CA1045545A - Biocidal compositions - Google Patents

Abstract

ABSTRACT

Biocidal compositions comprising certain quaternary ammonium compounds and 1,2-benzimothiazolin-3-ones. The combination has a greater biocidal activity than would be expected from either component alone. Also methods for protecting aqueous media against infection by micro-organisms by addition of the said compositions to the media, and methods for protecting paint films and wood against fungal attack using the said compositions as anti-fungal agents.

Description

- Dx.26110 ~ .-,, ., j 04S5~S

..i j ~hiB invention relates to blocidal compo6itions and '' more pRrtioularly to combinations and complexes of lunown biocide6 having improved effectivenes6.
UK Patent Speeirication No. 884,541 relates to a process for the protection of ~queou~ media against infectionby mioro-organisms by adding to the aqueou6 media a 1,2-benziso-thiazolin-3-one which may be substituted in the benzene ring .I by chlorine or brominet or salts thereof.

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Dx.26110 ~04SSgLS
Also known to have biocidal activity iare quaternary ammonium compvunds in which one at least of the substituent6 on the nitrogen atom i6 a long~chain alkyl group or an alkyl phenoxyethoxyethyl group.
It has now been ~ound that ,certain 192-benz isothiazolin~3-one - compound6 and quaternary a~m~nium compounds in admixture - are particularly e~fective bioci~esO
According to the pre~ent invention there are provided ::
. . . - _ .... - . . .
blocldal compositions comprising: -A(i) a quaternary ammonium compound of the general ~ormula R R1R2R3N + X Formula I
wherein R represents a C10_C22 alkyl group or a C1- Cg alkylphenoxyethoxyethyl group, or a phenoxyethyl group~

¦ R1 repre8ent6 a C10- C22 alkyl group, a C1- C4 alkyl ,~ ~5 group or a benzyl group~
., .~ R2 and R3 each independently represent a C1 - C~ alkyl group and -X~ repre6ents an anion ~, ~
or (ii~ a quaternary ammonium compound of the general formula .

.
~ - 4 x Formula II

wherein R4 represents an optionally substituted C~O - C22 alkyl group :~
represent6 an an~on,~
,~, , . ~ .
~ 3 _ . :

''~': ; , , ' ': ~
, , .. - . - , ., . ., .. ; . . . . . . . . .. .. . . .

lO~;L5545 Dxo261 10 ~- ~` and wherein the heterocyclic nucleu~ may be fu6ed with a benzene nucleus adjacent to the nitrogen atom and which may carry an amino substituent; and B a ~ubstituted or unsub~tituted 1~2~benz i60thiazolin-3-one oo~po~md having the gener~l formula ~ ao NE
~J\S/ Formula III
!
~herein the benzene nucleus may carry substi~uents 6elected from halo~en atom~ C1-C4 aIkyl groups, C1-Cl~ alkoxy groups, the nitro group and the cyano ~roup.
; 10 ~8 examples of the alkyl ~roups whioh are represented by R and R1 in the compou~ds of Formula I there may be mentioned the dodeoyl group and the hexadecyl group.
As an example of the alkylphenoxyethoxyethyl group which R represents there may be mentioned the p-octylphenoxyethoxy ethyl grou~.
The ~roups R2 and R3 ~ay be, for example, ethyl, propyl, isopropyl or the lsomeric butyl groups but are pra~erably methyl '' groups.
The groups R4 in the compound~ o~ Formula II may be~ ~or example~ a dodecyl group or a hexadeoyl group~ and as an example o~
j a substituent whioh ma~ be prese~t in the group R4 there may be mentioned a substituted or unsubstituted pyridinium radical as defined in ~ormula II.
As example~ of the anions which are represented by X there may be mentioned the ohloride, bromide, acetate or methosulp~ate ion~
Speoi~ic examples of the quatern~ry ammonium compounds o~
Formula I and II are dodec~l benæ~l dimethyl ammorium chloride~

:

;. ~. :. , ,: ~ :~: ,:.:, .,.,:: , : .,, , , , ,: . ; ,. . , : , .. . . . ...

