NZ244531A - Anaesthetising, sedating or euthanising marine animals using a solution of eugenol or isoeugenol - Google Patents

Description

NEW ZEALAND PATENTS ACT, 1953 No.: 244531 Date: 28 September 1992 COMPLETE SPECIFICATION METHODS FOR SEDATING, ANAESTHETISING OR EUTHANISING AQUATIC ORGANISMS We, NEW ZEALAND INSTITUTE FOR CROP & FOOD a New Zealand company, Canterbury Agriculture and Science Lincoln, Canterbury, New Zealand hereby declare the invention for which we pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following statement:- (followed by page 1A) 24 4 5 3 This invention relates to methods for sedating, anaesthetising or euthanising aquatic organisms.

The practice of catching fish or other aquatic organisms usually involves the organisms undergoing some stress. The organisms usually associate capture with predation and therefore struggle to escape immobilisation. This struggle can have a major impact on the post-mortem physical condition of the organism, j In aquaculture, the cultured organisms are usually individually handled during their life cycle. With excitable fish species, such as chinook (king) salmon (Oncorhynchus tshawytscha), great care must be taken to ensure that the animals are not bruised, scaled or in any other 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.

Chemical anaesthetics such as MS-222, benzocaine and phenoxyethanol and more recently etomidate and metomidate (Kreiberg, H, Bulletin of the Aquaculture Association of Canada, 92-3: 52-54 (1992)) are often used during the routine handling of cultured organisms. These anaesthetics cannot be used during harvesting due to their relative residual toxicity to the consumer. This restriction applies to the application of the majority of analgesic, sedative and anaesthetic chemical agents available.

Moreover, were it not for food safety considerations, cost, operator safety and potential abuse would eliminate many analgesic, sedative and anaesthetic chemicals from everyday use in harvesting operations.

Harvesting methods which are currently in use include: cold anaesthesia (Mittal AK, J Fish Bio, 519-520 (1978)); carbonic acid anaesthesia (Post G, The Progressive Fish-Cultuiist, 41,142-144 (1979)); nitrous oxide/carbonic acid anaesthesia (see Nakagawa et al., US 4,807,615); galvonarcosis (Cuny et al.,JFish Res Board Can, 35,1297-1302,1978) and anoxia/exhaustion/carbonic acid anaesthesia/cold anaesthetic combinations. Each of these methods has disadvantages in practice.

Cold anaesthesia generally requires the organisms to be removed (struggling) from the water and transported to the low temperature water bath. This method generally involves some handling damage and frequently results in severe struggling of the organisms. Graded or gradual temperature change is often considered impractical when processing large numbers of organisms on remote farm sites.

Carbonic acid anaesthesia generally results in severe struggling prior to anaesthesia taking hold. The advantage of this method is that the organisms can be contained for anaesthesia without removal from the water. 244531 Nitrous oxide/carbon dioxide anaesthesia is reputed to overcome the frantic struggling of the organisms which occurs when using carbon dioxide alone. In our experience, the severity of the struggle is reduced but not eliminated and may vary considerably with the species of the organism.

Galvonarcosis is difficult to use in sea water due to the high current densities required to produce the required electric current in the organism. This method works well in fresh water but is subject to misuse in its application by the use of excessive voltage gradients and excessive exposures. There is also a significant risk to the operators using this method.

Anoxia/carbonic acid anaesthesia/cold anaesthesia and exhaustion are used separately or in combination. These methods essentially suffocate the organisms. This is inhumane and generally results in poor quality tissue.

The inability to handle aquatic organisms without risking damage to physical appearance and subsequent post-mortem muscle quality is therefore a significant problem.

It is accordingly an object of the present invention to provide methods of handling aquatic organisms which go some way towards overcoming the above disadvantages or at least offer the public a useful choice.

Accordingly, in a first aspect the 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 OH K (wherein R is CHCHCH3 or CH2CHCH2) in an amount sufficient to sedate or anaesthetise said organism. 24 45 31 In a further aspect, the invention provides a method of euthanising an aquatic organism comprising the step of contacting said organism with a solution containing a compound of the formula OH OGH. (wherein R is CHCHCH3 or CH2CHCH2) in an amount sufficient to euthanise said organism.

In still a further aspect, the invention provides a method of harvesting an aquatic organism to substantially retain the pre-mortem flesh quality of said organism comprising the step of contacting said organism with a solution containing a compound of the formula OH OGH, (wherein R is CHCHCH3 or CHjCHCHj) in an amount sufficient to sedate, anaesthetise or euthanise said organism.

In yet a further aspect, the invention provides a method of transporting a live aquatic organism comprising the steps of: -4 24 45 3 (a) sedating and/or anaesthetising said organism by contacting the organism with a solution containing a compound of the formula OH OCH, (wherein R is CHCHCH3 or CH^CHCHj) in an amount sufficient to sedate or anaesthetise said organism as required; and (b) transporting said organism while sedated or anaesthetised.

In a further aspect, the invention provides a method of transporting an aquatic organism comprising the steps of: (a) contacting said organism with a solution containing a compound of the formula OH (wherein R is CHCHCH3 or CH2CHCH2) in an amount sufficient to induce a pre-rigor state in said organism; and (b) transporting said organism while in said pre-rigor state. r 244 531 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 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.

