Biodegradable plastics are mostly applied in packaging materials (e.g., shopping bags), waste collection bags, catering products, and agricultural applications (Shen et al., 2009). At the end of their commercial life these products are expected to degrade into harmless end products. Degradation takes place in composting plants or in anaerobic digestion plants (van der Zee, 2014) together with the bio-waste (food and yard waste) to give rise to compost. Compost is applied to soil as a fertilizer in order to increase organic matter. Degradation can also take place directly in soil where biodegradable plastic products are intentionally left after use (e.g., mulch films for weeds control). Since the agricultural soil is the medium for the production of food for humans and feed for farm animals, the absence of negative effects linked to applications of biodegradable plastics is of great concern to the interested parties. That is why all the testing protocols developed to characterize the biodegradable plastics and packaging include the assessment of ecotoxicity potentially originated from the biodegradation. Standards have been developed to specify the requirements for biodegradable plastics and packaging in composting (i.e., organic recycling), in home composting, and in soil. The most relevant standards on plastics that are biodegradable in different environments are shown in Table 1. All standards require (i) full biodegradation levels as a first tier of testing that prevents accumulation in soil and (ii) ecotoxicity testing as a second tier of testing in order to show environmental safety. The testing approach is similar in all cases: the plastic material is exposed to the matrix of interest (e.g., compost, soil) in a very high dose and let biodegrade. After a given time, a sample of the matrix is tested with biotests for ecotoxicity together with a control sample not exposed to the plastic item and a control sample exposed to a GRAS (Generally Recognized As Safe) substance, such as cellulose. No significant difference must be found between the test samples and control samples.

The standards on compostability (e.g., EN 13432) requires ecotoxicity assessment on compost samples where plastic material added at a 10% concentration has been composted for 3 months. The biological test is based on a germination and growth assay with two plant species and derives from the OECD 208 Guidelines for testing of chemicals (OECD, 2006).

Ecotoxicity requirements are also enforced by the standards on biodegradability in soil (the Italian and French national standards and the “OK Biodegradable soil” certification programme). Ecotoxicity tests must be carried out on soil samples where plastic material added at a 1% concentration has been degraded for 3 months. The prescribed concentration of plastics is much higher of the amount that is expected to end up normally in the environment. A 1% loading is much higher than the expected application loading of biodegradable plastics in soil. For example: a typical biodegradable plastic film for mulching is 1.5 × 10⁻⁵ m thick and has a density of 1250 kg m⁻³. This means 1.88 × 10⁻² kg m⁻² for one application. The soil depth where the plastic is typically used or remains after use is presumed to be 0.20 m, in agreement with the normal depth of soil tillage. Therefore, 1 m² of plastic film covering 1 m² of soil surface will typically be mixed with a volume of soil equal to 0.2 m³. This amount of soil weighs ~300 kg, considering a soil bulk density of 1500 kg m⁻³. Therefore, the typical loading of the plastic film in normal use will be ~0.0063% (1.88 × 10⁻² kg/300 kg × 100).

The ecotoxicity biotests required by the different standards are shown in Table 2.

The current approaches to the standardization of composting and soil biodegradation are considered satisfactory and suitable for the assessment of the environmental safety of biodegradable plastics (Fritz, 2014). The reason lies in the very high mineralization threshold levels (90%) required by these standards, which can be considered as an indication of total biodegradation and of no remaining residues (De Wilde, 2014). Additionally, the ecotoxicity tests applied to the compost or to the soil after the biodegradation of the plastic, make sure that products of biodegradation do not affect environment.

However, due to the growing volumes of biodegradable polymers and plastics, interest in their environmental safety is increasing and more research is carried out (Kapanen, 2012). A strong concern of the public opinion is about the environmental impact of persistent substances possibly released during polymer biodegradation and composting which could subsequently be spread into the environment during fertilization with compost, or directly diffused during their biodegradation in soil. It is therefore of interest for all the stakeholders to extend the range of the applied ecotoxicity tests in order to dispel any doubt and assure a smooth development of this promising industrial sector. This is the reason why some attempt has been made to apply biotests, used in other sectors of environmental sciences, in the assessment of biodegradable plastics safety.

