AU641477B2 - Control of fungal infections in aquaculture - Google Patents

Description

641477 S F Ref +-55951 FORM COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE: Class Int Class S Complete Specification Lodged: Accepted: Published: Priority: Related Art: S9r 0o- S S6 S r S

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S.i Name and Address S of Applicant: Address for Service: F.Hoffmann-La Roche AG 124 Grenzacherstrasse CH-4002, Basel

SWITZERLAND

Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia a g sa.

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*a Complete Specification for 'the invention entitled: S Control of Fungal Infections in Aquaculture The following statement is a full description of this invention, including the best method of performing it known to me/us 5845/3 RAN 4450/56 Abstract A method of controlling fungal infections of animals in aquaculture caused by the genus Fusarium spp. involves applying as a fungicidal or fungistatic agent a compound of 5 formula SH3 (cH2)1 C- CH2-CH-CH 2

*H

3 i -R methyl or X signifies methylene and R hydrogen, or a salt thereof to the animals in aquaculture. The invention also 25 concerns a fungicidal or fungistatic feed usable in such method.

RAN 4450/56 Control of Fungal Infections in Aquaculture C i, *Cr eec.

C Sr S. 15 The development of the commercial culture of animals in aquaculture, especially of crustacea, has been accompanied by the occurrence of infectious and non-infectious diseases.

Many of the important diseases suffered by, for example, penaeid shrimps, are caused by organisms which are ubiquitous and have been established in all the major penaeid culture areas of the world, e.g. in Japan, Korea,, Taiwan, the Philippines, Tahiti, Indonesia, Thailand, Malaysia, India° lilaysia, India, the Caribbean, Brazil, Mexico, Panama, Ecuador, Colombia, the U.S.AR Australia. France. Spain and Italy. With increasing density and production level of Sipenaeid shrimps diseases have broken out rapidly. Outbreaks of fungal infections, e.g. Lagenidium and Sirolpidium.

bacterial attacks, e.g. Vibrio and Aeromonas. and even viruses, e.g. Baculovirus, are frequent in hatcheries, most of these problems being due to insufficient control of the rearing systems and abs ice of sanitary procedures as in terrestrial husbandr- jdisinfection, regular dry-out, separate equipment foreach tank and separate rooms for maturation, spawning, hatching and larval rearing). The result is a weakening of the larvae and a lowering of their .1e. 0 C..

e eiC Pa/8.1.91 -2resistance to diasase. Although antifungal agents such as trifluralin and Malachite green, and antibiotics used sparingly in the tanks have achieved some success the need to have a dry-out every six to eight weeks of production in S 5 order to eliminate bacterial strains which have become increasingly resistant and pathogenic has not been avoided.

It has now been found that compounds of the formula

R

Wherein n signifies 0 or 1 .and X signifies oxygen and R methyl, Sor X signifies methylen and R hydr-Ten, oifai fen a d r and salts thereof are effective in co.. :olling fungal infections of animals in aquaculture caused by the genus Fusarium spp. either by reducing or eradicating an existing infection or preventing it breaking out (fungicidal S activity) or by preventing the spreading of an existing S infection (fungistatic activity). Accordingly, the present 25 invention provides a method of controlling fungal infections of animals in aquaculture caused by the genus Fusarium spp., characterized by applying as a fungicidal or fungistatic agent a compound of the formula I given above or a salt the compounds of formula I the compounds occur in optically active form. Formula I is accordingly intended to embrace the racemates as well as the separated optically active isomers. Moreover, the opportunity for a cis- and a trans- -arrangement of the methyl substituents of the morpholine ring (in the case when X signifies oxygen) is encountered, 3 o 1 1 1 giving rise to geometrical isomerism, and formula I is accordingly intended to embrace the separated geometrical isomeric forms (cis- and trans-forms) as well as mixtures thereof.

