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Title:
COMPOSITIONS AND METHODS FOR SEDATING, ANAESTHETISING AND EUTHANASING AQUATIC ORGANISMS
Document Type and Number:
WIPO Patent Application WO/2002/038145
Kind Code:
A1
Abstract:
The invention relates to compositions for use in sedating, anaesthetising or euthanasing aquatic organisms, comprising: an effective amount of isoeugenol or a salt thereof, wherein the isoeugenol or salt thereof is present as the trans isomer substantially free of the cis isomer, and one or more components chosen from surfactants, solvents and other excipients. Also provided are methods of sedating, anaesthetising or euthanasing aquatic organisms, comprising contacting the organisms with an effective amount of such a composition.

Inventors:
BELL DONALD JOHN (NZ)
JERRETT ALISTAIR RENFREW (NZ)
Application Number:
PCT/NZ2001/000249
Publication Date:
May 16, 2002
Filing Date:
November 07, 2001
Export Citation:
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Assignee:
AQUI S NZ LTD (NZ)
BELL DONALD JOHN (NZ)
JERRETT ALISTAIR RENFREW (NZ)
International Classes:
A22B3/08; A61K31/085; A61P23/00; (IPC1-7): A61K31/05; A61P23/00
Domestic Patent References:
WO1996027377A11996-09-12
WO1995017176A11995-06-29
WO1998054958A11998-12-10
Foreign References:
NZ244531A1996-05-28
Attorney, Agent or Firm:
Calhoun, Douglas C. (P.O. Box 949 Wellington 6015, NZ)
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Claims:
CLAIMS
1. An active composition for use in sedating, anaesthetising or euthanasing aquatic organisms, comprising: an effective amount of isoeugenol or a salt thereof, wherein the isoeugenol or salt thereof is present as the trans isomer substantially free of the cis isomer, and one or more components chosen from surfactants, solvents and other excipients.
2. An active composition as claimed in claim 1, wherein the isoeugenol or salt thereof comprises 99% by weight or more of the trans isomer of isoeugenol.
3. An active composition as claimed in claim 1 or 2, wherein the isoeugenol or salt thereof comprises 99.5% by weight or more of the trans isomer of isoeugenol.
4. An active composition as claimed in any one of claims 1 to 3, wherein the composition includes the surfactant polysorbate 80.
5. An active composition as claimed in any one of claims 1 to 4, wherein the composition includes PEG 400.
6. An active composition as claimed in any one of claims 1 to 5, wherein the composition comprises: about 50% by weight isoeugenol, wherein the isoeugenol contains greater than 99% by weight of the trans isomer; about 40% by weight Polysorbate 80; and about 10% by weight PEG 400.
7. 6 An active composition according to any one of claims 1 to 4, wherein the composition comprises: about 50% by weight isoeugenol, wherein the isoeugenol contains greater than 99% by weight of the trans isomer ; and about 50% by weight Polysorbate 80.
8. A method of sedating, anaesthetising or euthanasing an aquatic organism, comprising the step of contacting the organism with a composition comprising an effective amount of isoeugenol or a salt thereof, wherein the isoeugenol or salt thereof is present as the trans isomer substantially free of the cis isomer.
9. A method of harvesting an aquatic organism comprising the steps of : contacting the organism to be harvested with a composition comprising an effective amount of isoeugenol or a salt thereof, wherein the isoeugenol or salt thereof is present as the trans isomer substantially free of the cis isomer, in order to induce a sedated, anaesthetised or euthanased state in said organism; and harvesting the organism while in said sedated, anaesthetised or euthanased state.
10. A method of transporting an aquatic organism in a live or prerigor state comprising the steps of : contacting the organism to be transported with a composition comprising an effective amount of isoeugenol or a salt thereof, wherein the isoeugenol or salt thereof is present as the trans isomer substantially free of the cis isomer, in order to induce a sedated, anaesthetised or prerigor state in said organism; and transporting the organism while in said sedated, anaesthetised or prerigor state.
11. A method as claimed in any one of claims 8 to 10 wherein the isoeugenol or salt thereof comprises 99% by weight or more of the trans isomer of isoeugenol.
12. A method as claimed in any one of claims 8 to 10 wherein the isoeugenol or salt thereof comprises 99.5% by weight or more of the trans isomer of isoeugenol.
13. A method as claimed in any one of claims 8 to 10 wherein the active composition comprises: about 50% by weight isoeugenol, wherein the isoeugenol contains greater than 99% by weight of the trans isomer; about 40% by weight Polysorbate 80; and about 10% by weight PEG 400.
14. A method as claimed in any one of claims 8 to 10 wherein the active composition comprises: about 50% by weight isoeugenol, wherein the isoeugenol contains greater than 99% by weight of the trans isomer; and about 50% by weight Polysorbate 80.
15. The use of isoeugenol or a salt thereof, wherein the isoeugenol or salt thereof is present as the trans isomer substantially free of the cis isomer, in the preparation of a composition for sedating, anaesthetising or euthanasing aquatic organisms.
Description:
COMPOSITIONS AND METHODS FOR SEDATING, ANAESTHETISING AND EUTHANASING AQUATIC ORGANISMS FIELD OF THE INVENTION This invention relates to compositions for sedating, anaesthetising and/or euthanasing aquatic organisms and to methods of using such compositions.

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 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 ; and Jerrett, A. R.; Stevens, J.; Holland, A. J. 1996: Tensile properties of rested and exhausted chinook salmon (Oncorhynchus tshawytscha) white muscle. (Journal of Food Science (USA) Vol. 61, No. 3,527-532).

In aquaculture, the culture organisms are usually individually handled during their life cycle. With excitable fish species such as chinook salmon, 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 Aquacultural 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.

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 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.

The applicants'earlier investigations have identified a number of phenolic compounds which are effective aquatic sedatives and anaesthetics and which are classified as food grade. Two of these are the compounds eugenol and isoeugenol (see for example, WO 95/17176 and WO 98/54958). The compound isoeugenol is available commercially as a mixture of its two isomers, ie the cis and trans isomers, and, prior to the present invention, has been used in this form as an aquatic sedative j anaesthetic.

The applicants have now unexpectedly found that the trans isomer of isoeugenol possesses characteristics which confer on it advantages over isoeugenol containing the cis isomer, when used as an aquatic sedative/anaesthetic. It is upon this unexpected finding that the present invention is based.

SUMMARY OF THE INVENTION Accordingly, in a first aspect the present invention provides an active composition for use in sedating, anaesthetising or euthanasing aquatic organisms, comprising: an effective amount of isoeugenol or a salt thereof, wherein the isoeugenol or salt thereof is present as the trans isomer substantially free of the cis isomer, and

one or more components chosen from surfactants, solvents and other excipients.

As used herein,"substantially free of the cis isomer"means that the isoeugenol contains no more than 2% by weight of the cis isomer. Preferably, the isoeugenol contains no more than 1% by weight of the cis isomer, more preferably, no more than 0.5% by weight of the cis isomer.

Preferably, the surfactant is a Polysorbate surfactant, more preferably a Polysorbate 80.

Preferably, the composition further includes a polyethylene glycol (PEG), more preferably PEG 400.

In a preferred embodiment, the composition contains: about 50% by weight isoeugenol, wherein the isoeugenol contains greater than 99% by weight of the trans isomer; about 40% by weight Polysorbate 80; and about 10% by weight PEG 400.

