Note: Descriptions are shown in the official language in which they were submitted.
21~1622
W-833/5
PROCESS AND COMPOSITION FOR INHIBITING
MICROBIAL GROWTH
FIELD OF THE INVENTION
This invention relates to compositions and methods for controlling . ~ -
the growth of Trichoderma viride.
BACKGROUND OF THE INVENTION
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The formation of slimes by microorganisms is a problem that is
encountered in many aqueous systems. For example, the problem is not
15 only found in natural waters such as lagoons, lakes, ponds, etc., and
confined waters as in pools, but also in such industrial systems as cool-
ing water systems, air washer systems and pulp and paper mill systems.
All possess conditions which are conducive to the growth and reproduc-
tion of slime-forming microorganisms. In both once-through and recircu-
20 lating cooling systems, for example, which employ large quantities ofwater as a cooling medium, the formation of slime by microorganisms is
an extensive and constant problem.
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Airborne organisms are readily entrained in the water from cooling
towers and find this warm medium an ideal environment for growth and
multiplication. Aerobic and heliotropic organisms flourish on the tower
proper whila other organisms colonize and grow in such areas as the
5 tower sump and the piping and passages of the cooling system. The
slime formation not only aids in the deterioration of the tower structure in
the case of wooden towers, but also promotes corrosion when it deposits
on metal surfaces. Slime carried through the cooling system plugs and
fouls lines, valves, strainers, etc., and deposits on heat exchange sur-
10 faces. In the latter case, the impedance of heat transfer can greatlyreduce the efficiency of the cooling system.
In pulp and paper mill systems, slime formed by microorganisms is
commonly encountered and causes fouling, plugging, or corrosion of the
15 system. The slime also becomes entrained in the paper produced to
cause breakouts on the paper machines, which results in work stoppages
and the loss of production time. The slime is also responsible for un-
sightly blemishes in the final product, which result in rejects and wasted
output.
The previously discussed problems have resulted in the extensive
utilization of biocides in cooling water and pulp and paper mill systems.
Materials which have enjoyed widespread use in such applications in-
clude chlorine, chlorinated phenols, organo-bromines, and various or-
25 gano-sulfur compounds. All of these compounds are generally useful for
this purpose but each is attended by a variety of impediments. For ex-
ample, chlorination is limited both by its specific toxicity for slime-forming
organisms at aconomic levels and by the tendency of chlorine to react,
which results in the expenditure of the chlorine before its full
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biocidal function is achieved. Other biocides are attended by odor prob-
lems and hazards with respect to storage, use or handling which limit
their utility. To date, no one compound or type of compound has
achieved a clearly established predominance with respect to the applica-
5 tions discussed. Likewise, lagoons, ponds, lakes, and even pools, eitherused for pleasure purposes or used for industrial purposes for the dis-
posal and storage of industrial wastes, become, during the warm
weather, besieged by slime due to microorganism growth and reproduc-
tion. In the case of industrial storage or disposal of industrial materials,
10 the microorganisms cause additional problems which must be eliminated
prior to the materials' use or disposal of the waste.
Naturally, economy is a major consideration with respect to all of
these biocides. Such economic considerations attach to both the cost of
15 the biocide and the expense of its application. The cost performance
index of any biocide is derived from the basic cost of the material, its
effectiveness per unit of weight, the duration of its biocidal or biostatic
effect in the system treated, and the ease and frequency of its addition to
the system treated. To date, none of the commercially available biocides
20 has exhibited a prolonged biocidal effect. Instead, their effectiveness is
rapidly reduced as a result of exposure to physical conditions such as
temperature, association with ingredients contained by the system toward
which they exhibit an affinity or substantivity, etc., with a resultant restric-tion or elimination of their biocidal effectiveness, or by dilution.
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As a consequence, the use of such biocides involves their con-
tinuous or frequent addition to the systems to be treated and their addi-
tion to multiple points or zones in the systems to be treated. Accordingly,
the cost of the biocide and the labor cost of applying it are considerable.
