Short-Term Effect of Poly Lactic Acid Microplastics Uptake by Earthworms, Eudrilus eugeniae.

The uptake of polylactic acid (PLA) microplastics, the most commonly used biodegradable plastics by earthworms Eudrilus eugeniae was investigated by observing their weight changes, biomass and the microplastics concentration after feeding them with a mixture of PLA and cow dung for 16 days at the concentrations of 0%, 10%, 30%, 60%, and 80% w/w dry weight. The mortality rate of the earthworms for all the PLA concentrations during the 16 days of feeding period was 0%. However, the microplastic affected the earthworms' weight signicantly (P-value 0.00027), especially at the concentration of 80% of PLA. The earthworms had the lowest weight gain at 80%, followed by 60%, 30%, and 10% of PLA, respectively. The earthworms subjected to 80% and 10% of PLA had a similar pattern with the control. This was perhaps due to the high organic matter in the worm’s feed for the 10% PLA and the digestion mechanisms, which led the earthworms to preserve their energy and become less active in the 80% PLA, compared to all the other treatments. The PLA microplastics concentration factor (CF) in the vermicast was the highest at the 10 % PLA compared to other treatments, because Eudrilus eugeniae did not degrade the PLA. This study concludes that even though PLA is a biopolymer-based, it cannot be bio- assimilated by the earthworm.


Introduction
Despite the practice of reducing, reusing, and recycling, plastics waste still poses a major problem due to its increasing diversity of properties and quantity. In 2013, plastics' global packaging production was 78 million tonnes, of which, 98% were produced using new non-recycled material (Geyer et al. 2017). Although 14% of these plastics were collected for recycling, only 2% were eventually recycled (Zhu, Yao, and Liu 2006). About 32% of the produced plastics end up in oceans or on land. In 2018, plastics' global production had reached 8.3 billion metric tons, and 76% of them became plastic waste. Only 9% of plastics produced was recycled, and 12% was incinerated. Approximately 79% of the plastics waste produced end up accumulating in a land ll or leaked into the environment (Geyer et al. 2017). By comparing the data from 2013 and 2018, it is evident that although the recycling rate has increased, the total volume of plastic waste produced has also been increased more than a hundred times.
The low recovery of plastics waste happened due to some reasons. Recycling facilities are expensive to built, and segregation requires extensive workforce. Regardless of the damaging effect towards the environment, these facilities are unable to recycle all type of plastics waste. For example, China's recycling facilities were the main cause of its enormous environmental pollution (Katx, 2019). On that note, China declared that they would stop importing plastics waste from any developed countries from July 2017. Due to this policy, other countries like Malaysia, Vietnam, Thailand, Indonesia, Taiwan, South Korea, Turkey, India and Poland were the new dumping sited for the developed counties (Asia 2019; BBC Reality Check Team 2019). This is one of the evidences of a global plastic waste issue. Neither all plastics can be recycled, nor their quality is the same after recycling (Achilias et al. 2007; Hopewell, Dvorak, and Kosior 2009; Schyns and Shaver 2020). Camacho and Karlsson (2000) reported that recycled plastics (food packaging plastics) were ve times more hazardous contaminants (ethylbenzene and xylenes) than the non-recycled ones. Under these circumstances, bioplastics and biodegradable plastics have been promoted as an alternative to the non-degradable fossil-based Bioplastics are renewable and sustainable resources, known primarily by aliphatic bio-polyester such as polyhydroxyalkanoates (PHAs) or poly-3-hydroxybutyrate (PHB), polyhydroxy valerate (PHV), polyhydroxyalkanoate (PHH) and polylactic acid (PLA) (Altaf et al., 2007;Carus, 2012). There has been increasing demand on this type of plastics particularly the PLA (polylactic acid) and PHAs (polyhydroxyalkanoates). For example, the PLA production reached 140,000 metric tons of PLA per year in the USA by Blair facility alone. It was estimated that the global bioplastics production would reach 2.62 tonnes in 2023 (Rosevelt et al. 2013). This shows that PLA maybe one of the leading bioplastics for the future (Jamshidian et al., 2010). Due to the increase in the production of bioplastics, it is highly expected to contribute to plastic waste problem. Since most biodegradation testing of bioplastic are done in a controlled conditions for a start, it is highly impossible to ensure a complete degradation of these materials. Their In general, plastics are complex long-lasting materials. However, with prolonged exposure to weathering condition, plastics tend to shatter into smaller pieces, both in the open ocean or buried inside the soil. These fragmentations are known as microplastics with a size range starting from 5 mm to nanometres. These ne The mortality of earthworms caused by petroleum and bio-based microplastics were observed in a few studies. For example, according to Cao et al. (2017), 2% of polystyrene (PS) which is petroleum-based plastics mixed with the earthworms feed cause a signi cant inhibition to the growth of the earthworms. In the case of lowdensity Polyethylene (LDPE), the mortality rate of the earthworms was between 8-25% when the LDPE concentration was 28 % and 60%, as observed by Huerta Lwanga et al. (2017). Nevertheless, a contradictory result was observed by Zhang et al. (2018). In his study, exposure of the earthworms to LDPE lms resulted in zero per cent mortality. This result might be caused by selective behavior of the earthworms toward their feed.
Polylactic acid (PLA) is known as bio-based plastics. Even though the mortality rate was zero, the weight loss of worms subjected to PLA was evident . The earthworms were also unable to digest and consume the PLA as carbon source (Qi et al. 2018). Even though PLA is plant-based plastics, it shows resistance to degradation without exposure to hydrolysis degradation rst. However, the two studies were conducted using PLA 50 and PLA 96, and any research on the effect of commercial PLA towards the tness of Eudrilus eugeniae is yet to be conducted. Eudrilus eugeniae has a natural ability to colonize organic waste, with high endurance and handling resistance, possess tolerance to a wide range of environmental factors and capable of digestion and assimilation of organic matter (Kooch and Jalilvand, 2008).
The purpose of this research is to observe the possibility of Eudrilus eugeniae earthworms to degrade PLA microplastics, as well as the effect of different concentrations of PLA on their weight changes, growth rate and cast concentration factor (CF) when the speci c concentration of PLA microplastics was added into their feed.

