Technical Paper

Biocides

Defensive synergy
Combining preservatives enhances protection of adhesives and sealants

Contact:
Matthias Hentz
Schlke & Mayr GmbH
T +49 40 52100-291
matthias.hentz@
schuelke.com

Matthias Hentz
Many waterborne coating and adhesive systems
are susceptible to microbiological spoilage. The
common isothiozolinone mixture of MIT and BIT
provides very effective long-term action, but it can
be slow to become effective. Bis(3-aminopropyl)
dodecylamine (BDA) is shown to operate synergistically with MIT/BIT and offers an effective way to
overcome this weakness.

any waterborne systems including adhesives

and sealants are susceptible to microbiological contamination and spoilage and require an
in-can preservative offering protection not only during
shelf-life but also during manufacture. Due to their different physiochemical or biological characteristics, not all
available preservatives are appropriate and fit in with the
type of formulation to be used.
Failure of preservation could cause severe spoilage due
to microbiological growth, leading to discolouration, loss
of paint structure or even foul gases and odours in
many cases these batches or even the finished products
cannot be marketed. This waste of resources conflicts
with what sustainability of products stands for: safe and
ecologically sound operation throughout their life cycle.

Coatings biocides: current situation

Figure 1: Germ count reduction tests comparing MIT/BIT/BDA with MIT/BIT

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Where accepted, in-can preservatives based on heterocyclic N,S compounds, such as chloromethylisothiazolinone
(CMIT) in combination with methylisothiazolinone (MIT)
are often formulated with formaldehyde-releasing
agents. These agents are known for their broad efficacy, vapour phase activity and fast-acting properties and
guarantee a microbiologically safe in-can system. However, these products are increasingly avoided by many
industries.
On the other hand, combinations like benzisothiazolinone
(BIT) and methylisothiazolinone (MIT) are widely known
and accepted as soft in-can preservatives for systems
free of formaldehyde. A broad spectrum of efficacy
against bacteria, yeast and moulds and a favourable
ecotoxicity profile makes this combination state of the art
when it comes to systems free of VOC and formaldehyde
or formaldehyde releasers. Nevertheless, weaknesses
occur when considering their speed of action (Figure 1)
though they offer good long term protection.
A problem now and especially in the near future is the
forthcoming European Biocidal Products Regulation
(BPR). This will lead to major changes regarding the
availability of biocidal actives. Out of more than 700 molecules identified for registration in 2001, only 92 have so
far been notified and all finally approved and marketed
active substances may amount to not more than 20-30,
even being optimistic. Moreover, the need to satisfy certain ecolabel requirements makes extensive research on
possible active combinations based on registered biocidal actives essential.
Bis(3-aminopropyl)dodecylamine (BDA) has been known
for years as an effective biocidal active. Main characteristics are its fast speed of action combined with a broad
and balanced spectrum of effect against bacteria, yeasts
and moulds in low concentrations. From extensive research work, BDA was recognised as an excellent syn-

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Technical Paper
Biocides

Figure 2: New approach to the usage of an in-can preservative

ergist for todays standard soft preservatives based on

MIT combined with BIT. It is able to markedly reduce the
necessary contact time to only a few minutes.

Experimental procedure for

biocidal tests
Biocide mixtures with and without BDA (as shown in Table 1) were used in initial germ count reduction tests.
Dilutions of the preservatives were prepared using sterile
hard water according to the European standard for testing chemical disinfectants and antiseptics. 50 ml portions
of the final solutions were each inoculated with 0.5 ml
micro-organism suspension having an initial micro-or-

Results at a glance
The common isothiozolinone mixture of CMI and
BIT provides very effective long-term action, but its
weakness is that it can be slow to become effective.
The use of formaldehyde to overcome this is increasingly unpopular and will be banned by 2015 within
consumer products.
Bis(3-aminopropyl)dodecylamine (BDA) is considered to be a safe biocide with rapid action. Its mode
of attack on micro-organisms differs from that of
MIT/BIT, and it is shown experimentally that a combination of these three materials operates effectively
and synergistically.
It is also suggested that to minimise any risk of
infection, the biocides should be the first products
added to the process water during manufacture. This
approach creates an effective formaldehyde-free
system which complies with the Biocidal Products
Regulation (BPR).