Salt formation of the compounds of formula I is rendered possible by virtue of the basic nature of the nitrogen atom of the morpholine or piperidine ring. The salts of the compounds I contemplated are those with physiologically acceptable acids, in particular hydrohalic acids, e.g.

hydrochloric acid and hydrobromic acid; phosphoric acid; nitric acid: monofunctional and bifunctional carboxylic acids and hydroxycarboxylic acids, e.g. acetic acid. maleic acid, succinic acid, fumaric acid, tartaric acid, citric 15 acid, salicylic acid, sorbic acid and lactic acid: and sulphonic acids, e.g. 1,5-naphthalene-disulphonic acid.

SThe compounds of formula I and their salts, as well as S the use of these substances as fungicidal and fungistatic agents in the control of fungi pathogenic to plants, animals and humans, are described in the patent and scientific S literature, e.g. in U.S. Patent Specifications Nos.

4,202,894 and 4.241,058 (Bohnen and Pfiffner), European Patent Specification No. 24,334 (Pfiffner), Med. Fac.

25 Landbouww. Rijksuniv. Gent, 44/2, pages 487-497 (1979; S/Bohnen and Pfiffner), and "Antifungal Activity of Amorolfine (Ro 14-4767/002) in Vitro and in vivo" by Polak and Dixon, Recent Trends in the Discovery, Development and Evaluation of Antifungal Agents, R.A. Fromtling pages 555-573 (1987, J.R. Prous Science Publishers, In the last-mentionedarticle the in vitrofungistatic activity of amorolfine [INN, published as recommended by the WHO; hydrochloride salt of the cis-compound of formula I in which n is 1, X is oxygen and R is methyl] on various medically important fungi is reported. On the basis of the results given, inter alia the poor or non-existent activity against opportunistic moulds such as Fusarium spp. (see Table 1 on -4 page 561), the present finding of the p g excellent activity of the compounds of formula I in the control of fungi of the genus Fusarium spp. in aquaculture must be deemed to be very surprising.

Of the compounds of formula I and their salts the previously mentioned amorolfine (rac-cis-4-{3-[4-(l,1- -dimethylpropyl)-phenyl] -2-methyl-propyll-2.6-dimethylmorpholine hydrochloride), and particularly cis-4-[3-(4- -tert.butyl-phenyl)-2-methyl-propyl] -2,6-dimethylmorpholine (fenpropimorph, Corbel have been found to be especially suitable fungicidal or fungistatic agents in the method of the present invention. A further especially suitable compound of formul I is (RS)-1-[3-(4-tert.butyl- 15 phenyl)-2-methyl-propyl]-piperidine (fenpropidin).

In practice the compound of formula I or salt thereof is applied to the animals in their aqueous environment, e.g.

tankor pond, undiluted or.in association with inert solid or liquid diluting medium. Such inert solid or liquid diluting medium may take the form essentially of water, in S, which case the compound of formula I or salt thereof is conveniently dissolved or otherwise dispersed therein, if S necessary with the aid of wetting, emulsifying and/or 25 dispersing agents. In particular, however, the inert solid or liquid diluting medium may consist of feed for fish or crustacea, and in such medicated feeds the content of active °ingredient i.e. compound of formula I or salt thereof, is generally n the range 0.0001 to 0.1 percent by weight, preferably 0.003 to 0.01 percent by weight. Such a medicated feed for fish or crustacea is preferably in the form of pellets or microcapsules.

The rate at which the compound of formula I or a salt thereof is applied to the aquatic environment of the animals to be treated is generally in the range 1 to 50 vig/1, preferably in the range 10 to 25 ug/l.

