Journal of Fish Biology: 519-520) or carbonic acid ' anaesthesia (Post, G. 1979: Carbonic Acid Anaesthesia for Aquatic Organisms.
The S Progressive Fish Culturist 41 (3): 142-144) do induce anaesthesia but can also cause .
considerable trauma in the process. They are accordingly not appropriate for use in harvesting if the quality of the post-mortem flesh is to be maintained as near pre-mortem as is possible.
It is therefore apparent that a need exists for a readily available non-toxic anaesthetic suitable for use inter alia in the harvesting of aquatic organisms. The ideal chemical anaesthesia for harvesting would be cost-effective, have low irritant qualities and be suitable for use with animals intended for human consumption.
JP 46-23256 discloses a general class of aromatic compounds having the formula a' Ri where R1 and Rz are H, OH or an alkoxy group or R1 and R'- are the same lower alkylenedioxy group and R3 is an alkenyl group. These compounds are disclosed as being effective as sedatives/anaesthetics for fish, with some compounds of the class being toxic and others non-toxic.
Two of the compounds covered by the general formula above are 4-allyl-2-methoxyphenol ' and 4-hydroxy-3-methoxy-1-propenylbenzene which are known as eugenol and isoeugenol , respectively. These compounds are non-toxic food grade additives which are approved for human consumption. They are however known irritants (Martindale, the Extra Pharmacopoeia, 29th Edition, Pharmaceutical Press, London) which means that they could be expected to be unsuitable for use as aquatic anaesthetics during harvesting due SUBSTITUTE ~H'~~'T

WO 95117176 ~ PCT/NZ94/00148 to the tendency of irritants to induce the organisms to struggle, which in turn reduces post-mortem flesh quality.
JP 46-23256 discloses that eugenol and isoeugenol are effective as aquatic anaesthetics/sedatives at different concentrations. In particular, Table 1 of teaches that eugenol is 100% effective as a fish anaesthetic/sedative at concentrations of 25 mg 1-1 (equivalent to ppm) or above, but totally ineffective at a concentration of 12.5 mg 1-', and that isoeugenol is 100% effective at concentrations of or above 25 mg 1-1, partially effective at a concentration of 12.5 mg 1'1 and totally ineffective at a concentration of 6.25 mg 1-1.
It has now surprisingly been found by the applicants that both eugenol and isoeugenol are effective aquatic anaesthetics when employed at concentrations which are disclosed in JP 46-23256 as being totally ineffective. It has also been found that at such concentrations the compounds are substantially non-irritants. It is upon these unexpected findings that the present invention is based.
SUMMARY OF THE INVENTION
Accordingly, in a first aspect, this invention provides a method of sedating and/or anaesthetising an aquatic organism comprising the step of contacting said organism with a solution containing a compound of the formula off \
a (where R is CHCHCH3 or CH,CHCH,) in an amount of up to but less than 12.5 mg 1-'.
SUBSTITUTE SHEET

~~~r~r~~

In a further aspect, the invention provides a method of harvesting an aquatic organism while substantially retaining its pre-mortem flesh quality comprising the step of contacting said organism with a solution containing a compound of the formula aH
OCH~
B
(where R is CHCHCH3 or CH2CHCH2) in an amount of up to but less than 12.5 mg 1-1.
In yet a further aspect, the invention provides a method of transporting a live aquatic organism comprising the steps of:
sedating and/or anaesthetising said organism to be transported by contacting the organism with a solution containing a compound of the formula off a (where R is CHCHCH3 or CH2CHCH2) in an amount of up to but less than 12.5 mg 1-1; and transporting said organism while sedated and/or anaesthetised.
In each of the above methods, the compound may be either eugenol or isoeugenol.
SUBSTITUTE SHEET

In still a further aspect, the present invention provides a sedative and/or anaesthetic aqueous solution for use in sedating and/or anaesthetising an aquatic organism which includes a compound of the fornmla OH
OCH, H
(where R is CHCHCH3 or CH ZCHCHZ) in an amount of up to but less than 12.5 mg 1-' .
