Introduction

The world annual production of plastic bags (PB) has increased significantly since the 1950s. The increase in the production of PB is due to their massive use in different human activities and the development of technology [1]. The effect of PB products on the economy is significant since an average person consumes them on a daily basis, either for personal use (clothing, furniture, office supplies, kitchen utensils, among others) or through other productive activities, such as construction, communications, transportation, storage. For this reason, the plastics industry is considered one of the most dynamic productive activities at a national and international level [2].

However, the poor disposal of waste has led to the accumulation of PB in the environment, causing contamination and health risks. Some authors consider that there could be between 7000 and 35,000 tons of PB in the oceans, while others say that more than five trillion pieces of various plastic types are currently floating [3]. In addition to this observable massive accumulation, it has recently been shown that PB, once exposed to light and hydrodynamism, lose some of their characteristics due to mechanical, biological, thermal, thermo-oxidative degradation or hydrolysis. They are fragmented into smaller pieces, which are called meso, and microplastics, and can find their way in living beings, including humans, causing great damage [4].

Although the utilization of single-use plastic bag products such as PB comes at a great environmental cost, millions of these PB are consumed around the world every day. For this reason, different countries have taken measures to reduce or eliminate their consumption. Among the solutions that have been proposed to address the problem is the substitution of conventional PB by the so-called bioplastics, which are materials developed through mostly renewable sources, designed to be biodegradable and, at best, compostable [5]. It is generally understood that biodegradation is the process by which biodegradable substances are assimilated by microorganisms and reintroduced into the environment. These substances are transformed into simple compounds such as carbon dioxide (CO2) or methane (CH4), through a process known as mineralization [6].

The prompt biodegradability of PB derives from the fact that their design allows interaction with the environment, which can quickly modify them at a physical and chemical structural level, promoting the biodegradation process. Therefore, the degree of biodegradation of PB materials is highly influenced by their structure and chemical composition, the environmental conditions and the microbial communities that they are subjected to [7].

The evaluation of the biodegradability of plastic biobags has been a challenge. In order to systematize the evaluation techniques and criteria, a series of standards have been established [8]. Goel et al. (2021) discuss the diversity of standards and the need to change them, or to find one that is appropriate to determine the biodegradation of bags [9]. However other users consider that the European EN13432:2001, Australian AS 4736: 2006, international ISO 17088:2021 and American ASTM D6400-12 are among the best-known standards. They consider several evaluation criteria such as: (a) disintegration, where 90% of the material must disintegrate into fragments smaller than a specific size (usually 2 × 2 mm), within a given timeframe (usually 12 weeks); (b) chemical degradation, which indicates that the plastic must comply with a level of conversion of organic carbon into CO2, within a certain timeframe (in industrial composting, most standards define 90% degradation in 180 days at 58 °C); (c) ecotoxicity where the growth and development of organisms in a control soil (obtained from organic waste that does not contain the test material) is compared with a compost sample of the same organic waste that usually contains 10% of test material, added at the beginning of composting (there should not be significant differences in the growth and development of the organisms); and (d) chemical characteristics, the plastic should have concentrations of heavy metals (Cu, Zn, Ni, Cd, Pb, Hg, Cr, Mo, Se, As) below 50% of those prescribed for compost in the country where the product is sold. However, despite the abundance of criteria, it cannot be said that there is a test that consistently confirms biodegradability, and those that do exist cannot be considered as rapid biodegradability tests [9,10,11].

Within the aforementioned criteria for the evaluation of the biodegradability of a plastic bag, the “ecotoxicity” stands out. It is generally evaluated using plants or earthworms, observing their development when exposed to the material, as described in the guidelines of the Organization for Economic Cooperation and Development (OECD) number 207 and 208 [12, 13]. Although these organisms can be useful, their interaction with plastic is mainly through contact, which could reduce, to a certain extent, the data obtained and the information that determines the toxic effect of a given plastic on ecosystems, so it is necessary to look for alternatives to improve this criterion. Other studies have used the larvae of Uloma sp. demonstrating its ability to survive by degrading polystyrene, but its ability to degrade the diversity of PB that currently exist has not been demonstrated [14].

