Selecting Preservatives and Testing Microbial Resistance

Need for Preservatives

Microorganisms can breakdown adhesive or sealant products before their service life is complete or, in some cases, even before their service life has even begun. These microorganisms include bacteria, fungi, yeast, and mold.

Microorganisms exist everywhere, especially where water and appropriate nutrients are available for their growth and survival. They thrive primarily at 20 - 30°C and high humidity.

Given their relatively simple needs for life, microorganisms can attach and grow in many adhesive and sealant products that contain water or naturally-based ingredients.

Some good sources of food include:

Starch
Dextrin
Cellulose
Animal fats
Vegetable oils
Polymers that contain aliphatic hydroxyl and ester groups

Polycaprolactone
Polyester-based urethanes, and
Biopolymers

Synthetic polymer water emulsions that are especially susceptible to microbial contamination include polyvinyl acetate, polyvinyl alcohol, and ethylene/vinyl acetate.

Even RTV silicone sealants that do not inherently support microbal growth are subject to microbial degradation. External chemicals commonly found near construction sealants can migrate into the sealant, and these chemicals may support microbial growth.

The base polymer in an adhesive formulation is not the only component that the formulator needs to worry about when fighting microbial growth. Formulation additives are also often an excellent nutrition source and become a primary focus of biological attack. These include:

Ester plasticizers
Cellulosic rheology modifiers, and
Epoxy ester stabilizers among others

Microbial contamination can manifest itself in a number of ways. There are primarily two stages in which microbial infection can become dominant:

When the adhesive or sealant is in liquid form. The microbes can feed off the moist environment and nutrients supplied by additives and raw materials.
After the adhesive or sealant is applied and cured. The microbial attack can occur on the finished surfaces of the polymer film.

Microbial Growth in Polymer Emulsions

Microbial growth on polymer emulsions could lead to costly customer quality issues and down time for factory decontamination. Bacterial growth can also contribute to a decrease in indoor air quality and lead to human health problems.

The effects of microbial growth in polymer emulsions are listed in the table below.

Property Change Due to Microbial Infection	Impact
Viscosity change	Polymer dispersions can become thinner or thicker depending on the effect of increased concentration of acidic by-products. Phase separation can also occur. Viscosity increase and microbial infection can also restrict the flow within the factory equipment piping, filters, etc.
pH change	The metabolic by-products often are acidic in nature. The reduced pH will cause destabilization of the polymer dispersion and promote a corrosive environment both in the factory (surface of plant equipment) and once in service (corrosion of substrates).
Odor production	Bacteria are often sulfur-reducing. Other microbes have the ability to produce odors based on their biochemical reactions.
Gas production	Bacteria can produce hydrogen sulfide gas which leads to odor and gas production problems.
Color change	Microbes can change the color of the product before or after application. Sulfur-reducing bacteria generally blacken the polymer dispersion or the finished product.
Visible surface growth	Microbes lead to color and viscosity change (see above).
Corrosion	Corrosion of plant equipment and of substrates can occur from metabolic byproducts and acid production.
Change in properties (due mainly to reduction in molecular weight)	Breakdown of the polymer molecular weight and/or change of dispersion property characteristics can affect the end-use properties of the adhesive or sealant.

Effect of Microbial Growth in Polymer Emulsions

The adhesive and sealant emulsions that are most susceptible to microbial growth are listed below in order of susceptibility:

PVOH stabilized PVA
PVOH stabilized VAE
Cellulose stabilized VASE
Rubber emulsions (e.g., SBR, natural, and polyurethane)
Surfactant stabilized VAE or acrylic
Styrene acrylic
Acrylamides, N-methyl acrylamide, N-butyl methyl acrylamide

Most of the microorganisms encountered in industrial practice are in the range of 4-9 pH.

Fungal organisms are more prominent at acidic pH, and
Bacterial organisms are more prominent at neutral to slightly alkaline pH.

Polymer emulsions generally fall in the ideal pH range for microbial growth (table below).

Types of Polymer	pH
Ethylene vinyl acetate Polyvinyl acetate PVA/acrylic PVA/Versatate and PVA/Acrylic Polyurethane	Acidic (pH 3.5-6.5)
Acrylic Styrene acrylic Styrene butadiene Polyolefins	Alkaline (pH 7.0-9.5)

Now, let's explore how to prevent microbial degradation and the properties of an ideal preservative for adhesive and sealants...

How do Biocides Work?

Antimicrobial agents, collectively known as preservatives, biocides/ or fungicides, are added to certain adhesive and sealant formulations in order to inhibit the growth of microorganisms either during shipping and storage (in-can preservation) or after the product is applied (dry-film preservation). Each antimicrobial agent has a specific spectrum of activity depending on the microbial agent encountered and the susceptible material.