The formula given above covers two compounds, eugenol and isoeugenol. Eugenol (4-allyl-2-methoxyphenol) is a well known commercially available compound first prepared in 1919. Over the years, it has been found to be useful in perfumery, in the manufacture of vanillin, as an insect attractant and as a dental antiseptic and analgesic.

Eugenol is a colourless or pale yellow liquid which can be obtained by conventional extraction techniques from a variety of natural sources, including Oil of Cloves, Oil Anise, Oil of Bay and Oil of Camphor (Claisen, Ann, 418, 113 (1919)). It is disclosed in the Merck Index (Eleventh Edition) as being practically insoluble in water but soluble inter alia in alcohols.

Isoeugenol (4-hydroxy-3-methoxy-1 -propenylbenzene) is also well known. Its primary commercial use has been in the manufacture of vanillin.

Isoeugenol is an oily colourless or yellow liquid which can again be obtained by convention extraction techniques from natural sources such as ylang-ylang. Alternatively, it can be obtained from eugenol by heating with caustic potash (West, J. Soc Chem Ind, 59, 275 (1940)). It is disclosed in the Merck Index as being slightly soluble in water and miscible in alcohols.

It has now surprisingly been found by the applicants that despite their relative insolubility, it is possible to add eugenol and isoeugenol to water containing an aquatic organism and to have a graduated sedative, anaesthetic and finally euthanistic effect on N.Z. PATENT OFFICE - MAR 1996 RECEIVED the organism. It is this unexpected finding by the applicants upon which the present invention is based.

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 veiy 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.

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 compound(s) in water or can be a mixture in which the active compound(s) are dissolved in an appropriate alcohol such as ethanol.

The amount of active compound (eugenol or isoeugenol) it is necessary to add to water in practising the invention will of course depend upon the number, size and species of organism; upon the volume of water to which the compound is to be added; upon the desired effect which is intended to be induced in the organism; and upon the timeframe in which the effect is to be induced.

By way of illustration, where the compound employed is eugenol and a sedative/anaesthetic effect is desired, it is preferred that the amount of eugenol used is from 2-25 mg l"1, more preferably from 3-15 mg l"1, still more preferably from 6-12.5 mg l"1, most preferably about 12 mg l"1.

In the alternative, where the compound employed is isoeugenol and a sedative/anaesthetic effect is once again desired, it is preferred that the amount of isoeugenol used is from 1-25 mg l"1, more preferably from 3-125 mg l"1, still more preferably from 6 to 10 mg l"1, and most preferably about 8 mg l"1.

The use of eugenol and isoeugenol as aquatic sedatives/anaesthetics according to this invention will row be illustrated with reference to the following experiment 24 4 5 EXPERIMENT MATERIALS AND METHODS Experimental animals Thirty-nine female king salmon (Oncorhyrtchus tshawytscha) with a mean wet weight of 365g (s.& = 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.

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 Iicor LI-1000 logger fitted with LI-192SA Underwater Quantum Sensor. The light intensity ranged between 12 and 23 /*mol s'1 m"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 2 4 45 31 isoeugenol (4-hydroxy-3-methoxy-l-proenylbenzene) were dissolved in 200 mis 99.7% ethanol to aid dispersion in seawater. Five trials were undertaken for eugenol with nominal concentrations of 25.0,12.5,8.0,63 and 3.0 ± 0.5 mg l"1 seawater. Three trials were undertaken for isoeugenol with nominal concentrations of 25.0, 8.0 and 3.0 ± 0.5 mg l"1 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 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 veiy 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. -9-. 24 4 5 3 1 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 l'1 (t-test, P<0.05). At a nominal concentration of 8.0 mg l"1, the mean time to elimination of the "coughing" reflex was approximately 2.5 times shorter for the isoeugenol formulation. At 3.0 mg l'1 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 l"1 nominal concentration isoeugenol produced a loss of equilibrium and weak ineffectual swimming motions but did not produce full suppression of the "cough" reflex. Eugenol produced a similar effect at a nominal concentration of 63 mg l"1.

The apparent difference in activity between the two formulations is likely to be attributable to differences in the solubility of the active ingredients. In practice, there appears to be little gained from nominal dosages above 125 mg l'1 for eugenol and approximately 8.0 mg l"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 resinned apparently normal feeding within approximately two hours of exposure to 8 mg l"1 isoeugenol for approximately 10 to 30 minutes (Jerrett, Holland and Cleaver, unpublished results).

The above experiment illustrates the sedative/anaesthetic application of the compounds in practicing the invention. Turning now to the application of the active compounds (eugenol and isoeugenol) in euthanising aquatic organisms, this can be generally achieved by contacting the organism with a solution containing a higher concentration of the active compound than is used in the sedating/anaesthetising applications. However, once again, the actual amount of each compound required to induce the effect will vary depending upon the type, size and weight of the organism and 24 4 5 2 - 10 - . upon the length of time the organism remains in contact with the solution containing the active compound.