In a final aspect, the invention provides an active composition suitable for use as an aquatic sedative or anaesthetic which comprises, in admixture, an effective amount of a compound of the formula off OCH~
H
(where R is CHCHCH3 or (~'.HzCHCH2) and an amount of polyethylene oxide sorbitan mono-oleate as a surfactant.
According to an aspect of the invention, there is provided, a method of harvesting an aquatic organism to maintain a post-morterrr flesh quality which is substantially equivalent to its pre-mortem tlesh quality comprising the steps of:
sedating and/or anaesthetising the organism to be harvested by contacting the organism pre-mortem with a non-toxic solution containing isoeugenol or eugenol in _Ja_ an amount of up to but less than 12.5 mg 1-', the solution being free of Oil oi~ Cloves;
and slaughtering the organism while sedated and.~or anaesthetised.
According to another aspect of the invention, there is provided, the use of isoeu~enol or eugenol in the preparation of a non-toxic sedative and/or anaesthetic solution for the sedation and/or anaesthesia of aquatic organisms, wherein the solution when prepared contains isoeugenol or eugenol in an amount of up to but less than 12.5 mg 1-' and is effective to sedate and/or anaesthetise each aquatic organism it contacts, and the solution being free of Oil of ('loves.
According to another aspect of the invention, there is provided, the use of isoeugenol or eugenol in the preparation of a non-toxic solution for the transport of aquatic organisms under sedation andlor anaesthesia, wherein the solution when prepared contains isoeugenol or eugenol in an amount of up to but less than 12.5 mg l~' and is effective to maintain each aquatic organism it contacts ui a sedated andlor anaesthetised state during tr4insport, and the solution being free of Oil of Cloves.
According to another aspect of the invention, there is provided, a non-toxic sedative and anaesthetic aqueous sol~ltion for use in sedating andior anaesthetising an aquatic organism which includes isoeugenol or eugenol as the active sedative and/or anaesthetic agent, the solution containing said isoeugenol or eugenol in an amount of up to but less than 12.5 mg 1 ' and being effective to sedate and/or anaesthetise each aquatic organism it contacts, and the solution being free of Oil of Cloves.
According to a further aspect of the invention, there is provided, an active composition for use as an aquatic sedative or anaesthetic which comprises, in admixture, an effective amount of isoeugenol or eugenol and an amount of polyethylene oxide sorbitan mono-oleate as a surfactant.
BRIEF DESCRIPTION OF 'THE DRAWINCr While the present invention :is broadly defined above, it will of course be appreciated by those persons skilled in the art that it is not limited thereto but that it also includes embodiments of which the following description provides examples. In addition, the -Sb-invention will be better understood by reference to the accompanying Figure 1 which shows the response of king salmon to nominal concentrations of (a) eugenol and (b) isoeugenol.

WO 95/17176 ~ ~ ~ ~ i ~ ~ PCT/NZ94/00148 DETAILED DESCRIPTION OF THE INVENTION
As summarised above, the present invention is based upon the applicants' surprising finding that the food grade additives eugenol and isoeugenol can be utilised as aquatic sedatives and/or anaesthetics at concentrations which were previously thought to be S totally ineffective. This finding has important consequences for the aquaculture industry, in terms of both the transporting and harvesting of aquatic organisms.
The aquatic organisms to which the methods of the present invention are applied are the so-called primary aquatic organisms which are cold blooded animals living in water and respiring dissolved oxygen. The methods of the present invention are preferably applied to very valuable high grade marketable organisms from an economic point of view.
Examples of such organisms 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.
For use in the present invention, the active compounds eugenol and isoeugenol can be readily obtained from commercial sources. Alternatively, eugenol can be obtained by conventional extraction techniques from a variety of natural sources such as Oil of Cloves (Claisen, Ann, 418, 113 (1919)), and isoeugenol prepared from eugenol by heating with a caustic potash (West, J Soc Chem Ind, 59, 275 (1940)).
In the present invention, the active compound (eugenol or isoeugenol) can be used in pure form or in a mixture. Such a mixture can be a suspension or emulsion of the active compounds) in water or can be a mixture in which the active compounds) are dissolved in an appropriate alcohol such as ethanol.