In another preferred embodiment, the composition contains: about 50% by weight isoeugenol, wherein the isoeugenol contains greater than 99% by weight of the trans isomer; and about 50% by weight Polysorbate 80.

In a further aspect, the invention provides a method of sedating, anaesthetising or euthanasing an aquatic organism comprising the step of contacting the organism with a composition comprising an effective amount of isoeugenol or a salt thereof, wherein the isoeugenol or salt thereof is present as the trans isomer substantially free of the cis isomer.

In still a further aspect, the invention provides a method of harvesting an aquatic organism which comprises the steps of :

contacting the organism to be harvested with a composition as defined above in order to induce a sedated, anaesthetised or euthanased state in said organism; and harvesting the organism while in said sedated, anaesthetised or euthanased state.

In still a further aspect, the invention provides a method of transporting an aquatic organism in a live or pre-rigor state comprising the steps of : contacting the organism to be transported with a composition as defined above in order to induce a sedated, anaesthetised or pre-rigor state in said organism ; and transporting the organism while in said sedated, anaesthetised or pre- rigor state.

In a still further aspect, the invention relates to the use of isoeugenol or a salt thereof, wherein the isoeugenol or salt thereof is present as the trans isomer substantially free of the cis isomer, in the preparation of a composition for sedating, anaesthetising or euthanasing aquatic organisms.

While the invention is broadly as defined above, it also includes embodiments of which the following description provides examples.

BRIEF DESCRIPTION OF THE DRAWINGS While the invention is broadly as defined above, it also includes embodiments of which the following description provides examples.

In particular, the present invention will be better understood with reference to the accompanying drawings, in which: Figure 1 shows the time taken for Chinook salmon to (a) become handleable, (b) reach anaesthesia, and (c) recover, when exposed to formulations containing various concentrations of active ingredients of AQUI-STM (isoeugenol) (o), trans-

isoeugenol (A) and cis-isoeugenol (Cl). Means + SEM (n=7 unless otherwise stated). ** Significantly different to the other treatments at the same concentration (P < 0.05, Student's t-test); Figure 2 shows the time period between Chinook salmon becoming handleable and reaching anaesthesia for AQUI-STM (isoeugenol), TRANS (trans-isoeugenol) and CIS (cis-isoeugenol) formulations; Figure 3 shows the margin of safety at various concentrations for QUI-STEM (isoeugenol) (0), TRANS (trans-isoeugenol) (A) and CIS (cis-isoeguenol) ( formulations. The margin of safety equals time for first fish to reach anaesthesia minus the average time to sedation for the relevant anaesthetic concentration; Figure 4 shows a comparison of the effect of exposure to 65 mg/L TRANS and CIS anaesthetic formulations on heart function in chinook salmon. Each bar consists of six replicates (means + SEM). The heart rate raw data reduced to 1 sample/sec (from 40 samples/sec) and was smoothed using a running average (5 min) to determine the point at which the heart rate dropped below 20 bpm. Start and end of arrhythmia were determined from the reduced data. Heart rates 5 min after exposure were 91.8 3.8 (TRANS) and 92.4 2.7 (CIS) beats/min.

Water temperature ranged from 15.1 to 17.4°C during the experiment; Figure 5 is a photostability comparison plot showing the rates of formation of degradation product for trans-isoeugenol and isoeugenol (mixture of cis and trans isoeugenol), as described in Example 3; Figures 6 and 7, respectively, illustrate the changes in composition of trans isoeugenol and isoeugenol (mixture of cis-and trans-isoeugenol) when not exposed to light (left hand bars) and when exposed to light (right hand bars), again as described in Example 3; and Figure 8 is a photograph of the samples of trans-isoeugenol and isoeugenol at the end of the study described in Example 3.

DETAILED DESCRIPTION OF THE INVENTION As defined above, the invention primarily relates to active compositions suitable for use as aquatic sedatives, anaesthetics and euthanasing agents. These active compositions contain isoeugenol.

Isoeugenol can exist as two geometric isomers, cis and trans, because of the presence of the double bond in the side chain. The cis and trans isomers of isoeugenol are shown below. trans-isomer cis-isomer Isoeugenol is commercially available in a form which contains approximately 88% by weight of the trans isomer and 12% by weight of the cis isomer. This is the form in which isoeugenol is present in the commercially available aquatic anaesthetic known as AQUI-STM.

Previously, the contribution (if any) of each isomer to the activity of the mixture as an aquatic sedative and anaesthetic had not been assessed. It was therefore not known whether it was one isomer or both which is or are responsible for the sedative/anaesthetic effect. However, as the trans isomer is proportionally strongly dominant in the commercial form of isoeugenol, the assumption has been that the trans isomer is the"active"component. This led the applicants to predict that the pure trans isomer, no longer being diluted by the cis isomer, would give a faster anaesthetic action than a mixture of the isomers. They

viewed such a faster anaesthetic action as undesirable in a number of aquatic applications, as it would in practice reduce the time period between the organism becoming handleable without stress and becoming anaesthetised, which in turn increases the risk of overdose and death of the organism through hypoxia. Upon investigation, however, and to the applicants'surprise, this was not the case.

In particular, what the applicants have unexpectedly found is that exposure of fish to the trans isomer of isoeugenol resulted in a long, slow, anaesthetisation process, thus providing a large safety margin between fish becoming handleable and reaching anaesthesia. In contrast, the cis isomer was found to anaesthetise the fish more quickly, reducing the"window"of safe dosages compared with the trans isomer, and thereby increasing the risk of overdose and death through hypoxia (ventilatory collapse).

Accordingly, the applicants have therefore surprisingly found that when the trans isomer of isoeugenol substantially free of the cis isomer is used, an aquatic sedative/anaesthetic composition can be provided which possesses advantages over compositions which contain the cis isomer of isoeugenol in addition to or instead of the trans isomer. It is these compositions which are the focus of this invention.

The active agent for inclusion in the composition is the trans isomer of isoeugenol, or a salt thereof such as the sodium salt, provided substantially free of the cis isomer, ie with 2% or less of the cis isomer present. It is however preferred that the trans isomer is provided in a form which is greater than 99% pure, ie with less than 1% of the cis isomer present and more preferably greater than 99.5% pure.

The presence of the cis isomer may be detected using either nuclear magnetic resonance (NMR) spectroscopy or gas liquid chromatography (GLC). Proton NMR is a preferred method for detecting the presence of the cis isomer.

Isoeugenol in the form of the trans isomer substantially free of the cis isomer may be obtained from a mixture of the cis and trans isomers using methods known in the art, for example as described by Boedecker, Fr. And Volk, H., Ber., 64B, 61 (1931), and by Bruckner-Wilhelms, A. Ann. Univ. Sci. Budapest, Rolando

Botvos Nominate, Sect. Chim., 13, pp 101-5 (1972); and in Chemical Abstracts 80, 95415C (1974). These methods involve separating out the trans isomer as its sodium salt, acidifying, dissolving the product in solvent and distilling to obtain the pure isomer.

The compositions of the invention, in addition to the active agent, contain one or more surfactants, solvents or other excipients.

Where as is preferred the compositions contain a surfactant, the surfactant may be chosen from those available in the art which are suitable for combination with isoeugenol. It is however preferred that the surfactant be a Polysorbate surfactant, with Polysorbate 80 surfactants being particularly preferred.

