Plastic products are advantageously used in many applications in agriculture because they offer versatile low-cost solutions . Most plastics are not biodegradable and after use plastic products must be collected and disposed of in a suitable way to prevent a build-up in nature . In some cases, this is impossible or is not completely effective, and therefore dispersion into the ground takes place . This has been underestimated in the past, but now the contamination of natural environments by plastics and microplastics is recognized as being a worldwide problem requiring mitigating action .Where dispersion into the ground is highly likely, the biodegradability of products is seen as a useful solution for preventing a progressive build-up of non-biodegradable plastics in agricultural fields . The biodegradability of plastics is an innovation that has created a lot of interest in the industry that manufactures plastic products for agriculture, among farmers, and among all those appointed to look after the state of health of the environment .
Recently, the European standard EN 17033 on biodegradable mulch films was approved . The standard is the result of many years of discussion and in-depth analysis  and it specifies the requirements for biodegradable films that are intended to biodegrade in soil, manufactured from thermoplastic materials, to be used for mulch applications . The material of the film is considered to be biodegradable if it achieves a minimum mineralization percentage of 90% (absolute or relative to the reference material) in a test period lasting no more than 24 months. This very high threshold level (90%) is in line with other standards about biodegradability (e.g.: EN 13432 on the compostability of packaging ) and it is considered as an indicator of total biodegradation and of no remaining chemical residues. It must be understood that the 90% mineralization requirement is a very high level which, in practice, represents complete biodegradation, the rest being immobilized as biomass. This is the reason why the tests are prolonged for a maximum of two years, to allow the biomass to self-digest and convert into CO2, which can be measured and accounted for in the mineralization percentage. This is not an indication of a slow biodegradation rate, because it is known that biodegradation is curbed by the available surface area [11,12]. Additionally, in order to dispel any doubts about the release of adverse molecules into the soil, two more additional assessment steps are required: a control of constituents and ecotoxicity testing. Regulated metals are listed and their maximum allowable concentrations, which are established following the EU ecological criteria for the eco-label granted to soil improvers. Furthermore, substances of very high concern are not allowed. Ecotoxicity tests are performed in order to investigate possible adverse effects resulting from the degradation of the mulch film in soil at the end of the intended service life. Soil samples supplemented with mulch film in high concentrations are tested after biodegradation using the acute toxicity plant growth test, acute toxicity earthworm test, and nitrification inhibition test with soil microorganisms.
Compliance with the standard shows that the plastic mulch film is intrinsically biodegradable and safe for the environment. The agronomic benefits and limits of biodegradable mulch films have been demonstrated by several field trials carried out in the last 20 years .
The degradation, in soil, of mulch films made with biodegradable plastics, after crop harvesting has been followed, in terms of its “disappearance”, in several studies [, , , , , ]. Methods to measure mulch degradation after use have been developed to demonstrate complete degradation and that there is no impact on subsequent crops [20,21]. Physical degradation is a relevant effect, if an impact on the growth of subsequent crops is to be avoided. However if physical degradation is not accompanied by an ultimate biochemical degradation, the final result is the production of microplastics i.e. the conversion of visible to invisible pollution. The disappearance is a sign of physical degradation, but is not proof of biodegradation, which can only be inferred from the results of respirometry carried out in the laboratory, following the CO2 evolution or O2 consumption from plastic materials. Microplastics may cause direct or indirect adverse effects on soil flora and fauna .
Zumstein et al.  proved the biodegradation of plastic films in agricultural soils by showing that the microbial colonization on the tested plastic sample (biofilm formation is the phase preceding degradation [, , ]) was accompanied both by CO2 evolution and the formation of microbial biomass containing the carbon that was originally present in the film. Palsikowski et al.  followed the biodegradation in soil of biodegradable materials based on poly(lactic acid) (PLA) and poly(butylene adipate-co-terephthalate) (PBAT), both with a visual analysis and by monitoring the carbon mineralization. However, the two analyses were independent, and consequently, a direct correlation of the results was not possible. Likewise, in independent studies, Souza et al.  followed the disintegration and mineralization of samples of aged mulch films made with PBAT. Kijchavengkul et al.  correlated the visual analysis of degradation that occurred in soil with the mineralization data obtained under composting conditions, and so the relationship was not fully consistent in terms of the different environmental conditions. A correlation between mass loss and the mineralization level of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and Poly(butylene succinate-co-adipate) (PBSA) was determined under composting conditions by Salomez et al. . In other studies, the disappearance of plastic samples followed by visual analysis was correlated with the mass loss, both characteristics being evidence of the physical degradation in soil but not necessarily proof of biodegradation .