As mentioned, the high demand for plastic bag products and the urgent need to replace them with plastic biobags, make it necessary to develop new methods/tools/techniques that consider the criteria already established by existing standards but add more evidence and reduce time to improve decision-making as regards whether a bioplastic can be considered biodegradable or compostable. Zophobas morio and Tenebrio molitor, insects belonging to the Tenebrionidae family known to consume and digest PB, can be appropriate for such purpose. Various studies, such as those by Yang [15,16,17], Brandon [18], Peng [19] and Cunguan et al. [20] have shown the great capacity of these organisms to degrade plastics, because of the bacteria found in their digestive tract and their digestive enzymes. This is in accordance with what was reported by Yang et al. [21]. Another study carried out in Taiwan in 2017 showed that two T. molitor and Z. morio bacteria can use PB as a carbon source [22].

Because of the few methodologies and standards available and considering the scarce unification of criteria in terms of biodegradation and compostability of bioplastics, the purpose of this research was to evaluate a rapid technique to identify biodegradable, compostable and/or toxic PB with T. molitor and Z. morio, and to offer a technical development that permits to validate compliance with national and international biodegradability and compostability guidelines, for bags intended to be compostable made with bioplastics.

Methodology

This research was carried out in the Environmental Engineering Laboratory of the Institute of Engineering, UNAM and in the Environmental Biogeochemistry Laboratory of the Institute of Geography, UNAM. The work was divided into the following seven stages: (1) search and acquisition of the compostable bags to be tested, (2) acquisition, establishment and arrangement of the tests with the larvae of T. molitor and Z. morio, (3) maintenance of the larvae and evaluation of consumption, (4) evaluation of biomass and plastic toxicity, (5) evaluation of total organic carbon (TOC), (6) analysis of reflectance attenuated infrared (IR-FT) spectroscopy in the feces of the larvae and (7) statistical analysis experimental design of randomized blocks and through an analysis of variance (ANOVA) followed by a Duncan's new multiple range test (MRT).

Selection of the Plastic Bags to be Tested

Disintegration, aerobic biodegradation and ecotoxicity tests were carried out on three compostable bags (WCare, EHappy and EAlternative). The bags were selected based on three criteria: they had to be easily available, made of similar components, and have a compostability certificate. Table 1 shows the composition according to the technical data sheet of each selected bag.

Table 1 Percentage composition according to the technical data sheet of the selected compostable plastic bags
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Test Arrangement with T. molitor and Z. morio Larvae

Tenebrio molitor and Z. morio larvae were obtained from establishments committed to their reproduction. Third-stage larvae were preferably used in the case of T. molitor, while fourth-stage larvae were used in the case of Z. morio. The test was conducted in 500 mL transparent polypropylene containers (this is not biodegradable or compostable). Polypropylene containers were used because of their availability and low price. Although they are made of non-biodegradable/compostable plastic material, they have no influence on the results because the waythey are manufactured does not allow them to be easily consumed by the larvae. 30 Z. morio larvae or 80T. molitor larvae were placed in the respective container. The larvae were weighed together before being placed in the container for the experiment. The treatments were classified according to the brand of the plastic bag which were fed to the larvae and kept in environmental conditions which ranged between 24.5 and 26.5 °C, and between 45 and 55% humidity. Inside the main container holding each experimental unit, a thermostat associated with a ceramic infrared focus was installed for temperature control. Moreover, a hygrostat was connected to a humidifier (all equipment for terrariums) in order to make it possible to maintain the humidity levels.

The compostable PB were added in small squares of approximately 1 cm2, and 5 g of each bag was added to each container with Z. morio or T. molitor. One g of carrot cuttings was added weekly as water source for the larvae.

Four repetitions were performed with each selected bag and a control group (CT) was maintained with the same repetitions. Both species in the control group were fed 10 g of wheat bran. The test arrangement can be seen in Fig. 1. The data were recorded during the 56 days of the experiment and when the 5 g of bag were consumed.

Fig. 1
figure 1

Polypropylene containers with larvae and compostable plastic

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At the beginning of the experiment, 80 larvae of T. molitor and 30 of Z. morio were placed in each experimental unit, they were weighed and the plastic bag was added as food. Throughout the experiment, the amount of time it took them to consume the added plastic was evaluated by weighing the plastic available in each experimental unit. The set of larvae was also weighed to assess their weight loss or gain, and a count was made of the live larvae (the dead ones were removed). This activity was carried out on a weekly basis. At the end of the experiment, the weight of the larvae in each experimental unit, the weight of the plastic that was not consumed by the larvae and the total number of live larvae were determined.

Determination of the Percentage of Bag Consumption

The recording of the feeding and consumption of the compostable PB was carried out weekly. To quantify consumption, the amount of food (plastic bag) was weighed concurrently. The amount of food left at the end of the test in all treatments was also weighed. The added plastic was extracted weekly to carry out the weighing, and later returned to its respective experimental unit.

