ASTM WK62355

    New Test Methods for Test methods to determine bioassimilation of biodegradable plastic materials

    (What is a Work Item?)

    Developed by Subcommittee: D20.96 | Committee D20 | Contact Staff Manager


    1. Scope

    SCOPE Plastic packaging and disposable plastic products are omnipresent in the 21st Century. With the exception of incinerated waste - a major proportion of plastic material is still accumulated on the planet. Most plastic is recyclable in some form or other. Unfortunately, the collection and segregation of plastic waste is a luxury only available in a few Western countries. In the main, plastic waste finds its way into the environment. Due to their robust and durable molecular structure plastics do not easily degrade in the natural environment and hence the need for degradable plastics has become a major topic of research. Despite the best efforts of social reform; littering is still prevalent in all sectors of society. Bio-degradable plastics are designed to retain functionality as a commodity plastic for the required service life but degrade to non-toxic end products in a disposal environment. They are typically designed to bio-degrade while undergoing changes in chemical structure as a result of oxidation in air, thus causing the breakdown of the molecules into small short chain length intermediates that are then bio-assimilated. The true measure of a materials capability to biodegrade is evidence of bio-assimilation, the absorption of the remnants by living organisms. In nature chaos exists with a multitude of micro-organisms all capable of converting the inherent carbon in short chain length degraded materials. In a compost scenario mesophilic microorganisms, or microorganisms that thrive in temperatures of about 68 to 113 degrees Fahrenheit (20 to 45 degrees Celsius), begin physically breaking down the biodegradable compounds. Heat is a natural by-product of this initial process and temperatures quickly rise to over 104 degrees F (40 degrees C). Mesophilic microorganisms are replaced by thermophilic microorganisms (microorganisms that thrive in the increased temperatures) during the second stage, which can last from a few days to several months. The thermophilic microbes work to break down the organic materials into finer pieces. during the second stage, temperatures continue to rise and if not closely watched, the compost pile can get so hot that it can eventually kill off all the micro-organisms. Techniques such as aeration and turning over the compost pile help keep temperatures below about 149 degrees F (65 degrees C), as well as provide additional oxygen and new sources for the thermophilic microorganisms to break down. The third stage, which typically lasts for several months, begins when the thermophilic microorganisms use up the available supply of the compounds. At this stage, temperatures begin to drop enough for mesophilic microorganisms to resume control of the compost pile and finish breaking down the remaining organic matter into usable humus. Given that the breakdown of short chain length intermediates is facilitated by micro-organisms and the raise in temperature will accelerate that process according to Arrhenius Principle (doubling the chemical reaction for every 10 degree increase). It can be seen that the breakdown in the open environment and within compost is directly related. Some materials are more suited to the elevated temperatures found in compost, whilst others will bio-assimilate in ambient temperature. The fact that either material is consumed by micro-organisms demonstrates their bio-assimilation. This Specification, in common with many other standards relating to plastics, uses standardized laboratory procedures to accelerate and measure the processes that may be expected to occur in the real world, and produce data on the basis of which guidance may be given as to the likely behaviour of the plastic in the real world and as to the likely behaviour of the plastic as compared to other materials. Such guidance can only be approximate. However, the methods of accelerated weathering are identical to methods to confirm durability of airframes and automobiles. This specification therefore provides laboratory test methods that can be used to determine whether a plastic product that is intrinsically bio-degradable or contains a pro-oxidant/pro-degradant additive is abiotically degradable and whether it is non-ecotoxic and capable of bio-assimilation. It applies only to aerobic conditions likely to be found in the environment. Data from the tests prescribed by this Specification will show whether the material is degradable, biodegradable, bio-assimilated and not eco-toxic and will provide a basis from which a laboratory report can give guidance as to the approximate timescale for abiotic degradation in any specified environmental conditions. It provides specific limits for molecular weight, for CoD and elongation-at-break for contaminants in residual materials and for gel fractions. It also provides limits for eco-toxicity. Although this Specification provides time-limits for CO2 evolution tests in the laboratory (for reasons of time and cost) it does not provide timescales for biodegradation in the outdoor environment because the conditions to which the residues from abiotic degradation will be exposed in that environment are infinitely variable. It would however be expected that biodegradation in the outdoor environment would proceed more rapidly than in a glass vessel in a laboratory, because micro-organisms do not thrive in glass vessels, and the process in nature is synergistic. ABIOTIC DEGRADATION Sub-samples of the test-materials are exposed to three exposure-cycles designed to demonstrate abiotic degradation behaviour before, during, and following exposure to the outdoor environment: Cycle 1 to demonstrate stability in storage