The purpose of this work was to monitor physical degradation by following the “disappearance”, a phenomenon easily perceivable without technical means, and to verify in parallel the biochemical degradation, which can be detected as CO2 evolution.
Materials and methods
The products tested in this study were two commercial plastic mulch films used in agriculture. One was made of a conventional non-biodegradable plastic material while the other was made of a plastic material biodegradable in soil.
The conventional plastic mulch film used in this study was a 25 μm thick low density polyethylene (LDPE) black film.
The biodegradable mulch film was 15 μm in thickness. In general, conventional mulch films are thicker than biodegradable ones because they must be recovered at the end of the cycle without ripping. The biodegradable mulch film complies with the EN 17033 biodegradability requirements . It is made with Mater-Bi EF04P, a biodegradable plastic material produced by Novamont in the form of pellets, and certified “OK Biodegradable Soil” (TUV Austria). The Mater-Bi EF04P is converted into a film by means of film blowing with the addition of carbon black (about 2.8%). The carbon black is supplemented using a masterbatch based on a biodegradable polymer present also in the Mater-Bi EF04P.
Mater-Bi EF04P is made with biodegradable polyesters, starch, and a natural plasticizer. Polyesters are made with monomers that biodegrade in soil . The plasticizer is a biobased, biodegradable polyol. This substance biodegrades completely within 28 days at room temperature, under aqueous aerobic conditions (Organic Waste Systems, Belgium, study FDI/1, data not shown). Leaves of a maple tree (Acer saccharinum L.) were picked from the ground after they had fallen naturally. The dried leaves were brought to the laboratory for testing.
A filter paper (Whatman 42) was used as the reference material.
The plastic materials and filter paper were cut with a cutter into squares of 2.5 cm each side. The leaves were tested as such. The total organic carbon content (TOC) of each material was determined by another laboratory (elemental analysis carried out by Redox Snc, Monza, Italy). The carbon content and the main characteristics of the test and reference materials are shown in Table 1.
The biodegradation was determined by means of respirometric tests, in accordance with the ASTM D 5988–18 test method, based on the measurement of CO2 production . The tests were performed with two replicates instead of three as required by the standard method. This modification was necessary for technical reasons relating to the organization of our laboratory.
Soil was collected from an agricultural field at the Centro Sperimentazione ed Assistenza Agricola (CeRSAA), in Albenga (Italy). The soil is classified as sandy loam following the Soil Taxonomy USDA and routinely analysed by CeRSSA. The main characteristics of the soil as determined by CeRSAA are shown in Table 2.
The soil was sieved to a 5-mm particle size. The water content was measured as weight loss (105 °C), and the final soil moisture was adjusted to 14.3% (about 50% of the Water Holding Capacity). The final soil pH was determined in a mixture of soil and deionized water: ratio of 1:2.5 (w/v) . The pH was 7.66.
For each replicate, 1g of test material, in the form of film, was mixed with 200 g of soil in a 1000 ml hermetically-sealed glass jar. The test was set up with blank jars (without material) and with reference material jars (1g of filter paper). Two replicates were carried out for the test material, for blank and for reference, and incubated in the dark, at 28 ± 2 °C. The set-up of the test is shown in Table 3.
A 50 ml beaker filled with 30 ml of 0.5 M KOH, as a CO2 trapping solution, was placed in each jar. The amount of CO2 produced was measured by means of titration of the KOH solution, with 0.3 N HCl [35,36] with a Mettler Toledo (T50) potentiometric titrator. The measurement was made every 2–3 days during the first two weeks, when the mineralization rate was expected to be maximal, and weekly or biweekly thereafter.
The moisture content was kept constant by adding deionized water throughout the biodegradation test, whenever the KOH solution was titrated and replaced with a fresh one.
The net CO2 production evolved from the test materials was calculated by subtracting the average amount of CO2 produced in the blank soils from the amount of CO2 produced in the test material jars. The biodegradation percentages were calculated from the ratio between the net CO2 production and the theoretical CO2 production (ThCO2) based on the carbon content. The ThCO2 of both mulch films was calculated without taking into consideration the contribution of the carbon black, a source of inorganic carbon.
At regular intervals, coinciding with the KOH titration, all the reactors were emptied by pouring the soil into containers. The soil with any residues was photographed and visually analysed to find any residues. Finally the soil and residues were put back into their reactors and the test resumed.
