Biocidal compositions and treatments

Phosphonium salts such as THP and aldehydes such as formaldehyde exhibit synergistic biocidal activity.

The present invention relates to biocidal compositions comprising 
tetraorgano phosphonium salts and biocidal treatments therewith. 
Tetrakis hydroxymethyl phosphonium (herein referred to as "THP") salts and 
tris hydroxymethyl phosphine are described and claimed for use as biocides 
for water treatment, plant protection, and for pharmaceutical and 
veterinary treatments in GB 2145708, GB-A-2178960, GB-A-2182563, 
GB-A-2201592 and GB-A-2205310 the disclosures of which are hereby 
incorporated by reference. The use as biocides of certain analogues of 
THP, including the methyl, ethyl and allyl tris (hydroxymethyl) 
phosphonium salts, is described and claimed in British Application No. 
9001831.8. 
The low molecular weight phosphonium salts described in the aforesaid 
patents are quite distinct from the higher molecular weight quaternary 
surfactants which also exhibit biocidal properties. The quaternary 
surfactants differ markedly from the low molecular weight hydroxy methyl 
phosphonium biocides in their chemical, physical and biocidal properties. 
Thus both quaternary ammonium and quaternary phosphonium salts having one 
long (i.e. 8 to 20 carbon) chain are effective as biocides. The activity 
correlates with the surfactancy, declining as the length of the chain is 
reduced to 8 carbons. The quaternary ammonium salts, which are more 
effective as surfactants than the corresponding phosphonium salts are 
similarly more effective biocides. 
The typical quaternary biocide has one fatty alkyl group and three methyl 
groups, but in the case of phosphonium salts the methyl groups can be 
substituted by hydroxymethyl groups without substantially affecting the 
biocidal activity. They may also be substituted by at least one aryl group 
e.g. the benzalkonium salts, without loss of either surfactancy or 
biocidal activity. 
In contrast, the low molecular weight hydroxy-methyl phosphonium salts are 
not surfactants and yet are highly active biocidally. The pattern of 
biocidal activity is quite different, however. They are effective against 
bacteria and algae at very much lower concentrations than the quaternary 
surfactants and are very rapid acting. Unlike the surfactants the activity 
of the low mol wt. phosphonium salts is specific to the presence of 
hydroxy methyl groups. Although it is possible to substitute one hydroxy 
methyl group by a methyl, ethyl or allyl group, without losing activity, 
if more than one hydroxy methyl group is substituted, or if the size of 
the substituent is increased above 3 carbon atoms the biocidal activity 
falls sharply. The tetra methyl phosphonium salts, and the aryl 
tris(hydroxymethy) phosphonium salts are inactive. 
There is no nitrogen analogue to the low molecular weight phosphonium 
biocides. 
For all the foregoing reasons THP salts and the related low molecular 
weight hydroxy methyl phosphonium and hydroxy methyl phosphine biocides 
are seen by those skilled in this art to form a quite distinct group of 
biocides in no way analogous to quaternary surfactants and presumably 
working by an entirely different mechanism. The use of long chain alkyl 
phosphonium salts for water treatment is known from EP066544. 
A variety of aldehydes, such as formaldehyde, acrolein and glutaraldehyde 
are widely used as biocides. It is known that certain aldehydes are 
synergistic with quaternary surfactants. 
We have now found that combinations of tris hydroxy methyl phosphine, or 
low molecular weight hydroxy methyl phosphonium biocides, especially THP 
salts, with aldehyde biocides, especially formaldehyde, and also 
glutaraldehyde and acrolein, possess synergistic biocidal properties. 
The present invention provides biocidal compositions comprising (i) at 
least one organophosphorus biocide, which has the fomula 
EQU [R.sub.n P(CH.sub.2 OH).sub.3 ].sub.v.sup.n+ n[X].sup.v- 
wherein R is hydroxymethyl, methyl, ethyl or allyl, X is an anion such that 
the compound is at least sparingly soluble in water, n is 1 or 0 and v is 
the valency of the anion X and (ii) at least one biocidal aldehyde or 
polymer thereof. 
The invention also provides a method of killing, or inhibiting the growth 
or reproduction of microbes or other pests on or in a substrate or medium, 
which comprises contacting said substrate or medium substantially 
simultaneously with the compounds (i) and (ii) specified above. 
The preferred compounds for use are the THP salts, especially THP sulphate 
(THPS) and THP chloride (THPC), and tris(hydroxymethyl )phosphine. 