SUBSTITUTE SHEET

WO 95117176 PCT/NZ94lOOt48 Alternatively, the active compound be used in the form of a composition which includes a surfactant as described below. Such a composition is preferred.
Subject always to the upper limit of 12.5 mg 1-1, the amount of active compound employed in the methods of the invention may vary, depending on whether the active compound is eugenol or isoeugenol. Where the compound employed is eugenol, it is preferred that the amount of eugenol used is from 2-12.5 mg 1'', more preferably from 3-10 mg I'1, most preferably from 6-8 mg t''.
In the alternative, where the compound employed is isoeugenol, it is preferred that the amount of isoeugenol used is from 1-10 mg 1'1, more preferably from 3-9 mg 1'1, most preferably from 5-8.5 mg 1-1.
As stated above, it is the applicants preference that the active compound form part of an active composition which includes a surfactant. For use in the methods of the invention, this surfactant could in theory be any commercially available surfactant having suitable properties but is preferably polyethylene oxide sorbitan mono-oleate. The surfactant polyethylene oxide sorbitan mono-oleate is commercially available under the trade name Polysorbate 80, with it being particularly preferred that the form of Polysorbate 80 sold as Liposorb-''0-20 (Lipochemicals, Inc., USA) be used.
The applicants have surprisingly found that the use of Liposorl~ 0-20 results in a composition having superior properties in terms of solubility in water and in terms of stability in aqueous solution over time as compared to compositions including other surfactants (such as Tweets 60, Tween*65, Tweer~ 80 and Spai'~ 80 (all ICI) and Triton X).
In view of this unexpected finding, the active composition comprising eugenol and/or isoeugenol and polyethylene oxide sorbitan mono-oleate forms yet a further aspect of the invention.
While various proportions of active compound and surfactant can be employed in forming the composition, it is presently preferred that each be included as 50% of the volume of the final composition. The most preferred composition is 50% by volume of isoeugenol * Trade-Mark $UBSTiTUTE SHEET

2~~C~~~~
_g_ (Naarden International, Bussem, The Netherlands) and 50% by volume Liposorb-0-(Lipochemicals Inc, USA).
The invention will now be illustrated with reference to the following Examples.

MATERIALS AND METHODS
Experimental animals Thirty-nine female king salmon (Oncorhynchus tshawytscha) with a mean wet weight of 365g (s.d. = 78) were used in this experiment. These animals were sampled from a tank population of 299 animals which had been reared indoors in a 28m3 oval tank from smoltification. The rearing tank was supplied with filtered seawater on a flow-through basis with a typical turnover of one tank volume per 3.5 hours. Auxiliary aeration maintained the dissolved oxygen concentration typically between 85 to 95% of saturation.
The salmon were fed ad libitum a 3mm proprietary salmon diet (NRM New Zealand Ltd, Nelson, New Zealand) at a rate up to but not exceeding 2% of body weight per day. For two months prior to the experiment, the food had been distributed into the bubble stream of an air stone positioned immediately adjacent to the tank wall. This facilitated capture and reduced the likelihood of learned net avoidance behaviour.
Animal sampling and test conditions Each animal was caught in a shallow dip net as it came to the surface adjacent to the tank wall to feed. Once caught, each animal was transferred to an insulated 700 litre tank located within 1 metre of the rearing tank. A lid was then placed on the tank for 5 minutes to allow the fish to settle slightly before the anaesthetic doses were added.
Prior to each trial, the tank was thoroughly cleaned and rinsed with fresh water. It was then filled to a depth of 490mm with 500 ~ 251 of sand-filtered 34% seawater at a temperature of 14.4 + 0.5°C. Auxiliary aeration was used to ensure dissolved oxygen saturation at the beginning of each trial. A YSI Model 57 dissolved oxygen meter was used to confirm that dissolved oxygen concentrations remained above 90%
saturation during each trial.