It is also particularly preferred that the composition comprises a liquefiable macrogol, preferably a liquid macrogol. It is preferred that the macrogol be a polyethylene glycol (PEG). A preferred PEG is PEG 400.

The proportions in which the above components are included in the composition can vary. In one preferred embodiment, an active composition of the present invention includes 50% by weight of the isoeugenol (in the form of the trans isomer substantially free of the cis isomer), 40% by weight of the surfactant Polysorbate 80 and 10% by weight of PEG 400.

In another preferred embodiment, an active composition of the present invention includes 50% by weight of the isoeugenol (as the trans isomer substantially free of the cis isomer), and 50% by weight of the surfactant Polysorbate 80.

In alternative embodiments, the compositions of the invention may comprise isoeugenol (as the trans isomer substantially free of the cis isomer) in combination with a suitable organic solvent, such as ethanol.

In still further embodiments of the invention, the compositions may comprise isoeugenol (as the trans isomer substantially free of the cis isomer) in the form of a suitable salt, conveniently the sodium salt, in an aqueous solution buffered to a pH of less than 7 (for example about 6.5).

The aquatic organisms to which the methods of the present invention may be 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.

The methods of the present invention of sedating, anaesthetising or euthanasing aquatic organisms may be carried out by contacting the organism with a sedative/anaesthetic composition as described above comprising as the active compound the trans isomer of isoeugenol substantially free of the cis isomer.

Conveniently, this may be achieved by adding the active composition to the water in which the aquatic organism is living, to give a concentration of the active compound of the desired level. Alternatively, depending on the species of aquatic organism to which the method is to be applied, it may be practical to inject the aquatic organism with the active composition.

The amount of the active compound employed in the methods of the invention may vary, depending on whether the organism is to be sedated, anaesthetised or euthanased. It will be appreciated that higher concentrations will normally be used where a deep anaesthetic or euthanasing effect is desired to be achieved quickly. The amount of the active compound employed may also vary according to the species being treated, the age of the organism, body weight and the like. It will also be understood that a progressive sedative to anaesthetic to euthanasing effect can be induced by altering the time the organism is in contact with the active compound.

However, by way of example, a concentration of the active compound in the range of about 5 mg/L (or even lower) to about 25 mg/L will generally be suitable for

treating salmon, with the preferred concentration being dependent on the desired effect.

The invention will now be illustrated with reference to the following non-limiting examples.

EXAMPLES EXAMPLE 1 Raw Materials The cis and trans isomers of isoeugenol were prepared by Industrial Research Ltd. Isoeugenol was supplied by AQUI-S New Zealand Ltd, Lower Hutt, New Zealand (Lot No 96150029). Trans-isoeugenol was separated as the sodium salt, acidified and then extracted with ethyl acetate, substantially according to the method described by Boedecker Fr and Volk, H., Ber., 64B, 61 (1931). The solvent was dried over magnesium sulphate and then evaporated to yield a 99% + pure trans-isoeugenol. The cis-isomer was recovered from the fraction that was not separated as the trans salt. This fraction was a mixture of approximately 66% cis and 34% trans-isoeugenol. It was extracted into ethyl acetate and recovered by evaporation. The trans-isoeugenol sample contained trace quantities (by NMR) of ethyl acetate. The cis-isoeugenol sample contained approximately 12% ethyl acetate (by NMR) that could not be removed by evaporation.

Animals Thirty female guppies (Poecillia reticulata) were used in the study. The efficacy trials were carried out at the Seafood Laboratory, Crop zu Food Research Ltd, Nelson. Animals were monitored for sedation (failure to avoid obstacles), anaesthesia (the point at which fish could be held out of the water for 30 seconds without any movement), and recovery (up-right swimming and avoiding obstacles). Five animals were treated at each condition. Animals were withheld from treatment for a period of 7 days following their first treatment.

Anaesthetic Formulations Six anaesthetic formulations were evaluated as follows: 1. QUI-STEM (batch no: 9708585): the standard formulation containing 50% isoeugenol and 50% polysorbate 80.

2. AQUI-S PLUS : the standard plus formulation containing 50% isoeugenol, 40% polysorbate 80 and 10% PEG 400.

3. AQUI-S TRANS : 50% trans-isoeugenol and 50% polysorbate 80.

4. AQUI-S CIS : 50%"cis-isoeugenol"and 50% polysorbate 80. Actual formulation 29% cis-isoeugenol, 15% trans-isoeugenol, 6% ethyl acetate and 50% polysorbate 80.

5. AQUI-S PLUS TRANS: 50% trans-isoeugenol, 40% polysorbate 80 and 10% PEG 400.

6. AQUI-S PLUS CIS: 50%"cis-isoeugenol", 40% polysorbate 80 and 10% PEG 400. Actual formulation 29% cis-isoeugenol, 15% trans-isoeugenol, 6% ethyl acetate, 40% polysorbate 80 and 10% PEG 400.

Results Initial trials were carried out at formulation concentrations of 20 and 40 mg/L to establish the expected response with AQUI-S. This indicated that the response of guppy to the anaesthetic is slow compared to salmonids. Fish treated with 20 mg/L QUI-STEM did not reach anaesthesia after 60 minutes. The average time to anaesthesia for guppy treated with 40 mg/L of AQUI-S was 23 minutes compared to approximately 4 minutes for the treatment of salmonids. Time to anaesthesia for treatment at 20 mg/L QUI-STEM was approximately 60 minutes.

It was noted that the AQUI-S CIS formulation did not disperse readily in freshwater and produced a cloudy dispersion at the treatment concentrations of 20 and 40 mg/L.

Following the initial trials a series of trials was carried out for each formulation at a treatment concentration of 40 mg/L. The average results are summarised in Table 2.

Table 2: Response of Guppy to treatment with different anaesthetic formulations Formulation Time to Time to Time to sedation, anaesthesia, Recover, Minutes Minutes Minutes AQUI-STM 4. 3 (sd=1. 5) 22.6 (sd = 10.9) 5.0 (sd = 1.7) AQUI-Sw TRANS 8. 5 (sd = 3.5) 38.5 (sd = 18.3) 10.9 (sd = 6.5) AQUI-S CIS 4. 5 (sd = 1.6) N. E 8 (sd= 3.9) AQUI-STM PLUS 3.6 (sd = 1.1) 15.1 (sd= 4.0) 4.8 (sud= 1. 2) AQUI-ST PLUS TRANS 3.5 (sd = 1. 1) 18.9 (sd = 2.6) 4.1 (sd = 0.5) AQUI-S PLUS CIS 2.2 (sd = 1. 0) N. E. 21.4 (sd = 4.3) Notes 1. Treatment concentration = 40 mg/L ( 0.8) 2. N. E.-not effective within 60 minutes.

3. sd = standard deviation 4. Animal wt (av) = 1.64g 5. Water temperature = 23°C Conclusions 1. Guppy responded slowly to AQUI-ST in comparison to salmonids. Their response to AQUI-STM PLUS was significantly faster. The recovery times, following treatment with AQUI-STM AND AQUI-STM PLUS, were similar at 4.8 to 5 minutes.

2. The CIS and TRANS QUI-STEM formulations were not very effective as anaesthetics. This may have resulted from poor dispersion characteristics. In particular the AQUI-S CIS formulation did not disperse well in the freshwater.