The so-called “Flash test,” based on the kinetic measurement of bioluminescence by Vibrio fischeri, has been applied on compost (Tuominen et al., 2002) and on vermiculite (Degli-Innocenti et al., 2001) exposed to plastic materials. The conclusion of the studies was that the Flash test could be reliably used to verify the ecotoxicity of extracts from the biodegradation matrices (either vermiculite or compost).

A nitrification inhibition test was applied to soil extracts (Bettas Ardisson et al., 2014) after a biodegradation test based on the standard ISO 14238 (2012) for the measurement of nitrification activity. The conclusion was that the test approach was suitable for reaching conclusions on the effects of biodegradable plastics on soil.

No other biotests have been applied to our knowledge in this sector. The intention of this work was to verify the applicability of a larger number of bioassays for the determination of the potential ecotoxicity of soil after the biodegradation of plastics. In particular, the organisms included bacteria and protozoa (Vibrio fischeri and Dictyostelium discoideum, respectively), the green alga Pseudokirchneriella subcapitata, plants (the monocotyledon Sorghum saccharatum and the dicotyledon Lepidium sativum), and invertebrates animals (Daphnia magna, a freshwater crustacean, and the Oligochaeta earthworm Eisenia andrei). The array of ecotoxicological tests, including those routinely adopted by the Italian Institute for Protection and Environmental Research (ISPRA) and applied by territorial Environmental Agencies, was employed on soils where samples of biodegradable plastics had been biodegraded for 6 months. These tests are based on the OECD Guidelines or on other recognized protocols.

The soils after biodegradation of three Mater-Bi plastic films (i.e., RIC 1362, RIC 1619, and RIC 1620—see Section Materials and Methods), named for simplicity “Mater-Bi soil,” of cellulose (“cellulose soil”) and the control soil were all tested in parallel.

The results concerning the possible toxicity of the Mater-Bi soils and cellulose soils on Bacteria and Protists are reported in Figures 1, 2. All the elutriates obtained from the Mater-Bi soils and cellulose soils stimulated the light emission of the bacterium V. fischeri when compared with the lab control, although the enhancement never reach the value +20% indicative of a biostimulation effect (Figure 1). However, no changes were observed among the values from the control soil and Mater-Bi soils or cellulose soil.

Figure 2 shows the effects of the Mater-Bi soils and cellulose soil on the survival and reproduction rate of the social amoeba D. discoideum. The elutriates did not cause negative effects on the survival of the organisms (Figure 2A) but induced a slight decrease of the reproduction rate (Figure 2B). Such decrease was significant, with respect to lab control, for the amebae exposed to the elutriates of the soil RIC 1620 (Mater-Bi containing soil) and of the cellulose soil; however, the effects never reach the decrease of 20% with respect to the lab controls, a value usually considered as an indication of weak toxicity.

The results presented in Figures 3–5 concern the evaluation of the possible effects of Mater-Bi and cellulose soils on autotrophic organisms.

The data related to the effects on a monocotyledon S. saccharatum and the dicotyledon L. sativum demonstrate that the seeds of both plants correctly germinate in the Mater-Bi soils and cellulose soils without differences in the germination rate with respect to the control soil or lab controls (Figures 3, 4).

In the same way, when the alga P. subcapitata was growth for 72 h in the elutriates obtained from the different soils no negative effects were observed on its replication rate (Figure 5).

Figure 6 shows the results of the acute and chronic toxicity tests using D. magna as model organism. The data reported in Figure 6A clearly demonstrate that there were no effects on the motility rate of the organisms after 24 h of exposure to the elutriate containing the Mater-Bi and cellulose soils. These results were confirmed by the data on the effects of the elutriate on the reproduction rate of D. magna (Figure 6B): no effects were found in organisms exposed to the elutriates from Mater-Bi soils; however, the elutriates containing cellulose soils determine an increase in the reproduction rate of this cladoceran, although in very variable extent.

Finally, in Figures 7, 8 are reported the results concerning the exposure of the earthworm E. andrei to the Mater-Bi soils and cellulose soils. The results obtained in earthworms exposed to the control soil were comparable to an standardized artificial soil (OECD, 2004) during the period of exposure (data not shown).