The percentage of consumption of compostable bags was obtained by the difference in weight of the plastic material using Eq. 1:

$$\%\; \text{of\; consumption} = \frac{Pip-Pfp}{ Pip }\times 100,$$
(1)

where Pip is the initial weight of the plastic (g), Pfp is the final weight of the plastic (g).

Evaluation of Biomass Percentage (Gain or Loss) and Ecotoxicity of Compostable Bags

The recording of the gain or loss of biomass in larvae was carried out by weighing every 7 days all the larvae in each experimental unit, during the test period. The larvae of each experimental unit were withdrawn, weighed together, and then returned to their respective experimental unit.

Larval survival was recorded for both species every 7 days during the experiment. For both species, the organism-by-organism count of each experimental unit was carried out, recording only the live larvae.

Analysis of Biodegradation of Compostable Bags by Determining the Total Organic Carbon Present in the Feces of the Larvae

The larvae were separated according to their species and placed in 2 containers of approximately 10 L, in which no substrate or food was added in order to purge or clean their intestines. They were kept under these conditions for approximately 2 days, till feces from the previously consumed food were no longer observed.

The experimental units consisted of 500 mL polypropylene containers with 1 g of compostable plastic cut in small pieces (10 mm × 10 mm). For the test with T. molitor, 7 g of larvae were placed in each container, while for the test with Z. morio, 15 g of larvae were placed in each container. A control group which was fed 1 g of wheat bran was also set up. Figure 2 shows various experimental units.

Fig. 2
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Polypropylene containers with the different compostable PB and the larvae of T. molitor (above) and Z. morio (below) during the collection of feces for the TOC test

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Feces, generated by the organisms of each experimental unit, were removed by using a mesh with a pore size of approximately 1 mm, and these were placed in a glass with a lid. The test lasted for 7 days. The stool samples were cleaned with magnifying glass and tweezers to remove cuticle remains from the larvae and small pieces of plastic from the sample.

The TOC evaluation was carried out with a Shimadzu carbon analyzer, model SSM-5000ª. Fifty milligrams of each material were weighed and introduced to the equipment to obtain the total carbon (TC) and 50 mg of each material were weighed and introduced to the equipment to obtain the inorganic carbon (IC). The TOC was obtained by the difference between TC and IC averages. Figure 3 shows some alumina-porcelain capsules containing the larva feces.

Fig. 3
figure 3

Alumina-porcelain dishes with 50 mg of feces from Z. morio larvae

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To obtain the biodegradation percentage through TOC analysis (Btoc), the TOC contained in the tested compostable bags was previously evaluated, and Eq. 2 was used for the calculation.

$${B}_{toc}=\frac{{P}_{toc}-{H}_{toc}}{{P}_{toc}}\times 100,$$
(2)

where Ptoc percentage of average TOC obtained in the compostable bag to be tested (average of 3 repetitions). Htoc percentage of TOC obtained in the feces of the larvae that consumed the plastic to be tested.

According to several standardized methods, for example, ASTM D5338-15, 17088:2021 ISO 14855–1:2005, ISO 14855-2:2007, and NMX-E-273-NYCE-2019, plastic (carbon compound) is considered biodegradable. After a biodegradation process, all or a required minimum amount is transformed into CO2 and biomass, and the plastic (carbon compound) that comes out intact after a controlled biodegradation process is considered non-biodegradable. In this experiment, the fraction of carbon present in feces was considered non-biodegradable because it remained intact after digestion (the biodegradation process).

This is very important because within the terminology of standards, an organic material that can biodegrade in a short time, 6 months to a year, is considered biodegradable compared to other organic materials such as polyethylene or other traditional plastics, which can take more than 100 years to biodegrade. These materials are biodegradable because of their chemical structure, but not in the standard sense.

Analysis of the Chemical Composition of the Feces Generated by the Digestion of the Plastic Consumed by the Larvae

The analysis of the chemical composition of the humus (feces) generated by the larvae of T. molitor and Z. morio was carried out using the IR-ATR infrared spectroscopy method for the detection of polymers, and by means of inductively coupled plasma optical emission spectrometry (ICP-OES) for metals. The infrared spectroscopy analysis was carried out with an infrared spectrometer Thermo Scientific, model NICOLET 6700 FT-IR, using the fully attenuated reflectance (ATR), technique for which the ATR accessory is used. The analysis of metal content in the feces of the larvae was carried out with a Milestone microwave oven, Ethos Easy model, with a power of 1800 kW and a PRO-2 rotor, which was used to carry out the plastic digestion process. The metals were quantified with an atomic emission optical spectrometer coupled to an ICP-OES, brand Agilent Technologies 5100.