SUBSTITUTE SHEET

6 ~ ~ PCT/NZ94I00148 _9_ A Minolta CR-200B Chroma Meter was used to characterise the grey tank colour according to the L*a*b* International Colour System (CIE, 1976) as L* = 55.4;
a* _ -0/9 and b* =1.7. Light intensity (400 to 700nm) at the physical centre of the tank was measured using a Licor LI-1000 logger fitted with LI-192SA Underwater Quantum Sensor. The light intensity ranged between 1.2 and 2.3 ~cmol s-' tri 2 with the tank lid removed.
Dose-response trials Five fish were used in seven of the eight dose-response trials with the remaining trial using four (Figure la). For each trial eugenol (4-allyl-2-methoxyphenol) or isoeugenol (4-hydroxy-3-methoxy-1-propenylbenzene) were dissolved in 200 mls 99.7%
ethanol to aid dispersion in seawater. Five trials were undertaken for eugenol with nominal concentrations of 25.0, 12.5, 8.0, 6.3 and 3.0 t 0.5 mg 1-1 seawater. Three trials were undertaken for isoeugenol with nominal concentrations of 25.0, 8.0 and 3.0 ~
0.5 mg 1't seawater. Experimental timing was initiated from the addition of the anaesthetic-ethanol mixture. Mixing of the anaesthetic-ethanol solution and the seawater was achieved by aeration. After 15 seconds mixing, the aeration was stopped to avoid accelerated air-stripping of anaesthetics. Care was taken to disturb the fish as little as possible during the mixing procedure.
Dose-response criteria The responses of the fish to the nominal anaesthetic concentrations were judged against two criteria. The time taken from the addition of the anaesthetic dose to the point where the fish failed to avoid a hand placed in their path gave an empirical estimation of a sedative effect. Animals in this state could be removed from the water but would rouse themselves and slightly struggle if they were not returned to the water within 5 to 10 seconds. Ventilation was often exaggerated and the fish would tend to swim slightly "nose up" near the tank surface. Fish in this stage were characterised as "handleable" for the purposes of this experiment.
The time taken from the addition of the anaesthetic dose to the point where the animals were insensible to forced extension of the operculum and contact with the gill lamellae SUBSTITUTE SHEET

WO 95/17176 2 ~ PCT/NZ94/00148 gave an indication of an anaesthetic effect. In this state the fish exhibited a loss of equilibrium but weak swimming motions were often present. Ventilation was often erratic and exaggerated at this stage. Fish that had not reached this state would respond within 30 seconds of contact with the gill lamellae with a reflexive "cough".
RESULTS AND DISCUSSION
At all of the concentrations tested, the two anaesthetic formulations generated very similar patterns of behaviour when first introduced into the seawater. Some initial "coughing" was observed and was particularly pronounced with the higher concentrations indicating a transitory period of gill irritation in some of the animals.
In both anaesthetic formulations, increasing sedation was characterised by a slight increase in swimming speed, moderate ventilation and a progressive decrease in the distance at which the tank walls and water surface were perceived. At the point of "handleability", the animals were often unaware of the tank walls until contact was made.
A loud noise could elicit a transient startle response at this level of sedation.
Progression from the point of "handleability" to the loss of the "coughing"
reflex is characterised by a progressive loss of equilibrium and effective swimming motions. The fish were insensitive to loud noises and physical restraint. The erratic ventilation observed at this stage slowed and eventually ceased as anaesthesia deepened leaving the fish inert and apparently unresponsive to physical stimuli.
Where the two formulations differ is in the nominal concentration and rate at which the abolition of the "coughing" reflex occurs. Figures 1(a) and (b) illustrate this result. There appears to be no effective or statistical difference between the formulations at a nominal concentration of 25 mg 1-' (t-test, P < 0.05). At a nominal concentration of 8.0 mg 1-1, the mean time to elimination of the "coughing" reflex was approximately 2.5 times shorter for the isoeugenol formulation. At 3.0 mg 1u the eugenol formulation did not produce sufficient depth of anaesthesia to depress the "cough" reflex however the sedated, "handleable", state was easily achieved. At 3.0 mg 1-1 nominal concentration isoeugenol produced a LOSS Of equilibrium and weak ineffectual swimming motions but did not SUBSTITUTE SHEE'i' WO 95/17176 217 ~ ~ ~ ~ PCT/NZ94/00148 produce full suppression of the "cough" reflex. Eugenol produced a similar effect at a nominal concentration of 6.3 mg 1-'.