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DX fff 26110 ~LO~S5~5 - hexadecyl trimethyl a~nonium bromide, hexadecyl pyridinium chloride, f4-ar~noquinaldinium dodecyl acetate~f didecyl dimethyl ammonium chloride and dioctyl dimethyl ammo~it~ chloride. The preferred compound is a dodecyl (lauryl) benzyl dimethyl ammonium salt~
g As examples of 1,2-benz isothiazolin-3-one6 which ma~ be emplo~ed to~ether with the quaternary ammonium compounds of Formulae I and II there may be mentioned 1~2-benz isothiazolirt-3-one itself -~- and the 5- and 6-chloro and 5-methyl derivatives thereo~. -The molar ratio of substituted or unsubstituted 1,2-benz isotbiazolin-3 one as bereinbefore described, to qt~ternary ammonium compound in the compositions of the pre6ent invention may be varied widely, but will eenerally be in the rAngef 1.0 : 0.04 ~o 1.0 : 1Ø
The combination of the quaternary aMmonit~ compound and the benzisothiazolinone has a biocidal actiYity greater than that lfrhich would be expected as the sum of the aotivities of the indiYidual components.
It i-g preferred tbat the composition co~prising the quaternary a~monium compound and the benzisothiazolinone is in the form of a complex of the two compounds in which there is one benzisothiazolinone group for each quaternary ammoni-un group i~ the quaternary~ammom um compoùnd.
~I These comple~eff~ according to the present invention may be prepared by mixing a~ aqueous solution of an alkali metalt pre~erably odium, salt of~the benz i60thiazolone with an aqueous solution of 25f the quaternary a~moniuIf compound in 6toichiometric proportion~f. The oomplex~6eparat6s ~d may be~purified by~dissolving it i~ an organic solvent~ 6eparRting an~ insolubl6~impurities, wash;n~-the ~olution ;~
f ~ 5 -~, ~
~;
~;

, Dx.26110 ~6~45545 ! with water, drying the solution and removing the solvent by evaporation or distillation under reduced pressure.
'~he biocidal compositions of the present invention find I application as bactericides and un~icides, p~rticularly in aqueous ~edia, for example~ tYater-oil emulsions, in-can preservation of water-based paints and adhesives and in coolinO water systems; also ; as paint-film fungicides and in preventing fungal attack on woodO
The compositions are also effective in the presence of anionic compounds - for example, soap, which are not compatible with quaternary ammonium - 10 biocides alone.
Thus the present invention also provides a method Por p~r~cring~
aqueous media against infection by micro-organi~ms, and ~ontrolling or preventing the proliferation o~ micro-or~anisms in aqueous media already lnfeoted thereby~ which comprises adding to the aqueous media from 1 to 1,000 part~ per million of a biocidal composition comprising in admixture a quaternary amm~nium compound and a substituted or unsllbstituted 1,2-benzlsothiazolin-~one as hereinbefore described.
A further feature of the invention provides a method for the protectior. of paint ~ilms against fungal attack wherein there is added to the paint before its application to a surface, a biocidal composition comprising in admixture a quaternar~ ammonium compoumd and Q substituted or unsubstituted 1~2-benzisothiazolin-~-one a~
~herei~before desoribed, in amount to provide a concentration in the .. .
in the paint o~ 500 to 10,000 parts per million.

A still ~urther featura of the invention provides a method for the protection of wood against iungal attack wherein the wood (, iR treated with a solution of a composition comprisin~
. ',~ ' . ' ' .
~;
. ~ ~

.. . , . , , , . ~, ~ ", . , . .. , , ,, .. ", . ... ..

104554S DX . 26110 ~ in admixtule a quaternar~ ammonium - compound and a substituted or unsub&tituted 1,2-benzisothiazolin-3-one a6 hereinbefore described9 the solution containing frol~ 50 to 20,00~ paste per nillion of the compo&itionO
The treatment of the wood may be by dipping, brushin~ or ~praying with a solution of the composition~ in which case the solution may be aqueous or substantially aqueous. For example, a 50~ 601utio~ of the composition in propylene ~lycol may be added to ~ater to give a substantially aqueous solution of the complex having the desired stren~th. The wood fiO treated may then be I allowed to dry at normal temperatures or the dr~ing may be accelerated i by heatin~ the treated wood for some time at an elevated temperature, for example, up to 50C for 6everal hour60 Alternatively the wood may be treated by vacuum or pressure impre~nation, in which case the composition may be applied from a non aqueous rnedium, for example, a hydrocarbon mediu~l such as toluene~
petroleum spirit or mixtures thereof.
'rhe invention i8 illu~trated but not limited by the following Fxamples in which parts and percenta~efi are by wei6ht Ullle~8 otherwise 5tated~ the ratio of parts by wei~ht to parts by volume being that of the kilogram to the litre.
, ~a~ .
~To 6.o6 parts of 1~2-benzisothiazolin-~-one is added 40.62 parts of a solution of sodium hydroxide prepared ~y di3solving

2~056 p~rt~ o~ sodium hydroxide in 50 parts of wateri 7~
` 1,;:
.~r .~ !