Material
Twenty-ve plastic containers with a diameter of 8.5 cm, a height of 6.0 cm, closed lid was used for the feeding experiment. Virgin polylactic acid, Grade PLA4032D were imported from Nature Works, USA by Innovative Pultrusion Sdn. Bhd. The PLA was ground to the required size of between 40 to 106 µm. Earthworms Eudrilus eugeniae were provided by the ECO-PRO laboratory in the School of Industrial Technology Universiti Sains Malaysia (USM). The cow dung was obtained from a cow farm in Balik Pulau, Penang, Malaysia.

Media Preparation
Pre-composted cow dung was used as a the primary feeding media., according to the methods by (Coulibaly et al. 2011; Manyuchi and Whingiri 2014). The cow dung was homogenized by passing them through a 2 mm and 800 µm sieve to avoid selective feeding, as suggested by Sampedro and Whalen (2007) and immersed in water for 24 hours to remove insects and pressed inside a piece of cloth to remove excess water before usage. The PLA microplastics were mixed with cow the dung at ve different concentrations, as shown in Table 1. A synthetic nylon-based cloth was placed on top of the media, which was a mixture of cow dung and PLA to attract the earthworms to cast on the top of the media as well as to collect and retain the moisture. Table 1 The ratio of PLA microplastics and cow dung mixture to obtain the desired percentage of PLA. The third column on the right shows the initial feed given to earthworm for each PLA percentage.
Percentage of PLA (%) PLA (g) Cow dung (g) (dry weight)  Table 2). The earthworms were adapted to the new environment for seven days, with continuous monitoring of moisture content, feed, and mortality for every two days.

Feeding experiment
A plastic container with a diameter of 8.5 cm and 6.0 cm height, with six holes was used. The cow dung and PLA microplastics mixture was placed into the container before an earthworm was added. Five replicates were used per treatment. The container was labelled and placed in a dark, closed box with 16/8 ratio of light/ drank.
The experiment was carried out for 16 days. Every four days, the earthworms were measured for their weight and checked for their feed moisture. After 16 days, the earthworm was subjected to gut voiding for 48 hours by placing them into an empty container. Five milliliters of distilled water was sprayed every two days to maintain the moisture. The cast was collected from the containers, placed them into a zip-locked bag and kept at 4˚C for further analysis.

Statistical Analysis
Statistical analysis such as descriptive analysis, analysis of variance, and correlation analysis was performed using Excel 2016 (Microsoft o ce 2016). Descriptive analysis was present as mean, standard deviation, maximum and minimum values for different parameters. A one way and two ways Anova was perform to determine any signi cant differences between the parameters. Correlation analyses using Pearson Correlation Coe cient Formula was conducted to show the relationship between concentration of PLA and earthworms weight change.

The pH values for the vermicast
The average pH of all the treatments did not vary much from the control. However, the treatment with 60% PLA concentration signi cantly differed with p < 0.05 (Table 3). For degradation to happen, a drop in pH value was expected due to the formation of lactic acid in the vermicast. The pH of vermicast produced by earthworms was neutral, indicating that the PLA microplastics in the vermicast did not undergo any degradation process.
The moisture content, pH, and temperature are considered the most important environmental factors that in uence the biodegradation of plastic materials and affect the earthworms' health. In the biodegradation process, moisture content affects the microbial activities and hydrolysis stage, which is crucial for polymers degradation (Müller, 2002). Earthworms activities and behaviors are positively affected by moisture because the water content of their body is about 90%. Similarly, temperature and pH are also affecting the activities of both microbes and earthworms. pH affects the enzymes released by the microbes to break down organic matters. Earthworms are less tolerant to extreme acidic and alkaline soil condition, causing them to escape from their growth medium. This may lead to their death. Availability of certain nutrients such as carbon, nitrogen, potassium and calcium are strongly dependent on pH (Viljoen and Reinecke, 1989). Due to those reasons, the CD's moisture content and pH value were 67.16 ± 0.327 % and 6.7 ± 0.005, respectively, as the cow dung used had already been composted.