The reason for the apparent differences in activity between the two formulations is likely S to be attributable to differences in the solubility of the active ingredients. In practice, there appears to be little gained from nominal dosages above 12.5 mg 1'' for eugenol and approximately 8.0 mg 1-' for isoeugenol.
General observations in relation to recovering from the anaesthetics suggest that recovery is rapid when the fish are placed in clean, well aerated water. Typically, when anaesthetized using low to medium concentrations to the point of suppression of the "coughing" reflex, the fish will regain orientation and coordinated swimming within approximately 5 to 15 minutes. In one incidence, approximately 171 (mean weight 578g) king salmon resumed apparently normal feeding within approximately two hours of exposure to 8 mg 1-' isoeugenol for approximately 10 to 30 minutes (Jerrett, Holland and Cleaver, unpublished results).

This example reports the applicants observations when SO% by volume isoeugenol is admixed with 50% by volume of various surfactants.
ICI ~veen 60 Very viscous solution which solidifies in cold weather (below about 15°C). Gentle heating will liquefy the solid material, ie. placing the container in a water bath (40°C).
Surfactant mixes well with isoeugenol but will solidify again if mixing does not take place quickly. Generally forms the composition as a very viscous mixture. Forms a cloudy white solution in water. A white precipitate forms after 10 minutes and by 20 minutes an emulsion has formed on the bottom of the container with some formation of isoeugenol globules. Will resuspend on shaking.
JO
SUBSTITUTE SHEET

~r ICI Tween 65 Solid needing direct heating, ie. on a hot plate to liquefy. Mixes well with isoeugenol but does need to be mixed quickly to stop the surfactant solidifying. Forms the composition as a very viscous solution. Mixes with water to form a cloudy solution but does not .
S appear to mix totally. Separation takes place almost immediately. Globules formed on the sides of the bottle. Separates out into two distinct layers, one yellow (isoeugenol) and the other a white precipitate formed after two minutes. Isoeugenol globules form at the top and the bottom of the solution. Some resuspension will occur with prolonged shaking.
ICI Tween 80 Isoeugenol and Tween 80 do not mix together easily. Will eventually ( 10 rains) blend with gentle mixing. Mixes well on addition to water. After S minutes precipitate forms.
Some emulsion formed at ten minutes. Will resuspend with a good shake although some globules appear to be left on the side of the container.
ICI Span 80 Mixes well with isoeugenol but separates out immediately on addition to water.
Triton X
Solid at room temperature. When melted mixes well with isoeugenol and immediately forms a cloudy solution. Mixes well on addition to water. Separation occurs as very fine precipitate within 10 minutes. Isoeugenol globules formed after 3 hours.
Appeared after 5 hours that both the surfactant and isoeugenol had separated out.
Resuspension will occur but some globules are left on the side of the container.
Liposorb-0-20 Surfactant is less viscous and mixes relatively easily with isoeugenol. Will mix with a good shake.

Mixture goes into solution well on addition to water. No deposit is left on the side of the bottle. A fine precipitate is formed on the bottom of the container after about 15 SUBSTITUTE SHEET

WO 95117176 Q '/~ PCT/NZ94/00148 minutes but this resuspends very easily once disturbed. Three hours before any emulsion is seen on the bottom of the flask. It appeared the isoeugenol did not separate out from the surfactant even when the mixture was left overnight. Total resuspension occurred on shaking.
It can therefore be seen that Liposorb-0-20 confers superior properties upon the ultimate composition in terms of mixability, solubility in water and stability. These advantages are surprising given that Tween 80 (also a polysorbate 80) is not nearly so effective.

This example demonstrates the effect of the applicants preferred composition (50% by volume isoeugenol and 50% by volume Lipsorb-0-20) on a range of aquatic organisms.
In this example, the composition is referred to as AQUI-S and the amount of isoeugenol with which the organisms are contacted is one-half of the AQUI-S dosage.