5 The type of animals in aquaculture to be treated against iftagal infectious caused by Fusarium spp.is preferably crustacea. This class of aquatic animals embraces inter alia members of the genus Penaeids (Penaeus spp.), fresh water prawns (Macrobrachium spp.) and lobsters (Homarus spp.), and such species are particularly effectively treated according to the method of the present invention. More particularly, the Penaeus spp. treated according to the method of tie present invention are warm water or cold water shrimps, such as Penaeus aztecus, P. brasiliensis, P. brevirostris, P. californiensis, P. canaliculatus, P. caramote, P. carinatus, P. duorarum, P. esculentus, P. indicus, P. japonicus, P. kerathurus, P. latisulcatus, P. merguiensis, P. monodon, P. occidentalis, P. orientalis, 15 P. plebejus, P. schmitti, P. semisulcatus, P. setiferus, P. sinensis, P. stylirostris, P. trisulcatus. P. vannamei, S. Metapenaeus ensis and M. monoceros. In the case of Sfresh water prawns the method of the present invention is preferably directed to the control of fungal infections of Macrobrachium carcinus, M. nipponense and M. rosenbergii. and in the treatment of lobsters those preferably treated are of the species Homarus americanus, H. gammarus and H. vulgaris.

25 The method of the present invention can be applied for a curative or preventive purpose, but the control is preferably curative.

Moreover, although the method of the present invention can be utilized for the control of fungal infections in any development stage. e.g. egg, larval or post-larval stage, of the animals in aquaculture to be treated, it is preferably effected for the control of a fungal infection in the post-larval development stage of said animals.

The method according to the invention is preferably applied for controlling fungal infections caused by Fusarium 6 solani.

In principle the method can be carried out in any type of aquaculture, but is particularly effected in a hatchery, a growing pond, a nursery pond or a rearing pond. Furthermore, the frequency with which the treatment, i.e. the applications of the compound of formula I or a salt thereof to the animals in aquaculture, can be varied according to the experience gained by observing the results, but in general, and conveniently, the method is carried out at regular intervals, preferably every two to four weeks.

The present invention also provides a fungicidal or fungi'static feed for fish or crustacea, characterized in 15 that i ~t is intended for the control of fungal infections caused by the genus Fusarium spp. in aquaculture and contains as a fungicidal or fungistatic agent an effective Samount of at least one compound of formula I, as given above, or a salt thereof.

In such a medicated feed for fish or crustacea the percentage weight content of active ingredient will depend on the volume of the aqueous environment in which the fish o* r crustacea find themselves, e.g. tank or pond water, and the desired final concentration of the compound of formula I or salt thereof in said aqueous environment. For practical purposes the content of active ingredient in the fungicidal or fungistatic feed is generally in the range 0.0001 to 0.1 S percent by weight. The preferred range is 0.003 to 0.01 percent by weight. The feed is preferably in the form of pellets or microcapsules.

The present invention further provides the use of a compound of the formulalI, as given above, or a salt thereof for controlling fungal infections of animals in aquaculture caused by the genus Fusarium spp.

7 The invention is illustrated by the following examples: Example 1 in vitro Evaluation of the Fungicidal/Funqistatic Activity of cis-4-[3-(4-Tert.butyl-phenyl)-2-methyl-propyll-2.6dimethylmorpholine (fenpropimorph) against Fusarium solani 1. Isolation of Fusarium solani b C: 0 *i 6 0*C

C

000e .0 C The strain of Fusarium solani used in the tests was isolated from a shrimp of the species Penaeus japonicus affected with F. solani, i.e. suffering from an infection caused by Fusarium spp. The mould was cultured at 22 0 C on agar [DST Agar (Oxoid)] to which brine had been added to the extent 20 g NaCl/l.