Experimental Design

A randomized block experimental design was set up, and data analysis was performed with an ANOVA test, followed by a Duncan's new MRT. This experimental design was used to determine differences between the treatments with both species and among the different bags. The following aspects were evaluated: percentage of plastic consumption, percentage of biodegradability, percentage of living organisms biomass (weight loss or gain), and percentage of survival.

Results

The results obtained at the end of the experiment are shown herein below. First, the rate of consumption is presented, then the evaluation of biomass and ecotoxicity, followed by the percentage of biodegradation through TOC analysis, and the analysis of feces. Finally, the metal content in feces is shown.

Determination of the Bag Consumption Percentage by Larvae

Table 2 shows the results obtained from the consumption of the different bags by the two species of larvae. It is important to mention that the larvae of the treatment groups were initially fed 5 g of PB while the control group was fed 10 g of wheat bran, and that no additional food was supplied during the time of the experiment.

Table 2 Comparison of the average consumption percentage and recovery of compostable PB with each treatment using Z. morio and T molitor
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From the results shown in Table 2, it is observed that with Z. morio the control group and the EHappy group consumed 100% of the food provided in 28 days. However, it is important to highlight that the control group was given twice as much food, so there are differences between the rate of consumption of wheat bran and the EHappy bag. Similarly, the rate of consumption of the EHappy group is considerably different from the rate observed with the WCare and EAlternative groups, since the EHappy group consumed 5 g in half the time. The WCare and EAlternative groups show a similar behavior, with consumption being slightly higher in the WCare group

On the other hand, with T. molitor it is observed that the control group and EHappy group consumed 100% of the food provided in 28 days and 35 days respectively. As with the species Z. morio, it is important to highlight that the control group was given twice as much food as the EHappy, EAlternative and WCare groups, which suggests that there are differences between the rate of consumption of wheat bran and EHappy bag.

As observed with Z. morio, the EHappy group shows considerable differences with the WCare and EAlternative groups, since the larvae of the EHappy group consumed the 5 g in little more than half the time required by the larvae of the other groups. However, with T. molitor, differences were also observed between the WCare and EAlternative groups, with the larvae of the WCare group being more efficient in consuming plastic.

When performing the statistical analysis, no significant differences were observed between the bag consuming species (F 0.3083, theoretical F α 0.05 9.28), which indicates that both species were equally efficient at consuming the different bags. In contrast, there were significant differences between the different bags tested, an F value of 131.281 was obtained, which is greater than the theoretical F value (α 0.05) 10.1. Finally, the MRT test (13,979) indicated that there are significant differences between the means of the control group (CT) and the EAlternative and WCare groups, and between the means of the EHappy group and the EAlternative and WCare groups.

The results above suggest that the physical and chemical characteristics of each bag are the factors that determine the rate of consumption of the two species of larvae. Both species seem to have a similar affinity as regards their choice of polymer mixtures contained in the different bags.

Studies that show the rates of consumption of polymers such as PBAT, PLA and corn starch separately or as a whole are very scarce, so it is not possible to compare the results obtained in this work with others. However, the work carried out by Peng et al. [23], who evaluated the consumption of PLA using T. molitor fed on different diets for 24 days, showed that diets with 10%, 20% and 30% PLA mixed with wheat bran were completely consumed in fewer than 24 days, the diet with 50% PLA was completely consumed in 37 days, while 2.17 g the 100% PLA diet was consumed. These results showed that adding wheat bran improves PLA intake. As observed in the results, the consumption of the bags was very good, considering that they were not mixed with bran, however, the bag polymer content, mainly starch, may be related to their good acceptance.

On the other hand, some studies such as those of Peng [19, 24, 25], Yang [26,27,28], and Bulak [29], among others, have shown that there are differences in the rate of consumption (% of consumption) of the various types of PB such as: polystyrene, polyethylene and polypropylene. These works show very clear differences between the tested PB, reporting consumption ranging from less than 12% to up to 90% in 45 days. This is consistent with the results obtained, even though they are not the same types of polymers.

Evaluation of the Biomass Percentage (Gain or Loss) and Ecotoxicity of Compostable Bags

Table 3, 4 and 5 shows the percentage of biomass gain or loss per larva at the end of the treatment, comparing both species.