(i) Rainbow trout (Salrno gairdneri) Dosage l7mg 1-1 AQUI-S (8.5 mg 1-1 isoeugenol) Temperature 12.5°C
Time for sedation 12 to 16 minutes Time for anaesthesia 30 to 40 minutes. More mature fish were the ones that took longer.
Recovery Similar to salmon, as reported in Example 1.
(ii) Rock Lobster (Jasus edwardsii) Dosage 17 mg 1-1 AQUI-S (8.5 mg 1-1 isoeugenol) Temperature 5.6°C
pH 8.2 Dissolved Oxygen Saturated Salinity 32.84 Reaction to Introduction No apparent awareness Time to Sedation Juvenile 8 to 10 minutes Adult 10 to 12 minutes Time to anaesthesia Juvenile 12 to 15 minutes Adult 15 to 20 minutes Recovery Equilibrium regained andwalking/swimming motions regained within 5 minutes of introduction to fresh water. Tail curl was noticeable after 15 minutes of introduction to fresh water. Lobster still remained calm with no avoidance of handling for a further SUBSTITUTE SHEET

217~'~'~~

25 minutes. At about 1 hour after being removed from the anaesthetic the aggressive behaviour was returning. Examination the next day showed full regaining of aggressive behaviour. Adults and juveniles responded in a similar fashion.
Sedation in this case is defined as the point at which equilibrium is lost and there is no aggression to being handled although the animal is definitely still aware of being handled.
Anaesthesia is defined as the point at which the lobster can be handled with no reaction from the animal. In this state, the tail fans are spread out and there is no reflexive curling in of the tail. When the animal is placed on its back the tail is fully extended.
(iii) Paua (Haliotis iris) Dosage 17 mg I-1 AQUI-S (8.5 mg 1-t isoeugenol) Temperature 5.6°C
pH 8.2 Dissolved Oxygen Saturated Salinity 32.84 Reaction to Introduction Awareness of substance but no apparent adverse reaction. ie. no avoidance.
Time to Sedation Juvenile 8 to 10 minutes Adult 13 to 20 minutes Time to anaesthesia Juvenile 12 Adult 30 minutes Recovery Appeared to be fully recovered by 6 to 10 minutes after removal from the anaesthetic.
Sedation in this case is defined as the point at which the animal is very slow to right itself after being placed on its back.
Anaesthesia is defined as the point at which the animal has released its hold on the substrate and usually fails completely to right itself after being placed on its back.
(iv) Long Finned Eels (Anb~uillea dieffenbachi) Dosage l7mg 1-1 AQUI-S (B.Smg 1'1 isoeugenol) Temperature Initial temperature 13°C
Final temperature 4°C
Dissolved oxygen Saturated Time to anaesthesia 10 to 15 minutes Recovery 10 to 12 minutes SljP~'t'~-ri ~-rc ~t-i~~T
1 1 1 V 1 ~ v~

WO 95117176 ~ ~ PCT/NZ94100148 Anaesthesia is defined as the point at which the eel lost equilibrium and turned over onto its back. No reaction to being handled.
(v) Snapper (Pagrus auratus) Dosage l2mg 1'1 AQUI-S (6mg 1-1 isoeugenol) Temperature 18°C
Dissolved oxygen 7.4 mg 1-1 Time to sedation 20 to 30 minutes Time to anaesthesia 40 to 50 minutes Recovery 10 to 20 minutes Sedation is defined as the point at which equilibrium is lost and anaesthesia is the point at which there is no reaction from the snapper upon removal from the water.
This is for juvenile snapper weighing approximately 12 grams.
(vi) Yellow Eyed Mullet (Aldriclzetta forsteri) Dosage 17 mg 1'1 AQUI-S (8.5 mg 1'1 isoeugenol) Temperature 10°C
Time to sedation 9 to 10 minutes Time to anaesthesia 14 to 15 minutes Recovery 5 minutes Flounder (Paralichthys lethostigma) and spotty's (Pseudolabrus celidotus) in the same ~
tank as the mullett appeared to respond in much the same manner within the same time frame.