conditions and during subsequent useful life, it needs to be shown that the material is initially stable to thermal ageing, and that it will maintain a period of stability in which it will not abiotically degrade if it is not exposed to UV. Cycle 2to demonstrate that the material will abiotically degrade more rapidly than a control sample without pro-degradant additive, during sustained exposure to UV light. Cycle 3to demonstrate that: a. the material will degrade if exposed to UV light, but then occluded, or b. if the material is never exposed to sunlight it will thermally abiotically degrade when the stabilisers are depleted. The progression and relative extent of oxidation can be monitored by (a)Fourier-transform infrared spectroscopy (FT-IR) or (b) UV/visible spectroscopy at regular intervals in accordance with ISO 10640 (as indicated in or (c) molecular-weight loss. For film samples 250 m, the change in transmission peak-height corresponding to the degrading materials should be reported as a function of the thickness of the film. When a transmission measurement is not possible because of the thickness or opacity of the sample, the reflection spectra [attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FT-IR)] should be recorded, and the relevant peak-height changes may be presented as a function of an appropriate unchanging reference-peak. NOTE 1For sheet samples (250 m), abiotic degradation may take significantly longer to achieve as the rate of oxidation, and of the subsequent degradation, will be controlled by the rate at which oxygen can permeate the plastic. This may lead to degradation occurring in layers from the surface, exposing non-degraded material beneath. An adjustment may be necessary to account for the differences in mode of degradation including, but not limited to, duration of exposure and the collection/separation of suitably degraded material (for example, by removal of degraded material from the surface as may be expected by mechanical factors such as friction in the outdoor environment). For molecular-weight determination and progression to biodegradation testing, degradation and removal of surface layers shall continue until the entire material has undergone abiotic degradation to the required molecular weight. The decline in mechanical properties of the film can be monitored with ageing at regular intervals using Test Method D882 or D638 in which the end-point of degradation shall be as specified in Practice D3826. The decline in molecular-weight of the test sample shall be measured by high-temperature gel-permeation chromatography (HTGPC) or size-exclusion chromatography (SEC). Cycle 3b (as indicated in 10.1.6) shall continue until the weight-average molecular weight of the test sample residue is 5,000 Daltons or less. At this point, the residues from Cycle 3b ( are oxidized materials; as assessed by FT-IR-carbonyl optical density (as indicated in, that are biodegradable, and can then be submitted to the procedures designed to demonstrate biodegradation outlined in Tier 2. Cycle 1Accelerated Thermal AgeingTo demonstrate storage life (in the absence of uv light and at temperatures no higher than 30C) without degradation and loss of mechanical properties, test samples are exposed to accelerated thermal-ageing at three different temperatures, and the resulting data is analysed according to ASTM D7444-11. Test temperatures shall be between 30 and 70C. The storage life of the test sample is estimated according to the period under test until significant features caused by degradation of the material are observed. Two methods may be used alone or in combination as follows: (1) Carbonyl optical density (COD 1714 cm-1): is determined by FT-IR in accordance with the procedures outlined in ISO 10640 clause 5. It is calculated from the relationship: COD1714 cm-1 = (aged sample carbonyl peak height carbonyl peak height, prior to ageing) Sample thickness, t, m (a) The unaged sample peak height should be that of the same test sample, prior to the commencement of the ageing cycle, and not a different unaged sample. (b) The period of stability corresponds to the duration under test where the increase in COD in thermal ageing does not exceed 0.001 (2) Tensile elongation-at-break is determined in accordance with Test Method D882 or D638. The reduction in tensile elongation at break shall be 5 %. Cycle 2Accelerated UV WeatheringTo demonstrate abiotic degradation during prolonged outdoor exposure to sunlight and heat, the test material is exposed to accelerated UV weathering in accordance with Practice D5208 for not more than 250 light-hours. Cycle 3Short Accelerated UV Exposure Followed by Accelerated Thermal Ageing Cycle 3ainitial UV exposureTo demonstrate thermal degradation of the test material following the depletion of primary stabilizers/phenolic antioxidants during the life of the product or by brief exposure to sunlight or both, a test sample is exposed to a period of accelerated UV ageing in accordance with Practice D5208 and under the same conditions as Cycle 2 for a period sufficient to deplete the primary stabilizers/phenolic antioxidants in the test sample but not sufficient to cause oxidation. A period of 48 hours would be appropriate. NOTE 2For some applications, for example, agricultural mulching film and other plastic products intended to be exposed to sunlight for extended periods, special stabilizers found within them, such as hindered amine light stabilizer (HALS)-type UV stabilizers, may inhibit the onset of degradation, and therefore, brief UV exposure may not be sufficient to simulate the effects of normal stabilizer-depletion over time. This will require an adjustment with regard to the particular components and application. In the case of applications, such as netting or the edges of mulch-film intended to be buried, these should be tested by Cycle 1 only. Cycle 