ganism count of approximately 105 cfu/ml (cfu = Colony

Forming Units) and stirred.
The mixture of test strains contained Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 11229), Candida albicans (ATCC 10231) as well as a freshly prepared
mixture of Enterobacter gergoviae (ATCC 33028), Escherichia coli (ATCC 11229), Klebsiella pneumoniae (ATCC
4352), Kocuria rhizophila (ATCC 9341), Pseudomonas fluorescens (ATCC 17397), Pseudomonas putida (ATCC 12633)
and Staphylococcus aureus (ATCC 6538).
These solutions were streaked out onto tryptone soya
agar (bacteria) or Sabouraud dextrose 4 % agar (fungi)
after 5, 10, 30 and 60 minutes. The cultures were incubated for 48 hours at 37 C. The evaluation was made on
the basis of semi-quantitative assessment of the microbial growth of the streaks.
Within the test period of 60 minutes the conventional
MIT/BIT mixture showed no efficacy even in high use
concentration. With the triple combination of MIT/BIT/
BDA a distinct effect was observed within five minutes.
The graphs in Figure 1 display the germ count reduction
test results against Staphylococcus aureus and the germ
suspension described above.

Minimising contamination risks

during production
Built upon this test, schlke developed a completely new
approach for using an in-can preservative during the
manufacturing process of adhesives and sealants: the
recommended order of addition (see Figure 2).

MIT/BIT

MIT/BIT/
BDA

Methylisothiazolinone (MIT)

2.5 %

2.5 %

Benzisothiazolinone (BIT)

2.5 %

2.5 %

2.7 %

Bis(3-aminopropyl)dodecylamine (BDA)

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Table 1: Biocide
mixtures with and
without BDA

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Table 2: MIC (Minimum Inhibitory Concentration) values for different active combinations based on MIT, BIT and BDA and synergy levels
MIC [% of the product]

P. aeruginosa

S. aureus

C. albicans

2,5 % MIT, 2,5 % BIT, 2,7 % BDA

0.031

0.031

0.016

7.7 % BDA

1.136

0.55

1.136

7.7 % MIT and BIT

0.04

0.06

0.07

SI (Synergy Index)

0.802

0.573

0.242

The combination of BIT, MIT and BDA ("parmetol MBX") is

added as the first component to the process water within the manufacturing process of any adhesive or sealant system. The fast speed of action allows the process
water and all potentially contaminated raw materials to
be treated in order to avoid any spoilage due to microbiological growth.
Accordingly, the risk of microbiological contamination
will be reduced over the entire manufacturing process. A
fast-acting biocide with long term protection characteristics allows for a safer manufacturing process and a wellpreserved final product. Lower usage concentration due
to a distinct synergistic effect between BDA, MIT and BIT,
lowered risk of downtimes and product recalls lead to an
economisation of raw materials and energy consumption
and adds value to the customer.

Why the biocide combination

should show synergy
As mentioned above, the combination of MIT, BIT and
BDA was found to have a synergistic nature due to their
different ways of action against micro-organisms. On the
one hand two moderate electrophiles like MIT and BIT
are representatives of a group of molecules referred to
as heterocyclic N,S-compounds. Because of their electrophilic character, these molecules react favourably with
thiol groups which are part of the amino acid cysteine a
protein.

Table 3: Challenge test on a dispersion paint using a mixed inoculum of bacteria,

yeast and moulds, identified as B, Y, M respectively in the results
FaKo Test SM 020
Weekly inoculation with
1010 cfu/ml(inoculum)

Inoculation Cycles
0

Adhesive,
unpreserved

+++
B,Y,M

+++
B,Y,M

./.

0.10 % MIT/BIT
(2.5/2.5 %)

+++
B,Y,M

+++
B,Y,M

./.

0.15 % MIT/BIT
(2.5/2.5 %)

+ B,Y

+ B,Y

++ B,Y

++ B,Y

0.10 % MIT/BIT/BDA
(2.5/2.5 /2.7 %)

+B

+B

0.15 % MIT/BIT/BDA
(2.5/2.5 /2.7 %)

Bacteria | Yeast | Mould

- free of growth | + slight growth | ++ moderate growth | +++ heavy growth

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At least four vital enzymes within the reaction centre of

bacteria and moulds carry cysteine. If these enzymes are
interrupted, irreversible damage of the micro-organism
can occur. It has been proved that isothiazolinones are
actively transported into the cell and not only interrupt
but also cross-link cell molecules via functional groups
and destroy the metabolic balance and structure of a cell.
Apart from electrophiles, membrane-active molecules
such as BDA, a long chain alkylamine, are known. These
amines react with cell membranes and damage the outer cell wall, causing a change in the membrane permeability which finally results in an osmotic lysis. This lysis
then allows the other actives MIT and BIT to penetrate
the weakened cell wall.