Table 3 Comparison of the percentage of biomass gain or loss per Z. morio larva in all the groups after consuming 5 g of bag or after 56 days
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Table 4 Comparison of the percentage of biomass gain or loss per T. molitor larva in all the groups after consuming 5 g of bag or after 56 days
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Table 5 Comparison of the average percentage of biomass gain or loss, obtained by Z. morio and T. molitor, in each group
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According to the results presented in Table 3, it can be observed that the highest average weight gain per Z. morio larva obtained at the end of the test corresponds to the control group with a 9.22% increase in biomass (from 0.312 to 0.337 g), followed by the WCare group with a 7.89% increase in biomass (from 0.314 to 0.338 g). Finally, the EHappy and EAternative groups showed a slight decrease in weight − 2.34% of biomass reduction (from 0.311 to 0.303 g) and − 1.09% of biomass reduction (from 0.309 to 0.305 g), respectively. These results suggest that the WCare bag probably presents the highest digestibility (compostability) in the long term, being very similar to that of the control group.

On the other hand, according to the results presented in Table 4, it can be observed that the highest weight gain per T. molitor larva obtained at the end of the test corresponds to the control group with a 60.31% increase in biomass (from 0.052 to 0.083 g). In the treatment groups, it corresponds to the EAternative group with a 37.49% biomass gain (from 0.053 to 0.072 g), followed by the WCare group with a 31.72% biomass gain (from 0.054 to 0.071 g), and finally the EHappy treatment with a 27.41% gain in biomass (from 0.052 to 0.067 g).

When the statistical analysis was performed, no significant differences were observed between the treatments (F 7.69, theoretical F α 0.05 10.1). However, when performing the MRT test, a value of 18,857 was obtained, which represents a significant difference when compared with the means of all the treatments, only between the CT group and the EHappy group. This shows that this bag is statistically the least capable of generating biomass gains (compared to the control feed), an indicator of low biodegradability. It is also important to highlight that the EA and WC bags show similarity in terms of biomass gains, which indicates a possible similarity in terms of their biodegradability. However, the WCare bag was the only one that showed a biomass gain, in both species during the test, which suggests that this bag may present the best physicochemical characteristics for its biodegradation.

On the other hand, significant differences were observed between both species in terms of their biomass gain or loss (F 12.17, theoretical F α 0.05, 9.28). This indicates that T. molitor presents a higher efficiency to digest the different polymers tested compared to Z. morio. The results may be related to the microbiota contained in the intestines of each species, the digestive enzymes, and possibly their larval stage.

The results described above suggest that the physical and chemical characteristics of each compostable bag are the factors that determine their degree of biodegradation, because, although there were differences between the digestion efficiency of both species, the EHappy bag was the only one that showed differences compared with the control group.

There are few studies that evaluate the activity of T. molitor and Z. morio with polymers such as PBAT and PLA; however, some studies such as the one conducted by Bozek [30], where pure PLA was fed to T. molitor larvae, showed biomass losses (− 15%) after 21 days. In the study by Peng [23], who also fed T. molitor larvae with pure PLA, a biomass loss of − 13.75% was observed at the end of the trial. However, a reduction in the PLA content, and an increase in the wheat bran content led to biomass increases; for example, the 50/50 mixture showed a 15% increase in biomass, and its control group (pure wheat bran) generated a 27% increase in biomass, suggesting that certain contents of other polymers may aid digestion and assimilation. When comparing the results of the aforementioned studies with those shown in Table 3, it can be noted that the loss of biomass was only registered with Z. morio and for the EAlternative and EHappy bags, but to a lesser extent, which may be due to the mixture of polymers in the bags (PBAT, PLA and starch), said mixture could influence degradation and assimilation. However, the physicochemical characteristics (quality) and proportions of each polymer making up the bags seem to be important, since in the work of Peng [23], an increase in biomass was only observed with wheat bran (containing other compounds). In the present work, such large biomass losses were not observed, and gains were recorded without the addition of wheat bran.

Finally, Wu and Criddle [31], report that the consumption of some polymers, such as polystyrene, can help the maintenance of the larvae in terms of their energy expenditure, but may not lead to an increase in biomass, due to nutrient deficiency in this plastic. As a result, a slight loss of biomass may be experienced. This phenomenon may have occurred with the polymers present in EHappy and EAlternative, and only with the metabolism of the Z. morio larvae that fed on them.

Tables 6, 7 and 8 shows the results obtained for the percentage of survival at the end of the test comparing both species.