2. Tests for antifungal activity on mycelium and conidiospores a) Preparation of the antifungal solutions: The initial "mother solution" of concentration 200 x il/1 was prepared by transferring 2 x 10 of fenpropimorph into 1 ml of I Tween 20 (polyethoxysorbitane laurate) to which 25 dimethyl sulphoxide had been added up to a total volume of 10 ml. This mother solution was submitted to a first dilution (1-2 volumes) with a further quantity of ,ween 20 (0.5 ml)/dimethyl sulphoxide. The range of concentrations investigated was obtained by succesive dilutions (initially 1-100 volumes and subsequently 1-2 volumes) of themmther solution with filtered autoclaved sea water. The mean salinity and pH of this sea water was 32% and 8. respectively.

b) Preparation of the inoculum: The inoculum consisted of a portion of the aforementioned agar taken from the surface and containing F. s ani in a Petri disc (0 9 cm). The -8 inoculum is removed at the periphery of the culture of F. solani aged for one week. The microscopic examination of this inoculum confirmed F. solani in the form of mycelium and conidiospores.

c) Test procedure: For each concentration of fenpropimorph in the solution to be tested the inoculum was placed into ml of the antifungal solution. After a 24 hours incubation period at 22 0 C three rinsings of the inoculum were carried out with filtered autoclaved sea i!wter. The inoculum was then placed at the centre of a Petri disc (0 9 cm; volume of agar DST Agar 10 ml) and the whole incubated at 22 0

C

until the evaluation.

3. Determination of the Results i: The in vitro activity of fenpropimorph on mycelium and S' conidiospores of F. solani was estimated 15 days after *@04 termination of the 24 hours treatment by calculating an index of growth inhibition, expressed as a percentage activity.

3 1 11 1 1 1 i 1 1 1 1 1 1 1 1 1 1 1 0_ .;a 9 4. Results' v 1 -v These are presented in the following table: Concentration of Percentage activity fenpropimorph Test 1 Test 2 in vl/i (average total (average total surface area of surface area of control cultures control cultures 40.4 cm 37.2 cm 2 3.125 83.5 85.5 6.25 96 96.5 12.5 100 100 15 25 100 100 50 100 100 1 1 i o 1 o S *5:i: 06 0: 0S

S

*00 0@ 9* 5: 0 0* Conclusions from the in vitro study 0r 000*SS 0::

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0:- The activity of fenpropimorph remains very high even at the lower concentrations tested. On the basis of a "fungicidal" activity fenpropimorph displays a fungicidal activity at concentrations greater or equal to 12.5 v.1/1 on F. solani. At lower concentrations the activity may be considerd to be fungistatic. However, inspite of the absence of regrowth measurable 15 days after the antifungal treatment the inoculum seems to survive on application of Santifungal solutions of concentrations 12.5 and 25 :Il fenpropimorph/l in v,.ew of the appearance of some off-white points of regrowth. Consequently, a strictly fungicidal activity is realized at concentrations above 25 ±il fenpropimorph/l1.,, What is indicated above for activity on mycelium and conidiospores 'is also valid for activity on conidiospores of F. solani alone. On non-germinated conidiospores of i 4 O S*u'b *Oi: S. 0 00!i 0C 04 Sin 0 00 .5 a, 0 F. solani the strictly fungicidally effective concentrations of fenpropimorph are from 15 Ji/I. On germinated conidiospores, i.e. "young" mycelium, the activity of fenpropimorph is considerably higher.

Example 2 in vitro Evaluation of the Fungicidal/Fungistatic Activity of rac-cis-4-{3-[4-(1,1-Dimethylpropyl)-phenyll-2-methylpropyl}-2.6-dimethylmorpholine hydrochloride (amorolfine) against Fusarium solani Fusarium solani wasisolated and cultured as described in part 1 of Example 1. Analogous to the method described in 15 part 2a) of Example 1 antifungal soluti.ons of amorolfine were prepared, starting from a mother solution of concentration 100 x mg/1 and finishing with solutions in sea water of concentrations x, x/2. x/4 etc. mg/l.

20 300 ji of agar (DST Agar) to which had been added 20 g/1 of sodium chloride were added to the base of each of a series of 5 ml test tubes. In each case the agar was then cultured with 10 j1 of a suspension of conidiospores of Fusarium solani, titrating about 5 x 105 conidiospores/ml.