3. The response of the fish to the QUI-STEM PLUS formulations was more uniform compared to the response to the AQUI-S formulations. The standard deviations for the AQUI-S PLUS and AQUI-S PLUS TRANS formulations were significantly less than for the other formulations tested.

4. AQUI-S PLUS CIS was not effective as an anaesthetic.

5. AQUI-S PLUS and QUI-STEM PLUS TRANS were the most effective anaesthetic formulations with similar sedation, anaesthesia and recovery times.

EXAMPLE 2 This example describes the efficacy and response of chinook salmon to varying concentrations of AQUI-STM (50% active ingredient isoeugenol and 50% non-ionic surfactant carrier, ie polysorbate 80), TRANS (50% trans-isoeugenol and 50% polysorbate 80) and CIS (50% cis-isoeugenol and 50% polysorbate 80). Toxicity of the cis-isoeugenol and trans-isoeugenol formulations were compared by determining the pattern and time to heart failure and by measurement of key liver, heart and muscle enzymes that indicate toxicity.

Materials and methods Formulations and trial protocols Three formulations were supplied by AQUI-S New Zealand Ltd. Staff performing the efficacy trials were unaware of the formulation they were testing but were informed of the concentration. The active ingredient used in each formulation was assayed by gas chromatography and the results supplied by AQUI-S New Zealand Ltd. The active component in the formulation designated as AQUI-S in this study contained 10.2 % cis-isoeugenol, 89.1 % trans-isoeugenol and 0.7 %

eugenol. The active component in the formulation designated as TRANS contained 98.6 % trans-isoeugenol and 1.4 % cis-isoeugenol. The active component in the formulation designated as CIS contained 19.8 % trans- isoeugenol, 72.7 % cis-isoeugenol, 3.0 % eugenol, 4.0 % chloroform and 0.5 % unidentified hydrocarbon.

Definition of handleable, anaesthesia and recovery Handleable: For the purposes of this experiment, a fish was defined as handleable when it lost reactivity to external stimuli. This occurred when it stopped avoiding obstacles in its path and when it could be held underwater against the wall of the tank for 3 seconds without movement.

Anaesthetised: For the purposes of this experiment, a fish was defined as anaesthetised when it lost a key reflex activity, i. e. the fish did not respond within 3 seconds when the operculum is lifted and the gill lamellae touched.

Recovered: For the purposes of this experiment, a fish was defined as recovered from anaesthesia when it exhibited normal swimming behaviour and avoided obstacles.

Experimental Animals The female chinook salmon (Oncorhynchus tshawytscha) used in this study were obtained from The New Zealand King Salmon Company Ltd hatchery at Pupu Springs. The fish had been transported to the laboratory on 30th May 2000 where they were smoltified in 5.8 m3 round tanks supplied with sand-filtered seawater at 30 L/min and auxiliary aeration (typical dissolved oxygen content of 8.0 mg/L). In the first week the fish were fed proprietary (NRM Ltd, Nelson) extruded pellets after smoltification and were then fed a moist, alginate-bound diet. The fish to be used in the trial were reared in two tanks and were amalgamated into a 5.80 m3 round tank 2 weeks prior to experimentation. The fish were anaesthetized with AQUI-STM at 20 mg/L to facilitate transfer. For two weeks prior to experimentation the fish were only disturbed for routine cleaning of the tank (bi-weekly), for daily feeding, and were crowded once for pre- experiment sampling. Feeding was stopped 2 days prior to experimentation.

Tank volume determination The volume of water in each experimental tank was reduced from 1000 L to 500 L ( 0.1%) prior to each treatment. The 500 L level was individually measured for each tank by weight. A Mettler PE 6000 balance ( 0.05 g; calibrated June 2000) was used for Tank A and an AND FW-60K balance (+ 0. 01 kg; calibrated January 2000) for the remaining five tanks. Initially 500 kg of water was weighed into each tank. To convert weight to L, the density of seawater was measured by weighing 1 L of seawater in a volumetric flask (this was repeated five times). The flask of seawater was weighed using a Mettler PJ 6000 balance ( 0.05 g; calibrated June 2000). The mean weight of 1 L of seawater was 1023.3 0. 1 g.

This was repeated by weighing 200 mL of seawater in a volumetric flask using a more accurate balance (Sartorius MC1 LC 820). The mean weight if 200 mL of seawater was 204.50 0. 01 g (n = 5). The density of seawater from the 1 L measure was 1.0233 0. 0001 kg/L and from the 200 mL measurements was 1.0225 0.00005 kg/L. The average of these two densities (1.023 kg/L) was used to calculate the number of L in each tank and the volume adjusted with additional seawater to give a final tank volume of 500 L. Before a drain hole was made in each tank a bucket of seawater was removed (weight recorded) to decrease the level below that of where the hole was to be drilled. Once the drain was in place seawater was added to the tank until the water flowed out of the drain. The weight of seawater added and the amount that flowed out of the drain was recorded to give the volume for each tank in the 13 to 16°C temperature range.

Pre-exposure handling and fish condition Approximately 40 hours before the efficacy trials the main population of fish was anaesthetized with AQUI-STM at 20 mg/L to facilitate transfer of 42 fish to six round plastic tanks (1.818 m3, 1. 41 m dia.; Skellerup Industries Ltd, model Roto Open Top Black, Christchurch, NZ). Each tank was supplied with auxiliary aeration and contained 1000 L of sand-filtered seawater supplied on a flow- through basis at 10 L/min. Leftover fish from the main population were transferred to a 6.08 m3 round tank supplied with sand-filtered seawater at 30 L/min and auxiliary aeration (typical dissolved oxygen content of 8.0 mg/L). A second transfer of animals to the experimental tanks was required to complete the efficacy trials and was performed in the same manner as the first transfer.

Water Quality The water temperature was measured to 0.1 °C with an YSI 550 DO meter (YSI Incorporated, Yellow Springs, USA). This instrument was checked against a Zeal Thermometer (No. 9540032, Mercury in glass-solid stem, calibrated June 1995, GB) and was accurate to within 0.1 °C at the temperatures measured. The dissolved oxygen concentration was measured to 0.01 mg/L with the same unit (calibrated per the manufacturers instructions). Salinity was measured to 0.5 ppt using a handheld refractometer (S/mill Atago, Japan) calibrated as per the manufacturers instructions. The pH of the seawater from each tank was measured to 0.01 pH units with a PHC 2005-7 pH probe (Radiometer Copenhagen) connected to a PHM 92 pH meter (Radiometer Copenhagen) calibrated with pH 7.0 and pH 4.0 buffers (Radiometer Copenhagen SUMO 13 and SUMO 12, respectively) per the manufacturers instructions. Water temperature, salinity and pH were measured prior to each trial. Dissolved oxygen concentration was measured before each trial, and when water samples were taken during the trial (1 min after introduction of anaesthetic and just prior to fish being transferred to a recovery tank). The mean incoming seawater temperature from 1st to the 12th October 2000 was 13.4 0.2°C (measured using an in-line temperature probe-Omron E5LC, Japan).

Drug application A 1: 10 stock solution of anaesthetic in distilled water was prepared in 250 mL Schott bottles for each formulation and each concentration used. This emulsion was added to the rearing tanks to produce initial concentrations of 10 0.5 mg/L, 15 0.5 mg/L, 25 0.5 mg/L and 50 0.5 mg/L. Mixing was achieved by introducing the material into the up welling caused by aeration. The fish were disturbed as little as possible during this procedure. Experimental timing was recorded from introduction of anaesthetic.