Results and discussion
A conventional non-biodegradable black mulch film (made with LDPE), a biodegradable black mulch film (made with Mater-Bi EF04P), and dry leaves of the silver maple were tested for biodegradation in soil, using the standard test method ASTM D 5988–18 . The mulch films were tested as 2.5 cm × 2.5 cm pieces. Filter paper was used as a reference material, instead of microcrystalline cellulose, in order to have a solid material instead of a powder. The filter paper was also cut into 2.5 cm squares. The leaves were tested just as they were. The samples were tested for biodegradation by following the CO2 evolution. The tested materials were easily visible in the reactors and therefore it was possible to follow their disappearance, with a qualitative visual assessment. After 14, 119, 292, 364 days of incubation, the content of the reactors was visually examined and photographed (Fig. 1), and then reintroduced into the reactors to continue their incubation (except after the last analysis, at 364 days).
The samples of conventional non-biodegradable mulch films made with PE were visible throughout the duration of the test. At the end of the test, the PE samples were recovered, cleaned, dried, and weighed. The PE samples looked to be intact (Fig. 2) and the total mass recovered at the end was practically identical to the amount introduced at the beginning of the tests, showing no weight loss (Table 4). This result was in line with the known persistence of the PE mulch film .
Table 4. Amount of PE introduced into each reactor at the start of the test and amount recovered at the end of the test. The differences are negligible.
On the other hand, after 119 days of incubation the filter paper samples and the leaves had completely disappeared. On the basis of this examination, one can conclude that the paper and leaves had disappeared in the period spanning from day 14 to day 119.
After 292 days, the EF04P mulch film had totally disappeared in reactor R3, but few small residues were still present in reactor R4 (Fig. 3). At the end of the test (364 days), there were no residues visible in either reactor. Thus, the biodegradable EF04P mulch film had completely disappeared between day 292 and day 364.
In addition to the physical degradation, the process was monitored by measuring the CO2 evolved from the samples, i.e. the mineralization. As a result, it was possible to verify whether the visual disappearance was only the effect of fragmentation or whether partial or substantial mineralization had occurred. The CO2 evolution of the different reactors was measured following the standard test method ASTM D 5988–18 . The cumulative CO2 production is shown in Fig. 4.
The reactor R7, containing filter paper, was discontinued after day 70 because of technical problems.
For the moment it is interesting to note, for subsequent discussion, that the cumulative CO2 production curves of soil (blanks) and of soil supplemented with PE are practically identical. One PE replicate (R5) produced less CO2 than one of the two blank replicates (R2), while the other PE replicate (R6) had a slightly higher production.
The mineralization percentages (i.e. the ratio between evolved CO2/ThCO2, where ThCO2 is the theoretical evolution achievable in the event of total oxidation of the total organic carbon present in the tested material) are plotted in Fig. 5.
The replicates of the EF04P mulch film showed a rather marked difference starting from day 50–100, even if they returned to coincide at the end of the test, reaching practically the same level of mineralization.
At the end of the test, i.e. after 364 days, the mean mineralization levels were as follows: EF04P mulch film, 88.0%; filter paper, 98.4%; leaves, 80.2%; PE mulch film, 2.5% (see Fig. 6). The mineralization of the paper, used as reference material, was in line with the validity requirements of the standard method ASTM D 5988–18 (mineralization >70% after 180 days), indicating that the soil was biologically active and the test valid.
In this experiment it was possible to follow the degradation of different solid materials, both with a simple qualitative assessment, i.e. looking at the “disappearance” of samples, and with a respirometric technique, i.e. monitoring the CO2 evolution. The former approach can be easily used in field trials and by farmers when applying commercial films in their fields, and the latter is a specific methodological approach that shows the true ultimate biodegradation. It is interesting to understand the correlation between the rudimentary approach, based on visual analysis, and the quantitative approach.
LDPE film samples were recovered intact at the end of the test, showing no mass loss. At that moment, the final mineralization level was 2.5%. We consider this value to be ascribable to an accuracy problem of the respirometric test for such low values, i.e. we consider the mass loss as being more reliable than the CO2 measurement in this context. As a matter of fact, the individual replicates of PE are very similar to the blanks (see Fig. 4). The cumulative CO2 curves suggest no difference between the PE and blank replicates, which is not surprising given the known resistance of PE to biodegradation. Thus, we consider the biodegradation value of PE as noise that could be decreased by increasing the number of replicates. We used only two replicates for technical reasons. Still, a 2–3% variation is an acceptable fluctuation of the background signal in a biological test after 1 year of testing.