5 In other instances, the difficulty of access to the zone in which slime for-
mation is 0xperienced precludes the effective use of a biocide. For ex-
ample, if in a particular system there is no access to an area at which
slime formation occurs the biocide can only be applied at a point which is
upstream in the flow system. However, the physical or chemical condi-
10 tions, e.g., chemical reactivity, thermal degradation, etc., which existbetween the point at which the biocide may be added to the system and
the point at which its biocidal effect is desired render the effective use of
a biocide impossible.
Similarly, in a system experiencing relatively slow flow, such as a
paper mill, if a biocide is added at the beginning of the system, its bioci-
dal effect may be completely dissipated before it has reached all of the
points at which this effect is desired or required. As a consequence, the
biocide must be added at multiple points, and even then a diminishing
20 biocidal effect will be experienced between one point of addition to the
system and the next point downstream at which the biocides may be
added. In addition to the increased cost of utilizing and maintaining mul-
tiple feed points, gross ineconomies with respect to the cost of the
biocide are experienced. Specifically, at each point of addition, an ex-
25 cess of the biocide is added to the system in order to compensate for thatportion of the biocide which will be expended in reacting with other con-
stituents present in the system or experience physical changes which
impair its biocidal activity.
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SUMMARY OF THE INVENTION
The present inventors have discovered that a composition of 2-(2-
bromo-2-nitroethenyl) furan (BNEF) and another biocidal component is
effective as a biocide directed towards controlling Trichoderma viride.
DESCRIPTION OF THE RELATED ART
U.S. Patent No. 5,158,972, Whitekettle et al., teaches the use of 2-(2-
bromo-2-nitroethenyl) furan and glutaraldehyde to control the growth of
Klebsiella r neumoniae. U.S. Pat. No. 4,965,377, McCoy et al., teaches a
method for forming 2-(2-bromo-2-nitroethenyl) furan which proved effec-
tive as an antimicrobial agent.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to compositions and methods for
controlling the growth of fungi comprising a synergistic mixture of (a) 2- ~ ;
(2-bromo-2-nitroethenyl) furan (BNEF) and (b) at least one additional
component selected from:
(1 ) Dodecylguanadine hydrochloride (DGH), or
(2) Diiodomethyl-p-tolylsulfone (DMTS).
The present inventors have found these combinations particularly
effective against the Trichoderma viride species which is a common
fungal nuisance found in industrial cooling waters and pulping and
papermaking systems.
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21~ 622
This particular species of mold is a member of the Fungi Imperfecti
which reproduce by means of asexual spores or fragmentation of myce-
lium. It is commonly found on fallen timber and is a widely occurring soil
organism. Because of its ubiquitous nature, this mold continually con-
taminates open cooling systems and pulping and papermaking systems.
Contamination can take the form of airborne spores or fungal mats - a
mass of agglomerated hyphae bound together with bacterial cells and
cemented by gelatinous polysaccharide or proteinaceous material. The
slimy mass entraps other detritus, restricts water flow and heat transfer
and may serve as a site for corrosion.
These fungi are able to grow in environments hostile to other life-
forms. While they are strict aerobes, Trichoderma produce both hyphae,
the vegetative structure, and spores which require minimal metabolic
turnover and are able to withstand harsher environmental conditions.
Accordingly, by reason of demonstrated efficacy in the growth inhibition
of this particular species, one can expect similar growth inhibition attrib-
utes when other fungi are encountered. It is also expected that these
compositions will exhibit similar srowth inhibition attributes when bacterial
andalgal speciesareencountered. ~:
In accordance with the present invention, the combined treatment
may be added to the desired aqueous system in need of biocidal treat-
ment, in an amount of from about 0.1 to about 200 parts of the combined
25 treatment of BNEF and DGH to one million parts (by weight) of the aque-
ous medium. Preferably, about 5 to about 50 parts of the combined
treatment of BNEF and DGH per one million parts (by wei~ht) of the
aqueous medium is added. The BNEF and DMTS may be added in an
amount of frorn about 1 to about 500 parts per million parts (by weight) of
30 the aqueous medium.