Also useful are methyl tris(hydroxymethyl)phosphonium salts, ethyl 
tris(hydroxymethyl)phosphonium salts and allyl 
tris(hydroxymethyl)phosphonium salts. 
The anion X may be any convenient anion which provides a salt which is 
preferably soluble at least to a concentration of 0.5 gm per liter of 
water at 25.degree. C. such as chloride, sulphate or phosphate or less 
preferably sulphite, phosphite, bromide, nitrate, borate, acetate, 
formate, lactate, methosulphate, citrate or carbonate. However other 
anions may be used which provide salts of reduced solubility in water but 
are soluble in organic solvents e.g. alcohols or hydrocarbons. 
The other key ingredient in our synergistic compositions is a biocidal 
aldehyde or biocidally active polymer thereof, such as formaldehyde, 
acetaldehyde, propionaldehyde, butyraldehyde, succinaldehyde, 
isobutyraldehyde, glutaraldehyde, crotonaldehyde, acrolein, chloral, 
glyoxal, metaldehyde, paraldehyde, metaformaldehyde or trioxan. 
The phosphonium compound and the aldehyde may normally be present in the 
composition in a weight ratio of 20:1 to 1:20 especially 9:1 to 1:9 
preferably 3:7 to 7:3. 
It will be understood that THPC and THPS solutions as prepared usually 
contain a small amount of adventitious formaldehyde, typically about 2% or 
3% by weight based on the THP salt, which is usually stripped down to 
under 1% before the product is supplied for use. It will be understood 
that the composition claims of the present invention do not refer to 
adventitious formaldehyde, but to formaldehyde added to the composition in 
addition to the trace that is normally present, to obtain synergistically 
enhanced biocidal action. 
The biocidal components of our invention are useful for treating aerobic or 
anaerobic water systems contaminated or liable to be contaminated with 
microorganisms. For example they are effective against Pseudomonas 
aeruginosa and Legionella pneumophila in boiler water, cooling water, 
industrial process water, geothermal water, central heating and air 
conditioning systems, for killing algae in swimming pools, lakes, streams, 
canals and reservoirs and for treating cooling water in power stations and 
for marine engines. 
The biocides are also useful in killing sulphate reducing bacteria such as 
Desulphovibrio in the above systems, and especially in oil field produced 
water, injection water, drilling fluids or water for hydrostatic testing. 
They are also useful as preservatives in aqueous based formulations such 
as bitumen and tar emulsions, paper sizes, adhesives, paints, cellulosic 
pulps including pulp thin stock and backwash recirculating liquor. 
The biocides are useful in disinfectants including farmyard, domestic and 
surgical disinfectants. They may be used in the fumigation of grain silos, 
crops and crop storage areas. 
The biocides are useful for killing bryophites, including mosses and 
liverworts, lichens, and sessile algae in lawns and gardens and on paths, 
drives, roadways, walls and other structures and on railways, airports and 
industrial estates. 
The biocides are useful for protecting plants against fungi, bacteria, 
viruses and other microbial plant pathogens, by application to the plants 
and or to the soil in which they are growing or to be grown, or for use in 
a seed dressing. 
The biocides may be used at higher concentrations as total herbicides, to 
kill higher plants. 
The selective activity of the biocides is concentration dependent. 
Generally at concentrations of between 10 and 2,000 ppm preferably 20 to 
1,500 e.g. 30 to 1,000 especially 50 to 500 ppm the biocides show 
selective activity against lower organisms such as bacteria, algae, mosses 
and fungi, but exhibit very low toxicity towards higher plants, fish and 
mammals. At higher concentrations, e.g. greater than 0.2% up to 
saturation, preferably 0.5 to 75% e.g. 1 to 60% and at dosages of greater 
than about 2 kg per hectare e.g. 2.5 to 5 kg per hectare the biocides are, 
however, effective total herbicides. 
Mixed alkyl hydroxyalkyl THP salts may be prepared by adding an aqueous 
base to a tetrakis (hydroxymethyl) phosphonium salt, e.g. sodium hydroxide 
in a proportion of from 0.5 to 0.75 equivalents, to form tris 
(hydroxymethyl) phosphine and reacting the latter with alkyl halide such 
as methylchloride, preferably at an elevated temperature of e.g. 