Biocides have mostly low molecular weight molecules that kill or suppress the growth of microorganisms. Molecular aspects of the action of these agents are outside can be found in the literature.^[1]

They generally work by entering the microorganism’s cell membrane to damage and inhibit the cell’s genetic activity or to interact directly with the cell membrane to destroy its integrity.

Mechanism of Antimicrobial Agents

Preventing Microbial Degradation

Biocides are particularly effective when used proactively in a formulation, however, they can also be used for clean-up of contaminated water or equipment. Proper factory maintenance strategies can prevent microbial infection from the source and reduce the need for a biocide.

The various plant hygiene preservation strategies are summarized below.

Keep incoming tank loading lines clean and dry or full of product protected with biocide. Avoid piping configurations that allow for the buildu-p of stagnant areas of emulsion.
Avoid leaving residues in storage tanks prior to filling with additional product, whenever possible.
Agitate storage tanks, if possible, and/or use materials in storage tanks on a first-in, first-out basis.
Avoid stagnant water. Avoid water in loading and packaging lines. Purge or drain all lines prior to use.
Check for contamination of process water and other key raw materials, including emulsions. This can be done by streaking samples of raw material onto appropriate growth media and incubating the growth media to observe the level of microbial growth (streak testing).
Clean and sanitize tanks, lines, hoses, and any surface that may come in contact with polymer emulsions. Cap hoses if possible. Inspect the cleaned areas visually after cleaning and sanitizing is completed, if possible.
Keep the manufacturing area clean and dry.
Conduct sterility testing in the manufacturing area on a monthly basis. Include visual check of the top of the storage tank each month.
Conduct streak testing on the outgoing product to confirm that they are free from microbial contamination.

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There are various preservation strategies for the formulator to use to protect his or her formulation. These include:

Checking and treating the water supply
Checking raw materials
Improving plant design and hygiene
Using a broad spectrum biocide

Properties of an Ideal Preservative for Polymer Emulsions

In an adhesive or sealant formulation, the main function of a biocide is to kill or inhibit the growth of microorganisms. In this respect they are used either to:

Prevent a potential problem
Correct a problem that already exists

In either case, the biocide must meet certain requirements to be an effective product. Some of the basic properties required for all biocides that are used in polymer emulsions are mentioned below.

Broad-spectrum activity against bacteria, molds, yeasts
Stability over a wide pH range
Stability at high temperature, not volatile
Resistance to redox agents
Water-soluble at low concentrations with correct partition characteristics
Compatibility with the polymer emulsion type and formulation components
No effect on viscosity
Low toxicity/ecotoxicology (generally free from heavy metals, formaldehyde, chlorophenols, etc.)
Relative regulatory approvals
Cost-effectiveness

Classification of Preservatives

Biocides can be classified in many different ways. The most practical method of classification is by how they work. The classes of biocides that will be discussed mainly are In-can and Dry-film preservatives.

In-can preservatives –
- Biocides work either with the product during manufacture and storage to increase shelf life or with the product after application to a substrate to prevent premature failure.
- In-can preservatives inhibit microbial growth in water-based products during the manufacturing process and product storage.

Dry-film preservatives –
- Dry-film fungicides inhibit mildew and/or algae growth in an applied adhesive or sealant. In dry film, the biocide additives are somewhat different than they are for in-can preservatives.
- Fungicides and mildewcides are used in both aqueous and solvent-borne adhesives and sealants to inhibit fungal and algae growth in the dry adhesive film.
- The primary requirement for a dry-film biocide is low water solubility for it is necessary that the active ingredient does not migrate out of the adhesive or sealant with time. Due to the requirement for fungicidal and algaecide activity, combinations of biocides are generally used.
- Dry film preservatives are used in both aqueous and solvent-based systems.

Biocide Chemical Families

Biocides are complex chemicals, typically with long chemical names. Formulators know many biocides by their general chemical classification or trade name.

A variety of chemicals can be used to stabilize polymers against biological attack. A general chemical classification scheme is listed in the table below.