3baccelerated thermal ageing following initial UV exposureAt the end of Cycle 3a, the test sample shall be removed from the UV exposure apparatus and begin thermal ageing in accordance with Practice D7444 and under the conditions used in Cycle 1. The sample shall begin Cycle 3b ageing within 24 h of completion of Cycle 3a. Longer intervals may not produce the same results on all materials. A sample of residue from Tier 1 should be dissolved in an appropriate non-reactive solvent and the gel phase, if any, separated by filtration, the gel dried and the amount of gel reported as weight-fraction of total sample. This should be regarded as a non-degradable fraction of polymer. The gel may be subjected to further oxidative degradation and extent of subsequent reversion to soluble and degradable material reported. The acceptable amount of gel is less than 5% wt. Biodegradation Proposed method to monitor biodegradation of bio-degradable plastic by in vitro evolution of carbon dioxide. Background Carbon dioxide evolution is an accepted method for monitoring the biodegradation of oxo degradable plastics. Nevertheless, this approach, which relies on incubating the oxidised plastic fragment in a soil or compost medium, is both time consuming and expensive to conduct. An alternative carbon dioxide evolution approach is outlined in this document. The method involves incubating oxidised fragments of biodegradable plastic in the presence of the soil borne organism Rhodococcus rhodochorous, (although in practice any suitable organism could be substituted-for example a marine based organism such as A. borkumensis), in a glass vial and periodically sampling head space for the carbon dioxide evolved by Gas Chromatography (GC) over a pre- determined timescale. GC head space analysis is common practice within industry and has the advantage of simplicity and speed over the conventional reactors vessel approach. Proposed method. Terrestrial and (Marine). Preparation of Rhodococcus rhodochorous (A. borkumensis) cell culture. Cultivate Rhodococcus rhodochorous (ATCC-29672) (A. borkumensis) in minimal media at 27C, shaking at 120rpm. Add 1 ml of Rhodococcus rhodochorous (A. borkumensis) overnight culture to 50 ml of Tryptic Soy Broth (TSB). Add sterile glass beads, and vortex the solution. Determine the cell concentration by optical density measurement at 600nm, and calculate the dilution required to achieve a cell concentration of 108 cells per milliliter of culture solution. Prepare 50ml of diluted cells at the required concentration of 108cells/ml in minimal media. Plastic sample preparation Take 0.5g of oxidized plastic fragment and vortex with sterile glass beads. Pass the residue through a sterile metallic sieve to obtain homogeneous sample. Weigh out 5mg of the residue into glass vials. Sterilize by covering with 70% ethanol solution and evaporate to dryness. Add 0.4ml of the Rhodococcus rhodochorous cell culture to the plastic in the glass vial and incubate at 30C whilst shaking at 120rpm. Prepare 5 replicate vials for each GC analysis point. Control and blank samples (permutations of media/plastic/Rhodococcus rhodochorous) can be tested in parallel, adopting the same approach as necessary. Carbon dioxide analysis For each time point with withdraw 50ul of sample from the head space of the vial with a high precision syringe. Inject the sample into a GC analyzer fitted with suitable column and hydrogen/air carrier gas. The concentration of carbon dioxide may be analyzed by reduction to methane in a flame ionization detector and determined against known standards. Since this is a closed method procedure, a finite number of head space analysis can be undertaken from the sample before exhaustion. It is proposed that the test be conducted over 28 days with up to 8 sampling intervals. Alternatively, a longer duration test may be scheduled but with an accordingly larger sampling interval to accommodate the sampling points. From the concentration of carbon dioxide evolved calculate and report the extent of biodegradation observed with respect to the original sample. Cell Proliferation test. As a supplementary analysis, the concentration of Rhodococcus rhodochorous (A. borkumensis) under the various conditions can be determined by a spectrophotometric turbidity measurement, performed at a wavelength of 600nm in combination with a micro plate reader following the manufacturers procedures. Prepare vials of oxo-biodegradable, control and blank samples in triplicate for each time interval of interest-as per the previous approach for carbon dioxide analysis. Incubate the samples at 30C whilst shaking at 120rpm. At each measurement point (ideally coincident with the carbon dioxide analysis) remove a sample aliquot (5-10ul), dilute as necessary and transfer to a micro plate reader. Measure the optical density at 600nm (OD600) and apply the necessary baseline correction for the media and plastic. Plot the data to assemble the growth curve for each sample. Eco-Toxicity The impact of the degraded plastics on the quality of soil and water is important, in particular with regard to any toxicity that the residual plastics may cause in the soil or water. Degraded residues from the abiotic tests are tested as follows: For aquatic toxicity, in accordance with OECD 202 and/or 203 and For terrestrial toxicity, in accordance with OECD 207 and/or 208.


    compostability: marine pollution: Terrestrial micro-organisms:marine organisms:bio-assimilation:


    No existing Standards provide methodology to assess bio-assimilation of degraded bio-degradable materials.

    The title and scope are in draft form and are under development within this ASTM Committee.

    Citing ASTM Standards
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    Work Item Status

    Date Initiated:

    Technical Contact:
    Michael Stephens

    Draft Under Development