40.degree.-60.degree. C. Alternatively, improved yields may be obtained by 
reacting the alkylhalide with tris (acetoxymethyl) phosphine prepared by 
the method of Mironova et al, Zhur Obshch, Khim. 37, No 12, pages 
2747-2752. The reaction may be carried out by heating at temperatures up 
to 140.degree., e.g. 120.degree. C. in suitable solvent such as toluene, 
for 2 to 20 hours e.g. 10 to 15 hours or with an acid catalyst, preferably 
acetic acid, for from 1 to 8 hours, e.g. 3 to 5 hours. 
The invention provides compositions containing the aforesaid biocides. In 
particular for use in water treatment and in agriculture we have found 
that the biocides are synergistic with surfactants. 
The surfactant may for example consist substantially of an at least 
sparingly water-soluble salt of sulphonic or mono esterified sulphuric 
acids, e.g. an alkylbenzene sulphonate, alkyl sulphate, alkyl ether 
sulphate, olefin sulphonate, alkane sulphonate, alkylphenol sulphate, 
alkylphenol ether sulphate, alkylethanolamide sulphate, alkylethanolamide 
ether sulphate, or alpha sulpho fatty acid or its esters each having at 
least one alkyl or alkenyl group with from 8 to 22, more usually 10 to 20, 
aliphatic carbon atoms. 
The expression "ether" hereinbefore refers to compounds containing one or 
more glyceryl groups and/or an oxyalkylene or polyoxyalkylene group 
especially a group containing from 1 to 20 oxyethylene and/or oxypropylene 
groups. One or more oxybutylene groups may additionally or alternatively 
be present. For example, the sulphonated or sulphated surfactant may be 
sodium dodecyl benzene sulphonate, potassium hexadecyl benzene sulphonate, 
sodium dodecyl dimethyl benzene sulphonate, sodium lauryl sulphate, sodium 
tallow sulphate, potassium oleyl sulphate, ammonium lauryl monoethoxy 
sulphate, or monoethanolamine cetyl 10 mole ethoxylate sulphate. 
Other anionic surfactants useful according to the present invention include 
alkyl sulphosuccinates, such as sodium di-2-ethylhexylsulphosuccinate and 
sodium dihexylsulphosuccinate, alkyl ether sulphosuccinates, alkyl 
sulphosuccinamates, alkyl ether sulphosuccinamates, acyl sarcosinates, 
acyl taurides, isethionates, soaps such as stearates, palmitates, 
resinates, oleates, linoleates, and alkyl ether carboxylates. Anionic 
phosphate esters and alkyl phosphonates, alkyl amino and imino methylene 
phosphonates may also be used. In each case the anionic surfactant 
typically contains at least one aliphatic hydrocarbon chain having from 8 
to 22, preferably 10 to 20 carbon atoms, and, in the case of ethers, one 
or more glyceryl and/or from 1 to 20 oxyethylene and/or oxypropylene 
and/or oxybutylene groups. 
Preferred anionic surfactants are sodium salts. Other salts of commercial 
interest include those of potassium, lithium, calcium, magnesium, 
ammonium, monoethanolamine, diethanolamine, triethanolamine, alkyl amines 
containing up to seven aliphatic carbon atoms,and alkyl and/or 
hydroxyalkyl phosphonium. 
The surfactant may optionally contain or consist of nonionic surfactants. 
The nonionic surfactant may be, e.g. a C.sub.10-22 alkanolamide of a mono 
or di-lower alkanolamine, such as coconut monoethanolamide. Other nonionic 
surfactants which may optionally be present, include tertiary acetylenic 
glycols, polyethoxylated alcohols, polyethoxylated mercaptans, 
polyethoxylated carboxylic acids, polyethoxylated amines, polyethoxylated 
alkylolamides, polyethoxylated alkylphenols, polyethoxylated glyceryl 
esters, polyethoxylated sorbitan esters, polyethoxylated phosphate esters, 
and the propoxylated or ethoxylated and propoxylated analogues of all the 
aforesaid ethoxylated nonionics, all having a C.sub.8-22 alkyl or alkenyl 
group and up to 20 ethyleneoxy and/or propyleneoxy groups. Also included 
are polyoxypropylene/polyoxethylene copolymers, 
polyoxybutylene/polyoxyethylene copolymers and 
polyoxybutylene/polyoxypropylene copolymers. The polyoxyethylene 
polyoxypropylene and polyoxybutylene compounds may optionally be 
end-capped with, e.g. benzyl groups to reduce their foaming tendency. 