Chemical Family	Characteristics
Formaldehyde/formalin and formaldehyde donors (e.g. Triazines)	Environmental concerns but still used due to low cost
Heavy metals (silver, mercury, etc.)	Some restricted due to toxicity and environmental concerns
Organosulfur: Isothiazolinone-based	Combination and separate use of 5-chloro-2-methyl-4-isothiazolin-3-one (CIT) and 2-methyl-4-isothiazolin-3-one (MIT) and 1,2 benzisothiazolin-3-one (BIT)
Organosulfur: Pyridine derivatives	Zinc pynithiones have low solubility in water
Others	Organic acids and their salts, nitrogen and phenolics compounds, glutaraldehyde, cyanobutane
Blended formulations of the above	Allows custom formulation for a specific product, processing range, and microorganism

Various Selected Chemical Types of Biocides

Optimal Preservative Selection

The choice and concentration of biocide depends on:

The type of microorganisms encountered
The chemical makeup of the formulation
The adhesive or sealant manufacturing processes, and
Their use in the service environment

The table below lists the principal features considered important in the choice of preservative.

Feature	Characteristics
Efficacy	Economical Broad-spectrum antimicrobial activity Unaffected by manufacturing processes or the physical condition of the product Effective over the product shelf life and/or service life
Safety, Health, and Environment	Safe at practical use levels and handling Appropriate regulatory approvals Acceptable environmental profile
Compatibility	Compatible and stable with a wide range of ingredients Water-soluble or dispersible in the product composition Effective over a wide pH range

Features Considered Important in Choosing a Biocide

Factors Influencing the Choice of Biocide

Although biocides have the lowest incorporation level of any additive in adhesives or sealants, they must be cost-effective. And, any biocide additive must be easy & safe to use, and acceptable to the environment. This final requirement is a source of consternation in certain regions where strict environmental regulations are limiting the use of what were once common biocide agents.

Other desirable features of a biocide include:

Rapid biocide activity
Lack of a strong or offensive odor
Water solubility
Heat stability, and
Microbial tolerance issues

Formulation and processing factors that are important in selecting a biocide and their influence on product performance are described in the following table.

Factor	Influence
Chemical composition of formulation ingredients	Most susceptible: Fillers such as china clay and calcium carbonate; natural materials such as starch and dextrin. Susceptible: Cellulose derivatives, polyvinyl alcohol solutions. Less susceptible: Polymer emulsions are generally attacked through their stabilizing system components rather than the polymer itself. Least susceptible: Concentrated or solid surfactants and defoamers.
Compatibility	Acid/base interactions can cause compatibility problems. This is generally seen as coagulation or gel formation.
pH	Preservatives are active over specific pH ranges. Polymer emulsion-based adhesives are prone to yeast and mold growth at pH of 3-7 and bacteria at pH of 6-8. Over pH of 9 and under a pH of 2, emulsion adhesives are less prone to attack.
Solids	Very low solids products have shown a particular sensitivity to attack.
Climate	Polymer emulsion adhesives designed for export to very warm countries are particularly susceptible to microbial attack. This is due to condensation of dilute films onto the product’s surface which are readily attacked.
Processing	High-temperature processes require the preservative to be heat stable or to be added after heat processing.
Redox state	Excess oxidizing agents can have deleterious effects on certain preservatives. This is also true for reducing agents.
Nucleophiles	Heterocyclic preservatives can be decomposed by nucleophilic attack. Primary, secondary and tertiary amines can deactivate some isothiazolinone preservatives. Sulfur nucleophiles like mercaptans shown the same effect.
Physical form	Liquid preservatives are easier to incorporate than powders and reduce exposure of factory personnel.
Solubility	The preservatives used should be soluble in the aqueous phase of the product.
Versatility	A broad spectrum of antimicrobial activity is required.
Cost	The high cost of some preservatives can be offset by their greater efficiency.

Factors Influencing the Choice of Biocide^[1]

Common Biocides for Polymer Dispersions

Some of the common preservatives currently used in polymer dispersions and characteristics important in their selection (e.g., target organisms, effect of oxidizing and reducing agents, and effect of pH and temperature) are shown in the table below.

Biocide	Target Organisms	Affected by Oxidizing Agents	Affected by Reducing Agents	Effective pH Range	Effective Temperature (°C)
1,3-Benzisothiasolin-2-one (BIT)	Bacterial & Fungal	Yes	Yes	2-14	<100
Methychloroisothiazolinone (CMIT)/Methylisothiazolinone (MIT)	Bacterial & Fungal	No	Yes	3-8	<40
1,2-Benzisothiaxolin-3-one (BIT)/Methylisothiazolinone (MIT)	Bacteria	Yes	Yes	3-10	<60
Methylisothiazolinone (MIT)	Bacteria	No	Yes	Up to 10	<45
Formaldehyde releasing biocides	Bacteria	Ammonia	Casein	*	**
Note: pH and temperature will determine the amount of formaldehyde release *Increased temperature increases formaldehyde release

Characteristics of Selected Common Biocides Used in Polymer Dispersions

Concentration of Biocide in an Emulsion

Antimicrobial agents are used in amounts ranging from ppm to 1% by weight. Additives or certain processing conditions can degrade the biocide originally present in the polymer emulsion. Therefore, the raw materials may contain little or no biocide by the time they are ready to use by the formulator, and they could be highly susceptible to biological contamination.

