Note: Descriptions are shown in the official language in which they were submitted.
~02~ ~~~
-1-
AP/5-17654/=
Method of controlling sea lice
The present invention relates to the compound S-6-chloro-2,3-dihydro-2-oxo-1,3-
oxazol-
[4,~-b]pyridin-3-ylmethyl-O,O-dimethylphosphorothioate known under the common
name
Azamethiphos for controlling sea lice in fish belonging to the group of the
salmonids. The
invention further relates to a method of controlling sea lice in salmonids, to
the use of the
above compound for controlling sea lice in the commercial farming of salmon
and trout,
and to the use thereof for the preparation of compositions for controlling sea
lice.
For the sake of simplicity, the common name Azamethiphos will be used
throughout this
specification instead of the chemical name cited above.
For some years, salmon and trout have been among the most popular edible fish
with
consumers on account of their fine taste and low fishbone content. Demand has
increased
to such a degree that traditional fishing in open waters has for long been
unable to satisfy
it. In recent years, therefore, fish farms have been established, especially
in the Northern
European countries, with the object of artificially breeding these fish.
The term "salmon" within the scope of this invention will be understood as
comprising all
representatives of the family Salmonidae, especially of the subfamily
Salmonini and,
preferably, the following species: Salmon salar (Atlantic salmon); Salmon
trutta (brown or
sea trout); Salmon gairdneri (rainbow trout); and the Pacific salmon
(Oncorhynchus): O.
gorbuscha; O. keta; O. nekra; O. kisutch; O. tshawytscha and O. mason. Also
comprised
are artificially propagated species such as Salvelinus species and Salmo
clarkii.
Preferred objects of the present invention are the Atlantic and Pacific salmon
and the sea
trout.
In present-day salmon and trout farming, juvenile fish are transferred in the
smolt stage
from fresh-water tanks to sea water cages. These latter are cubic, rectangular
or also round
cages having a metal frame which is covered with a fairly fine-meshed net.
These cages
are lowered into the sea until they are 9/10 submerged and then so anchored
that they are
_2_
accessible from the top.
In another variant, the fish are kept in sea water tanks of different shape.
The cages are
moored in sea inlets such that a constant flow of water passes through them in
order to
ensure a sufficient supply of oxygen. A constant flow of salt water in the sea
water tanks is
also maintained along with a supply of oxygen. In this artifical environment
the fish are
fed and, if necessary, provided with medication until they mature sufficiently
for
marketing as edible fish or are selected for further breeding.
Extremely intensive cage stocking is maintained in these fish farms. The fish
density
reaches orders of magnitude of 10 to 25 kg of fish/m3. In this pure
monoculture, the
exceedingly high fish densities coupled with the other stress factors cause
the caged fish to
become in general markedly more susceptible to disease, epidemics and
parasites than
their free-living cospecifics. In order to maintain healthy populations, the
caged fish must
be treated regularly with bactericides and permanently monitored.
Besides infectious diseases, the prime threat in commercial salmon farming is,
however,
attack by parasites. In particular, two representatives of the class of
Crustaceae
(crustaceans) cause substantial losses in yield. These parasites are popularly
known as sea
lice. The one species is Lepeophtheirus, L. salmonis; the other is Caligus, C.
elongatus.
They are easily recognised. The former has a brown, horseshoe-shaped dorsal
shell; the
latter is also bxown, but much smaller. These sea lice injure the fish by
feeding on the
scales, epithelium and the mucosa. When infestation is severe, these parasites
also damage
underlying demos. If, moreover, infected salmon are kept in cooler waters,
then they are
normally no longer able to protect themselves from these pests. As a
consequence,
secondary infections and water-logging will occur, even if the sea lice are
removed. In
extreme cases, severe wounding resulting from infestation by these parasites
leads to
further tissue damage caused by ultraviolet radiation (McArdle & Bullock,
1987) or to the
death of the fish from osmotic shock or the secondary infections (Saward et
al., 1982;
Tally, 1988a).
Sea lice are meanwhile widely prevalent and encountered in all fish farms.
Severe
infestation kills the fish. Mortality rates of over 50%, based on sea lice
infestation, have
been reported from Norwegian fish farms (Needham, 1978, cited in Steward et
al., 1982).
