Digestion

Enumerating microplastics present in biota, excised tissues or environmental samples can be challenging because the plastic may be masked by biological material, microbial biofilms, algae and detritus [12]. To isolate microplastics, organic matter can be digested, leaving only recalcitrant materials (Table 8.1). Traditionally, digestion is conducted using strong oxidizing agents; however, synthetic polymers can be degraded or damaged by these chemical treatments, particularly at higher temperatures. Environmentally exposed plastics, which may have been subject to weathering, abrasion and photodegradation, may have reduced structural integrity and resistance to chemicals compared to that of virgin plastics used in these stress tests [133]. As such, data ascertained using caustic digestive

Target tissues of animals exposed to microplastics (a) under laboratory conditions and (b) in the environment. Total number of studies 120

FIGURE 8.3 Target tissues of animals exposed to microplastics (a) under laboratory conditions and (b) in the environment. Total number of studies 120.

TABLE 8.1 Optimized Protocols for Digesting Biota or Biogenic Material to Isolate Microplastics

Treatment

Exposure

Organism

Reference

HNO, (22.5 M)

20°C (12 h) + 100°C (2 h)

Blue mussels

Claessens et al. (2013) [51]

HNO, (22.5 M)

20°C (12 h) + 100°C (2 h)

Blue mussels oysters

Van Cauwenberghe & Jansen (2014) [54]

HNO, (22.5 M)

20°C (12 h) + 100°C (2 h)

Blue mussels lugworms

Van Cauwenberghe et al. (2013) [23]

HNO; (100%)

20°C (30 min)

Euphausiids

copepods

Desforges et al. (2015) [16]

HNO, (69%—71 %)

90°C (4 h)

Manilla clams

Davidson 6c Dudas (2016) [61]

HNO, (70%)

2 h

Zebrafish

Luet al. (2016) [105]

HNO, (22.5 M)

20°C (12 h) + 100°C (15 min)

Brown mussels

Santana et al. (2016) [63]

HNO; (65%) HC104 (68%) (4:1)

20°C (12 h) + 100°C (10 min)

Blue mussels

De Witte et al. (2014) [52]

HNO; (65%) HC104 (68%) (4:1)

20°C (12 h) + 100°C (10 min)

Brown shrimp

Devriese et al. (2015) [38]

CH,0, (3%)

72 h

Corals

Hall et al. (2015) [32]

KOH(10%)

2-3 weeks

Fish

Foekema et al. (2013) [75]

KOH(10%)

60°C (12 h)

Fish

Rochman et al. (2015) [58]

KOH(10%)

2-3 weeks

Fish

Lusher et al. (2016) [89]

H,0, (30%)

60°C

Blue mussels

Mathalon 6c Hill (2014) [53]

H,0, (30%)

20°C (7 d)

Biogenic matter

Nuelle etal. (2015) [137]

H,0, (15%)

55°C (3 d)

Fish

Avio etal. (2015) [81|

H,0, (30%)

65°C (24 h) + 20°C (<48 h)

Bivalves

Li etal. (2015) [57]

NaCIO (3%) NaClOj (10:1)

20°C (12 h) 20°C (5 min)

Fish

Collard et al. (2015) [82]

Proteinase К

50°C (2 h)

Zooplankton

copepods

Cole et al. (2014) [12]

Assumptions: “Overnight” Given as 12 h; “Room Temperature” Given as 20°C.

agents should be interpreted with caution, and the likely loss of plastics from the digestive treatment carefully considered.