In these cases, additional biocide must be added to further preserve the product.

Since the biocide will become depleted with time, the level of biocide present in a formulation must be well above the minimum inhibitory concentration necessary to prevent biological growth.

Factors that can decrease the concentration of a biocide in an emulsion and affect its shelf life are mentioned below:

Dilution of the emulsion
Changes in pH
Use of high temperatures during formulation
Addition of reducing agents such as sulfites or bisulfites
Addition of oxidizing agents such as peroxides, persulfates or bleach
Addition of good nucleophiles such as ammonia, amines, or mercaptans
Addition of certain metal salts
Addition of biologically contaminated formulating ingredients
Addition of starches, modified celluloses, polyvinyl alcohol, sugars, and other naturally occurring polymers

A worrisome event that often plagues formulators is that the microorganism can mutate to become resistant to the selected biocide. This has resulted in the practice of biocide rotation.

Many suppliers will switch biocides every 6 months to 2 years even if there have been no outbreaks. The belief is that by switching products the microorganism is not allowed to become resistant to any specific biocide.

After you have selected a biocide/preservative for your formulation, its recommended to test if it would perform well.

Regulations Driving Future Trends

Environmental and regulatory concerns dominate the current state of biocide development. Compliance with VOC limits in the United States, the Biocidal Products Directive (BPD) and REACH legislation in Europe are driving much of the product development occurring today.

The proliferation of regulations around the world will continue to dramatically limit the availability of chemistry choices. As a result, many suppliers have reduced their R&D efforts regarding the development of new biocides and are focusing on acquisitions of other companies and chemical variations and blends of current products.

Controlled release of biocides at the right place and at the right time is the ultimate objective of technology to control microorganisms.

To this end, research has begun in biocide microencapsulation technology. A timely and targeted release improves the effectiveness of the biocide, broadens their application range, and ensures optimal dosage. Although this new technology offers great potential to the adhesive and sealant industries, little commercial development has been carried out to date in this field.

Adhesives Professionals - Stay Alert!

Update your knowledge on preservatives for adhesives and sealants by taking live course: Biocides in Waterborne Adhesives: How to Select in Today's Regulatory Environment? by Edward Petrie.

Biocides in Waterborne Adhesives: How to Select in Today's Regulatory Environment?

Testing Microbial Resistance of Formulations

Unfortunately formulation chemists are not microbiologists. They often lack suitable knowledge to select an optimal biocide, and they are unaccustomed to testing methodology to verify that a selected biocide and concentration level will provide the required performance. That is why a close working relationship between the formulator and the biocide supplier is required.

The biocide suppliers have a deep understanding of chemical compatibility and the spectrum of microbiological activity that can occur.

A biosusceptibility (bio-challenge) test should be run on the completely formulated product to confirm that it is adequately protected against microbial attack.
The measurement of minimum inhibitory concentration (MIC) is necessary to quantify biocide efficiency. MIC values are presented as ppm of biocide required to inhibit the growth of a given organism in a given product.
Accelerated testing is often used when determining MIC. A high level of humidity and temperature is used and microbial growth is easily evident in a 4-6 week time period.

MIC is the lowest preservative concentration at which growth of a test organism is inhibited under laboratory conditions. However, using MICs to measure bacterial resistance is arguable since much higher concentrations of biocides are used in practice.

ASTM D4783 describes test methods for the resistance of adhesives in containers to bacteria, yeast, and fungi. Tests that have also been developed for the paint industry have been used to measure the performance of a preservative in adhesives or sealants.
ASTM D2574 can be used to test the efficiency of an in-can biocide, and
ASTM D3456 can be used to test the susceptibility of applied coatings to microbial attack.

Testing can be especially difficult for those new to biostability issues. The table below provides a general outline of a test methodology that is applicable to most adhesive and sealant formulations.