The concentration of PLA microplastics in the vermicast
The PLA microplastics were recovered from the vermicast after 16 days of feeding. The concentration of the microplastics in the cast was signi cantly higher in the 10% of PLA (Fig. 1) than all the other treatment. A signi cant effect was observed between the CF and different PLA concentration, as shown in Table 4. This may be related to the digestion of organic matter, leading to an increase in the cast's microplastics concentration, compared to the organic matter. The concentration factor (CF) is expressed as the microplastics' ratio in the vermicast to microplastics in the feed. The cast concentration factor (CF) was the highest in 10% PLA, which means the most e cient for PLA accumulation. It is perhaps because the organic matter ratio to the microplastics was the highest compared to the rest. After ingestion, an increase in the concentration of microplastics in the cast occurred due to the organic matter's consumption in the earthworms' guts. In contrast, the PLA was not digested and therefore excreted in its initial form. Microplastics' concentration in the cast by a factor of 0.9 leads to a higher risk of incorporating microplastic into the soil, which may leach to the groundwater. This shows that earthworms were not able to degrade the PLA microplastics after digestion. A similar result was observed by Lwanga et al. ( 2016) when L. terrestris cast concentration factor was the highest when the worms were subjected to the lowest concentration of LDPE microplastic. Compared to 10%, the other treatment showed gradually lower cast concentration factor. The earthworms in this experiment were starved for 48 hours. After the starvation, they were fed with PLA for 16 days and starved again for the following 48 hours. During the 16 days of feeding, the earthworms subjected to 10% and 80% of PLA had a similar weight gain curve with the control. However, their weight changes signi cantly depending on the PLA concentration, with a P-value of 0.00107 (Table 5). It was positively correlated with the PLA concentration, as shown in Table 6. During the 16 days, the earthworms showed a steady weight increase for all treatment (Fig. 2). That may be because PLA microplastics were the contributing factor towards the earthworm's weight. A second gut voiding was conducted after the 16 days of feeding to conclude the effect of PLA on the earthworm's net weight, as in Table 7. ANOVA test shows a signi cant difference between the different concentration of PLA with time on the earthworm's weight gain within the same group, with a P-value of 1.64E-40 (Table 5). After the 16 days of feeding, the weight gained by the earthworms in the different concentration of PLA used was in the sequence was 0% > 60% > 80% >10% > 30%, respectively. Then, the earthworms were subjected to 48 hours of guts voiding, and the weight of earthworms after the second gut voiding was compared to the weight after the rst starvation. The result indicates that the weight gain change sequence was 0% > 60%> 10% > 30% > 80% PLA. Since the control with 0% PLA results in the highest weight gain among all other PLA treatments, it shows that the cow dung was the main source of organic matters for the earthworms (Table 7). Therefore, it can be concluded that the net weight gain was due to the assimilation of the cow dung, and not the PLA microplastics that was mixed in the feed. This relationship became more signi cantly different by the gut voiding pre-treatment, and the P-value increased to 0.00028 (Table 5). This result shows that gut content impacts the earthworms' weight exposed to cow dung and PLA. These studies have shown that although the LDPE is a petroleum-based plastic, the worms could break them upon digestion. In contrast, although the PLA is plant-based, the worms could not break them after digestion.
The result is contrary to the assumption that plant-based plastic is biodegradable, while their petroleum-based counterpart is not.
Our study concluded that the PLA microplastics eaten by the earthworms were indigested particles and cannot be used as carbon sources. Therefore, the plant-based PLA microplastic is not biodegradable using earthworms, Eudrilus eugeniae. That indicated the biodegradable plastics might not necessarily be a better alternative for conventional plastics.

Conclusion
In this study, different concentration of PLA microplastics was added in the feeding media for Eudrilus eugeniae. The mortality was 0% for all treatments, even though they were able to ingest the microplastics and egest in their cast. The worms were unable to bio-assimilate the microplastics since, perhaps because their feed content is a simpler form of organic matter, which is easier to digest than the microplastic. The PLA has affected the earthworm's weight, and may even be the digestive system or the guts load. They were able to adapt to the existing PLA microplastics in the media within the short-term exposure, even if they could not degrade and used it as the source of their organic matter. In conclusion, PLA had impacted the earthworms' weight, and the CF had indicated the presence of a high concentration of PLA in the vermicast. It is not biodegradable with respect to using Eudrilus eugeniae earthworms as an agent for degradation.

Declarations
Ethics approval and consent to participate All institutional and national guidelines for the care and use of laboratory animals were followed Consent for publication Not applicable

Availability of data and materials
The data that support the ndings of this study are available from the corresponding author upon reasonable request

Competing interests
The author declares no con ict of interest  Figure 1 Cast concentration factor (CF) for different concentration of PLA microplastics.

Figure 2
The effect of the different concentration of PLA microplastics on the earthworm weight over time. The weight was measured every four days during the 16 days of the feeding experiment.