8.2.2.3.1 Nitric Acid

Nitric acid (HNO-) is a strong oxidizing mineral acid, capable of molecular cleavage and rapid dissolution of biogenic material [134]. When tested against hydrochloric acid (HC1), hydrogen peroxide (H,0,) and sodium hydroxide (NaOH), HNO, resulted in the highest digestion efficacies, with >98% weight loss of biological tissue [51]. The optimized protocol involved digesting excised mussel tissue in 69% HNO. at room temperature overnight, followed by 2 h at 100°C. Desforges et al. [16] also tested HN03, HC1 and H,0, in digesting zooplankton and similarly identified nitric acid as the most effective digestion agent based on visual observations; here, the optimized digestion protocol consisted of exposing individual euphausiids to 100% HNO. at 80°C for 30 minutes. Adaptations of nitric acid protocols have been successfully used to isolate fibers, films and fragments from a range of organisms [23,38,54,61,64,105]. While largely efficacious in digesting organic material, a number of studies observed that oily residue and/or tissue remnants remained postdigestion [15,51,63] which have the potential to obscure microplastics. In response, De Witte et al. [52] proposed using a mixture of 65% HNOs and 68% perchloric acid (НСЮ4) in a 4:1 v/v ratio (500 mL acid to 100 g tissue) to digest mussel tissue overnight at room temperature followed by 10 minutes boiling; this resulted in the removal of the oily residue. Recovery rates for 10 and 30 mm PS microspheres spiked into mussel tissue and subsequently digested with nitric acid range between 93.6% and 97.9% [51]. However, the high concentrations of acid and temperatures applied resulted in the destruction of 30 x 200 mm nylon fibers and melding of 10 mm polystyrene microbeads following direct exposure. Researchers have found that polymeric particles, including polyethylene (PE) and polystyrene (PS), dissolved following overnight exposure and 30 minutes boiling with 22.5 M HN03 [81,135]. Polyamide (PA, nylon), polyester (PET) and polycarbonate have low resistance to acids, even at low concentrations; furthermore, high concentrations of nitric, hydrofluoric, perchloric and sulfuric acid are likely to destroy or severely damage the majority of polymers tested, particularly at higher temperatures. The absence of synthetic fibers in biota digested using HNO, is likely a reflection of the destructive power of the acid [57].

8.2.2.3.2 Other Acids

Formic and hydrochloric acids (HC1) have also been suggested as digestive agents. With scleractinian corals (Dipsastrea pallida), formic acid (3%, 72 h) has been used to decalcify polyps to assist in the visualization of ingested blue polypropylene shavings [32]. HC1 has also be trialed as a digestant of microplastics from pelagic and sediment samples; however this non-oxidizing acid proved inconsistent and inefficient in digesting organic material [12].

8.2.2.3.3 Alkalis

Strong bases can be used to remove biological material by hydrolyzing chemical bonds and denaturing proteins [136]. Excised fish tissues, including the esophagus, stomach and intestines, have been successfully digested using potassium hydroxide (KOH, 10%) following a 2-3 week incubation [75,89]. The protocol has been adapted for the dissolution of gastrointestinal tracts of fish and mussel, crab and oyster tissues, either directly or following baking (450°C, 6 h), by incubating tissues in 10% KOH at 60°C overnight [58,135]. This latter method has proved largely efficacious in removing biogenic material, being well suited to the dissolution of invertebrates and fish fillets, but proving less applicable for fish digestive tracts owing to the presence of inorganic materials; as with HNO,, an oily residue and bone fragments may remain following digestion. Another strong base, sodium hydroxide (NaOH; 1 M and 10 M), has been successfully applied to remove biogenic material (e.g., zooplankton) from surface trawls, with 90% efficiency based on sample weight loss [12]. Foekema et al. [75] suggest polymers are resistant to KOH, and Dehaut et al. [135] showed no demonstrable impact on polymer mass or form, except in the case of cellulose acetate (CA). Testing the rapid KOH digestion protocol achieved a 100% microplastic recovery rate [135]. Tabulated data confirm PA, PE and polypropylene (PP) are resistant to 10% KOH, but polycarbonate (PC) and PET are degraded. Cole et al. (12 tested 40% NaOH (60°C) on a range of polymers and observed deformation of PA fibers, yellowing of polyvinyl chloride (PVC) granules and melding of polyethylene particles; similarly. Dehaut et al. [135] noted PC, CA and PET were degraded using this protocol. Notably, the compiled chemical resistance data indicate PE and PVC are resistant to NaOH, even at concentrations of 50% at 50°C. That Cole et al. [12] observed changes in polymers supposedly resistant to the given treatment highlights the necessity for comprehensive testing of applied treatments prior to use on biota.