Evaluating the level of synergy

Accordingly, MIC values tests were carried out related to
the DGHM (German society for Hygiene and Microbiology) procedures and synergism was determined by a
method explained in Kull et al [1] applying the formula:

(1)
Where:
QA = concentration of compound A in % of product, acting
alone, which produced an end point (MIC compound A);
Qa = concentration of compound A in % of product, in the
mixture, which produced an end point;
QB = concentration of compound B in % of product, acting
alone, which produced an end point (MIC compound B);
Qb = concentration of compound B in % of product, in the
mixture, which produced an end point.
If SI (the Synergy Index) is greater than one, antagonism
between the products is indicated. If the sum is equal
to one, additivity is indicated. But when SI is less than
one, synergism is shown, i.e. the lower the SI the greater
the synergy shown. According to the data in Table 2, the
combination of MIT, BIT and BDA acts synergetic.

Further tests confirm that

synergy exists
Further testing was carried out to prove the performance
of this combination. This is done by applying a challenge
test to determine the susceptibility of a waterborne formulation to microbiological spoilage and accordingly the
preservation efficacy of the in-can preservative system
used.
In separate batches, different concentrations of the test
preservatives are added to the unpreserved samples. An
unpreserved sample is tested as a growth control. Two
days after incorporation of the preservatives the test
batches are inoculated with a current germ load (0.2 ml,
titre 1010 cfu/ml) with a further weekly inoculation of the
test batches.
The bacterial inoculum was similar to that used in the
tests described previously, but specifically consisted of:
Kocuria rhizophila (ATCC 9341), Staphylococcus aureus
(ATCC 6538), Enterobacter gergoviae (ATCC 33028), Escherichia coli (ATCC 11229), Klebsiella pneumoniae (ATCC
4352),Pseudomonas aeruginosa (ATCC 9027), P. fluorescens (ATCC 17397), and P. putida (ATCC 12633).

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Technical Paper
Biocides
The yeasts used were Candida albicans (ATCC 10231) and
Aspergillus brasiliensis (ATCC 16404) with Penicillium funiculosum (ATCC 36839) used as the test mould.
The test batches were both inoculated and streaked out
on agar plates once a week (tryptone-soya agar for bacteria (TSA) and Sabouraud dextrose agar (SA) for yeasts
and moulds). The first streak (sterility test) was done on
agar plates with and without neutraliser TLSH to detect
as much pre-contamination as possible.
After three days of incubation at 25 C the microbial
growth of the streak cultures is evaluated. For safety reasons negative streaks are observed for another two days
and evaluated again. The preserving effect of the various
product concentrations is judged semi-quantitatively by a
system from via + to +++. The microbial growth is classified in relation to bacteria, yeasts and
moulds. Generally the test is performed
for a maximum of six weeks, i.e. six inoculation cycles, but stopping after +++
growth becomes evident.
The results were clear: 0.15 % of the MIT,
BIT and BDA combination withstood the
entire challenge test whereas MIT and
BIT alone at the same concentration
showed moderate growth of bacteria
and moulds after three cycles. These
results are summarised in Table 3.

Future-proof protection
The combination of benzisothiazolinone
(BIT), methylisothiazolinone (MIT) and
bis(3-aminopropyl)dodecylamine (BDA)
has been shown to feature a synergistic
effect due to their different modes of
action against micro-organisms, characterised by their different chemical
nature: heterocyclic N,S-compounds
and long chain alkylamines.
This synergism leads to a lower level of
usage allows for fast action due to the
presence of BDA and due to the synergetic combination of MIT/BIT and BDA
for sound in-can preservation properties thus forming an alternative product that does not add to VOC or AOX
content.
Free of formaldehyde may be the future path of adhesive and sealant preservation and a broad compliance to
eco label requirements is already necessary. Consuming less resources and
still maintaining production process and
finished products safe and ecologically
sound is the way to guarantee sustainability along the entire supply chain.
Several tests demonstrated that this
sustainable approach of process and
product hygiene is a distinct alternative
to commonly used biocide combinations such as BIT and MIT or formaldehyde and formaldehyde releasers. The
combination of MIT, BIT and BDA is a

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ECJ

future-oriented, forward-looking solution for todays and

tomorrows requirements.

REFERENCE
[1] Kull F. C. et al, Applied Microbiology, Vol. 9, 1961, pp 538-541.

BIBLIOGRAPHY
[1] 
Siegert W., Gckel A., Carstens S., International Journal for Applied
Science, June 2011, pp 35-40.
[2] 
Roden K., The Preservation of Personal Care Products, Society of
Cosmetic Scientists (Singapore), 2009.
[3] 
Paulus W., Directory of Microbicides for the Protection of Materials: A Handbook, Kluwer Academic Publishers, 2005.