The data demonstrate that there are no toxic effects of the studied soils on the survival rate of worms (Figure 7). Data concerning the number of the cocoons produced (Figure 8A) and the value of the number of juveniles earthworms (Figure 8B) demonstrate that the reproduction rate (Figure 8C) is similar to the value observed in the animals maintained in the control soil.

Plastic contamination of terrestrial and aquatic ecosystems is nowadays considered one of the major environmental issues. In fact, the plastic global production was about 299 million tons in 2013 (PlasticsEurope, 2015) and while efforts to proper recovery and recycling are increasing still a fraction of plastic waste is not recovered and represents a contamination to some environments (UNEP, 2014). Due to the important role of plastic products in our society, in the last two decades different biodegradable plastics were produced with the aim to substitute, at least in part, the plastic products; this, together with a substantial effort toward recovery and recycling, should allow to reduce the negative environmental effects due to plastic waste accumulation in the different ecosystems (World Economic Forum, 2016).

However, not all the biodegradable plastics are “environmental friendly” and this depends on their chemical composition: in some case it was found that degradable plastics were toxic for edaphic and/or aquatic organisms (Fritz, 2014), thus their use no giving all the expected environmental advantages. In particular, few studies have been devoted to investigate the possible biological effects of plastic degradation products in organisms characteristic of the different trophic levels in the food chains of the edaphic and aquatic ecosystems.

In this research, the environmental compatibility of the biodegradable plastic Mater-Bi was studied evaluating the potential toxicity of its degradation products on a wide range of model organisms using well known standardized guidelines or, in some cases, scientific literature protocols.

Mater-Bi is a plastic material based on corn starch and biodegradable polyesters widely used for package (Bastioli, 2005). Same Mater-Bi grades are certified as “OK Biodegradable Soil” by Vinçotte (Belgium). The certification programme established by Vinçotte requires a biodegradation level of more than 90% after 2 years in soils (under controlled physical-chemical and microbiological conditions) and no ecotoxicity in soil exposed to the test material.

Ecotoxicity is assessed in soil samples after degradation of the plastic material introduced at high concentration in comparison with control soils. The ecotoxicity test is a seedling and plant growth test based on the OECD 208 (OECD, 2006) standard test method.

These characteristics have encouraged a wider employ of this biodegradable plastic also for other uses such as in the agricultural field as mulch films (Kyrikou and Briassoulis, 2007).

For this reasons it was considered of great importance to clarify the possible toxicity of this new biodegradable plastic with a larger array of biotests.

With the aim to clarify this important aspect, Mater-Bi was degraded in soils under controlled conditions for 6 months at a 1% concentration, following the Vinçotte certification programme testing scheme.

The selected model organisms for the evaluation of soil quality were bacteria and protozoa (V. fischeri and D. discoideum, respectively), the green alga P. subcapitata, plants such as the monocotyledon S. saccharatum and the dicotyledon L. sativum, and invertebrates animals such as the D. magna, a freshwater crustacean, and the Oligochaeta earthworm E. andrei.

The ecotoxicological tests on the alga and plants demonstrated that the Mater-Bi biodegradation products are not harmful for these autotrophic organisms. It should be noted that a large part of the bioassays used are chronic tests i.e., the more sensitive at organism level.

Similarly, no effects were found when the soil toxicity was evaluated using as model organisms two invertebrates such as D. magna and the earthworm E. andrei: also in this case the endpoints evaluated, both mortality (acute toxicity) and reproduction (chronic toxicity) ensured the higher sensitivity of the employed bioassays.

The data concerning the possible effects of the plastic degradation products on the bacterium V. fischeri using the Microtox test indicated that there are no negative effects on these organisms. These results are in line with previously reported data indicating that the Mater-Bi degradation products in soils do not affect the nitrification process depending on the microbiological activity (Bettas Ardisson et al., 2014).