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The combined treatment is added, for example, to cooling water
systems, paper and pulp mill systems, pools, ponds, lagoons, lakes, etc.,
to control the formation of fungal microorganisms, which may be con-
tained by, or which may become entrained in, the system to be treated. It
5 has been found that the compositions and methods of utilization of the
treatment are efficacious in controlling the fungal organism, Trichoderma
viride, which may populate these systems. It is thought that the com-
bined treatment composition and method of the present invention will also
be efficacious in inhibiting and controlling all types of aerobic microorgan-
1 0 isms.
Surprisingly, it has been found that when the ingredients aremixed, in certain instances, the resulting mixtures possess a higher de-
gree of fungicidal activity than that of the individual ingredients compris-
15 ing the mixture. Accordingly, it is possible to produce a highly efficaciousbiocide. Because of the enhanced activity of the mixture, the total
quantity of the biocidal treatment may be reduced. In addition, the high
degree of biocidal effectiveness which is provided by each of the ingredi-
ents may be exploited without use of higher concentrations of each.
The following experimental data were developed. It is to be
remembered that the following examples are to be regarded solely as
being illustrative and not as restricting the scope of the invention.
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2~ 2~622
DESCRIPTION OF THE PREFERRED EMBODIMENT
The combined treatments were added in varying ratios and over a
wide range of concentrations to a iiquid nutrient medium which was sub-
5 sequently inoculated with a standard volume of a suspension of sporesfrom Trichoderma viride. Growth was measured by determining the
amount of radioactivity accumulated by the cells when 14C-glucose was
added as the sole source of carbon in the nutrient medium. The effect of
the biocide chemicals, alone and in combination, is to reduce the rate
10 and amount of 14C incorporation into the cells during incubation, as
compared to controls not treated with the chemicals. Additions of the
biocides, alone and in varying combinations and concentrations, were
made according to the accepted "checkerboard" technique described by
M.T. Kelley and J. M. Matsen, Antimicrobial A~ents and ChemotheraPv.
15 9:440 (1976). Following a two hour incubation, the amount of radioactiY-
ity incorporated in the cells was determined by counting (1 4C liquid scin-
tillation procedures) for all treated and untreated samples. The percent
reduction of each treated sample was calculated from the relationship: :
20 Cont!ol 14C!cPm) - Treated 14C(cpm) x 100 = % reduction
Control 14C(cpm)
Plotting the % reduction of 14C level against the concentration of
each biocide acting alone results in a dose-response curve, from which
25 the biocide dose necessary to achieve any given % reduction can be
interpolated.
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Synergism was determined by the method of calculation described
by F.C. Kull, P.C. Eisman, H.D. Sylwestrowicz and R.L. Mayer, APPlied
Microbioloqv, 9,538 (1961) using the relationship:
QA QB
--+--= synergism index (Sl) ~ ~;
. . .
wherein~
Qa = quantity of compound A, acting alone, producing an end point
Qb = quantity of compound B, acting alone, producing an end point
QA = quantity of compound A in mixture, producing an end point
QB = quantity of compound B in mixture, producing an end point
The end point used in the calculations is the % reduction caused
by each mixture of A and B. QA and QB are the individual concentrations
in the A/B mixture causing a given % reduction. Qa and Qb are deter~
mined by interpolation from the respective dose response curves of A
and B as those concentrations of A and B acting alone which produce the . ~:same % reduction as each specific mixture produced.
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2121622
Dose-response curves for each active acting alone were deter-
mined by linear regression analysis of the dose-response data. Data
were fitted to a curve represented by the equation shown with each data
set. After linearizing the data, the contributions of each biocide compo-
5 nent in the biocide mixtures to the inhibition of radioisotope uptake weredetermined by interpolation with the dose-response curve of the respec-
tive biocide. If, for example, quantities f QA plus QB are sufficient to
give a 50% reduction in 1 4C content, Qa and Qb are those quantities of
A are B acting alone, respectively, found to give 50% reduction in 1 4C
10 content. A synergism index (Sl) is calculated for each combination of A
and B.
Where the Sl is less than 1, synergism exists. Where the Sl=1,
additivity exists. Where Sl is greater than 1, antagonism exists.
