A study protocol for chemical analysis and toxicity testing of virgin and recycled microplastics and associated chemicals

Plastics can contain a variety of different chemicals, which are either intentionally (IAS) or non-intentionally (NIAS) added substances. Recycled plastics can contain especially NIAS, which might vary in amount and characteristics, possibly compromising the applicability of the plastics. As plastics can eventually degrade into microplastics, these substances can be released into their environment and upon human exposure, pose a threat to human health. Therefore, simple methods for the comprehensive monitoring of these chemicals are needed to guarantee the safe use of plastics. The purpose of this study was to set up methods for analyzing and toxicity testing of chemicals present in secondary microplastics of virgin and recycled origin. Accordingly, the chemical compounds of virgin and recycled polypropylene (PP), low-density polyethylene (LDPE), and high-density polyethylene (HDPE) microplastics were extracted using water, methanol, and chloroform as solvents, and the extracts were analyzed with nuclear magnetic resonance (NMR) and total reflection X-ray fluorescence (TXRF) methods. In addition, two cytotoxicity assays were applied to study the toxicity of the chloroform extracted virgin and recycled microplastics in human adenocarcinoma Caco-2 cells. The combination of NMR and TXRF methods allowed extensive analysis of the released chemicals showing that chloroform was the most efficient solvent for extraction. The results showed that microplastics milled from mechanically recycled plastics released more organic compounds and inorganic elements compared to microplastics milled from virgin plastics. In addition, the chloroform extracted microplastics decreased cell viability dose dependently and the observed effect was more prominent with the recycled microplastics compared to their virgin counterparts. In conclusion, these results suggest that chemicals tend to accumulate in recycled plastics, and therefore, these chemicals need to be monitored to guarantee the safe use of recycled plastics. This study showed that chloroform extraction is efficient in releasing substances accumulated in plastics for chemical analysis and toxicity testing.