1045S45 DX, 26i10 The rnixture is warmed to effect solution and there i~ then added, with stirring, 28.10 parts of a 48.596 aqueous solution of lauryl benzyl dimethyl ammonium chlorideO An immediate phase separation occurs and the two-phase system i8 warmed for 2 hours at 90-100C.
The lower layer (20.09 parts) is separated and is dried overnight in a vacuum oven at room temperature. The product is dissolved in 250 parts of benzene and the solution i8 heated under reflux using a Dean and Stark separator to . .
- 10 remove water from the refluxin~ mixture. The benzene solution is cooled and filtered. The filtrate is washed with four separate portions of 50 parts of water and is then dried over anhydrous magnesium sulphate. The drying agent is removed by filtration and the benzene solution is evaporated to dryness under vacuum. The residual product, 16.7 parts, containg 0.1~6 of chloride ion and it~i infra-red spectrum indicates the prosence of both benzisothiazolin-3-one and quaternary ammonium components.
.- . ~ .
Comparison between the bactericidal activity o~ 1.2-.
benzisothiazolin-3-one ~BIT) and the complex from the latter compound and lauryl benzyl dimethyl ammonium chloride (LBDAC).
1 ml. of an overnight broth culture of the baoterium .. . . .
, Pieudomonas aeru~inosa is introduced into each of 100 ml 25 Yolume6 of test solutio;ns of the biocides in 250 mlO conical flasks, at concentrations oIn 1000, 500, 250 and 100 p.p.m.
'., ' respectiYely.
, ` , . :; . , - .
~ . . , ,.,,, :
,8~

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s Dx. ~6110 ;iS9LS
.
The test solutions are prepared from a soIution of 1.0 g.
of the biocide in 100 ml. methanol/water 1 : 1 v/v by dilution with water to give a test solution of the desired strength.
Thus a test solution containins 1000 p.p.m. of biocide also contains 5~ methanol, and lower concentrations of bioclde proportionally less methanol.- ~ach culture is then incubated on a rotary shaker at 25 C and samples are removed a~ter 2 '~ hours and 6 hours in order to determi~e the number o~ surviving - cells. Result~ are as follows:-Concentration Survivins_~ells/ml.,after:
~,' of biocide (p p.m.) 2 hours 6 hours BIT 1000 8.0 x 106 0 5 ~ 3.0 x 107 0 250 ~ 3.0 x 107~ 3.0 x 107 100 ~ 3.0 x 107~ 3.0 x 107 Equimolecular1000 0 0 complex of BIT and LBDAC 500 0 0 ~~ ~ 100 1.0 x 103 0

3; ~
, 20 Control , ~ ` (no biocide) 7 3.0 x 1077 3.0 x 107 Control (5~ aqueous methanol) ~73.0 x 107;7 3.0 x 107 It is clear from tkle above that the complex has a ~ bigher bacter1cidal actlvitg than the same amount of 1.2 be~zisothiazolin-~-one alD~e~

Dx.26110 , ' The 5,~6 aqueous methanol control represents the highest concentration of methanol in any of the tests, and shows that ,, the methanol has no detectable effect on the resultfi.
, ~ ample .
~` 5 Comparison of the fungicidal activity of 1.2-benzi60thiazolin-3-orle (BIT) and the complex from the latter compound and lauryl ~enzyl dimethyl ammonium chloride (LBDAC~.
Four 1 cm. square nylon meshes contaminat~d with overnig~ht growth of Aspergillu6 ~G~ are introduced into 100 ml. Yolumes of test solutions of the biocides in 250 ml. conical flasks, prepared as de6cribed in Example 2. Each culture i8 then incubated at 25C on A rotary shaker. Two nylon squares are ~' removed from eaoh fla6k after a contact time of 3 hours and the remainlng two squares after 6 hours. On removal from the solution each nylon 6quare ~ gorously shaken in 10 ml.
, of sterile water for 5 minute6 and tran6ferred on to a malt agar medium in a Petri dish. The latter is incubated for 3 days at 25C, after which the 6urvival or kill of the fungal .,.. '1 : ~ myoelium i6 determined accordi~g to the presence or absence ; 20 of fungal ~rowth on the malt agar medium from the nylon mesh.
Pesults are as follows:-~' , ' .
,. ~.