The data in the following tables come from treating Trichoderma
viride, a common nuisance fungal ~ype found in industrial cooling waters
and in pulping and papermaking systems, with varying ratios and concen-
trations of BNEF and DGH. Shown for each combination is the % reduc-
20 tion of 14C content (% I), the calculated Sl, and the weight ratio of BNEFand DGH.
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TABLE I ~
DGH vs. BNEF ~ .
ppm ppm Ratio
DGH1 BNEF2 DGH:BNEF %I Sl
2.5 0 100:0 91
1.25 0 100:0 83
0.63 0 100:0 38
0.31 0 100:0 0
0.16 0 100:0 0
0.04 0 100:0 0
0 40 0:100 89
0 20 0:100 81
0 5 0:100 55
0 1.25 0:100 12
0 0.63 0:100
0 0. 31 0:100 0
2.5 40 1:16 98 1.99
2.5 20 1:8 98 1.61
2.5 5 1:2 96 1.34
2.5 1.25 2:1 95 1.28
2.5 0.63 4:1 95 1.27
2.5 0.31 8:1 94 1.27
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12
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TABLE I (Cont'd)
DGH vs. BNEF
ppm ppm Ratio
5 DGH1 BNEF2 DGH.BNEF %I Sl
1.25 40 1 :32 97 1.44
1.25 20 1:16 96 1.05
1.25 5 1 :4 93 0.76
10 1.25 1.25 1: 1 91 0.68
1.25 0.63 2: 1 91 0.67
1.25 0.31 4:1 90 0.66
0.63 40 1 :64 95 1.25
0.63 20 1 :32 91 0.87~
15 0.63 5 1:8 84 054b
0.63 1.25 1 :2 79 0.43
0.63 0.63 1: 1 73 0.44
0.63 0.31 2:1 60 0.52*
0.31 40 1:129 91 1.28
20 0.31 20 1 :65 82 1.02
0.31 5 1:16 62 0.79
0.31 1.25 1 :4 42 0.68
0.31 0.63 1 :2 39 0.56
0.31 0.31 1:1 21 0.83
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TABLE I (Cont'd) :
DGH vs. BNEF :- ~
ppm ppm Ratio -
DGH1 BNEF2 DGH:BNEF %I Sl
0.16 40 1:250 91 1.18
0.16 20 1:125 84 0.87
0.16 5 1:31 69 0.51
0.16 1.25 1 :8 37 0.64
0.16 0.63 1:4 29 0.55
0.16 0.31 1:2 22 0.52
0.04 40 1:1000 88 1.29
0.04 20 1 :500 81 0.93~
0.04 5 1:125 59 0.66~ :
0.04 1.25 1:31 12 1.59
0.04 0.63 1 :16 9 0.99 :
0.04 0.31 1 :8 0 1.02 - : ~ -
TABLE ll
DGH vs. BNEF
ppm ppm Ratio
DGH1 BNEF2 DGH:BNEF %I Sl
2.5 0 100:0 91
1.25 0 100:0 66
0.63 0 100:0 19
0.31 0 100:0 0
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14
TABLE ll (cont'd)
DGH vs. BNEF
ppm ppm Ratio
5 DGH1 BNEF2 DGH:BNEF %i Sl
0.16 0 100:0 0
0.04 0 100:0 0
0 40 0: 100 90
10 0 20 0: 100 85
0 5 0: 100 53
0 1.25 0:100 16
0 0.63 0:100 11
0 0.31 0:100 0
15 2.5 40 1:16 98 1.88
2.5 20 1 :8 98 1.46
2.5 5 1:2 97 1.17
2.5 1.25 2:1 96 1.10
2.5 0.63 4:1 95 1.10
20 2.5 0.31 8:1 94 1.10
1.25 40 1 :32 98 1.39
1.25 20 1:16 97 0.98
1.25 5 1 :4 93 0.68*
1.25 1.25 1: 1 82 0.68*
25 1.25 0.63 2:1 85 0.63*
1.25 0.31 4:1 89 0.58*
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TABLE !l (cont'd)
DGH vs. BNEF
ppm ppm Ratio
5 pGH1 BNEF2 DGH:BNEF %I Sl
0.63 40 1 :64 94 1.28
0.63 20 1 :32 91 0.87*
0.63 5 1 :8 81 0.55*
0.63 1.25 1:2 63 0.55*
0.63 0.63 1 :1 56 0.54*
0.63 0.31 2:1 51 0.54*
0.31 40 1:129 94 1.19
0.31 20 1 :65 89 0.80*
0.31 5 1:16 76 0.47*