The paper disappeared in the period between day 14 and day 119, when its mineralization went from 20.5% to 85.6% (see Fig. 1). In the same period, the leaves also disappeared, but reached a lower mineralization level (67.9% on day 119). The CO2 evolution continued during the subsequent test phase, when the leaves were not visible anymore. Thus, either the crumbled leaves continued to be mineralized into CO2, or the biomass formed in the first 119 days underwent self-digestion, with a progressive release of CO2.
It is interesting to consider the two replicates of the EF04P mulch film more carefully. On day 119, on visual inspection, replicate R4 showed a higher amount of residues than replicate R3. This qualitative but, still, clear evidence was in line with the concurrent mineralization %, which was only 33.1% for R4, and 50.8% for R3. The mineralization course of R3 remained higher also as the test continued, and likewise, the physical degradation was also more advanced, as was noticeable during the visual analysis. On day 292, very tiny pieces of black film were still present in the R4 soil, whereas the R3 soil was free of particles that were visible to the naked eye. At the end of the test, the replicates were at the same level of mineralization (R3, 89.0%. and R4, 87.0%) and both were free of visual contaminants. We believe that the reason for this variation between the replicates was the high load of film introduced into each reactor. The applied loading (1 g of materials in 200 g of soil, i.e. 0.5% w/w) is much higher than the expected application of mulch films in soil. The normal depth of soil to which the plastic fragments can distribute after rototilling is about 0.30 m. Therefore, the plastic film covering 1 m2 of soil surface will typically be mixed with a volume of soil equal to 0.3 m3. This amount of soil weighs approximately 450 kg, considering a soil bulk density of 1500 kg/m3. The typical mass of 1 m2 of biodegradable plastic mulch film is about 19 g (with a thickness of 15 μm and a density of 1250 kg/m3). Therefore, the typical loading of a plastic film under normal conditions is approximately 0.0042%, i.e. 2 orders di magnitude less than the dose tested in this work. Under these conditions, pieces can crumple and tangle together in one reactor, making intimate contact between the surface of the samples and the soil (which is necessary for biodegradation to happen) more difficult. Thus, by chance, the samples in R4 could have formed clusters of two or more samples, thereby slowing the overall degradation. This hypothesis needs further testing, to be confirmed.
The mineralization of maple leaves plateaued at a mineralization level of about 80% in one year. In a study carried out in a European Project named “EU project AIR2 CT93 1099” , the mineralization of different leaves was measured in soil at 20 °C. Birch leaves, oak leaves, and pine needles reached a plateau at 56.2%, 55.8%, and 61.9%, respectively, in one year.
Air-dried leaf litter collected without field exposure from Norway spruce, mountain ash, European beech, and sycamore maple showed mineralization levels of about 55%, 85%, 30%, and 50% respectively, when mixed with quartz sand and incubated at 20 °C for 716 days .
The disappearance of biodegradable mulch films applied in the fields is a common experience both for experimenters and farmers. After crop harvesting, the mulch film is mixed with the soil and then it disappears. Physical degradation is a strong indication of biodegradation but it could be a consequence of a limited biodegradation still sufficient to cause the embrittlement of the material and its fragmentation into “invisible” pieces mixed in the soil. In other words, the physical degradation might produce microplastics, i.e. persistent particles.
In this experiment we showed that the disappearance of paper, leaves, and a biodegradable mulch film is accompanied by a substantial evolution of CO2. Paper, leaves, and biodegradable mulch film were still easily visible, with a mineralization level of about 20%, 30%, 40%, respectively. At longer times, when the materials were no longer visible, the mineralization level was of about 70%–80%. The biodegradable plastic mulch film showed disintegration and mineralization comparable with paper and leaves, as expected considering its compliance with the European standard EN 17033. The CO2 evolution shows that physical degradation is accompanied by an ultimate biodegradation, i.e. the material is not converted into plastic particles invisible to the naked eye but truly converted into CO2, at levels comparable with that of natural matter (leaves) and products based on natural polymers (filter paper). At the same time, the conventional mulch film made with PE showed no fragmentation, no loss of mass, and a negligible mineralization level, which was very likely due to the fluctuation in the basic (endogenous) respiration.
To conclude, the biodegradable mulch film, when in contact with soil, undergoes a biodegradation process whose evident consequence is disappearance, which however is the epiphenomenon of complete biodegradation, and is similar to what happens to paper and tree leaves. This in turn suggests that, like leaves, biodegradable mulch film does not build up in soil.
Source: Disintegration and mineralization of mulch films and leaf litter in soil
Authors: Maurizio Tosin, Marco Barbale, Selene Chinaglia, Francesco Degli-Innocenti