Efficacy study experimental method The air stone in the experimental tank was left in place for one minute after the introduction of anaesthetic to ensure adequate mixing, and then removed. A few drops of an anti-foaming agent (anti-foam 1520 emulsion, Dow Corning) were added to the experimental tank immediately after the introduction of the anaesthetic to aid in making observations. Times to sedation and anaesthesia

were monitored and individual measurements were recorded to the nearest 30 sec. Due to the depth of the tanks a 300 mm dia. aluminium net was used to hold the fish against the side of the tank and remove them from the water for determination of the presence or absence of the gill cough reflex. This net was strung with fine, black, knotless mesh netting that was strung taut across the frame to produce a slightly yielding membrane. Once all seven fish had reached the state of anaesthesia or 60 min had passed (whichever came first), the fish were removed from the experimental tank and transferred to a recovery tank.

The recovery tanks were identical to the experimental tanks except that they were filled with-1000 L of fresh seawater. The air stone was removed immediately prior to fish being transferred in order to make observations and record the time taken for fish to recover. Each trial was recorded using a black and white underwater video camera.

Determination of active ingredient in treatment water C&FR Seafood Unit Physiology Standard Method &num 008 (Determining the concentration of AQUI-S with GIBBS Reagent) was used to confirm the concentration of each anaesthetic formulation in the efficacy and toxicity trials.

In the efficacy trials duplicate treatment water samples were collected at the beginning (1 min after the introduction of the anaesthetic into the up-welling caused by aeration when the anaesthetic was thoroughly mixed) and at the end of each treatment for most tanks. Samples were taken mid-water as it was determined that the taking of samples at the surface resulted in highly variable concentrations. Each sample was measured in triplicate and the mean of all the results for each tank was recorded (see individual dose trial tables in the Results section). As this method was only used as confirmation of the anaesthetic concentration, for graphing purposes, all concentrations are reported as 10,15, 25 or 50 mg/L.

Toxicity trial methods After the efficacy trial had been completed, more salmon from the main population were transferred into the experimental tanks in order to carry out the toxicity trial. Salmon were anaesthetised with 20 mg/L AQUI-S to aid in the transfer. Eight fish were transferred to each of the six experimental tanks with

each tank being supplied with auxiliary aeration and contained 1000L of sand- filtered seawater on a flow-through basis (10 L/min).

For the toxicity trial only the CIS and TRANS anaesthetic formulations were used. Salmon were anaesthetized in the rearing tank using the anaesthetic AQUI-STM at a concentration of 25 mg/L. After an exposure time of 30 min one fish was transferred to a Perspex holding trough (60 x 290 x 100 mm) in the laboratory. Seawater was introduced to the holding tank via a plenum at one end and exited via a second plenum at the other end in a flow-through manner.

This arrangement ensured that the water delivered to the fish was bubble free and that the chamber was held at constant depth. Influent water entered the chamber through a short length (17 mm) of soft rubber hose (9 mm dia.). This tube was inserted into the mouth of the fish-10 mm to irrigate the gills.

Fish were kept under anaesthesia (Stage IV to Stage V) in the holding tank by maintaining the anaesthetic concentration (either the CIS or TRANS formulation) of the irrigating seawater solution at 65 mg/L. This was achieved by providing a stirred reservoir of anaesthetic stock solution at the appropriate concentration.

This solution was delivered to a stirred seawater/anaesthetic mixing chamber (Microlene W1OPR Filterpure housing) at a rate of 0.69 mL min-1 (0. 003 ml/min SEM) via a peristaltic pump (Masterflex Console Drive: pump head 70 13-20; C- Flex tubing L/S 13). The flow rate of the seawater was controlled by an AlOHS- PC Platon flow-meter with the flow set at 0.385 L min-1 3%. The irrigating seawater flow and the anaesthetic stock solution flow rates were confirmed by repeated weight/time measurements.

Temperature of the irrigating seawater was monitored with a Type T thermocouple temperature probe (RS Components 219-4674, Auckland, NZ) in the influent plenum (the difference between the inflow and outflow water was negligible). The temperature was not controlled and came in at the ambient temperature.

Measurement of dissolved oxygen in the influent and effluent water The oxygen consumption rate was monitored using a dissolved oxygen flow-thru probe (MI-730 Microelectrodes, Inc.). Water from either the influent or effluent

flow was diverted through the electrode for measurement by using a pneumatic valve. This was set on a cycling time switch so that the oxygen level in the effluent water was measured for 10 min and the influent water for 5 min. The probe was calibrated in situ. The influent water was delivered from a 700 L elevated, aerated, constant head reservoir. The water was oxygen saturated with this being confirmed by measurements (YSI 550 DO meter, Yellow Springs Instruments). A second reservoir of seawater was sparged with oxygen free nitrogen gas to act as the zero oxygen standard.

ECG measurement A single 0.2 mm stainless steel needle electrode was inserted in the ventral midline of the salmon, ensuring that the needle was close to, but not touching the heart. An earth electrode was placed in the Perspex holding tank with the fish. The electrodes were connected to a UFI 2122i bio amplifier (UFI, Morro Bay, California) set to provide a gain of 500 with a high pass filter set to a roll off frequency of 10 KHz and a low pass roll off frequency of 10 Hz.

Measurement recording All measurements were recorded using a PowerLab 4sp data acquisition system (ADInstruments Ltd, NSW, Australia) connected to a Toshiba laptop running Chart V4.01 software (ADInstruments). The Type T thermocouple probe was connected to a T-type signal-conditioning pod (ADInstruments) prior to connection to the PowerLab unit. The dissolved oxygen probe was connected to an oxygen adaptor (Microelectrodes, Inc., Mass. USA) prior to the PowerLab. The amplified ECG signals from the bio amplifier were processed via the Chart software to produce a heart rate output. The sampling rate was set at 40 samples/sec.

Blood sampling and pH measurement Just prior to complete heart failure, the fish was removed from the holding box and a blood sample was taken. A 0.5 mL sample of venous blood was taken from the ventral aorta using a heparin-rinsed syringe (Terumo lml syringe, 11/2 inch needle, 21 gauge; rinsed with 5 mg Li/heparin per 50mL dist. water). The blood was then transferred into 1.5 ml Eppendorf tubes. Blood pH was measured immediately after extraction using a combined pH electrode (Model: pHC 4000-8,

Radiometer Inc., Copenhagen; calibrated as directed) connected to a pH/mV meter (PHH92 Lab Meter, Radiometer, Copenhagen).

Enzyme and glucose measurement Once the blood sample had been taken it was spun down in a bench-top centrifuge (MSE Microcentaur) for 2 min at 5000 rpm. The plasma was extracted and the pellet discarded. Creatine kinase (CK), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and glucose were all measured in the plasma using a Reflotron'D IV (Roche Diagnostics NZ Ltd).

Reflotron Check and Precinorm universal control system were used to verify correct operation of the machine.

White muscle pH measurement White muscle cut-surface pH measurements were taken from the left fillet of each fish. The pH measurements were made on the surface of the D 1 block exposed by a transverse section of the fillet as described by Jerrett, et al., (J.