~' `, ' ' , ~ ' ~ , ~

. ~

~ 45~45 Dx.26110 Concentr~tion Survival of fun ~ s after contact of biocide ti~e of:
(p.p.m.) ~ h~ 6 hour~
BIT 1000 j - -500 _ _ 250 _ _ ' . CoMplex 1000 - - ~
5C0 _ _ ~.
. , .
~. 250 .
. 10 100 _ _ ~` Control - (no biocide) ~++ +++
. Control ~5 Aqueous methanol) +++ ++~

., .
. 15 In the above qualitative assessment +++ indicates vigorous growth, while - indicates total kill. It is clear :~
. `! that the complex has a higher fungicidal activity than 1.2-~ benzisothiazolin-3-one a1one.

¦ ~ 20 : Comparison of the bacteriostatio and fungistatic activity :, 1: -o~ 1.2-benzisothiazolin-3-one (BIT) and the complex ~rom the latter compound and lauryl benzyl-dimethyl ammonium chloride (LBDAC). .
. 1.2 Bénzisothiazolin 3-one and the complex are each dissolved in a 1:1~v~v methanol/water solvent, and the solutions are~:~added to nutrient:agar and to malt agar to : provide conoentratlons of 20, 40, 60, oO, 100 and 200 p.p.m.
of the active ingredien~. Ihe test bacteria, ~scherichia ooli, D~,261 10 1~4S5~5 Stap~loco cus aureus and Pseudomonas g~s~gy~ are streak i~oculated on to the 6urface of nutrient agar in Petri dishes at the concentrations indicated aboYe. I'he te6t fungi Altenaria tenuis, Pullularia pullulans, ~h~~1Ym ~lobo~um, Pol~tictus ver~ic~lor,r~2~,hoderm~ viride and A~e~ n ~er are similarly inoculated (point inoculum) on to the malt agar medium. The nutrient agar plates are - incubated for 1 day at 37C and the malt agar plates are incubated for 7 days at 28C, and then examined for the . _ .,,, -. . -- . .
presence or absence of growth~ Results are as follows:-Minimal inhibitory concentr _ o , against Ps. aeru~inos~ E. coli Staph.aureus ~ BI'r 40 20 20 ', , 15 Complex ~00 20 20 '~ ' Minimal inhibitory concentration (p.~.m.) ~, against ,; A,tenuis P. Ch. P- rr. A
t~ 3~ ~lobosum versicolor viride ni~e.r 20 BIT ~ 20 ~0~0 < 20 ho 200 ` ComplexC 20 ~ 20< 20 ~ 20 40 60 LBDAC 80 ~ 20200 < 20 200 ~200 : , ' ' From the above it can be seen that the ~ungistatic ~` ~ activity of the complex`is, in general, fiomewhat greater than that of either component of the complex used alone.
~ ~ .
,. . ~ ~ ' :

45~ Dx. 26110 am~le ~
Comparison of the fungicidal activity of 1~2 - benziso-, thiazolin-3-one (BIT) alone and together with the cationic ,-~ compounds lauryl benzyl dimethyl ammonium chloride (LBDAC), cetyl trimethyl ammonium bron~de (CThB) and cetyl pyridinium chloride (CPC), thenonionic adduct of nonylphenol with 8 mols ~ of ethylene oxide and the anionic sodium dioctyl ;~ - sulphosuccinate (SDSS).
~ ~ ~ Sterilised 1 cm. square nylon meshès on malt agar are .. . .
~' 10 seeded with a spore suspension of Asper~illus nige~ and incubated for 24 hours at 25C, after which time the mesh .... .
~ ~ is covered with fungal mycelium while no spore formation has ,.; . :
commenced. Each test biocidal composition is prepared at -, the required concentration in a 100 ml. volume of water in a 250 ml. conical flask. Four fun~us-seeded squ~res are tran6~
ferred into each flask and the flasks are then rotated on an , , .
orbital shaker. Two meshes are removed from each flask after i ~ 3 hours and the remaining t~lo meshes after 6 hours of incubation.
,', .
~, Each mesh is vigorously shaken in 15 ml. of sterile water : 20 before being transferred on to a malt agar plate. The latter ' is inoubated for 3 days at 25C after which the nylon meshes ~i are examined for the presenae or absence of fun~al growth.
A oomplete kill of the fungal myceliu~ is indicated by the absence of any growth on or around the nylon mesh on the malt 25agar p}ate. ~ ~ ;
p ' . ; .
~ Resulta are ae followa:-,,~1;, I