After 4 days incubation at 22 0 C in such a way that the mycelium could occupy the whole surface of the agar and produce conidiospores 3 ml of antifungal solution of known concentration were transferred into each tube. After 24 hours incubation ato22 0 C three drainings and 2 rinsings with filtered autoclaved sea water are effected. The third rinsing was followed by one of two procedures: in the procedure without subcul,turing 3 ml of Sabouraud bouillon (a special nutrient medium for the culturing of Fusarium spp.) were added to each tube; in the procedure with subculturing a withdrawal of mycelium and conidiospores is effected on the surface of the i 11 agar in each tube. Each inoculum so withdrawn, not quantified precisely, was deposited in a fresh tube at the surface of 300 pl of agar.

The evaluation of the tests was effected 15 days after the 24 hours treatment. In the procedure without subculturing the activity was qualified as truly fungicidal and denoted with if no regrowth of fungus could be detected in the macroscopic examination of the agar. The activity was deemed to be of a fungistatic nature and denoted with if regrowth of the fungus could be detected, albeit less rapid than that of the non-treated fungus in the control tubes. In the case of there being no difference between the regrowth- Of the treated and untreated (control) fungus this was 15 denoted In the procedure with subculturing any antifungal activity was denoted in the case of tihel( o 'absence of/macroscopically detectable regrowth and denoted for the opposite situation. The results are presented in the following table: 0 *0* I i 1 3' 0 1 1 25 S. L 1 1 1 1 1 1 1 1 1 1 1 12 a.

~9 S Oa *0 0 0 0*0 9* 0v S Og Ya a.

*0

S.

S

.000 a 0000AO .5 a ta..

"a a.

a .61.0 a a.

a. a Table 2 .Concentration of -amorolfine in 0 1.25 2.5 5 10 20 40 80 160 mg/i (x 160) Fungicidal/fungistatic activity (procedure without subculturing) 15 Fungicidal/fungistatic activity (procedure with subculturing) The conclusions drawn were that in the procedure without subculturing the minimum concentration of amorolfine in the aqueous medium which allowed in vitro an activity of fungicidal nature lay between 5 and 10 mg/i. whereas in the procedure with subculturing, which perhaps eliminated a 25 residual anti-fungal effect due to the accumulation of product in the agar not eliminated in the rinsings this minimum fungicidal concentration was higher and lay between 10 and 20 mg/V.

Example 3 in vitro Evaluation of the Fungicidal/Fungistatic Activit y of RS'-1-r3-4-Tert.butvl-Dhenv)-2-methvl-propyl1- Diperidin (fenpropidin) agrainst Fusarium solani Fusarium solari was isolated and tests for antifungal activity on, mycelium and conidiospores and the determination -13of the results were carried out,for fenpropidin by analogous procedures to those described in Example 1 hereinbefore for fenpropinmorph. The appropriate results are presented in the following table: *0 S 15 e, si a* Concentration of Percentage activity fenpropidin Test 1 Test 2 in il1/1 (average total (average total surface area of surface area of control cultures control cultures 40.4 cm 37.2 cm 6.25 84 86 12.5 95 94 25 100 100 50 100 100 Conclusions from the in vitro study The activity of fenpropidin remains very high even at the lower concentrations tested. On the basis of a ss* "fungicidal" activity fenpropAdin displays a fungicidal activity at concentrations greater or equal to 25 1/1 on SF. solani. At lower concentrations the activity may be considered to be fungistatic. However. inspite of the absence of regrowth measurable 15 days after the antifungal treatment the inoculum seems to survive on application of antifungal solutions of concentrations 25 and 50 pIl fenpropidin/l in view of the appearance of some off-white points of regrowth. Consequently, a strictly fungicidal Sactivity is realized at concentrations above ca. 25 il fenpropidin/l.