Table 6 Comparison of the percentage of survival of Z. morio larvae in all groups after consuming 5 g of bag or after 56 days
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Table 7 Comparison of the percentage of survival of T. molitor larvae in all groups after consuming 5 g of bag or after 56 days
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Table 8 Comparison of the average percentage of survival in each group using Z. morio and T. molitor
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The results shown in Table 6 suggest that there seem to be no differences in the survival of Z. morio larvae between the EHappy, CT and WCare groups, with approximately 94.17% and 95% of the larvae placed at the beginning of the experiment remaining alive and therefore, probably no toxic effect or any other effect of the plastic on the larvae is observed. In addition, no great difference was shown between the group fed the EHappy bag in which the 5 g of plastic was completely consumed in 21 days, and the group fed the WCare bag that did not consume all of the plastic but continued for 56 days and the control group that consumed 10 g of wheat bran in 35 days. However, the group fed the EAlternative bag showed survival differences with respect to the other treatments because it did not consume all the plastic and the survival decreased considerably to 85.83% by the 56th day.

On the other hand, the results obtained with T. molitor in Table 7 show that there seem to be differences in the survival of larvae between the control group and all the treatment groups, which suggests that T. molitor is probably more sensitive to the consumption of compostable plastics than Z. morio, since the survival in the control group was 98.75% on average, while it was 91.87% in the EHappy group and 89.37% and 83.75% in the WCare and EAlternative groups respectively. Likewise, the results show that there seem to be differences between the treatments with respect to survival, being most noticeable the difference between the EHappy and WCare groups with 91.87% and 89.37%, respectively vs the EAlternative group with 83.75%. In the same way, the larvae of the EAlternative treatment had the lowest survival, which suggests a possible toxic effect or some physical characteristic in the structure of the plastic that produces mortality.

In the evaluation of the survival percentage, no significant differences were observed between the survival of both species (F 0.2320, theoretical F α 0.05 9.28), which means that both species showed similar survival, being affected in the same way by the compostable bags which were fed to them. In contrast, significant differences were observed between the different bags provided as food and the survival of the organisms, since an F-value of 17.986 was obtained, this being greater than the theoretical F value α 0.05 10.1. Finally, the MRT test (6,553) indicated that there are significant differences between the means of the EAlternative group and the other groups.

As regards other experiments where some compostable polymers were fed to Tenebrionid larvae, the study by Peng [23] reported a survival of T. molitor larvae of 82% when the larvae were fed 100% pure PLA. The same study recorded a survival of T. molitor of 93%; with a 10% PLA diet and a survival of 91% with 100% wheat bran diet. These results are similar to those shown in Table 6. Therefore, it is possible that the effect of the EAlternative bag could only be due to the lack of nutrients, and not so much to a toxic effect, since in the case of the WCare and EAlternative bags the survival was evaluated at 56 days, while in the case of the EHappy bag, the survival was evaluated at 28 days, when the larvae had eaten up the 5 g.

Other studies in which plastics were fed to Tenebrionidae larvae include that of Peng [24], where PVC only was fed to T. molitor larvae, reporting a survival of 80% up to 35 days. In said work, losses are attributed mainly to the lack of nutrients. Peng [25], reported survival rates of 83% to 95% in periods of 33 to 56 days, during which Z. atratus larvae were fed with polystyrene and low-density polyethylene, attributing deaths mainly to malnutrition. Finally, Yang [27] reported a survival of T. molitor fed with polystyrene greater than 85% at 32 days. These results may suggest that this phenomenon could have been observed in the present study and is not directly related to bag toxicity, but to a lack of nutrients.

Analysis of the Biodegradation of Compostable Bags by Determining the TOC Present in the Feces of the Larvae

The results obtained from the percentage of biodegradation achieved by the digestion of the organisms are presented in Tables 9, 10 and 11.

Table 9 Percentage of biodegradation of compostable bags achieved by the digestion of Z. morio larvae as determined by TOC analysis
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Table 10 Percentage of biodegradation of compostable bags achieved by the digestion of T. molitor larvae as determined by TOC analysis
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Table 11 Comparison of the average percentage of biodegradation of compostable bags in each treatment, by determining the TOC present in the feces of Z. morio and T. molitor
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As can be seen in Table 9, the compostable bag that presented a greater biodegradation due to the digestive activity of Z. morio was WCare with 43.49%, followed by the EAternative bag with 15.01%, EHappy with 11.22%, and finally the CT group. with 9.53%. These results indicate that there seem to be significant differences in almost all treatments, suggesting that the physical and chemical characteristics (proportion of polymers) of the WCare bag make it more easily degradable and assimilable by Z. morio compared to the other bags. On the other hand, the fact that the CT group that was fed wheat bran presented the lowest biodegradation according to the TOC analysis may be mainly related to the chemical composition of the food and the microbiota present in the intestine of Z. morio.