Compositions of our invention may contain an amphoteric surfactant. 
The amphoteric surfactant may for example be a betaine, e.g. a betaine of 
the formula: --R.sub.3 N.sup.+ CH.sub.2 COO.sup.-, wherein each R is an 
alkyl, cycloalkyl, alkenyl or alkaryl group and preferably at least one, 
and most preferably not more than one R, has an average of from 8 to 20, 
e.g. 10 to 18 aliphatic carbon atoms and each other R has an average of 
from 1 to 4 carbon atoms. Particularly preferred are the quaternary 
imidazoline betaines of the formula: 
##STR1## 
wherein R and R.sup.1 are alkly, alkenyl, cycloalkyl, alkaryl or alkanol 
groups having an average of from 1 to 20 aliphatic carbon atoms and R 
preferably has an average of from 8 to 20, e.g. 10 to 18 aliphatic carbon 
atoms and R.sup.1 preferably has 1 to 4 carbon atoms. Other amphoteric 
surfactants for use according to our invention include alkyl amine ether 
sulphates, sulphobetaines and other quaternary amine or quaternised 
imidazoline sulphonic acids and their salts, and other quaternary amine or 
quaternised imidazoline carboxylic acids and their salts and Zwitterionic 
surfactants, e.g. N-alkyl taurines, carboxylated amido amines such as 
RCONH(CH.sub.2).sub.2 N.sup.+ (CH.sub.2 CH.sub.2 CH.sub.3).sub.2 CH.sub.2 
CO.sub.2, and amino acids having, in each case, hydrocarbon groups capable 
of conferring surfactant properties (e.g. alkyl, cycloalkyl, alkenyl or 
alkaryl groups having from 8 to 20 aliphatic carbon atoms). 
Typical examples include 2 tallow alkyl, 1-tallow amido alkyl, 
1-carboxymethyl imidazoline and 2 coconut alkyl N-carboxymethyl 
2-(hydroxyalkyl) imidazoline. Generally speaking any water soluble 
amphoteric or Zwitterionic surfactant compound which comprises a 
hydrophobic portion including a C.sub.8-20 alkyl or alkenyl group and a 
hydrophilic portion containing an amine or quaternary ammonium group and a 
carboxylate, sulphate or sulphonic acid group may be used in our 
invention. 
Compositions of our invention may also include cationic surfactants. 
The cationic surfactant may for example be an alkylammonium salt having a 
total of at least 8, usually 10 to 30, e.g. 12 to 24 aliphatic carbon 
atoms, especially a tri or tetra-alkylammonium salt. Typically 
alkylammonium surfactants for use according to our invention have one or 
at most two relatively long aliphatic chains per molecule (e.g. chains 
having an average of 8 to 20 carbon atoms each, usually 12 to 18 carbon 
atoms) and two or three relatively short chain alkyl groups having 1 to 4 
carbon atoms each, e.g. methyl or ethyl groups, preferably methyl groups. 
Typical examples include dodecyl trimethyl ammonium salts. Benzalkonium 
salts having one 8 to 20 C alkyl group two 1 to 4 carbon alkyl groups and 
a benzyl group are also useful. 
Another class of cationic surfactants useful according to our invention are 
N-alkyl pyridinium salts wherein the alkyl group has an average of from 8 
to 22, preferably 10 to 20 carbon atoms. Other similarly alkylated 
heterocyclic salts, such as N-alkyl isoquinolinium salts, may also be 
used. 
Alkylaryl dialkylammonium salts, having an average of from 10 to 30 
aliphatic carbon atoms are useful, e.g. those in which the alkylaryl group 
is an alkyl benzene group having an average of from 8 to 22, preferably 10 
to 20 aliphatic carbon atoms and the other two alkyl groups usually have 
from 1 to 4 carbon atoms, e.g. methyl groups. 
Other classes of cationic surfactant which are of use in our invention 
include alkyl imidazoline or quaternised imidazoline salts having at least 
one alkyl group in the molecule with an average of from 8 to 22 preferably 
10 to 20 carbon atoms. Typical examples include alkyl methyl hydroxyethyl 
imidazolinium salts, alkyl benzyl hydroxyethyl imidazolinium salts, and 2 
alkyl-1-alkylamidoethyl imidazoline salts. 