The extent of the damage depends on the time of year and on environmental
factors, for
example the salinity of the water (Rae, 1979) and average water temperature
(Tally,
~~~~~~r
-3-
1988b). In a first phase, sea lice infestation is seen in the appearance of
the parasites
attached to the fish and later - even more clearly - from the damage caused to
skin and
tissue. The most severe damage is observed in smolts which are just in the
phase in which
they change from fresh water to sea water. The situation is made even worse by
the
specific conditions in the fish farms, where salmon of different age groups
but of the same
weight class are kept together; where fouled nets or cages are used; where
high salt
concentrations are to be found; where flow through the nets and cages is
minimal and the
fish are kept in a very nan-ow space.
Fish farmers who are confronted with this parasite problem have to suffer
substantial
financial losses and to carry additional expense. On the one hand, their fish
are debilitated
and damaged by the lice, resulting in lower rates of growth increase, and on
the other,
secondary infections have to be controlled with expensive medicaments and
labour-intensive measures. The fish can often no longer be sold, as the
consumer will
reject the damaged hoods. This parasitic infestation can pose a threat to the
salmon
farmer's livelihood.
The worst damage is caused by Lepeophtheirus, as even a few parasites cause
widespread
tissue damage. The life cycle of Lepeophtheirus consists substantially of two
free-swimming larval stages (naupilus and copepodid stages), four chalimus
stages, a
pre-adult stage and the actual adult stage (Institute of Aquaculture, 1988).
The chalimus,
pre-adult and adult stages are host-dependent.
The most dangerous stages, because they cause the greatest damage, are all
those
parasiticising on the fish, especially the actual adult stages.
Pest control agents which can be used to combat sea lice are commercially
available, for
example Trichlorfon (dimethyl-2,2,2-trichloro-1-hydroxyethylphosphonate),
which
requires concentrations of 300 ppm in sea water, and Dichlorphos (2,2-
dichloroethenyl
dimethyl phosphate), which is effective from a concentration of 1 ppm. The
shortcoming
of these compounds is, however, the high concentrations in which they have to
be used
and the ecological problems associated therewith, which are of even greater
consequence
on account of the high half-life times.
Surprisingly, in Azamethiphos, S-6-chloro-2,3-dihydro-2-oxo-1,3-oxazo1~4,5-
b]pyridin-3-
ylmethyl-O,O-dimethylphosphorothioate, a representative of an entirely
different class of
2
-4-
compounds, a substance has been found which, while having very low toxicity to
fish, is
even more effective and, in particular, whose photolytic and hydrolytic
degradability is
more rapid by about a factor of 3 as compared with the known sea lice control
agents and,
furthermore, which can be successfully used against all pre-adult and adult
stages of sea
lice on fish.
Thus, for example, in direct comparison with the known control agents
mentioned above,
Azamethiphos is still fully effective (100% control) in sea water having a
salinity of
23-33% at the low concentration of 0.1 ppm, i.e. with 10 times less than
Dichlorovos and
3000 times less than Trichlorfon. Furthermore, the in vitro test in the
temperature range
from 4 to 15°C shows that Azamethiphos is 100% effective at a
concentration of 0.1 ppm
even within 1 hour, whereas it is necessary to use the 20-fold amount of
Dichlorvos and
the more than 1000-fold amount of Trichlorfon. Moreover, Azamethiphos is
markedly
more effective against the very resistant chalimus stages, so that the number
of
applications can be reduced.
A further advantageous property of Azamethiphos is that, at the proposed
concentrations,
other marine animals such as lobsters, oysters, crustaceans (except the sea
lice), fish and
marine plants do not suffer injury. The degradation products of Azamethiphos
are in any
case non-injurious to marine fauna and flora.
Azamethiphos and its preparation and activity against representatives of
insects and
arthropods is disclosed in German Offenlegungsschrift 2 131734. There are,
however, no
hints in scientific or patent literature that Azamethiphos might also be
outstandingly
effective against representatives of the class of the Crustaceae.
Compared with other organophosphorus insecticides, Azamethiphos is very
readily
soluble in water and can therefore be used undiluted. More easy to handle,
however, are
compositions containing the compound in dilute form. Suitable diluents for
fish and other
marine animals and plants are non-toxic substances which may be liquid or
solid and,
directly prior to the use of this invention, also water.