What is indicated above for activity on mycelium and conidiospores is -also valid for activity on conidiospores-'r$ 14 F. solani alone. On non-germinated conidiospores of F. solani the strictly fungicidally effective concentrations of fenpropidin are from 25 il/1. On germinated conidiospores, i.e. "young" mycelium, the activity of fenpropidin is considerably higher.

SExample 4 In a shrimp farm the shrimps are maintained in an extensie or iteensive manner and in both cases are fed during the growing phase 2 or 3 times a day, depending on their growth stage and weight. The shrimps eat slowly and more or less continuously. Shrimps which are in the growth stage of just a few days after the attainment of post-larval 15 stage or those weighing 2-5 g require quite a different amount of feed compared with shrimps weighing 10 g or more, and in addition the pellet diameter of pelleted feed must .o also be chosen according to the growth stage or weight of Sthe shrimps to be fed therewith. Thus. for a shrimp weighing 0.5-2 g a pellet diameter of 1-2 mm is usually acceptable, whereas a pellet diameter of 3-5 mm is generally recommended for shrimps weighing 10 g or more.

0 8, O .i A typical shrimp feed in pelleted form with a pellet diameter appropriate for the growth stage or size of shrimps S" t be fed to shrimps in intensive culture has the following composition: a 0 15 Percent by weight Fish meal 16 SShrimp head meal Squid meal Soybean meal 31 Cereal products or by-products 22-24 Fish oil 4 Soybean lecithin 1 Cholesterol 0.2 Binder 1-3 Dicalcium phosphate 2.25 Vitamin mix Trace mineral mix 0.05 100 s The pellet ed eed is administered by hand or automatic feeder. Shrimps in the post-larval stage normally require an amount of feed which is approximately 25-50% of their body S weight 2 or 3 times a day. This amount may be decreased to about 3% for shrimps in extensive culture. The most effective way to determine the amount feed necessary is to feed the shrimps according to their demand, i.e. by placing sufficient feed in a feeding tray or on a feeding platform.

checking the amount remaining, if any, a few hours thereafter, and decreasing or increasing the next amount of feed appropriately. The required daily feed allowance is also indicated by the activity and behaviour of the shrimps. For example, underfeeding is indicated if one or two hours after &too' feeding a significant number of shrimps tend to remain at the edge of the pond or tank.

For the treatment of fungal infections caused by S Fusarium spp. the feed composition given above was augmented with 0.0001-0.1% preferably 0.003-0.01 by weight of a compound of the formula I hereinabove or a salt thereof, e.g. cis-4-[3-(4-tert.butyl-phenyl)-2-methyl-propyl] -2,6-dimethylmorpholine (fenpropimorph), rac-cis-4-{3- 16 -[4-(1,1--dimethylpropyl)-phenyl] -2-methyl-propyll-2,6dimethylmorpholine hydrochloride (amorolfine) or (RS)-1-3-(4-tert.butyl-phenyl)-2-methyl-propyl]piperidin (fenpropidin). An amount of such medicated feed was administered ffonce or twice during a day according to the demand of the shrimps. and this was repeated after three weeks, and once again after a further three weeks.

it was consequently established that shrimps provided with such medi4cated feed remained substantially free of fungal infections caused by Fusarium spp.

0 *0 0 0 0 0 *wo

Claims (22)

1. A method of controlling fungal infections of animals ,in aquaculture caused by the genus Fusarium spp., characterized by applying as a fungicidal or fungistatic iagent a compound of the formula R CH, r I CH (CH 2 0 DCH 2 -H CH 2 CH 3 "3 R wherein n signifies 0 or 1 and X signifies oxygen and R methyl, or X signifies methylene and R hydrogen, or a salthereof to theefto e animals in aquaculture.

2. ,A method according to claim 1, characterized in that in formula I X signifies oxygen and R methyl. 2

3. A method according to claim 2. characterized in that .the compound of formula I is cis-4-[3-(4-tert.butyl-phenyl)- -2-methyl-propyl] -2.6-dimethylmorpholine.

