To remove biological material from field-collected samples, Cole et al. [9] developed a digestion protocol employing a serine protease (Proteinase K). Material was desiccated (60°C, 24 h), ground and homogenized by repeatedly drawing samples through a syringe. Next, samples were mixed with homogenizing solution (400 mM Tris-HCl buffer, 60 mM EDTA, 105 mM NaCl, 1% SDS), acclimated to 50°C, enzymatically digested with Proteinase К (500 mg/mL per 0.2 g DW sample) and mixed with sodium perchlorate (NaC104,5 M). Ultrasonication was demonstrated to have a deleterious effect on digestion efficiency, owing to protein precipitation in the media. With marine samples, the Proteinase К method proved to have a digestion efficacy of >97%, and the method was used to isolate fluorescent polystyrene microspheres (20 mm) ingested by marine copepods. The authors note that additional enzymes could be used depending on the chemical makeup of the organism or samples in question (e.g., chitinase with chitinous invertebrates). The enzyme pepsin causes no damage to polymers, but proved only partially effective at digesting biogenic material [100]. Enzymes have been successfully applied recently in the isolation of microplastics from the intestinal tracts of turtles with Proteinase К [102]; mussel tissue with Corolase 7089 (AB enzymes) [103] and herring digestive tracts with Proteinase К and H,0, [104]. In contrast to chemical digestion techniques, enzymes ensure no loss, degradation or surface change to plastics present [9] and are less hazardous to human health.

Table 7.2 summarizes the effects of enzyme digestion on Mytilus trossulus [108].

Effect of Temperature

The performance of digesting solutions across different temperatures was systematically investigated by Karami et al [106]. In the first phase, the efficiency of different oxidative agents (NaCIO or H,0,), bases (NaOH or KOH) and acids (HC1 or HNO,; concentrated and diluted [5%]) in digesting fish tissues at room temperature (RT, 25°C), 40°C, 50°C or 60 °C were measured. In the second phase, the treatments that were efficient in digesting the biological materials were evaluated for their compatibility with eight major plastic polymers (assessed through recovery rate method for the water samples). The isolation of microplastics from Asian clams was based on previous studies [98,107].

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