Thus, in accordance with the present invention there are provided methods and compositions for sedating/anaesthetising aquatic organisms. Further, and most importantly, the active agents which are responsible for producing the sedating/anaesthetising effect are food grade additives which are non-carcinogenic (Finding of the WHO Expert Committee on Food Additives reported in Martindale, The Extra Pharmacopoeia, 29th Edition), and which are non-irritating to the organism at the concentrations employed.
Those persons skilled in the art will therefore appreciate the advantages of the present invention as well as the many applications to which the methods and compositions of the present invention can be put. As a first example, the methods and compositions can be employed in the harvesting of aquatic organisms for ultimate human consumption. This is particularly so in the case of organisms such as fish which otherwise struggle violently SUBST~TUTF SHEET

21'~~' ~2~

to avoid capture, having a major impact on the post-mortem quality of the tissue.
However, when sedated and/or anaesthetised in accordance with the present methods this struggling is at least much reduced, if not eliminated. Further, any residual concentration of eugenol or isoeugenol in the tissue of the organism following harvesting does not detract from the suitability of the flesh for human consumption.
Additionally, where the aquatic organism is a shellfish, sedation/anaesthetisation of the shellfish greatly eases the extraction of the flesh from the shell.
A further application of the sedation and/or anaesthetic methods and compositions is in the transportation of live aquatic organisms. This is once again particularly the case with fish which are to be transported live to overseas markets and where the natural undamaged appearance of the fish is critical to the market price obtained.
Still a further application of the invention is in the transport of aquatic organisms to a market where the organism is to be sold in a pre-rigor state. By "pre-rigor"
it is meant a state in which the tissue of the organism remains alive for a prolonged period following administration of the eugenol/isoeugenol but in which the organism is no longer capable of control of its musculature and from which the organism will not recover.
The organism is therefore in a state of "living death".
The advantage of transporting the organism in this pre-rigor state is that the organism need not be transported in its aquatic environment. Instead, the organism can be transported "dry", which represents a considerable reduction in expense over that associated with the transportation of live organisms in their aquatic environment.
Additionally, as the tissue of the organism remains alive until the organism has reached its market, the flesh remains "fresh" and able to command a market premium over flesh from organisms euthanised before transport.
Other applications of the present methods and compositions will be readily apparent to the skilled worker in this art.
SUBSTITUTE SHEET

WO 95/17176 ~ PC"T/NZ94/00148 In addition, the use of lesser amounts of eugenol and isoeugenol as compared to the amounts previously disclosed as being effective has significant advantages. In particular, the applicants have found that, while effective in sedating/anaesthetising the organisms, the higher concentrations of eugenol and isoeugenol disclosed in JP 46-23256, do tend to irritate the organism and cause it to exercise more vigorously until such time as the sedative/anaesthetic effect takes hold. This is undesirable in terms of the effect this activity can have on the post-harvest flesh quality of the organism.
Equally, with the higher concentrations, the risk of the flesh becoming "tainted" by the residual levels of eugenol and isoeugenol is much increased. Again, this is undesirable, particularly where the flesh is to be consumed raw.
Finally, as a matter of simple economics, it is preferable to use lesser amounts of the active compounds to reduce overheads. In a large scale aquaculture operation, this can lead to considerable savings.
It will be appreciated that the above description is provided by way of example only and that the present invention is limited only by the lawful scope of the appended claims.
SUBSTITUTE SHEET

Claims

CLAIMS:

1. A method of harvesting an aquatic organism to maintain a post-mortem flesh quality which is substantially equivalent to its pre-mortem flesh quality comprising the steps of:

sedating and/or anaesthetising the organism to be harvested by contacting the organism pre-mortem with a non-toxic solution containing isoeugenol or eugenol in an amount of up to but less than 12.5 mg l-1, the solution being free of Oil of Cloves;
and slaughtering the organism while sedated and/or anaesthetised.