4. A method according to any preceding claim, characterized by applying the compound of formula I or a salt thereof in undiluted form.

5. A method according to any one of claims 1 to 3. characterized by applying the, compound of formula I or a o salt thereof in association with inert solid or liquid diluting medium.

6. A method according to claim 5, characterized in that the diluting medium consists of feed for fish or crustacea wherein the concentration of active ingredient is in the range 0.003 to 0.01 percent by weight. 18

7. A method-according to any preceding claim, characterized in that the compound of formula I or a salt thereof is applied at a rate of from 1 to 50 ig/l of aquatic environment of the animals to be treated.

8. A method according to any preceding claim, characterized in that the animals to be treated are crustacea.

9. A method according to claim 8, characterized in that the crultacea are members of the genus Penaeids (Penaeus spp.), fresh water prawns (Macrobrachium spp.) or lobsters (Homarus spp.).

10. A method according to claim 9, characterized in that the crustacea are warm water or cold water shrimps of the S species Penaeus aztecus, P. brasiliensis, P. s brevirostris, P. californiensis, P. canaliculatus, P. caramote, P. carinatus, P. duorarum, P. esculentus, P. 20 indicus, P. japonicus, P. kerathurus, P. latisulcatus, P. merguiensis, P. monodon, P. occidentalis, P. orientalis, P. plebejus. P. schmitti, P. semisulcatus. P. setiferus, P. sinensis, P. styliostris, P. trisulcatusP. vannamei, Metapenaeus ensis and M. monoceros. O "11i.

A method according to claim 9. characterized in that the crustacea are fresh water prawns of the species Macrobrachium carcinus, M. nipponense and M. rosenbergii.

12. A method according to claim 9, characterized in that the crustacea are lobsters of the species Homarus americanus, H. gammarus and H. vulgaris.

A method according to any preceding claim, ,characterized in that the control is curative. 19

14. A method according to any preceling claim, characterized in that it is effected for the control of a fungal infection in the post-larval development stage of animals in aquaculture. o

15. A method according to any preceding claim, characterized in that the species of fungus causative of the fungal infection to be controlled is Fusarium solani.

16. A method according to any preceding claim, characterized in that it is effected in a hatchery, a growing pond, a nursery pond or a rearing pond.

17. A method according to any preceding claim, characterized in that it ise carried out at regular intervals every two to four weeks.

18. A fungicidal or fungistatic feed for fish or crustacea, characterized in that it is intended for the 20 control of fungal infections causcd by the genus Fusarium spp. in aquaculture and contains as a fungicidal or 'fungistatic agent an effective amount of at least one compound of the formula 25 C, H C H-CH-CH X CH-. wherein n signifies 1 or 2 and X signifies oxygen and R methyl, or X signifies methylene and R hydrogen, or a salt thereof.

19. A fungicidal or fungistatic feed according to claim 18, characterized in that in formula I X signifies oxygen and R methyl.

20 A fungicidal or fungistatic feed according to claim 18 or 19. characterized in that it contains from 0.003 to 0.01 percent by weight of the compound of formula I or of a sal~t thereof. .0* 0 fK @0 0 0 000 0 0 00 *0 0 0 0006 be 0 0* 00 00 0 000 0 *00000 0 6000 0e 00 0 be 0 0~0 00 *0 0 qee.

21. A method according to claim 1, substantially as described herein with particular reference to Example 4.

22. A fungicidal or fungistatic feed according to claim 18, substantially as described herein with particular reference to Example 4. s Dated 19 July, 1993 F. Hoffmann-La Roche AG Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON OS 0O S 0 0 000 0 0 S 0 S 0000 0* 0 00 S. 0 S S* 0 0 S. S @000' 0S S S S. 0 *00 OSSO 0 OSSS @0 0 5S 05 0 [Pdiv21\OO1 1 2:KEH21o1 21 of 1