0.31 1.25 1 :4 28 1.19
0.31 0.63 1 :2 25 0.92*
0.31 0.31 1:1 26 0.65*
0.16 40 1:250 93 1.12
0.16 20 1:125 88 0.77*
0.16 5 1:31 73 0.45*
0.16 1.25 1:8 34 0.82*
0.16 0.63 1:4 22 0.77*
0.16 0.31 1:2 23 0.49*
0.04 40 1:1000 92 1.15
0.04 20 1 :500 87 0.73*
0.04 5 1:125 53 1.00
0.04 1.25 1:31 19 1.36
0.04 0.63 1:16 12 0.96
0.04 0.31 1 :8 9 0.62
:` 212~2
16
Asterisks in the Sl column indicate synergistic combinations in
accordance with the Kull method supra, while:
indicates a product with 33% actives D~H and
2 indicates a product with 10% actives BNEF
In Tables I and ll, differences seen between the replicates are due
to normal experimental variance.
In accordance with Tables l-ll supra., unexpected results occurred
more frequently within the product ratios of DGH to BNEF of from about
4:1 to 1:500. Since the DGH product contains about 33% active biocidal
component, and the BNEF product contains about 10% active biocidal
component, when based on the active biocidal component, unexpected
results appear more frequently within the range of active component of
DGH:BNEF of about 13:1 to 1:150. At present, it is most preferred that
any comm0rcial product embodying the invention comprises a weight
ratio of active component of about 1:1 DGH:BNEF.
The data in the following tables come from treating Trichoderma
viride, a common nuisance fungal type found in industrial cooling waters
and in pulping and papermaking systems, with varying ratios and concen-
trations of BNEF and DMTS. Shown for eacl1 combination is the % re-
duction of 14C content (%I), the calculated Sl, and the weight ratio of
BI~IE~ and DMTS.
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2~21622
17 .
TABLE lll
DMTS vs. BNEF
ppm ppm Ratio
DMTS BNEF DMTS:BNEF %l Sl
6 0 100:0 96
3 0 100:0 93
1.5 0 100:0 90
0.75 0 100:0 77
0.38 0 100:0 60
0.19 0 100:0 54
0 40 0: 100 91
0 20 0: 100 83
0 5 0:100 71
0 2.5 0:100 62
0 1.25 0: 100 52
0 0.63 0: 100 38
6 40 1:6.7 98 2.14
6 20 1 :3.3 97 1.86
6 5 1.2:1 96 1.64
6 2.5 2.4:1 96 1.65
6 1.25 4.8:1 96 1.66
6 0.63 9.5:1 96 1.62
3 40 1:13.3 92 2.21
3 20 1 :6.7 94 1.44
3 5 1:1.7 93 1.11
3 2.5 1.2:1 94 0.97
3 1.25 2.4:1 93 1.04
3 0.63 4.8: 1 94 0.96
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18
TABLE lll (Cont'd)
DMTS vs. BNEF
ppm ppm Ratio
5 DMTS BNEF DMTS:BNEF %I Sl
1.5 40 1 :26.7 86 2.64
1.5 20 1:13.3 89 1.38
1.5 5 1 :3.3 92 069*
1.5 2.5 1: 1.7 91 0.65*
1.5 1.25 1.2:1 89 0.70*
1.5 0.63 2.4: 1 40 0.66*
0.75 40 1 :53.3 94 1.12
0.75 20 1 :26.7 91 0.86*
0.75 5 1 :6.7 85 0.66*
0.75 2.5 1 :3.3 83 0.65*
0.75 1.25 1:1.7 80 0.74*
0.75 0.63 1.2:1 77 0.83*
0.38 40 1:105.3 94 1.06
0.38 20 1 :52.6 91 0.74*
0.38 5 1:13.2 81 0.63*
0.38 2.5 1 :6.6 78 0.57~
0.38 1.25 1 :3.3 71 0.79*
0.38 0.63 1:1.7 61 1.41
0.19 40 1:210.5 91 1.25