2. A method according to claim 1 wherein the solution contains isogenenol.

3. A method according to claim 2 wherein said isoeugenol is present in an amount of from 1 to 10 mg l-1.

4. A method according to claim 2 wherein said isoeugenol is present in an amount of from 3 to 9 mg l-1.

5. A method according to claim 2 wherein said isoeugenol is present in an amount of from 5 to 8.5 mg l-1.

6. A method according to claim 1 wherein the solution contains eugenol.

7. A method according to claim 6 wherein said eugenol is present in an amount of at least 2 mg l-1.

8. A method according to claim 6 wherein said eugenol is present in an amount of from 3 to 10 mg l-1.

9. A method according to claim 6 wherein said eugenol is present in an amount of from 6 to 8 mg l-1.

10. A method according to any one of claims 1 to 9 wherein said solution further includes a polyethylene oxide sorbitan mono-oleate surfactant.

11. The use of isoeugenol or eugenol in the preparation of a non-toxic sedative and/or anaesthetic solution for the sedation and/or anaesthesia of aquatic organisms, wherein said solution when prepared contains isoeugenol or eugenol in an amount of up to but less than 12.5 mg l-1 and is effective to sedate and/or anaesthetise each aquatic organism it contacts, and the solution being free of Oil of Cloves.

12. The use of isoeugenol or eugenol in the preparation of a non-toxic solution for the transport of aquatic organisms under sedation and/or anaesthesia, wherein said solution when prepared contains isoeugenol or eugenol in an amount of up to but less than 12.5 mg l-1 and is effective to maintain each aquatic organism it contacts in a sedated and/or anaesthetised state during transport, and the solution being free of Oil of Cloves.

13. The use of claim 11 or claim 12 wherein said solution contains isoeugenol.

14. The use of claim 13 wherein said isoeugenol is present in an amount of from 1 to 10 mg l-1.

15. The use of claim 13 wherein said isoeugenol is present in an amount of from 3 to 9 mg l-1.

16. The use of claim 13 wherein said isoeugenol is present in an amount of from 5 to 8.5 mg l-1.

17. The use of claim 11 or claim 12 wherein said solution contains eugenol.

18. The use of claim 17 wherein said eugenol is present in an amount of at least 2 mg l-1.

19. The use of claim 17 wherein said eugenol is present in an amount of from 3 to 10 mg l-1.

20. The use of claim 17 wherein said eugenol is present in an amount of from 6 to 8 mg 1-l.

21. The use of any one of claims 11 to 20 wherein said solution further contains a polyethylene oxide sorbitan mono-oleate surfactant.

22. A non-toxic sedative and anaesthetic aqueous solution for use in sedating and/or anaesthetising an aquatic organism which includes isoeugenol or eugenol as the active sedative and/or anaesthetic agent, said solution containing said isoeugenol or eugenol in an amount of up to but less than 12.5 mg -l and being effective to sedate and/or anaesthetise each aquatic organism it contacts, and the solution being free of Oil of Cloves.

23. A solution according to claim 22 which contains isoeugenol as the active sedative and/or anaesthetic agent.

24. A solution accordind to claim 23 wherein said isoeugenol is present in an amount of from 1 to 10 mg 1-l.

25. A solution according to claim 23 wherein said isoeugenol is present in an amount of from 3 to 9 mg 1-l.

26. A solution according to claim 23 wherein said isoeugenol is present in an amount of from 5 to 8.5 mg 1-l.

27. A solution according to claim 22 which contains eugenol as the active sedative and/or anaesthetic agent.

28. A solution according to claim 27 wherein said eugenol is present in an amount of at least 2 mg 1-l.

29. A solution according to claim 27 wherein said eugenol is present in an amount of from 3 to 10 mg 1-l.

30. A solution according to claim 27 wherein said eugenol is present in an amount of from 6 to 8 mg 1-1.

31. A solution according to any one of claims 22 to 30 which further includes a polyethylene oxide sorbitan mono-oleate surfactant.

32. An active composition for use as an aquatic sedative or anaesthetic which comprises, in admixture, an effective amount of isoeugenol or eugenol and an amount of polyethylene oxide sorbitan mono-oleate as a surfactant.

33. A composition according to claim 32 wherein the surfactant is Liposorb-0-20.

34. A composition according to claim 32 in which said surfactant is 50% by volume of the total composition.

35. A composition according to claim 34 which is 50% by volume isoeugenol and 50% by volume Liposorb-0-20.