Finally, an extremely sensitive bioassay using as model organism the social amoeba D. discoideum was used. This amoeba lives in the soil pore water and it is cultivated in lab in an aqueous medium. This ecotoxicological test is therefore adequate to study the biological effects of contaminants present in the soil using the elutriate extracts. This test was found to be more sensitive than the classical bioassays used by the environmental agencies as demonstrated in previous studies devoted to investigate the toxicity of waters and sediments of a freshwater ecosystem (Sforzini et al., 2008). Our results demonstrated no effects on the survival rate of the protozoa but the chronic test on the reproduction, that in 24 h analyses the second/third amoebae generation, shown a slight decrease up to 10% with respect to controls, a value that, being lower than 20%, is not usually considered an indication of toxicity.

The variability of the data observed in some biotests (in particular in the reproduction tests of D. magna and E. andrei) should suggest to use more replicates in future application of these bioassays to biodegradable plastics testing.

Altogether, the data obtained in this study demonstrate that the soils exposed to high amount of Mater-Bi have no noxious effects on edaphic organisms; in particular, mono and dicotyledon plants results, indicate that Mater-Bi plastic products are innocuous for agricultural uses. It should be noted that the bioassay using as model organism the earthworm E. andrei is a 28 d test for the evaluation of the survival rate but it lasts 56 d when the endpoint is the reproduction. This latter chronic test evaluates the number of cocoons produced but also the number of newborn worms that develop in the soils; this ecotoxicological test has proved to be sensitive to the effects of low concentrations of different toxic chemicals (Van Gestel et al., 1992; Spurgeon et al., 1994). The use of this test confirmed that Mater-Bi degradation poses no risk for edaphic biodiversity.

In this study, we have used four bioassays for aquatic model organisms i.e., bacteria, algae, protozoa, and crustacean. The elutriates obtained from the Mater-Bi soils were no toxic for the organisms used for freshwater ecotoxicological analysis: these results are of great relevance from an ecotoxicological point of view. In fact, these data indicate that there is no risk that toxic chemicals possibly released during plastic degradation may contaminate surficial or underground waters with the consequence to enlarge the environmental problems.

The importance to use an adequate control for the different bioassays should be also emphasized. It appears relevant to associate to the usual “lab control” and to the control soil without plastic item an additional control soil in which an amount of a natural, no toxic product with a composition similar to the bioplastics is added to the sample. In this case cellulose, a sugar polymer, sharing similar molecular characteristics with Mater-Bi, a bioplastic with a relatively high biodegradability, and sugar content due to the presence of starch. This cellulose sample could show some differences from the control due to the high amount of nutrient compounds (in this case sugar) added to the matrix. It is known that eutrophic conditions could cause negative effects directly to the different model organisms used in the tests. Moreover, the excess of sugar and other nutrients in the soils may change, during the experiment, the structure of the community, that may alter the survival/reproductive capacity of a particular organism. Our study demonstrates that when limited changes in the performance of the organisms exposed to Mater-Bi soils were found, these effects were similarly observed in the organisms exposed to the soil added with cellulose.

When considered altogether these data suggest that the ecotoxicological approach used in this study could represent a standard for future investigation on the environmental impact of plastic degradation products by means of (i) the use of a standardized, detailed protocol for bioplastic degradation, (ii) the evaluation of a large battery of ecotoxicological tests using acute and chronic endpoints, (iii) the use of microorganisms, plants, and animals, (iv) the use of model organisms suitable to test the toxicity of soils and elutriates following international accepted protocols. These rules adopted in this study may represent the basis for the lab analysis of the environmental compatibility of new bioplastics and other biotechnological materials.

The results reported in this study demonstrate that Mater-Bi is a bioplastic that seems to meet an adequate standard of environmental safety. Although a large battery of ecotoxicological tests, using both acute and chronic end points, was used no noxious effect was found. The use of model organisms typical of soil and freshwater allows us to exclude possible negative effects on the biodiversity of the edaphic ecosystem and of the related freshwater (both groundwater and surficial) compartments.

SS was the responsible of the research activity and expert of soil ecotoxicological tests; LO was the responsible of the freshwater ecotoxicological tests; SC supported the different aspects of the research activity i.e., from soil and elutriate preparation to the test performance; AV was the research coordinator.