For ease of handling, the size of the commercial packs is such that they can
be added
undiluted to specific volumes of water. The size of the packs is governed by
the average
dimensions of the cage, so that packs can, for example, be provided for
addition to 10 m3,
50 m3, 100 m3, 500 m3 or 1000 m3 of water. Thus, for example, for addition to
a cage of
520 m3, it would be possible to combine suitable unit dose packs, in this case
(1 x 500)
and (2 x 10).
For use in actual practice, foils are also suitable which contain the pest
control agent in a
readily water-soluble matrix.
The unit dose packs contain Azamethiphos, undiluted or in cc>njunetion with
non-toxic
diluents, in a formulation ready for addition to specific volumes of water.
The packs
contain Azamethiphos in a concentration of 0.005 to 2 ppm, more conveniently
from 0.01
to 1 ppm and, most preferably, from 0.05 to 0.5 ppm, when they are added to a
specific
volume of water.
Useful concentrations are in the range from 0.005 to 2 g AS/m3, preferably
from 0.01 to
1 g AS/m3, more particularly from 0.05 to 0.5 g AS/m3 (AS = active substance).
The dilute fornmlations of this invention are prepared by mixing the active
substance with
liquid and/or solid formulation assistants by stepwise mixing and/or grinding
such that the
ready for use formulation so obtained exerts an optimum antiparasitic
activity.
The formulation steps can be extended to include kneading, granulating (to
granular
formulations) and, if appropriate, compressing (to pellets, tablets).
Suitable formulation assistants are, typically, solid carriers, solvents and,
where
appropriate, surfactants, which are non-toxic to marine flora and fauna.
The following formulation assistants are used for preparing the formulations:
solid carriers
such as kaolin, talcum, bentonite, sodium chloride, calcium phosphate,
carbohydrates,
cellulose powder, cotton seed meal, polyethylene glycol ether, if necessary
binders such as
gelatin, soluble cellulose derivatives, if desired with the addition of
surface-active
compounds such as ionic or nonionic dispersants; also nattu~al mineral fillers
such as
calcite, montmorillonite or attapulgite. To improve the physical properties it
is also
possible to add highly dispersed silicic acid or highly dispersed absorbent
polymers.
Suitable granulated adsorptive carriers are porous types, for example pumice,
broken
brick, sepiolite or bentonite; and suitable nonsorbent carriers are materials
such as calcite
or sand. In addition, a great number of pregranulated materials of inorganic
or organic
nature can be used, e.g. especially dolomite or pulverised plant residues.
-6-
Suitable solvents are: aromatic hydrocarbons, preferably the fractions
containing
8 to 12 carbon atoms, e.g. xylene mixtures or substituted naphthalenes,
phthalates such as
dibutyl phthalate or dioctyl phthalate, aliphatic hydrocarbons such as
cyclohexane or
paraffins, alcohols and glycols and their ethers and esters, such as ethanol,
ethylene glycol,
ethylene glycol monomethyl or monoethyl ether, ketones such as cyclohexanone,
strongly
polar solvents such as N-methyl-2-pyrrolidone, dimethyl sulfoxide or dimethyl
form-
amide, as well as vegetable oils or epoxidised vegetable oils such as
epoxidised coconut
oil or soybean oil; and water.
Depending on the type of formulation, suitable surface-active compounds are
non-ionic,
cationic and/or anionic surfactants having good emulsifying, dispersing and
wetting
properties. The term "surfactants" will also be understood as comprising
mixtures of
surfactants.
Suitable anionic surfactants can be both water-soluble soaps and water-soluble
synthetic
surface-active compounds.
Suitable soaps are the alkali metal salts, alkaline earth metal salts or
unsubstituted or
substituted ammonium salts of higher fatty acids (Cto-C22)> for example the
sodium or
potassium salts of oleic or stearic acid, or of natural fatty acid mixtures
which can be
obtained, for example, from coconut oil or tallow oil.
Frequently so-called synthetic surfactants are used, especially fatty
sulfonates, fatty
sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates.