Another class of cationic surfactant for use according to our invention 
comprises the amido amines such as those formed by reacting a fatty acid 
having 8 to 22 carbon atoms or an ester, glyceride or similar amide 
forming derivative thereof, with a di or poly amine, such as, for example, 
ethylene diamine or diethylene triamine, in such a proportion as to leave 
at least one free amine group. Quaternised amido amines may similarly be 
employed. 
Alkyl phosphonium and hydroxyalkyl phosphonium salts having one C.sub.8-20 
alkyl groups and three C.sub.1-4 alkyl or hydroxyalkyl groups may also be 
used as cationic surfactants in our invention. 
Typically the cationic surfactant may be any water soluble compound having 
a positively ionised group, usually comprising a nitrogen atom, and either 
one or two alkyl groups each having an average of from 8 to 22 carbon 
atoms. 
The anionic portion of the cationic surfactant may be any anion which 
confers water solubility, such as formate, acetate, lactate, tartrate, 
citrate, chloride, nitrate, sulphate or an alkylsulphate ion having up to 
4 carbon atoms such as a methosulphate. It is preferably not a surface 
active anion such as a higher alkyl sulphate or organic sulphonate. 
Polyfluorinated anionic, nonionic or cationic surfactants may also be 
useful in the compositions of our invention. Examples of such surfactants 
are polyfluorinated alkyl sulphates and polyfluorinated quaternary 
ammonium compounds. 
Compositions of our invention may contain a semi-polar surfactant, such as 
an amine oxide, e.g. an amine oxide containing one or two (preferably one) 
C.sub.8-22 alkyl group, the remaining substituent or substituents being 
preferably lower alkyl groups, e.g. C.sub.1-4 alkyl groups or benzyl 
groups. 
Particularly preferred for use according to our invention are surfactants 
which are effective as wetting agents, typically such surfactants are 
effective at lowering the surface tension between water and a hydrophobic 
solid surface. We prefer surfactants which do not stabilise foams to a 
substantial extent. 
Mixtures of two or more of the foregoing surfactants may be used. In 
particular mixtures of non-ionic surfactants with cationic and/or 
amphoteric and/or semi polar surfactants or with anionic surfactants may 
be used. Typically we avoid mixtures of anionic and cationic surfactants, 
which are often less mutually compatible. 
Preferably the organo phosphorus compound and the surfactant are present in 
a relative weight concentration of from 1:1000 to 1000:1, more usually 
1:50 to 200:1, typically 1:20 to 100:1, most preferably 1:10 to 50:1, e.g. 
1:1 to 20:1 especially 2:1 to 15:1. 
Effective doses of the mixture of organophosphorus compound, aldehyde and 
surfactant are typically from 2 ppm to 2000 ppm more usually 20 ppm to 
1,000 ppm e.g. 50 ppm to 500 ppm especially 100 to 250 ppm. 
Compositions for use in water treatment may additionally or alternatively 
contain other biocides, oxygen scavengers, dispersants, antifoams, 
solvents, scale inhibitors, corrosion inhibitors and/or flocculants. 
Compositions according to our invention for use in controlling bryophites, 
lichens or fungal or microbial plant pathogens, contain an effective 
amount of a biocide as aforesaid, together with a horticulturally or 
agriculturally acceptable diluent, carrier and/or solvent therefor. 
The organo phosphorus compounds may be present as a solution in water at 
effective concentrations up to saturation. They will usually be supplied 
as concentrates at about 50 to 80% by weight concentration, e.g. 75% by 
wt. before mixing with the aldehyde but will normally be diluted to a 
concentration of from 0.01 to 10% by wt. before application. Where damage 
to higher plants is to be avoided it is preferred to use concentrations 
below 1% w/w biocide, preferably below 0.2%. Alternatively the biocides 
may be admixed with or absorbed upon inert, particulate, non-phytotoxic 
solids such as talc or dissolved in organic solvents or suspended in or as 
dispersions or emulsions. Thus the compostions of the invention are 
preferably in the form of emulsifiable concentrates in organic solvents 
such as alcohols hydrocarbons, and amides such as dimethyl formamide 
including cyclic amides such as N-methyl pyrrolidone, the concentrate also 
containing a surfactant e.g. as specified above. They may be used in 
conjunction with other moss killers or biocides, such as herbicides, 
fungicides, bactericides, insecticides and weedkillers, or with 
surfactants, wetting agents, adhesives, emulsifiers, suspending agents, 
thickeners, synergists, hormones, plant growth regulators or plant 
nutrients. 