The results obtained with T. molitor (Table 10) show that WCare bag was again the compostable bag that presented a greater biodegradation with 48.23%, followed by the EAlternative bag with 15.04%, CT with 13.75%, and finally EHappy with 10.75%. These results indicate that there seem to be significant differences in almost all treatments, with results very similar to those obtained with Z. morio. These results suggest that the physicochemical characteristics of the WCare bag are also the most suitable for T. molitor to easily digest the polymers present.

Significant differences (F 466.551, F theoretical α 0.05 10.1) were obtained in the statistical analysis, while the MRT test generated a value of 0.042. When compared with the means of all the treatments, significant differences were observed between the WCare group and the other groups (EHappy, EAlternative and CT). These results confirm that the WC bag is the one that presents the best biodegradation characteristics by beetles of the Tenebrionidae family, and the EHappy bag could be the one with the worst physical and chemical characteristics, which limits its digestion (biodegradation) by the two species (T. molitor and Z. morio).

Regarding the low percentages of biodegradation obtained with wheat bran (CT control group), with both species, these results may be mainly due to the chemical composition of this product. According to Chaquilla-Quilca [32], the composition of wheat bran consists of approximately 60% total dietary fiber (mainly composed of cellulose, hemicellulose and lignin), 20% starch, 14% high biological value protein, 5% lipids and the rest ashes and moisture. Said composition allows the larvae to satisfy their physiological requirements allowing an increase in biomass, however, it is likely that they have certain difficulties assimilating the polymers contained in the dietary fiber. According to Ortiz [33], lignin forms a matrix that surrounds cellulose and hemicellulose, and lignin degradation is a prerequisite for the hydrolysis of the other components of plant biomass, which are the main source of carbon and energy for biomass microorganisms. On the other hand, hydrophobicity, the random, complex structure, and lack of common hydrolyzable bonds, make lignin resistant to degradation by most cellulose-degrading microorganisms. Its biodegradation is carried out by a restricted number of microorganisms, mainly fungi, which are not part of the microbiota of the larvae. This could be a possible explanation for the increase in biomass, but a limited decrease in TOC.

On the other hand, no significant differences were observed between both species (F 0.8005, theoretical F α 0.05 9.28), indicating that T. molitor and Z. morio can be considered statistically equally efficient for polymer degradation. However, it is possible that both species metabolize the plastic polymers, carbohydrates, fats and wheat bran protein differently, since differences were observed in terms of the percentage of biomass generated by both species, suggesting that T. molitor can possibly build more biomass and Z. morio uses it mainly as energy source. It is possible that these results may be related to the larval stage of the organisms and the requirements of their metamorphosis, and to differences in terms of the microbiota present in the intestines of each species.

The results obtained could not be compared with other works because TOC measurement in the feces of the larvae is not commonly used as a technique to determine the biodegradation of polymers. According to Wu and Criddle [31] who wrote in Chapter 5 of Characterization of biodegradation of plastic bags in insect larvae, “the measurements most used to determine this parameter are: gravimetric determination of weight loss, gel permeation chromatography (GPC), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), proton nuclear magnetic resonance (1H NMR), differential scanning calorimetric analysis (DSC), microscopic observations, water contact angle (WCA), labeling isotopic, among others”. However, the measurement of the TOC percentage in the feces of the larvae seems to be an appropriate technique since the difference between the organic carbon present in the polymer fed to the larvae and the organic carbon found in their feces partially reflects the carbon that is assimilated and used by organisms, in quantitative terms. This is so, because in this system there are no other forms of losing carbon since organic carbon enters the bag in a controlled manner. It is either assimilated there, leading to a decrease at the exit of the digestive process (which occurred), or on the contrary, if the material is very recalcitrant and cannot be assimilated, the amount of carbon leaving the system should be approximately the same as the one entering it.

Analysis of the Composition of T. molitor and Z. morio Feces

Figures 4, 5 and 6 show the comparison of the IR spectra of each compostable bag, and the IR spectra obtained by the digestive activity of both species when consuming each bag.