Food Sci., 61: 527-532 (1996)) using a combination pH surface electrode (Model: 450-C Sensorex, CA, USA) calibrated as directed using pH 7.0 and pH 4.0 buffers (Radiometer Copenhagen SUMO 13 and SUMO 12, respectively). This electrode was connected to a model PHM 202 pH meter (Radiometer, Copenhagen).

Statistical methods and data analysis Graph plots were generated using SigmaPlot 2000 for Windows Version 6.0 (copyright 1986-2000 SPSS Inc.). In the efficacy trials the dose-response curve samples were plotted as means standard error of the mean (SEM). Student's t-test was used to test differences between sample means. A confidence level of 95% (P < 0.05) was used.

In the toxicity trial, heart rate and dissolved oxygen data was initially acquired at a sampling rate of 40 samples/sec in order to accurately resolve the ECG signal.

These large data sets were reduced to 1 sample/sec and were exported as text files to SigmaPlot for analysis and graphing. The effects of anaesthetic exposure on heart rate were characterised by three measures for each experiment. The heart rate 5

min after exposure to the formulation, time to onset and cessation of arrhythmia was taken directly from the data set. For the purposes of this experiment,"arrhythmia" was defined as a rapid, cyclical change in heart rate of more than 30 beats/min. The point of incipient heart failure was defined as a sustained drop in the mean heart rate to below 20 beats/min. This value was determined from the data generated after applying a 5 min running average to the reduced data-set in SigmaPlot.

RESULTS Efficacy trial AQUI-STM FORMULATION (Active ingredient: 89.1% trans-isoeugenol, 10.2 % cis-isoeugenol, 0.7% eugenol) AQUI-STM 10 mg/L: Fish had no adverse reaction to the introduction of anaesthetic.

Fish did not swim round the tank after the anaesthetic had been introduced; instead they sat upright on the bottom of the tank and were ventilating rapidly and coughing and using their pectoral fins vigorously. Patches of oil were visible on the water surface possibly indicating a lack in dispersion. Water temizerature 13. 80C Salinity 34 % o PH 8. 13 D. O. before 7. 36 mg/L D. O. 1 min 7. 68 mg/L D. O. end/L Anaesthetic conc. 9.49 0.96 mg/L (n = 6) Confirmation Fish weight (mean SEM) 93.7 5.6 g Fork length (mean SEM) 204 3 mm Time to sedation 6. 8 min (1 fish was never sedated) Time to anaesthesia Fish never reached anaesthesia Exposure time 60 min Time to recover 20. 7 1. 3 min

QUI-STEM 15 mg/L: Fish showed no adverse reaction to the introduction of anaesthetic. Anaesthetic action was gentle and fish began to lose equilibrium quickly. Gentle recovery.

Watertemperature 13. loC Salinity 35 % o pH 8. 09 D. O. before 8.16 mg/L D. O. 1 min 8. 25 mg/L D. O. end L Anaesthetic conc. 18.10 0.38 mg/L (n =7) confirmation Fish weight (mean SEM) 90.2 5. 6 Fork length (mean SEM) 203 4 mm Time to sedation25. 4 ~ 5. 6 min Time to anaesthesia 1/7 anaesthetised at 56.5 min Exposure time 60 min Time to recover 31. 3 3.1 min

AQUI-STM 25 mg/L: Fish showed no adverse reaction to the introduction of anaesthetic. Fish rapidly lost equilibrium. Fish had quite a long exposure to the anaesthetic and thus only one fish recovered. Water temperature 13. 50C Salinity 35 % o pH 8. 11 D. O. before 7. 94 mg/L D. O. 1 min 8. 01 mg/L D. O. end 7. 96 mg/L Anaesthetic conc. 24.32 0.34 mg/L (n = 12) confirmation Fish weight (mean ~ SEM 96.1 ~ 7.8 Fork length (mean SEM) 210 6 mm Time to sedation 9. 9 ~ 0. 8 min Time to anaesthesia 35. 3 2.3 min Exposure time 42 min Time to recover 1 7 recovered at 46 mm (6/7 did not recover) QUI-STEM 50 mg/L Fish showed no adverse reaction to the introduction of anaesthetic. Fish rapidly lost equilibrium. Fish reached anaesthesia within minutes of each other and therefore all recovered. Watertemperature 14. 1oC Salinity 35 # pH 8. 13 D. O. before 7. 81 mg/L D. O. 1 min 7. 85 mg/L D. O. end 7. 81 mg/L Anaesthetic conc. 48.06 0.88 mg/L (n = 9) confirmation Fish weight (mean SEM) 82.3 i 4. 2 Fork length mean SEM) 198 3 mm Time to sedation 3 min Time to anaesthesia 6. 0 0.7 min Exposure time 8. 5 min Time to recover 26 1.4 min (7/7 fish recovered)

TRANS FORMULATION (Active ingredient 98.6% trans-isoeugenol, 1.4% cis- isoeugenol) TRANS 10 mg/L: No adverse reaction to introduction of anaesthetic. Fish swam slowly around the tank. Very little coughing and anaesthetisation appeared to be gentler than for AQUI-S at the same concentration. Ventilation was not as rapid and they did not use their pectoral fins as much as in the AQUI-S treatment. Could smell the anaesthetic more. Less oil on the surface of water

Watertemperature 13. 90C Salinity 34 # pH 8. 12 D. O. before 7. 78 mg/L. D. O. 1 min 7. 60 mg/L D. O. end 7. 54 mg/L Anaesthetic conc. 9.90 0.26 mg/L (n = 6) confirmation Fish weight (mean SEM) 106.4 11.8 g Fork length (mean SEM) 207 ~ 6 mm Time to sedation 3/7 fish 33.7 4.8 min; 4/7 fish were never sedated) Time to anaesthesia Fish never reached anaesthesia Exposure time 60 min Time to recover 13. 1 0. 9 min TRANS 15 mg/L: No adverse reaction to introduction of anaesthetic. Fish were slightly agitated (first trial of the day). Fish ventilated rapidly and coughed. Watertemperature 13. 30C Salinity 35% o pH 8. 12 D. O. before 8. 22 mg/L D. O. 1 min 8. 12 mg/L D. O. end 7. 94 mg/L Anaesthetic conc. 16.59 0.33 mg/L (n = 12) confirmation Fish weight (mean SEM) 87.7 ~ 5. 8 Fork length (mean SEM) 202 ~ 5 mm Time to sedation 6/7 fish 27.6 4.0 min; 1/7 fish was never sedated) Time to anaesthesia 1/7 fish reached anaesthesia at 59 min Exposure time 60 min Time to recover 28. 6 ~ 1. 6 min

TRANS 25 mg/L: No adverse reaction to introduction of anaesthetic. The majority of fish reached anaesthesia after #35 min, however, 2 fish took another 10 min to become anaesthetised and this extra exposure time resulted in none of the fish recovering. Watertemperature 13. 90C Salinity 35 # pH 8. 12 D. O. before 8. 00 mg/L D. O. 1 min 8. 01 mg/L D. O. end7. 87 mg/L Anaesthetic conc. 24.04 0.27 mg/L (n = 12) confirmation Fish weight (mean SEM) 110.6 6.0 g Fork length (mean SEM) 218 4 mm Time to sedation 7. 6 0.4 min Time to anaesthesia 35. 3 2.5 min Exposure time 46 min Time to recover 7 7 did not recover