8.2.2.3.4 Oxidizing Agents

Hydrogen peroxide (H202) and peroxodisulfate potassium (K2S208) are oxidizing agents. Mathalon and Hill [53] used H202 (30%) at 55°C-65°C to digest mussel soft tissue, and although largely effective, the authors noted “flakes of debris” remained. Li et al. [57] also applied this method, but incubated samples in an oscillating incubator, and then at room temperature for 24-48 h. Avio et al. [81] similarly tested alternate treatments in digesting intestinal tracts of mullet (Mugil cepbalus); while H202 was an efficacious digestant, Avio et al. [81] identified that direct application of H202 resulted in only a 70% retrieval of spiked microplastics, with losses linked to H,02 foaming. A number of studies noted excessive foaming might obscure samples or lead to sample loss [51,53,81]. A density separation of stomach contents with hypersaline (NaCl) solution followed by digestion of isolated material with 15% H202 resulted in a much improved 95% recovery rate for spiked microplastics. Dehaut et al. [135] trialed 0.27 M K2S2Os with 0.24 M NaOH in digesting biological tissues; while largely efficient in digesting biogenic material (<99.7% mass reduction), the authors noted its expense and highlighted issues with crystallization of the digestive solutions and incomplete digestion causing blockages during filtration. Avio et al. [81] observed that 15% H202 had no visible impact on PE or PS microspheres, although slight modifications to FTIR spectra were observed. Conversely, Nuelle et al. [137] identified some visual deformities to exposed plastic and quantified a 6.2% loss in size for PP and PE particles (<1 mm). Resistance data indicate 30% H202 should have little or no effect on PE or PP, again highlighting the importance of thorough testing of protocol applicability. Tabulated data indicate PA and PE are also prone to damage or dissolution from 30% H202. K2S208 resulted in no changes in the mass or appearance in the majority of exposed polymers, but caused complete dissolution of cellulose acetate [135]; chemical resistance data are currently unavailable, and this requires further testing.

8.2.2.3.5 Sodium Hypochlorite

Sodium hypochlorite (NaCIO) is used as an endodontic irrigant, with a near linear dose- dependent dissolution efficiency for biological tissue [138]. Collard et al. [82] digested fish stomach contents, in an overnight exposure with ~3% NaCIO; filtered digestants were subsequently washed with 65% HNO, and digested in a 10:1 NaCIO and HN03 solution for 5 minutes. The technique caused no visible degradation of a range of polymers (PET, PVC, PE, PP, PS, PC or PA). Resistance chart data indicate 15% NaCIO would degrade PA, although no data are provided for the ~3% NaCIO concentration applied by Collard et al. [82].

8.2.2.3.6 Enzymes

Enzymatic digestion has been mooted as a biologically specific means of hydrolyzing proteins and breaking down tissues [12]. To remove biological material from field- collected samples, Cole et al. [12] 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 (NaCl04, 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 [137]. It has recently been reported that enzymes have been successfully applied in the isolation of microplastics from: intestinal tracts of turtles with Proteinase К [139], mussel tissue with Corolase 7089 (AB enzymes) [140] and herring digestive tracts with Proteinase К and H,02 [141]. In contrast to chemical digestion techniques, enzymes ensure no loss, degradation or surface change to plastics present [12] and are less hazardous to human health. The trade-off is a protracted method, necessitating increased researcher time when considering large-scale field sampling and monitoring.
  • 8.2.2.3.7 Filtering Digestants

Following digestion, chemical agents can be filtered to retain any recalcitrant materials (e.g.. undigested tissue, inorganic residue, microplastics). Viable filters include 0.2 and 0.7 mm glass fiber filters [61,63], 5 mm cellulose nitrate filters [54], 5 mm cellulose acetate membranes [82], 50 mm mesh [12] and 250 mm mesh [77]. Larger pore size facilitates rapid filtering but will result in the loss of smaller plastics [12]. Glass fiber filters can shed and might be considered a source of contamination; smoother filters (e.g., membrane filters) are typically easier to scrape and less prone to fragment (personal observations of the authors). Microplastics on filters can be visualized directly (see Section 8.2.3), transferred to slides [63] or extracted. Collard et al. [82] suggest placing filters in methanol solution, ultrasonicating (50 Hz), centrifuging (5000 rpm, 5 min, 20°C) and then removing pelleted plastic by pipette; while this method was suitable for a range of polymers, the methanol caused a 25% weight reduction in tested PVC particles.

Density Separation

Although most commonly utilized in studies of water and sediment samples, density separation has been used in four biotic studies. Three studies used NaCl to separate less- dense particles [53,57,81] while Collard et al. [82] used a centrifuge. Following settlement of denser materials, the supernatant is filtered and the resulting material examined under a microscope. Density separation can be useful in studies following digestion. Saturated salt solutions, such as NaCl (aq), allow the separation of less-dense particles where there are large amounts of inorganic matter (e.g., sand, chitin, bone) that have not been dissolved (Lusher, personal observations). Density separation has been recommended by the MSFD (EU) for Europe. NaCl is recommended because it is inexpensive and nonhazardous; however, the use of NaCl could lead to an underestimation of more-dense particles (>1.2 g/cm3). Nal and ZnCl, solutions have been considered as viable alternatives to NaCl (aq) [142]. Their high density makes them capable of floating high-density plastics, including PVC.

 
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