:.,: : :

.,' ', ' , ' . , . . ' ,, . ' ~ ' . .:

DX.26110 - 10455~S
Survival of fun~us after contact time of Biocide (~.p.~ k~ 6 hours Control (no biocide) +~ ~++
BI~ (1000) -~++
BIT~ (100) ++,+ +~
BIT (100) ~ LBDAC (20) - -BIT (100) + LBDAC (10) +
BIT ( 100) ~ CTAB (20) ~ ~ -BIT (100~ + CTAB (10) . ~ -., ~ - - . :
BIT (100) + CPC (20) BIT (100) + CPC (10) ++ +
BIT (100) + adduct . (10) ~+~ ~++
BIT ~100) + SDSS (10) ~+~ +++
LBDAC t20) +~+ ~+
CTAB (20) ~++ +++
CPC (20~ ~ +~+ ~+~ :
Adduct (10) +~+ ~++ : :
SDSS (10) +~+ . +~+
,~: . '' ~ ' 20 ~ In this ¢ase the BIT was used in the form of a solution of ~ l ; ~ BIT (33.3~) in ethylene diamine t24.~h) and water (42.7~o) : ~l (percentages by weight).
, In the above table +~+, ~+ and ~ indicate vigorous growth, -. ~ i ~ . , : , ~
. moder~te growth and slight growth respectively, and - indicate~ :.
a total kill~

;, ~ .~ .
; j ,. , - .

Dx. 26110 1~5545 The above tests show that, in the presence of the cationic compounds lauryl benzyl dimethyl ammonium chloride, cetyl trimethyl ammonium bromide a~d cetyl pyridinium chloride, 1.2-benzisothiazolin-3-one is more effective as a fungicide ~ 5 than when used alone. It is also apparent that the non-ior~c - nonylphenol ethylene oxide adduct and the anio~ic sodium dioctyl sulpho6uccinate surfactants do not haYe a similar efEect with the 1.2-benzisothiazolin-3-one.
The experiment described below shows in greater detail ; 10 the effect obtained by using 1.2-benzisothiazolin-3-one and lauryl benzyl dimethyl ammo~ium chloride in combination. All other details are as described above.
~; 5urvival of~ un~us ~fter contaot time of:
1 hour ~ E~ 4 hours 6 hours BIT~1000 ~++ + +
500 +++ ~++ ++~ ~++

,, ~ ,1 100 +++ +,,,+
i l 50 ++~ ~++ ++~ +++
~1 LBDAC 50 ++-~ +++ +++ +t+
.~ , " 20 +++ ~ +~ +++
' " 10 +++ +~+ ~+ +++
,, , :
BIT(100) ~ LBDAC(20) *++ +
BIT(100) + LBDAC(10) +++ +++
BIT (50j + LBDACt10) ~ +
~The BIT in this case was used in the form of a solution in agueous ethylene diamQne as described~in the first part of this Example.

~5~5 DX. ~6110 - Example 6 Comparison of the bactericidal activity of 1.2-benr~iso-thiazolin-3-one (BIT) alone and together with lauryl benzyl - dimethyl ammonium chloride (LBDAC), the adduct of nonylphenol with o mols of ethylene oxide and sodium dioctyl sulphosuccinate (SDSS).
Test biocide solutions are prepared at the required concentration in 100 ml. volumes of water in 250 ml. conical ~; flasks. rThe control consisted of 100 ml~ of sterile water.
A 1 ml. suspension of a 24 hour culturè of Pseudomonas ~ -aeru~inosa is transferred into each flask, and the flasXs are then incubated at 25C in an orbital shaker. r~be numbers ., .
- of surviving bacteria ~re determined after 1, 2 and 6 hours.
Resiults are as follo~G:-~urviK, I ~f b~c-~ri~ Icount per ml.) after:
, Biocide ( ~ 1 hour 2 hours 6 hour~ -/ BIrP~ ~1000)~ 300 x 1057300 x 105 ~-10 '' ~BIrr~ (1C0~7 3O x 105~300 x 105 ~ 300 x 105 , . , LBDAC ~10) 30 x 105 42 x 10 120 x 103 '~ 20 Adduct (10) ~300 x 105~30G x 105 ~ 300 x 105 i ~ .
SDS~ ~10) ~3G0 x 105~300 x 105 ~ 300 x 105 BIT~100) + LBDACt10) ~10 x 10 57 x 103 C 10 , l ~ BIrr~100) ~ Adduct(10) >300 x 1~ > 300 x 105 ~ ~300 x 105 ~` BIT(100) + SVSS (10) ~ 300 x 105 ~ 300 x 105~ ~300 x 105 Control > 300 x 105>3O x 105 ~ 300 x 10 The BIT in this ~ase was used i~ the form of a solution t ~ iD aqueous~ethyleDe diamine~as described in the first~part of Example 5~