TRANS 50 mg/L: Fish showed a slight reaction to the introduction of anaesthetic (two fish coughed). Fish rapidly lost equilibrium. Five of the 7 fish reached anaesthesia by 7.5 min. An extra 8.5 mins was needed for the last two fish to become anaesthetised and therefore only 1 fish recovered. Water temperature 14. 1oC Salinity 34% o pH 8. 12 D. O. before 7. 80 mg/L D. O. 1 min 7. 87 mg/L D. O. end 7. 81 mg/L Anaesthetic conc. 49.80 0.28 mg/L (n = 12) confirmation Fish weight (mean SEM) 97.2 8. 1 Fork length (mean SEM) 209 ~ 5 mm Time to sedation 4 min Time to anaesthesia 9. 1 1.5 min (6/7 fish anaesthetized by 13 min) Exposure time 16 min Time to recover1/7 recovered after 30 min; 6/7 did not recover

CIS FORMULATION (Active ingredient: 72.7% cis-isoeugenol, 19.8% trans- isoeugenol, 3% eugenol, 4.5% other) CIS 10 mg/L: Fish had no adverse reaction to the introduction of anaesthetic. Fish did not swim round the tank after the anaesthetic had been introduced; instead they sat upright on the bottom of the tank and were ventilating rapidly and coughing and using their pectoral fins vigorously. Fish would often dart across tank. Patches of oil were visible on the water surface possibly indicating a lack in dispersion. Recovery to upright swimming was rapid even though fish were still sedated. Watertemperature 14. 00C Salinity 34 % 0 pH 8. 10 D. O. before 7.83 mg/L D. O. 1 min 7. 88 mg/L D. O. end 7. 70 mg/L Anaesthetic conc. 10.29 0.34 mg/L (n = 12) confirmation Fish weight (mean SEM) 92.8 6. 2 Fork length (mean SEM) 198 5 mm Time to sedation 5/7 fish 33.0 10.3 min; 2/7 fish were never sedated) Time to anaesthesia Fish never reached anaesthesia Exposure time 60 min Time to recover 13. 5 1.5 mm

CIS 15 mg/L: Fish had no adverse reaction to the introduction of anaesthetic.

Fish were ventilating rapidly and coughing and using their pectoral fins vigorously. Recovery was rapid once fish swam off bottom. Fish had a high tail beat frequency and were quick to swim upright even though they were still quite heavily sedated Water temperature 13. 20C Salinity 34 # pH 8. 12 D. O. before 8. 17 mg/L D. O. 1 min 8. 25 mg/L D. O. end 8. 16 mg/L Anaesthetic conc. 17.30 0.21 mg/L (n = 12) confirmation Fish weight (mean SEM) 82.1 i 4. 6 Fork length (mean SEM) 196 5 mm Time to sedation 22. 5 3. 0 min Time to anaesthesia 3/7 fish reached anaesthesia 52.0 ~ 2. 1 min) Exposure time 60 min Time to recover 18. 6 1.3 min

CIS 25 mg/L: Fish had no adverse reaction to the introduction of anaesthetic.

Fish reached anaesthesia quite quickly, however, even though their exposure time was 24 min only 3/7 fish recovered. Two fish that died started to ventilate (gulped a few times) but then stopped. Water temperature 13. 80C Salinity 35% o pH 8. 11 D. O. before 8. 02 mg/L D. O. 1 min 8. 02 mg/L D. O. end 7. 98 mg/L Anaesthetic conc. 26.47 0.23 mg/L (n = 12) confirmation Fish weight (mean ~ SEM) 103.9 ~ 7.7 Fork length (mean ~ SEM) 214 ~ 4 mm Time to sedation 7. 0 0.8 min Time to anaesthesia 17. 6 1.5 min Exposure time 24 min Time to recover 3 7 recovered 21. 7 2.3 min) ; 4/7 did not

CIS 50 mg/L: Fish showed no adverse reaction to the introduction of anaesthetic. Fish rapidly lost equilibrium. Six out of seven fish were anaesthetised by 8.5 min, however the last fish did not reach anaesthesia until 12 mins and therefore over-exposing the fish to the point that only 2 fish recovered. Watertemperature 14.2°C Salinity 35 # pH 8. 11 D. O. before 7. 75 mg/L D. O. 1 min 7.84 mg/L D. O. end 7. 79 mg/L Anaesthetic conc. 47.22 0.67 mg/L (n = 12) confirmation Fish weight (mean ~ SEM 110.4 6.4 g Fork length (mean SEM) 217 4 mm Time to sedation 3. 2 ~ 0. 4 min Time to anaesthesia 7. 6 ~ 0. 8 min (6/7 anaesthetised in 8.5 min) Exposure time 12. 0 min Time to recover 2/7 recovered after 20 min and 30 min; 5/7 did not

Toxicity trial-Measurement of plasma enzymes Baseline fish TRANS CIS (AQUI-STM) n = 5 n = 5 n = 4 Weight (g) 64.8 1.41 102.38 6.27 102.02 3.77 Length (mm) 183 183 ~ 2 212 ~ 3 214 ~ 3 Blood pH 7.50 ~ 0. 05 7.67 ~ 0. 01 7.59 0. 03 Flesh pH 7. 82 ~ 0. 03 7.94 ~ 0. 01 7.93 0. 01 CK (units/L) 429 17 999 146 787 ~ 301 (n=3) GPT (units/L) 20 7 46 10 (n=4) 76 ~ 17 GOT (units/L) 450 43 542 152 577 92 Glucose 1.83 ~ 0. 26 1.25 ~ 0. 21 1.58 0. 34 (mmol/L) CK = Creatine kinase; GPT = glutamate pyruvate transaminase ; GOT = glutamate oxaloacetate transaminase. Values are the Mean SEM.

Results summary-Efficacy As the concentration of each formulation of anaesthetic increased the exposure time required for fish to become handleable and to reach anaesthesia decreased (Figures la a & b).

# Full anaesthetisation was reached only at concentrations of 25 mg/L and 50 mg/L for all three formulations (Figure lb).

# Fish exposed to the CIS formulation at a concentration of 25 mg/L reached the state of anaesthesia twice as fast as fish exposed to the other formulations (Figures lb & 2).

If fish were exposed to anaesthetic of any formulation for-5 min after they had reached anaesthesia, their ability to recover was severely limited (Figure lc).

Fish exposed to the CIS formulation recovered faster than fish exposed to AQUI-STM and the TRANS formulation at 15 and 25 mg/L (Figure lc).

The course of recovery for fish exposed to the CIS formulation was very different to the other treatments. Fish ventilated lightly but very rapidly, and tail beat frequency when they began to move was much higher. When fish began to swim they did not swim on their sides for a period and then regain equilibrium, they almost immediately swam upright although they remained sedated for some time.

Results Summary-Margin of Safety The margin of safety for the TRANS formulation is up to 2.5 times greater than that for the CIS formulation (Figure 3) Results Summary-Toxicity Plasma enzyme assays did not usefully discriminate differences between the formulations.

Times to onset of arrhythmia, end of arrhythmia and time to a mean heat rate below 20 beats/min all suggest that the CIS formulation was more supportive of cardiac function than the TRANS formulation (Figure 4).

This was consistent with CIS being less of a general depressant and more of a narcotic than the TRANS formulation and consistent with the efficacy study observations.

Discussion As trans-isoeugenol is the dominant isomer in isoeugenol (89.1% trans, 10.2% cis, 0.7% eugenol) it was assumed that trans-isoeugenol was the"active" component. One would predict that the pure trans isomer would give a faster anaesthetic action than a mixture of the isomers. Surprisingly, this was not the case.