The fatty sulfonates or sulfates are usually in the form of alkali metal
salts, alkaline earth
metal salts or unsubstituted or substituted ammonium salts and generally
contain a Cg-C22-
alkyl radical which also includes the alkyl moiety of acyl radicals, e.g. the
sodium or
calcium salt of lignosulfonic acid, of dodecylsulfate, or of a mixture of
fatty alcohol
sulfates obtained from natural fatty acids. These compounds also comprise the
salts of
sulfated and sulfonated fatty alcohol/ethylene oxide adducts. The sulfonated
benzimida-
zole derivatives preferably contain 2 sulfonic acid groups and one fatty acid
radical
containing about 8 to 22 carbon atoms. Examples of alkylarylsulfonates are the
sodium,
calcium or triethanolamine salts of dodecylbenzenesulfonic acid,
dibutylnaphthalene-
sulfonic acid, or of a condensate of naphthalenesulfonic acid and
formaldehyde. Also
suitable are corresponding phosphates, e.g. salts of the phosphated adduct of
p-nonyl-
~~2~~~~:
phenol with 4 to 14 moles of ethylene oxide.
Non-ionic surfactants are preferably polyglycol ether derivatives of aliphatic
or
cycloaliphatic alcohols, or saturated or unsaturated fatty acids and
alkylphenols, said
derivatives containing 3 to 30 glycol ether groups and 8 to 20 carbon atoms in
the
(aliphatic) hydrocarbon moiety and b to 18 carbon atoms in the alkyl moiety of
the
alkylphenols.
Further suitable non-ionic surfactants are the water-soluble adducts of
polyethylene oxide
with polypropylene glycol, ethylenediaminopolypropylene glycol and
alkylpolypropylene
glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts
contain
20 to 250 ethylene glycol ether groups and 10 to 100 propylene glycol ether
groups. These
compounds usually contain 1 to 5 ethylene glycol units per propylene glycol
unit.
Representative examples of non-ionic surfactants are
nonylphenolpolyethoxyethanols,
castor oil thioxilate, polypropylene/polyethylene oxide adducts,
tributylphenoxypoly-
ethoxyethanol, polyethylene glycol and octylphenoxypolyethoxyethanol. Fatty
acid esters
of polyoxyethylene sorbitan, e.g, polyoxyethylene sorbitan trioleate, are also
suitable
non-ionic surfactants.
Cationic surfactants are preferably quaternary ammonium salts which contain,
as N-substi-
tuent, at least one C8-C22alkyl radical and, as further substituents,
unsubstituted or
halogenated lower alkyl, benzyl or hydroxy-lower alkyl radicals. The salts are
preferably
in the form of halides, methylsulfates or ethylsulfates, e.g.
stearyltrimethylammonium
chloride or benzyl bis(2-chloroethyl)ethylammonium bromide.
The surfactants customarily employed in the art of formulation are described
e.g. in the
following publications:
"Mc Cutcheon's Detergents and Emulsifiers Annual", MC Publishing Corp.,
Ridgewood,
NJ USA, 1981",
Helmut Stache, "Tensid-Taschenbuch"(Handbook of Surfactants), 2nd. ed., C.
Hanser
Verlag MunichNienna 1981.
Suitable binders for water-soluble granules or tablets are chemically modified
polymeric
natural substances which are soluble in water or alcohol, for example starch,
cellulose or
protein derivatives (e.g. methyl cellulose, carboxymethyl cellulose, ethyl
hydroxyethyl
cellulose, proteins such as zero, gelatin and the like) as well as synthetic
polymers such as
polyvinyl alcohol, polyvinyl pyrrolidone and the like. Tablets also contain
fillers (e.g.
starch, microcrystalline cellulose, sugar, lactose and the like), glidants and
disintegrators.
The application of the compositions of this invention to the parasites can be
made by
introducing the compositions in the form of solutions, emulsions, suspensions
(drenches),
powders or tablets to the cage, where they are rapidly dissolved and
distributed by the
movement of the fish and the water. Concentrated solutions can also be diluted
before
addition to cages containing larger volumes of sea water. Concentration
problems in the
pens do not arise, as the fish thresh about vigorously in expectation of feed
each time the
cage is opened and so ensure rapid dilution.