By way of illustration, where the organism is a fish (such as a king salmon) and is to be euthanised in a commercially appropriate timeframe (a matter of minutes only), the animal will be contacted with a solution containing the active compound in an amount of at least 50 mg l"1. Unreported observations by the applicants have shown that a concentration of the active compound in the range above successfully euthanised salmon fry within 2 minutes.

Similar physiological effects have also been noted for other aquatic organisms including cockles, mussels, oysters, star fish, polychaete worms, chitons, limpets and crabs. It is therefore believed that eugenol and isoeugenol are effective over the wide range of phylogentically diverse aquatic organisms previously identified.

Thus, in accordance with the present invention there are provided methods for selectively sedating, anaesthetising and euthanising aquatic organisms. Further, and most importantly, the active agents which are responsible for producing the sedating, anaesthetising or euthanising effects 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).

Those persons skilled in the art will therefore appreciate the advantages of the present invention as well as the many commercial applications which the methods of the present invention have. As a first example, the methods 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 to avoid capture, having a major impact on the post-mortem quality of the tissue. However, when sedated or anaesthetised in accordance with the present methods this struggling is at least much reduced, if not eliminated. Further, any residual concentration of the active compound 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 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 the aquatic organisms to a market where the organism is to be sold in a pre-rigor state. By "pre- 11-. tl H ^ v) j rigor" it is meant a state in which the tissue of the organism remains alive for a prolonged period following initial contact with the solution containing the active compound 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".

This pre-rigor state can be induced using a solution containing an amount of the active compound sufficient to anaesthetise the organism but which is less than is required to immediately euthanise the organism. This amount will once again be readily determinable by the skilled worker having regard to the variables discussed previously.

The advantage of transporting the organism ir, 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 will be readily apparent to the skilled worker in this art While the above represent general applications of the invention in which a wide range of concentrations of active compound can be employed, the use of the preferred lower amounts (less than 12.5 mg l"1 of eugenol and isoeugenol as sedatives/anaesthetics) has significant advantages. In particular, the applicants have found that, while effective in sedating/anaesthetising the organisms, higher concentrations of eugenol and isoeugenol do tend to irritate the organism and cause it to exercise more vigorously until such time as the sedative/anaesthetic effect takes hold This may be 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 increased. Again, this is less than desirable, 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 and to ease environmental concerns. In a large scale aquaculture operation, this can lead to considerable savings.

Finally, 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. 244531

Claims (15)

WHAT WE CLAIM IS:

1. 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 OCH. (wherein R is CHCHCH3 or CHjCHCH^ in an amount sufficient to sedate or anaesthetise said organism.

2. A method as claimed in claim 1 wherein said compound is eugenol (4-allyl-2-methoxyphenol).

3. A method as claimed in claim 1 wherein said compound is isoeugenol (4-hydroxy-3-methoxy- 1-propenylbenzene).

4. A method of euthanising an aquatic organism comprising the step of contacting said organism with a solution containing a compound of the formula OH OCH, [wherein R is CHCHCH3 or CHjCHCHj) in an amount sufficient to euthanise said organism.

5. A method as claimed in claim 4 wherein said compound is eugenol (4-allyl-2-methoxyphenol).

6. A method as claimed in claim 4 wherein said compound is isoeugenol (4-hydroxy-3 -methoxy-1 -propenylbenzene). 24 4 531 - 13-

7. A method of harvesting an aquatic organism to substantially retain the pre-mortem flesh quality of said organism comprising the step of contacting said organism with a solution containing a compound of the formula OH OCH, (wherein R is CHCHCH3 or CHjCHCHj) in an amount sufficient to sedate, anaesthetise or euthanise said organism.

8. A method as claimed in claim 7 wherein said compound is eugenol (4-allyl-2-methoxyphenol).

9. A method as claimed in claim 7 wherein said compound is isoeugenol (4-hydroxy-3-methoxy-l-propenylbenzene). of:

10. A method of transporting a live aquatic organism comprising the steps (a) sedating and/or anaesthetising said organism by contacting the organism with a solution containing a compound of the formula OH OCH, (wherein R is CHCHCH3 or CHjCHCHa) in an amount sufficient to sedate or anaesthetise said organism as required; and (b) transporting said organism while sedated or anaesthetised.

11. A method as claimed in claim 10 wherein said compound is eugenol (4-allyl-2-methoxyphenol). patent N.z. 5 - MAR 1996 244531 - 14-

12. A method as claimed in claim 10 wherein said compound is isoeugenol (4-hydroxy-3-methoxy-1 -propenylbenzene).

13. A method of transporting an aquatic organism comprising the steps of: (a) contacting said organism with a solution containing a compound of the R (wherein R is CHCHCH3 or CHjCHCHj) in an amount sufficient to induce a pre-rigor state in said organism; and (b) transporting said organism while in said pre-rigor state.

14. A method as claimed in claim 13 wherein said compound is eugenol (4-allyl-2-methoxyphenol).

15. A method as claimed in claim 13 wherein said compound is isoeugenol (4-hydroxy-3-methoxy-l-propenylbenzene). formula OH By the authorised agents A » PARK & SON N.Z. PATENT C. 5 - MAR 1996 RECEIVED