. j : .., Dx.26110 ~O~SS45 The benzisothiazolone-lauryl benzyl dimethyl ammonium chloride combination9 in the ratio 100 ppm : 10 ppm sho~s activity after 6 hours whilst the other combinations do not, and is AS active as 1000 ppm of the benzisothiazolone alone.
ample 7 Comp~rison of 1.2-benzisothiazolin-3-one (BIT) and its complex with lauryl benzyl dimethyl ammonium chloride (LBDAC) ~ as paint film fungicides.
; ~ The test biocides are mixed into an e~ulsion paint to ~ provide the concentrations (w/w) in wet paint as shown below.
. .
A commercially availablé fungicide, 2 -(4'-thia~olyl) - benzimidazole, ; is used as a control. The paint in the can is then stored at 50 C for 2 weeks in order to simulate prolonged storage.
The treated paint is applied to one side Qf a filter paper "
5.5 cm. in diameter, and after 24 hours a second coat of paint ; 15 is ~pplled to the si~me ~ide.
A Gecond set of filter papers coated with two layers of treated paint is subjected to a weathering procedure consisting of a 24 hour leaching period in a standard spray cabinet (BS 3900 F4) and 24 hour heatin~ in an oven at 65C~
The filter papers are then transferred on to malt asar in a Petri dish (paint side uppermost) and inocùlated with one of . j .
; the two test fungi Pullularia pullulans and Altern_ria tenuis.
... .
The inoculi~ consists of a visible suspension of the test organi m sprayed over the painted filter paper and surrounding border of agar in the Petri dish. The inoculated paint films are incubated at 25C for 5 days, after which the filter papers are examined ~or the presence or absence of fungal growth. Re`sults are a~ follows:- ;

17 _ Dx 26110 ~ 5545 Minimal inhibitor~__evel of _iocide in ~int ,~ ~
; Compound (p.p.m.) Storage Weathering Alt. tenuisP. Pullula=6 is P. Pullulanus .. , . ,.~_ .. _ .. _~ -Complex of BIT and LBDAC (1000,3000)1000 1000 3000 1000 BIT(500,1000, 2000, 3) 500 500 ~000 3000 2-(4'-thiazolyl) benzimidazole (1000,3000) 3000 1000 3000 1000 ....~ ._ ... _ _ . :
: `:
Since only one tllird of the weight of the complex is BI~, it i6 clear that the BIT i8 more active as a paint film fungicide when in the form of its complex with LBDAC than when used ~, J alone, ~, ~( Example 8 ;~ 15 The aotivity of the 1,2-benzisothiazolin-~-one (BIT)/ lauryl , benzyl dimethyl ammonium chloride ~LBDAC) complex as an antifungal agent in prevent1ng the growth ofcellulc1ytic fungi on wood. ~`
Malt agar plates were spra~ed with a suspension of ~
versiaolor or Chr~sospori~um li~norum- Three cubical blocks of beeoh wood (sides 1 cm.) were soaked for 5 minutes in a 0.3~ w/v aqueou6 ~olution of BIT/LBDAC oomplex, (i.e. containing 0.3 g. complex in 100 ml.
solution; prepared by adding a 50~ solution of compIex in propylene .~,.;
glycol to water), dried at 50C for 4 hours and eqiulibrated at room temperature for 20 hours.
A PY 900 tpoly (vin~l chloride))soreen mesh whioh had previously beer. soaked in~70~ ethanol for 36 hour~ and allowed to dry was txansferred on ~o the innoculated agar surfaoe ir~ each plate. The three biocide-treated beeohwood blocks were placed on the screen mesh and innoculated t 25C for three week~. The~blocks were then e~a~ined for the presence 1?~ 4 5 5 4 S Dx~26110 , or absence of fun~al growth.
Results are as follow~s:- -,~ ~ Growth o~ beech blocks of Polxstictus ~,ersicolor~
' 5 Control (no biocide) ~ *~
BIT/LBDAC complex ~1 .
~, 0,3~ solution :i -.. - - -' :'' ' -Key: ~+~ indicates YigOrOUs growth ~ oderate ; 10 _ " no " ttotal kill) .; . , , :
' It is clear from theæe re~ults that the BI~/IBDAC complex '~ at tho,above-mentioned ooncentration presents the ~rowth o~ the fungi on beeoh wood.
, .. . . .
.' ~a~ . -'''~ 15 ; The effeot of BIT/IBDAC complex in retarding the oxidation ~ , of nitrite by Nitrobacter in a model cooli~g tower system.
,, ,l A model cooling tower 6ystem WRS constructed, consisting i, of a wooden frame 6upporting six wooden slat6~ the fxa~e being mous~ted over a reservoir holding 10 litre6 of water containing 20 sodium nitrite as a corrosion inhibitor~ The water is then ~, co~tRmsinated with 1 g. o~ oooling tower 61ime lunown to harbour Nitrobacterl and the w~ter ln the reservoir is directed to the top o~ the wooden slats by means~of a Churchill pump, so that the water tri¢klea down~oYer the~slats and returns to the reservoir. If no 27 biocide is added to the water the'wooden slats become colonised'by a ,, . , ~
?~ ~ ~
~, Nitrobacter - containi~lg slime which rapidly ovidi~es nitrite to ' nitrabe.