Fig. 4
figure 4

Comparison of the IR spectra of the EAltenative bag (a), and after the digestive activity (feces) of Z. morio (b) and T. molitor (c)

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Fig. 5
figure 5

Comparison of the IR spectra of the EHappy bag (a), and after the digestive activity (feces) of Z. morio (b) and T. molitor (c)

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Fig. 6
figure 6

Comparison of the IR spectra of the WCare bag (a), and after the digestive activity (feces) of Z. morio (b) and T. molitor (c)

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As can be seen in comparative Figs. 4, 5 and 6, a decrease in the signal of the most representative peaks of the IR spectra was evidenced, in the 3 compostable bags tested. Said peaks are 1780 cm−1, 1504 cm−1, 1459 cm−1,1410 cm−1, 1390 cm−1, 1268 cm−1, 1110 cm−1, 1019 cm−1, 873 cm−1, 727 cm−1, PBAT characteristic [34]. For PLA 1453–1450 cm−1, 1381 cm−1, 1210 cm−1, 1190 cm−1, 1160 cm−1 and 1083 cm−1, 869 cm−1, 757 cm−1 [35,36,37,38,39], and the peak 2919–2920 cm−1 (PLA possibly) showed decreased signal or was no longer detected. Finally, the 3320–3310 cm−1 peak was no longer detected, and a decrease in the 934 cm−1 peak characteristic of corn starch was observed [40,41,42,43]. These results suggest that there was a partial biodegradation of the polymers present, which was corroborated with the TOC analysis seen above. In the same way as in the TOC analysis, the bag that presented the greatest change was the WC bag, followed by the EAlternative bag and, finally, the Ehappy bag, indicating that they all present a certain degree of biodegradation once they pass through the digestive tract of the organisms. However, they all show different susceptibility, being Ehappy the most resistant to the process.

When comparing the results obtained through IR analysis in this work, with the results of other works, the biodegradation of the polymers present in the tested bags was confirmed. Some works where the biodegradation of PB is reported by a reduction in the signal or a change in the IR spectrum measured in insect feces, are those of Wang [44], Rana [45], Ghatge [46], Bulak [29], Yang and Wu [39], Yang et al. [47] and Peng [19, 23,24,25], all being consistent with the results obtained in terms of their interpretation.

Analysis of Metals by Mass Spectrometry (ICP-OES)

Table 12 shows the results of the evaluation of metals detected after PB (EHappy, EAlternative and WCare) digestion by the larvae of Z. morio and T. molitor. Likewise, a comparison is made with the maximum permissible limits (LMP) established by the standards NOM-004-SEMARNAT-2002 and UNE EN 13432:2001, and those detected in the intact bags.

Table 12 Comparison of the metals detected in the compostable bags evaluated, and after their digestion by Z. morio and T. molitor
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According to Table 12, a minimal increase in the amount of Cr, Cu, Pb and Zn is observed after the digestion of the compostable bag. When compared with the maximum permissible limit (LMP), it is observed that they are within the acceptance criteria and can be considered a useful method for the evaluation of metals after a biodegradation process. It is also important to highlight that the amount of metals detected before and after the larval digestion process is similar or higher, which suggests that the metals are not accumulated in the organisms.

Conclusions

The two similar species of the Tenebrionidae family, Z. morio and T. molitor, present similar characteristics in terms of consumption, survival, and biodegradation of compostable PB. According to the methods of analysis used, both species are potential, adequate and viable tool that can be used for the implementation of a rapid method to evaluate biodegradation, ecotoxicity and metal content in compostable PB. It can also be a support method for other methodologies that evaluate the same parameters in compostable bags, making them more efficient and effective. In the case of this research, only the use of this method in the evaluation of compostable bags or disposable items made with mixtures of PBAT, PLA and corn starch is mentioned.

The physical and chemical characteristics and the proportions of the different polymers (PBAT, PLA and corn starch) that make up the compostable bags influence the consumption speed and the affinity of T. molitor and Z. morio for feeding on them.

The T. molitor species shows a greater capacity to digest and assimilate all the bags tested, which suggests that its microbiota and digestive enzymes are more efficient for biomass gain. On the other hand, it is possible that Z. morio uses the energy obtained from the polymers, mainly for maintenance of its vital functions and not for tissue construction.

The toxic effect of the compostable bags on T. molitor and Z. morio was not observed.

A reduction in the TOC content was observed in the three compostable bags evaluated, when they were consumed and excreted by T. molitor and Z. morio, indicating that there was a biodegradation process.

The biodegradation process was corroborated by observing a decrease in the signal in the representative peaks of the infrared spectrum, in the three compostable bags when the feces of both species were analyzed.

It was possible to detect metals in the feces of the larvae of T. molitor and Z. morio, in quantities similar to, or greater than, those detected in the intact compostable bags, suggesting that there is no accumulation phenomenon.