0.19 20 1:105.3 88 0.82
0.19 5 1:26.3 78 0.60
0.19 2.5 1:13.2 69 0.78
0.19 1.25 1 :6.6 61 1.01
0.19 0.63 1:3.3 54 1.34
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212~ ~22
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TABLE IV
DMTS vs. BNEF
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DMTS BNEF DMTS:BNEF %l Sl
6 0 100:0 95
3 0 100:0 92
1.5 0 100:0 88
0.75 0 100:0 79
0.38 0 100:0 58
0.19 0 100:0 34
0 40 0: 100 86
0 20 0: 100 76
~- 15 0 5 0:100 54
0 2.5 0:100 38
0 1.25 0: 100 29
0 0.63 0:100 17
6 40 1 :6.7 97 2.31
6 20 1 :3.3 95 2.32
6 5 1.2:1 96 1.95
6 2.5 2.4:1 96 1 96
6 1.25 4.8:1 95 1 98
6 0.63 9.5:1 95 1.96
3 40 1:13.3 97 1.46
3 20 1 :6.7 96 1.24
3 5 1:1.7 95 1 07
3 2.5 1.2:1 94 1 07
3 1.25 2.4:1 94 1 07
3 0.63 4.8:1 94 1 07
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2~21 622
TABLE IV (Cont'd)
DMTS vs. BNEF
ppm ppm Ratio ~: I5 DMTS BNEF DMTS:BNEF %I Sl
1.5 40 1 :26.7 96 1.07
1.5 20 1:13.3 94 0.84
1.5 5 1 :3.3 92 0.68~
1.5 2.5 1:1.7 91 0.65~ -
1.5 1.25 1.2:1 89 0.69
1.5 0.63 2.4:1 88 0.72
0.75 40 1 :53.3 g4 0.93
0.75 20 1 :26.7 93 0.64
0.75 5 1 :6.7 85 0.54~
0.75 2.5 1 :3.3 81 0.60*
0.75 1.25 1 :1.7 80 0.57
0.75 0.63 1.2:1 81 0.52
0.38 40 1:105.3 93 0.84
0.38 20 1 :52.6 89 0.60
0.38 5 1:13.2 77 0.54
0.38 2.5 1 :6.6 62 0.93
0.38 1.25 1 :3.3 51 1.43
0.38 0.63 1:1.7 63 0.79
0.19 40 1:210.5 93 0.78
0.19 20 1:105.3 86 0.63
0.19 5 1:26.3 75 0.43
0.19 2.5 1:13.2 54 0.94
0.19 1.25 1:6.6 46 1.12
0.19 0.63 1:3.3 41 1.21
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Asterisks in the Sl column indicate synergistic combinations in
accordance with the Kull method supra.
.
In Tables lll and IV, differences seen between the replicates are
5 due to normal experimental variance.
In accordance with Tables III-IV supra., unexpected results occurr-
ed more frequently within the product ratios of DMTS to BNEF of from
about 2.4:1 to about 1:105.3 as 100% actives. Since the DMTS product
10 contains 40% active biocidal component and the BNEF product contains
10% active biocidal component, unexpected results appear more fre-
~uently within the range of active component (100% actives basis) of
DMTS:BNEF of about 9.6:1 to about 1:26.3. At present, the most pre-
ferred ratio comprises a weight ratio of active component of about 1:1
15 DMTS:BNEF.
I
While this invention has been described with respect to particular
embodirrlents thereof, it is apparent that numerous other forms and modi-
fications of this invention will be obvious to those skilled in the art. The
20 appended claims and this invention generally should be construed to
cover all such obvious forms and modifications which are within the true
spirit and scope of ~he present invention.
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