Initi~lly9 the rate of los~of nitrite ~rom the ~ystem was i' ;, :, , ~ , i:
19 - ?
~ 5~

: `\
~ Dx.26110 ~:)4S5~5 determaned in the absence of biocide by removing water samples ,-~nd - analysinF for nitrite content. 'Ihe nitrite level was then adiusted to a hieher concentration before 150 ppm. of BIT~LBDAC complex was added to the ~later. The ~coolirgtower' water was then circulated - --for the necessary period until the concentration of nitrite was appreciably reduced, the water in the ~ystem ~as drained o~f and fresh nitrite - containin~ water was added. Be~orè the next biocide - for test w~s added~ the rate of loss of nitrite in the biocide-~ree ~ystem was checke3d, to ensure that the nitrite-oxidising actiYity had normalised, i.e. all biocides were compared In the same system consecutively.
Re~ults are as ~ollows:-B ide and concentration Circulation Ni~trate leveladded to water initially _me ~ detected ln water (ppmi) Control (no biocide) 0 965 i 2 450 I LBDAC, 250 ppM. 49 4 i 3 L~96 Il 8 470 Poly(hexamcthylene biguanide) 0 ô40 , bydrochloride, 85 ppmO 4 705 1 I ~ 30 11 260 ~ 18 125 -3~ 5 o ; BII/LBDAC co~plex, 150 ppm. 2 690 ; ` ~ ; ; 16 655 DX.26110 ~LS~

These results indicate that under the experimental conditions the BIT/LBDAC complex is markedly more effective than either LBDAC alone or poly(hexamethylene biguanide) hydrochloride (a ~ell-known bactericide) ~e~eas these two compounds ~aintained the initial level of nitrite concentration for approximately 10 days and 4 days respectively, there ~Jas no significant fall in the nitrite level of the water treated with BIT/LBDAC complex a~ter 58 days.
~`~~~~ ~ am~le 10 - .
The efficiency o~ BIT/LBDAC complex in retarding the oxidation of nitrite by Nitrobacter in a cooling tower.
An all-wood cooling tower system~ 25,000 gallons in capacity ~ th a ~aximum temperature of ~2C. was lsed for this trial.
Before the biocide was added the cooling water wa~ bled so that addition of nitrite to ~ive a concenkration of 500 ppm the total dissolved solids were not more than 1,000 ppm. The biocide (BIT/
L~DAC complex) was added at the distribution troughs at th~ top of the tower to give an initial concentration of 150 ppm. active ingredient .
The single dose of biocide controlled microbial activity and hence ~atisfactorily prevented lo~æ of nitrite from the system for a period up to ei~ht weeks. Comparison with quaternary ammonium compounds showed that BI'~/LBDAC complex provides protection for àt lcast 7 times longer than 250 ppm. of LBDAC alone, In this test, coMbinations of BIT with LBDAC or Dequadi~
were co~pared for bactericidal activity, _ 21 -. . , .; . .,: . ., :, .: :., . :- . .. . : . . ... . .:, . ... .
. . , . ". . , . , . ... , . . . ,.: . .: : . .

.. . . .