The TRANS formulation (98. 6% trans, 1.4% cis) which contained 9.5% more of the"active"component compared with the AQUI-S formulation did not have a 9.5% increased activity as an anaesthetic. The activity (time to handleable and anaesthesia) was similar to AQUI-STM (Example 2, Fig. 1) or was slower (guppy data from Example 1: AQUI-S TM formulation of trans). Using the CIS formulation (Example 2,72.7% cis, 19.8% trans, 3% eugenol, 4% chloroform, 0.5% unidentified hydrocarbons) with low levels of the"active"component it was expected that the anaesthetic action would be much slower. But the time to anaesthesia at a concentration of 25 mg/1 was half that of AQUI-STM and the TRANS formulation. If fish were exposed to any formulation for longer than 5 min after they had reached anaesthesia, the chance of recovery was minimal.

Thus, exposure to the TRANS formulation of the anaesthetic resulted in a long, slow anaesthetisation process providing a large safety margin between fish becoming handleable and reaching anaesthesia (Figure 3). In contrast, the CIS formulation anaesthetised the fish more quickly, reducing the"window"of safe dosages compared with the TRANS formulation. This would increase the risk of overdose and death through hypoxia (ventilatory collapse).

It was also noted that on exposure to the CIS formulation fish became agitated compared with little reaction upon exposure to AQUI-STM or the TRANS formulation. Chinook salmon exposed to the CIS formulation typically ventilated at a faster rate and the anaesthetic action was not as smooth, with more coughing and agitated swimming. The"startle"reflex was quite pronounced in fish exposed to the CIS formulation at 10 mg/L. In comparison, there was no adverse reaction to the introduction of AQUI-STM or the TRANS formulation.

Agitation of animals on exposure to an anaesthetic is not a desirable property and is not supportive of the concepts of rested harvesting.

EXAMPLE 3 An investigation into the thermal and photostability of trans-isoeugenol (TIE) and isoeugenol (IE) was carried out.

Materials and Methods The trans-isoeugenol used in the study was sampled from batch 3/10B manufactured on the 3rd of October 2000 by Fish Transport Systems Ltd (97.6% isoeugenol (trans 99.5%, cis 0.5%), 0.6% eugenol by NMR). The isoeugenol used in the study was sampled from a 200L drum of commercially prepared isoeugenol, Lot # 00150036-2 of 2 manufactured by PT Djasula Wangi on the 17th of February 2001 (97.6% isoeugenol (trans 87.5%, cis 12.5%), 1.1% eugenol by NMR).

Two 50g samples of each material were stored in sealed glass bottles over a one- month storage in a 40°C oven. The oven was also equipped with an artificial light source generated by UV and Cool White fluorescent tubes. The light source was validated with a chemical actinometric system. Foil protected one of each of the samples from exposure to the light sources over the course of the study.

Each week the samples were briefly removed from the oven so that 0.228mL of material could be removed for each NMR analysis. NMR analyses were conducted in duplicate.

NMR spectra were obtained using a 500MHz spectrophotometer at Industrial Research Limited, from samples prepared according to AQUI-S New Zealand Limited's (ANZ) standard operating procedures.

The integrals resulting from each of the NMR analyses were entered into ANZ's standard integral template. These integrals represent the area under the peaks assigned to a particular compound. The standard error of the NMR methodology is 0. 5% w/w.

The aim of the study was to identify differences in the stability of each material.

Results Degradation Product An unidentified degradation product was observed for all samples that were exposed to the light source during the study. Figure 5 refers to the peak integral

for the unidentified degradation product as a function of time. The integral represents the NMR response of the singlet found at 3.57ppm.

This figure shows that the rate of formation of the integral at 3.57ppm, for this unidentified degradation product, is faster in isoeugenol than trans-isoeugenol.

Over the period of study the rate of formation of the degradation product was 1.45 times faster for isoeugenol, than that of trans-isoeugenol.

Thermal vs Thermal and Photo Stability No significant stability differences were observed in samples that were not exposed to the light source in the oven (left hand bars). However significant stability effects were observed in samples that were exposed to the light source (right hand bars).

Figures 6a, 6b, 6c, 7a, 7b and 7c show that when exposed to light: The percentage of trans-isoeugenol decreases, refer to Figures 6a and 7a The percentage of cis-isoeugenol increases, refer to Figures 6b and 7b The integral of eugenol increases, refer to Figures 6c and 7c This trend was observed for both trans-isoeugenol and isoeugenol. However in all cases the isoeugenol samples showed faster rates of degradation than those observed for the trans-isoeugenol samples. Thus: The rate of degradation of trans-isoeugenol was found to be 1.3 times faster for the isoeugenol sample.

*The rate of formation of cis-isoeugenol was found to be 1.3 times faster for isoeugenol.

*The rate of formation of the eugenol integral was found to be 1. 1 times faster for isoeugenol.

Sample Colours Figure 8 shows a photo of the samples as they appeared at the end of the period of study. A significant discoloration occurred in the samples that were exposed to the light source throughout the trial.

This photograph also shows that a significant difference exists between the extent of discolouration of isoeugenol and trans-isoeugenol when exposed to light. The isoeugenol sample is significantly darker than that of trans-isoeugenol.

INDUSTRIAL APPLICATION Those persons skilled in the art will appreciate the advantages of the present invention and the many applications to which the methods and compositions of the invention can be put.

As a first example, the methods and compositions can be employed in the harvesting of aquatic organisms for 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, anaesthetised or euthanased using the present composition, this struggling is at least much reduced, if not eliminated.

The compositions of the invention also have particular application in the transportation of live aquatic organisms, particularly where fish are to be transported live to overseas markets and where the natural undamaged appearance of the fish is critical to the market price obtained.

Another application is in the transport of aquatic organisms to a market where the organism is to be sold in a pre-rigor state, that is, a state in which the tissue of the organism remains alive for a prolonged period following administration of active agent but in which the organism is no longer capable of control of its musculature and from which the organism will not recover.

Another application is for animal husbandry for grading, vaccination or other health purposes.

Other applications of the present methods and compositions will be readily apparent to the skilled worker in this art.

The particular advantage offered by the present compositions which contain the trans isomer of isoeugenol but which are substantially free of the cis isomer is the long, slow anaesthetisation process, particularly for excitable fish. This provides a large safety margin between the fish becoming handleable and reaching anaesthesia. In practice, this reduces the risk of overdose and death through hypoxia (ventilatory collapse).

Moreover, the applicants have found that on exposure to cis isoeugenol, fish tend to become agitated, in comparison with exposure to trans isoeugenol which causes little reaction. Agitation of fish on exposure to an anaesthetic is not a desirable property and is not supportive of the concepts of rested harvesting.

Therefore, in at least substantially eliminating the cis isomer from their compositions, the applicants avoid this difficulty.

It has also been found that isoeugenol comprising a mixture of the trans and cis isomers degrades more rapidly on exposure to light than isoeugenol in the form of the trans isomer. Accordingly, active compositions according to the invention containing the trans form of isoeugenol substantially free of the cis isomer offer greater storage stability than compositions containing a mixture of the isomers.

Such enhanced stability represents a further advantage of the present compositions.

It will be appreciated that the above description is provided by way of example only and that the present invention is not limited thereto.