The antiparasitic compositions normally contain 0.1 to 99 %, preferably 0.1 to
95 %, of
Azamethiphos, and to 99.9 % to 1%, preferably 99.9 to 5% by weight, of a solid
ox liquid
adjuvant, and 0 to 25 %, preferably 0.1 to 25 %, of a surfactant.
Whereas commercial products are preferably formulated as concentrates, the end
user will
normally employ dilute formulations obtainable by diluting the commercial
product with
water.
The compositions can also contain further ingredients such as antifoams,
preservatives,
viscosity regulators, binders, tackifiers and fertilisers or other chemical
agents to obtain
special effects.
Formulation Examples (throughout percentages are by wei ht
F1. Emulsifiable concentratesa) b) c)
Azamethiphos 25 % 40 % 50
%
calcium dodecylbenzenesulfonate5 % 8 % 6
%
castor oil polyethylene
glycol
ether (36 mol of ethylene5 % - -
oxide)
tributylphenol polyethylene
glycol
ether (30 mol of ethylene- 12 % 4
oxide) %
cyclohexanone - 15 % 20
%
xylene mixture 65 % 25 % 20
%
~o~o~~~
_9_
Emulsions of any required concentration can be produced from' such
concentrates by
dilution with water.
F2. Solutions a) b) c) d)
Azamethiphos 80 % 10 % 5 % 95 %
ethylene glycol monomethyl20 % - - -
ether
polyethylene glycol 400 - 70 % - -
N-methyl-2-pyrrolidone - 20 % - -
epoxidised coconut oil - - 1 % 5 %
ligroin (boiling range - - 94 % -
160-190)
These solutions are suitable for application in the form of microdrops.
F3. Granulates a) b)
Azamethiphos 5 % 10
kaolin 94 % -
highly dispersed silicic acid 1 %
attapulgite - 90 %
The active substance is dissolved in methylene chloride, the solution is
sprayed onto the
carrier, and the solvent is subsequently removed by evaporation under vacuum.
Such
granular formular formulations can be mixed with the feed.
F4. Dusts a) b)
Azamethiphos 2 % 5 %
highly dispersed silicic1 % 5 %
acid
talcum 97 % -
kaolin - 90 %
Ready for use dusts are obtained by intimately mixing the carriers with the
active
substance.
- 10-
F5 -Water dispersible powder a) b)
mixture c)
(I) Azamethiphos 25 % 50 75
% Jo
sodium ligninsulfonate 5 % 5 % -
oleic acid 3 % - 5
%
sodium diisobutylnaphthalenesulfonate- 6 l0 10
%
octylphenol polyethylene
glycol ether
(7-8 mol of ethylene oxide)- 2 % -
highly dispersed silicic 5 % 10 10
acid %
kaolin 62 % 27 -
%
a) b) c) d)
(Ia) Azamethiphos 5% 10% 20% 50%
sugar 95% 90% 80% 50%
d)
(Ib) Azamethiphos (tech. 53.8%
ca. 53.8%)
condensate of fom~aldehyde
and the sodium salt of
naphthalenephenolsulfonic
acid 4%
fatty alcohol sulfates and
alkylarylsulfonates 2%
kaolin 16l0
sodium aluminium silicate 24.2l0
(Ic) Azamethiphos 50%
sodium lauryl sulfate 0.5%
dispersant H granulate 2%
kaolin 16%
silica
31.5%
The active substance is thoroughly mixed with the adjuvants and the mixture is
thoroughly
ground in a suitable mill, affording wettable powdeis which can be diluted
with water to
give suspensions of any desired concentration.
-11- ~~2~a~
F6. Emulsifiable concentrate a) b) c)
Azamethiphos 10 % 8 % 60 %
octylphenol polyethylene glycol
ether
(4-5 mol of ethylene oxide) 3 % 3 % 2 %
castor oil polyglycol ether
(36 mol of ethylene oxide) 4 % ~ % 4 %
cyclohexanone 30 % 40 15
%
xylene mixture 50 % 40 1~ %
%
Emulsions of any required btained from
concentration can be o this concentrate
by dilution
with water.
F7. Dusts a) b)
Azamethiphos 5 % 8 %
talcum 95 % -
kaolin - 92 %
Ready for use dusts are obtained by mixing the active ingredient with the
carrier, and
grinding the mixture in a suitable mill.