The compositions of our invention may be applied to lawns, flower or 
vegetable beds, arable land, meadowland, orchards or woodland, or 
hydroponic beds, or to the seeds, roots, leaves, flowers, fruit and/or 
stems of plants, or to paths, roads, walls, wood-work, brickwork or 
similar invasible surfaces. 
The composition may be of value, inter alia, in controlling moss or sessile 
algae in lawns or on paths or walls, as seed dressings, as sprays for 
controlling fungal, bacterial or viral infections on leaves, flowers and 
fruit, such as mildew, botrytis, rust, fusarium, mosaic diseases or wilt, 
for application to soil or to the roots of seedlings (e.g. of brassica 
seedlings to inhibit club root) and in the control of numerous fungal, 
vital, protozoal and bacterial diseases of plants, including fungal 
blights such as potatoe blight, cankers such as apple canker, scabs, root 
rot, and base rot of bulbs. The compositions are especially effective in 
protecting cereal crops including wheat, barley, rye, oats, rice maize 
millet and sesame against a broad spectrum of plant diseases. 
Other crops of importance which may be protected according to our invention 
include sugar cane; root vegetables including carrots, parsnips, turnips, 
beetroot, sugar beet, radishes, swedes and mangolds; brassicas including 
cabbages, broccoli, cauliflower and sprouts; grazing land; pulses 
including peas, broad beans, French, beans, runner beans, navy beans, 
kidney beans and lentils; curcubaceous plants including cucumbers, 
marrows, gourds and squashes, oilseed rape, timber, rubber, cotton, 
coffee, cocoa, jute, tomatoes, potatoes, yams, tobacco, bananas, coconut 
palm, olives, alliums including onions, shalots, leeks, garlic, chives and 
spring onions, ground nuts, peanuts, sorghum, oil palm, roses, hemp, flax, 
lucerne, alfalfa, tea and fruit, including citrus fruit, apples, plums, 
peaches, nectarines, mangoes, pears, cherries, grapes, berries, currants, 
dates, figs, avocados, almonds, and apricots. 
The mixtures of tetra organophosphonium compounds and aldehydes are more 
effective biocides, against a variety of microorganisms and pests, than 
the individual compounds. In the case of formaldehyde the formulation may 
be prepared substantially in advance and stored prior to use. In the case 
of certain of the other aldehydes however, it is preferred to prepare the 
composition in situ by adding the aldehyde and the organophosphonium 
biocide separately to the locus to be treated or to mix them as required 
for use, since the two compounds are chemically incompatible, if mixed 
together and stored for long periods. 
The invention is illustrated in the following Examples THPS means bis 
[tetrakis(hydroxymethyl) phosphonium] sulphate. "Empigen" is a registered 
trademark of Albright & Wilson Limited. "Empigen" BAC is a fatty alkyl 
dimethyl benzyl ammonium chloride.

EXAMPLE 
1. METHOD 
1.1 Biofilm generation 
Mild steel biostuds were allowed to foul over a 3 week period with a mixed 
microbiological enrichment of sulphate-reducing bacteria (SRB), aerobic 
bacteria and anaerobic bacteria from North Seal oil production platform 
water injection systems. This was accomplished using a recirculating 
biofilm generator specially designed for the purpose. The device was 
constructed of PVC pipe, containing a row of mild steel studs, mounted so 
that their exposed faces were flush with the interior of the pipe. The 
culture medium was circulated by means of a centrifugal pump and any gas 
build up was removed from the system using a bleed valve. Sterile anaerobi 
fresh nutrient/seawater medium was bled into the system daily to maintain 
the growth of the biofilm; 75% of the fluid volume of the device being 
replaced each day. The device was run for 3 weeks to allow a table biofilm 
to develop. Check studs were removed regularly to confirm the stability of 
biofilm build up. 
1.2 Biocide test regimes 
The biocides tested were are follows: 
______________________________________ 
WEIGHT % ACTIVE 
BIOCIDE COMPONENT FRN w/w 
______________________________________ 
1 THPS 0.1551 11.63 
Empigen BAC 0.0233 1.17 
36.6% Formaldehyde 
0.2869 10.50 
Water 0.5347 
Total actives 23.30 
2 THPS 0.2951 22.13 
Empigen BAC 0.0233 1.17 
Water 0.6816 
Total actives 23.30 
3 36.6% Formaldehyde 
0.6323 22.13 
Empigen BAC 0.0233 1.17 
Water 0.3444 
Total actives 23.30 
______________________________________ 
The three products were challenged against sessile populations using the 
standard static method text described in section 1.3. This test is 
designed to determine the concentration necessary to kill aerobi, 
anaerobic and SRB bacteria after a contact time of one hour. 
