Mold Resistance Testing for Packaging Materials: Standards, Methods, and Evaluation

Mold resistance testing (also known as fungal resistance or mildew resistance testing) evaluates whether packaging materials such as plastic films, paperboard, foams, adhesives, and labels can withstand fungal growth during humid storage and long-distance transport. This article explains the key testing standards (ISO 846, ASTM G21, IEC 60068-2-10, GB/T 2423.16, JIS Z 2911), typical sample types, test organisms, procedures, rating criteria, and industry applications, helping manufacturers and suppliers select the appropriate test for quality control and regulatory compliance.

Technical Overview

Mold testing for packaging materials is a laboratory evaluation used to determine whether packaging can support or resist the growth of fungi under warm and humid conditions. During ocean freight, monsoon-season storage, or extended warehouse dwell times, the relative humidity inside shipping containers and cartons can remain above 85–90% for weeks, creating an environment in which airborne mold spores germinate on susceptible surfaces. Fungal growth on packaging leads to visible staining, musty odor, loss of mechanical strength, corrosion of enclosed metal components, contamination of food and pharmaceutical products, and costly customer rejections or returns.

Mold testing answers two distinct questions: first, whether the material itself provides nutrients that support fungal growth (intrinsic resistance); and second, whether antifungal or antimicrobial treatments incorporated into or coated onto the material are effective (fungistatic efficacy). Results are typically expressed as visual growth ratings on a numerical scale, often supplemented by measurements of changes in mass, tensile strength, or surface appearance after exposure. Accredited third-party laboratories conduct these tests under controlled temperature, humidity, and inoculation conditions in accordance with internationally recognized standards.

• ISO 846 – Plastics: Evaluation of the action of microorganisms (fungal growth resistance and inhibitory effect)
• ASTM G21 – Determining the resistance of synthetic polymeric materials to fungi
• IEC 60068-2-10 / EN 60068-2-10 – Environmental testing, Test J: Mould
• GB/T 2423.16 – Chinese national mold test standard, harmonized with IEC 60068-2-10
• ASTM D2020 – Mildew (fungus) resistance of paper and paperboard
• JIS Z 2911 – General Japanese test methods for fungus resistance of materials

Testing Samples

Plastic Films and Flexible Packaging

Polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), polyamide (PA), ethylene vinyl alcohol (EVOH) barrier films, multilayer laminates, pouches, shrink films, and blister materials are commonly tested. Pure polyolefins are generally nutrient-poor and intrinsically resistant, but plasticizers, slip agents, masterbatch carriers, residual monomers, adhesives, and printing inks can act as carbon sources that support fungal growth, which is why finished structures rather than raw resins are often evaluated.

Paper, Paperboard, and Corrugated Boxes

Cellulose-based substrates such as kraft paper, corrugated fiberboard, folding cartons, molded pulp trays, and paper labels are inherently susceptible to mold because cellulose is a direct nutrient for many fungi. Testing frequently follows ASTM D2020, JIS Z 2911, or the general procedures of ISO 846, and is often applied to verify the performance of anti-mold coatings, sizing agents, and humidity-controlled storage practices for export packaging.

Foam and Cushioning Materials

Expanded polyethylene (EPE), expanded polystyrene (EPS), polyurethane (PU) foams, EVA inserts, and sponge materials used as protective cushioning are evaluated for fungal susceptibility. Polyester-type PU foams are particularly vulnerable to microbial attack, while polyether-type foams and polyolefin foams tend to be more resistant; foam samples may also be tested to confirm the efficacy of incorporated biocides.

Adhesives, Tapes, Labels, and Printing Inks

Starch-based, protein-based (casein, animal glue), and polyvinyl alcohol adhesives are nutrient-rich and highly supportive of fungal growth. Water-based inks, natural rubber-based pressure-sensitive tapes, and label adhesives are also frequently included in mold test programs, either as standalone samples or as components of finished packaging assemblies.

Biodegradable and Bio-Based Packaging

Materials such as polylactic acid (PLA), polyhydroxyalkanoates (PHA), starch blends, cellulose films, and bagasse fiber trays are intentionally more susceptible to microbial attack. Fungal resistance testing per ISO 846 helps distinguish between degradation behavior in composting environments (covered by biodegradability standards such as EN 13432) and stability during the intended service life of the packaging.