F8. Granulate
Azamethiphos 10 %
sodium ligninsulfonate 2
carboxymethyl cellulose 1 %
kaolin 87 %
The active substance is mixed and ground with the adjuvants, and the mixture
is
subsequently moistened with water. The mixture is extruded, granulated and
then dried in
a stream of air.
F9. Granulate
Azamethiphos 3 %
polyethylene glycol 200 3 %
kaolin 94 %
-12-
The finely ground active substance is uniformly applied, in a mixer, to the
kaolin
moistened with polyethylene glycol. Non-dusty coated granulates are obtained
in this
wanner.
F10. Suspension concentrate
Azamethiphos 40 %
ethylene glycol 10 %
nonylphenol polyethylene glycol
ether
(15 mol of ethylene oxide) 6 %
sodium ligninsulfonate 10 %
37 % aqueous fom~aldehyde 0.2
solution
silicone oil in the form of
a 75 %
aqueous emulsion 0.8 %
water 32 %
The finely ground active substance is homogeneously mixed with the adjuvants,
giving a
suspension concentrate from which suspensions of any desired concentration can
be
obtained by dilution with water.
The use of Azamethiphos for the preparation of a composition for controlling
Crustaceae,
especially sea lice, which parasiticise on fish, is an object of this
invention.
Biological Examples
1. Toxicity to salmon lice (in vitro test)
a) Collecting and cultivating the salmon lice
Adult and pre-adult stages of the salmon louse are gently removed with broad
forceps
from naturally infected Atlantic salmon which have been kept in fish farms,
separated
according to stage and sex, and kept in sea water tanks at 10°C and
under continuous
aeration. The sea water used for cultivating the lice comes from the fish farm
from which
the infected salmon have been taken. The tests themselves are carried out over
4$ hours
after collecting the lice.
b) In vitro test for determining the contact action of the control went
Plastic containers are filled with 50 ml of sea water (10°C). Into each
container are put 5
female and 5 male adults as well as 5 pre-adult salmon lice. The sea water is
rapidly
~~~~~~t~e
-13-
decanted through a sieve and replaced by SO ml of the test solution (sea water
of 10°C
containing the test compound). The lice are treated in this solution for 1
hour, as this
corresponds more or less to the conditions in the fish pens. Each container is
then flushed
with fresh sea water and the lice are kept in fresh sea water. The test is
evaluated by
making a mortality count of the lice in accordance with sex, stage and
concentration of
test compound. The count is repeated hourly until there are no more lice
surviving. All test
are carried out in triplicate.
bl) Ranae-finding test
The lice are treated according to b) with active substance concentrations of
0.001 to 1.0
ppm for a period of 1 hour, and the mortality rate is determined by counting
the dead
parasites. Mortality is found to be 100% at 0.1 ppm after 1 hour, and at 0.01
ppm after 2
hours. Below 0.01 ppm, the parasites survive for longer than 24 hours.
b2) Effect of temperature and salinity on the toxicity of the active substance
The effect of temperature is determined at values from 4° to
16°C at an active substance
concentration of 0.01 ppm and a treatment time of 1 hour. The following
results are
obtained:
Temperature [°C] 4 8 10 12 16
parasite mortality [%] 68 60 57 65 78
Above and below 10°C a slight increase in mortality is observed. In
contrast, an increase
in the salinity from 23% to 30%C at the same concentration of active substance
and for
the same treatment time has no significant effect on mortality.
2. Toxicit~gainst salmon lice (in vitro test)
Five naturally infected Atlantic salmon are taken from the pen and transferred
to well
aerated sea water tanks. They remain there for 48 hours for acclimatisation
and feed is
withheld for 24 hours before the addition of test compound. A group of 5
salmon is treated
for 1 hour at a concentration of 1.0 ppm of test compound, and a second group
of 5 salmon
is treated at a concentration of 0.1 ppm. The fish are kept for 24 hours in
fresh sea water
(without test compound) and a count is then made of dead and still living
parasites. An
untreated group of fish is also included in the evaluation. The test is
carried out in
2~2Q~~z~
- 14-
triplicate.
The evaluation shows that all adult and pre-adult stages are killed at both
concentrations
of 0.1 and 1.0 ppm. Thus in the in vitro and in vivo tests, matching results
are obtained
with the test compound.