1.3 Static biocide tests 
Individual fouled studs were suspended in separate 125 ml vessels 
containing seawater/biocide solution at the appropriate concentration. The 
biocide solutions were prepared using sterile., anaerobi seawater and 
required concentrations of biocide. 
1.4 Enumeration of bacteria 
After one hour of exposure, the studs were removed to 10 ml volumes of 
anaerobic diluent containing 1 g of sand. Vortex mixing was used to 
disrupt the biofilm and produce a homogenous suspension. The initial 
dilution was then serially diluted to 10.sup.-8 in further 10 ml aliquots 
of anaerobic diluent. Control studs, exposed to seawater without biocide, 
were treated in an identical manner to ensure comparability with the 
biocide treated studs. 
The dilution series were then used to inoculate selective enumeration 
media: lactate based broth media for enumeration of SRB; anaerobic 
seawater yeast/peptone agar (in an anaerobic cabinet) for anaerobi 
bacteria; seawater nutrient agar for aerobi bacteria. Enumeration series 
were incubated at 30.degree. C. Aerobic and anaerobi bacteria were counted 
after 5 days incubation and SRB numbers were determined after a full 
incubation period of 28 days. 
2. RESULTS 
______________________________________ 
Contact 
Conc Time Surviving bacteria per stud 
Biocide 
(ppm) (hrs) SRB Anaerobes 
Aerobes 
______________________________________ 
Control 
0 1 1.1 .times. 10.sup.5 
2.3 .times. 10.sup.5 
2.4 .times. 10.sup.5 
1 1.1 .times. 10.sup.5 
3.1 .times. 10.sup.5 
2.9 .times. 10.sup.5 
100 1 2.5 .times. 10.sup.5 
2.9 .times. 10.sup.4 
1.0 .times. 10.sup.3 
250 1 2.5 .times. 10.sup.1 
2.0 .times. 10.sup.4 
0 
1 500 1 0 5.0 .times. 10.sup.2 
0 
1000 1 0 0 0 
1500 1 0 0 0 
100 1 4.5 .times. 10.sup.4 
8.5 .times. 10.sup.4 
3.5 .times. 10.sup.3 
250 1 2.4 .times. 10.sup.1 
1.3 .times. 10.sup.4 
0 
2 500 1 0 4.0 .times. 10.sup.3 
1.0 .times. 10.sup.2 
1000 1 0 5.0. .times. 10.sup.2 
0 
1500 1 0 0 0 
100 1 4.5 .times. 10.sup.3 
3.0 .times. 10.sup.4 
1.8 .times. 10.sup.3 
250 1 1.1 .times. 10.sup.5 
4.3 .times. 10.sup.4 
1.9 .times. 10.sup.3 
3 500 1 1.5 .times. 10.sup.6 
4.5 .times. 10.sup.5 
2.3 .times. 10.sup.4 
1000 1 4.5 .times. 10.sup.4 
1.0. .times. 10.sup.4 
5.0 .times. 10.sup.2 
1500 1 1.1 .times. 10.sup.3 
1.7 .times. 10.sup.4 
4.0 .times. 10.sup.2 
______________________________________ 
3. COMMENTS 
3.1 The control levels of SRB, anaerobes and aerobes are all high 
indicating the presence of the high concentration of viable bacteria in 
the biofilms. 
3.2 All biocides contain the same total active level. 
3.3 The results for biocide 3 (Formaldehyde based) show that this system is 
not very effective. 
3.4 The results for biocide 2 (THPS based) show it to be fairly active. 
3.5 The results for biocide 1 (THPS/Formaldehyde based) show it to be more 
effective than biocide 2 despite having a lower THPS content (the deficit 
being made up with less active formaldehyde). It is therefore apparent 
that synergism is occurring. 
EXAMPLE 2 
A composition comprising equal proportions of a glutaraldehyde and THPS 
showed increased activity against bacteria compared with glutaraldehyde 
and increased activity against fungi compared with THPS at equivalent 
total biocide concentrations. In each case the activity was substantially 
greater than the mean of the activities of the separate biocides.