Wood and Wood-Based Packaging

Wooden pallets, crates, plywood cases, and veneer packaging can develop surface mold even when treated against quarantine pests. Anti-mold treatments, kiln-drying claims, and protective coatings for wood packaging are verified through fungal exposure tests conducted under high-humidity conditions.

Testing Items

Depending on the objective of the assessment, mold-related testing of packaging materials typically covers the following items, which can be selected individually or combined into a test program:

• Fungal growth resistance test – visual growth rating after 28 days of incubation with a mixed spore suspension
• Fungistatic and antifungal efficacy test – verification of anti-mold agents, coatings, and antimicrobial masterbatches
• Effect of fungal attack on material properties – changes in tensile strength, elongation, bursting strength, mass, and color after exposure
• Mixed-spore challenge test – inoculation with a defined consortium of mold species relevant to transport and storage environments
• Yeast and mold enumeration – microbial limit counting on packaging surfaces for hygiene-sensitive applications
• Simulated transport humidity exposure – evaluation of mold development under cyclic or sustained high-humidity conditions
• Protective system verification – performance validation of desiccants, barrier films, and anti-mold packaging systems

Testing Methods

Test specimens are typically cut into pieces of approximately 50 mm × 50 mm or prepared as strips or finished packages, with at least three to five replicates per condition. Specimens are conditioned, often surface-sterilized by ethanol wiping or ultraviolet exposure to remove adventitious contamination, inoculated with a standardized fungal spore suspension, and incubated on agar media or in humidity chambers for the specified duration.

Agar Plate Method (ISO 846 Method A / ASTM G21)

Specimens are placed on mineral-salts agar in petri dishes and inoculated by spraying or brushing with a mixed spore suspension containing five representative fungal species. Incubation is carried out at 28 ± 1 °C with relative humidity of at least 85–95% for 28 days. Because the agar supplies only mineral nutrients and no carbon source, any fungal growth observed must be supported by the packaging material itself or by contaminants and additives on its surface. Positive virulence controls and negative sterility controls are run in parallel to confirm test validity.

Humidity Chamber Method (IEC 60068-2-10 / EN 60068-2-10 / GB/T 2423.16)

This method is widely used for packaging of electronic and electrotechnical products. Inoculated specimens are suspended in a humidity cabinet maintained at 28–30 °C and relative humidity above 90%, typically for 28 days, with extended durations of 56 or 84 days available for long-duration assessments. The test verifies that packaging materials do not support visible mold growth even in the presence of adventitious nutrients such as dust, fingerprints, and processing residues, and evaluates any resulting effects on appearance or function of the packaged item.

Inhibitory Effect Method (ISO 846 Method B)

To assess antifungal treatments, specimens are placed on a glucose-containing nutritive medium so that abundant fungal nutrients are available. The comparison of growth on treated specimens versus untreated references, including observation of inhibition zones, demonstrates whether the fungistatic additive is present, effective, and capable of migrating to the surface. Related guidance for evaluating fungistatic compounds in plastics is provided by ISO 16869.

Paper and Board Methods (ASTM D2020 / JIS Z 2911)

ASTM D2020 evaluates the mildew resistance of paper and paperboard by exposing inoculated specimens in a high-humidity chamber and rating mycelial development on the surface. JIS Z 2911 provides generalized fungus resistance procedures applicable to paper products, textiles, coatings, and other materials, using spore suspensions of designated mold species applied to the test surface followed by incubation under warm, saturated-humidity conditions.

Test Organisms and Standard Conditions

The most commonly used reference fungi in packaging mold testing include:

• Aspergillus brasiliensis (formerly Aspergillus niger)
• Aspergillus flavus
• Penicillium funiculosum
• Trichoderma virens (formerly Trichoderma viride)
• Chaetomium globosum
• Paecilomyces variotii
• Aureobasidium pullulans

Standard incubation conditions are 24–30 °C (most commonly 28 ± 1 °C), relative humidity of 85–95% or higher, and a primary test duration of 28 days, with extended durations applied when long-term storage performance must be demonstrated. Spore viability of the inoculum is verified before and after the test to ensure the biological validity of the procedure.