5S4S ~
.
Results are as follows:
Biocide Control (no biocide) 1~3 x 107 BI~, 200 ppm. 8-o x 108 LBDAC, Z0 ppm. ,B.3 x 103 Dequadin, 20 ppm. 1.0 x 103 BI~ (100) ~ LB~AC (10) < 10 ~ -... . ~ .
BIT (100) ~ Dequadin (10) 2.o x 102 All conce~trations re~er to active ingredient.
The synergi~m shown by BIT and LBDAC acting together i~ clearly seen. A lower order of ~ynergism is also shown between BIT and Dequadin.
Example 12 In this test, oombi~ation~of BIT and Domiphen~ BIT and Dequadin, and BIT and Phemeride were tested ~or fungicidal ac~ivity against ~ J~ , the test method being that described in Example 5.
Results are as follows:
~ Survival of ~un~us after contact times ~ hourfi ~ , '' ' Co~trol (no biocide) ~t ~++
BIT, 100 ppm. ~ +~
Dequadin, 20 ppm. ~+~ ~+
Domdphen~ 20 ppm. +++ ~++
Phemerideg 20 ppm. ~+~ ~+~ j;
BIT (100) ~ Dequadin (10) +~
BIl' (100) + Domiphen ~iiO) ~ ~+ ~ ~ ¦
BIT ~100) ~ Phemeride t10) +~ +

!:

~45545 ~2611Q

These result~ indicate the presence of synergi6tic activity between BIT and Dequadin, BIT and Domiphen9 and to a lesser extent betwe~n BIT and Phemeride~
Dequadin, Domiphen and Phemerid~ (Registered Trade Marks) are all quaternary ammonium compounds having phar~aceutical applications, and have the following structures:-2 ~ 2 : ~ CN3 ~ 2X- De~u~din C ~ - N~ C~I C~ O ~ . X~ Domiphen X~ . CN~ ~ 2CN2CCH2CN2 ~ - CN2 C(C~l3~3 Phe~eride ' ' ~

~;

I ' .
I ~.

-: ; : : . ; ~ : . : .

Claims (7)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. Biocidal compositions comprising:
A (i) a quaternary ammonium compound of the general formula R R1R2R3N+ X- Formula I
wherein R represents a C10-C22 alkyl group, a C1-C9 alkyl-phenoxy-ethoxyethyl group, or a phenoxyethyl group;
R1 represents a C10-C22 alkyl group, a C1-C9 alkyl group or a benzyl group;
R2 and R3 each independently represent a C1-C4 alkyl group and X- represents an anion;
or (ii) a quaternary ammonium compound of the general formula Formula II

wherein R4 represents an optionally substituted C10-C22 alkyl group;
X- represents an anion, and wherein the heterocyclic nucleus may be fused with a benzene nucleus adjacent to the nitrogen atom and which may carry an amino substituent; and B a substituted or unsubstituted 1,2-benzisothiazolin-3-one compound having the general formula Formula III

wherein the benzene nucleus may carry substitutents selected from halogen atoms, C1-C4 alkyl groups, C1-C4 alkoxy groups, the nitro group and the cyano group;
wherein the molar ratio of substituted or unsubstituted 1,2-benzisothiazolin-3-one to quaternary ammonium compound is in the range 1.0:0.04 to 1.0:1Ø

2. Biocidal compositions as claimed in Claim 1 wherein the quaternary ammonium compound and the substituted or unsubstituted 1,2-benzisothiazolin-3-one are in the form of a complex of the two compounds in which there is one benziso-thiazolinone group for each quaternary ammonium group in the quaternary ammonium compound,

3. Biocidal compositions as claimed in Claim 1 or 2 wherein the quaternary ammonium compound is a dodecyl benzyl dimethyl ammonium salt.

4. A process for the preparation of the biocidal compositions claimed in Claim 2 which comprises mixing an aqueous solution of an alkali metal salt of the 1,2-benziso-thiazolin-3-one with an aqueous solution of the quaternary ammonium compound in stoichiometric proportions, and collecting the complex which separates,

5. A method for protecting aqueous media against infection by micro-organisms and controlling or preventing the proliferation of micro-organisms in aqueous media already infected thereby, which comprises adding to the aqueous media from 1 to 1000 parts per million of a biocidal composition as claimed in Claim 1.

6. A method for the protection of paint films against fungal attack wherein there is added to the paint before its application to a surface a biocidal composition as claimed in Claim 1, in amount to provide a concentration in the paint of 500 to 10000 parts per million,

7. A method for the protection of wood against fungal attack wherein the wood is treated with a solution of a biocidal composition as claimed in Claim 1, the solution containing from 50 to 20000 parts per million of the composition.