Test	Methodology
Microbial challenge testing for efficiency	The microbiological effectiveness of a biocide is tested by repeated sequential challenges with a specific acclimatized microbial. Once a satisfactory biocide is found the next step is to test its stability in the formulation.
Biocide active ingredient compatibility	Some simple tests can determine compatibility to ensure that there are no undesirable side reactions, color change, odor, gelation, precipitation, etc. These tests involve incorporating the biocide at various levels and storing the product for prolong periods of time at high and low temperatures and observing any changes.
Chemical stability in the product formulation	Quantitative analytical tests are used to measure the level of active ingredient during the product’s lifetime.
Quality control testing	Checking of dosing accuracy Checking of biocide stability Checking of biocide active ingredient with supplier’s certificate of analysis Where regulatory limits are provided, the biocide type and level need to be stated on safety data sheets

General Test Methodology Employed to Assure that a Product is Adequately Protected from Microbial Growth

Many biocide suppliers are developing services to assist in selecting and formulating with biocides. For example, Dow Biocides has developed an advanced testing methodology called Taunovate. Some features of this methodology are as follows:

Rapidly determines the microbial status of a formulation, as well as effectiveness of the biocide product or products
Can be used with any industrial standard test method including ASTM and IBRG (International Biodeterioration Research Group) tests
Allows a wide variety of biocide options to be tested quickly including different concentrations of the existing biocide, other biocide products, and combinations of biocides.

Commercially Available Biocides for Adhesives and Sealants

Many of the biocides used in adhesive and coating systems contain sulfur. Isothiazoline-based biocides are the most popular and this class contains multiple chemistries. Many commercial biocides are made of blends of two or more biocides to improve range and efficiency.

Products in use:

3:1 ratio of 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT) and 2-methyl-4-isothiazolin-3-one (MIT) is frequently used. CMIT/MIT has broad spectrum efficacy versus bacteria, algae, and fungi.
1,2-benzisothiazolin-3-one (BIT) products have been used in a limited range of industrial applications requiring long-term preservation for bacterial control.
Recently, a new microemulsion technology was introduced using 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT).

Several commercial biocide agents that are used for in-can preservation and dry-film protection are indicated in the table that follows. This list is only an example of the commercial biocides in use today. It is far from complete.

Company	Active Ingredient	Trade Name
In-Can Biocide Agents:
Lonza	1,2-Benzisothiazolin-3-one in dipropylene glycol	Proxel™ GXL
Lanxess	Benzisothiazolone	Veriguard 19
Lanxess	Aqueous dispersion of approx. 25 % 1.2-dibromo-2.4-dicyanobutane.	Tekatamer 38 A.D.
Lanxess	1, 2-dibromo-2.4-dicyanobutane (24%) and isothiazolinones (0.08%)	BIOCHEK® 430
Lanxess	1, 2-dibromo-2.4-dicyanobutane (19%) and 1, 2-benzisothiazolin-3-one (6%)	BIOCHEK® 410
Lanxess	Tetrahydro-3,5-dimethyl-2H-1,3,5-thidiazine-2-thione	N-521 Dispersion N-521 PAC 24 N-521 P
Dow	1-(3-Chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride combined with a stabilizer (sodium bicarbonate).	DOWCIL™ 75
Lanxess	o-phenylphenol, tetrahydrate sodium salt	P1
Dow	o-phenylphenol	Dowcide 1
Lanxess	Blend of 15% o-phenylphenol and 5% dazomet.	Veriguard OD
Troy	4,4-dimethyloxazolidine (DMO)	Nuosept™ 101
Dow	5-chloro-2-methyl-isothiazolone and 2-methyl-4-isothiazolinone	Kathon LX 1.5%
Dry-Film Biocide Agents:
Lonza	Zinc pyrithione	Zinc Omadine®
Lanxess	2-(4-thizolyl)benzimidazole	METASOL® TK-100
Dow	Diiodomethyl-p-tolysolfone	AMICAL™ 48
Dow	10,10’oxybisphenozyarsine (OBPA)	Durotec 7603
Troy	3-iodo-2-propynylbutyl carbamate (IPBC)	Troysan Polyphase AF-1
Lanxess	o-phenylphenol (OPP) + dodecylguanidine HCL	Veriguard 13
Lanxess	Tetrachloroisophthalonitrile + p-[(diiodomethyl) sulfonyl] toluol	Veriguard 67

Biocide Agents for Adhesives and Sealants

By combining different types of biocides (e.g., a fast acting biocide with a long-term preservative) the biocide supplier can target a broader antimicrobial spectrum and offer optimal protection.

Also, certain combinations of active biocide ingredients have been shown to provide synergy – providing performance at lower concentrations than a higher concentration of either active ingredient alone.

Biocides Used in Adhesives and Sealants

References

Cresswell, M.A. and Holland, K., “Preservation of Aqueous-Based Synthetic Polymer Emulsion and Adhesive Formulations”, Chapter 9 in Preservation of Surfactant Formulations, Morpeth, F.F., ed., Blackie Academic, Glasgow, 1995.

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