Evaluation and Rating Criteria

At the end of incubation, specimens are examined with the naked eye and under low-magnification microscopy, and growth is documented photographically. Typical rating scales (for example the 0–4 scale of ASTM G21) are applied as follows:

• 0 – No growth visible
• 1 – Trace growth, covering less than 10% of the test surface
• 2 – Light growth, covering approximately 10–30% of the surface
• 3 – Moderate growth, covering approximately 30–60% of the surface
• 4 – Heavy growth, covering more than 60% of the surface

ISO 846 uses a similar 0–5 intensity scale that distinguishes growth visible only microscopically from growth visible to the naked eye. Ratings of 0 or 1 are generally considered acceptable for most commercial packaging applications, while ratings of 2 or higher indicate susceptibility and the need for material or process changes. Supplementary measurements such as tensile strength per ISO 527 or edge crush strength per ISO 3037 may be performed before and after exposure to quantify functional degradation.

Applications

Mold resistance testing of packaging materials is applied across many industries in which humidity exposure, product sensitivity, or long supply chains create fungal contamination risks. Typical applications include:

• Food and beverage packaging – supporting hygiene control programs and preventing mold contamination of dry goods, baked products, and shelf-stable foods
• Pharmaceutical packaging – demonstrating material stability and microbial quality for primary and secondary packaging in regulated supply chains
• Electronics and semiconductor packaging – qualifying cushioning, barrier films, and shipping containers using humidity chamber mold tests aligned with IEC 60068-2-10
• Export and ocean container shipments – validating anti-mold programs, desiccant strategies, and protective coatings for goods transported through tropical climates
• Footwear, leather goods, textiles, and furniture packaging – preventing mold damage to moisture-sensitive consumer products during multi-week transit
• Biodegradable packaging development – balancing end-of-life biodegradability with adequate shelf-life and transport resistance
• E-commerce and long-term warehousing – qualifying packaging intended for extended storage in uncontrolled environments

Test reports from accredited third-party laboratories are routinely used for supplier qualification, incoming material inspection, failure analysis after mold-related customer complaints, and claims substantiation for anti-mold treated packaging.

FAQ

Why does mold grow on packaging materials?

Mold requires moisture, a suitable temperature, oxygen, and a nutrient source. Under high humidity, paper and natural fibers serve as direct nutrients, while plastics can support growth through additives, adhesives, inks, coatings, or surface contamination such as dust, grease, and fingerprints.

Which standard should be selected for my packaging material?

ISO 846 and ASTM G21 are the primary methods for polymeric materials, IEC 60068-2-10 and GB/T 2423.16 are preferred for packaging of electronic products, and ASTM D2020 or JIS Z 2911 are suited to paper and fiber-based substrates. The intended market, product type, and customer specification usually determine the final choice.

How long does mold testing take?

The standard incubation period is 28 days. Including spore suspension preparation, sample preconditioning, and evaluation, a typical test program is completed in five to six weeks, with extended 56- or 84-day options available for long-duration claims.

What samples and quantities are required?

Flat specimens of approximately 50 mm × 50 mm are standard, with three to five replicates per test condition plus reference and control samples. Finished items such as boxes, pouches, foam inserts, or labels can often be tested intact; specific requirements should be confirmed with the testing laboratory before submission.

What test result is considered acceptable?

A growth rating of 0 (no growth) or 1 (trace growth) is generally acceptable. Ratings of 2 or higher indicate that the material supports fungal colonization, and corrective actions such as antifungal treatment, material substitution, or improved moisture protection are recommended.

Can the effectiveness of an anti-mold treatment be verified?

Yes. The inhibitory effect method of ISO 846 and comparative testing of treated versus untreated specimens demonstrate whether a fungistatic agent is effective and whether it remains active at the material surface. This is commonly performed during supplier qualification and periodic quality verification.

Do synthetic plastics need mold testing if they are not naturally biodegradable?

Yes, in many cases. Even when the base polymer is inert, additives, plasticizers, adhesives, coatings, and surface contamination can support growth. For export packaging and electronics applications, mold testing is frequently a contractual or customer-specified requirement regardless of polymer type.