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Potassium Hydroxide pellets (Thermo Fisher Scientific UNILAB CAS No. 1310-58-3) were dissolved with Milli-Q (Millipore) water to form a 10% w/v aqueous solution. Absolute ethanol AR grade (Thermo Fisher Scientific CAS No. 64-17-5) was used undiluted. Stainless steel (316 grade) filters (547 μm pore size - 55 mm Ø, Plain Weave; 263 μm pore size - 19 mm Ø, Plain Weave; and 26 μm pore size - 19 mm Ø; Twill Weave) were punched from woven mesh. Polypropylene filters (30 μm - 47 mm Ø Millipore) were sourced from Merck.

Irregular polyethylene (PE) particles were cut into nominal 1 mm lengths from a green laboratory rope using a scalpel. Irregular polystyrene (PS) particles (1–2 mm) were purchased from Sigma Aldrich. Polyester (PES) fibers (1–5 mm) were abraded from a fluorescent safety vest using commercial sandpaper. Commercial rayon thread (Gütermann CA 02776) was cut into nominal 1 mm lengths and individual fibers were then teased apart. All polymers had their chemical composition confirmed using Fourier Transform Infrared Spectroscopy (FTIR) with a PerkinElmer Spectrum 100 universal ATR (16 scans at 4 cm^–1 resolution, wavenumber range = 4000–600 cm^–1). Spectra were searched against a commercial library (NICODOM) following Kroon et al. (2018).

Local fish shops provided adult farmed barramundi (Lates calcarifer) samples for analysis. These were either viscera removed from the fish frame (n = 3), or whole fish frames with viscera intact (n = 6). Frames and viscera were transported to the laboratory then frozen at −20°C for storage until processing. Samples were defrosted at ambient room temperature (RT; ∼22°C) and fish frames were dissected to extract the viscera. The swim bladder was removed from all samples. Viscera were then either analyzed whole (n = 6) (Experiment A and C) or dissected further to separate the stomach and intestine (n = 3) (Experiment B). Visual assessment of Experiment C samples revealed they contained extremely large solid lipid deposits when compared with samples prepared for Experiment A and B. Therefore, some deposits that were not well attached to the viscera were removed to ensure samples were comparable between the three experiments (see Supplementary Figure S1). All tissues were weighed individually and placed in glass vessels for digestion.

To examine the applicability of EtOH resuspension to both RT and elevated temperature (40°C) digestion methods, two different aqueous KOH digestions were conducted (Table 1). For the RT method (Foekema et al., 2013), the whole viscera (Experiment A), or the individual stomach and intestine (Experiment B), were digested with 10% (w/v) KOH solution with a final concentration of 1:4 (tissue weight/KOH volume) (Supplementary Table S1) at ambient RT for 14 days. In Experiment B, the stomach and intestine from each sample were analyzed separately. Sample vessels (1 L glass Schott bottles or a 1 L beaker) were gently swirled by hand once a week throughout the digestion period.

For the elevated temperature method (Karami et al., 2017) (Experiment C), viscera were digested in 10% (w/v) KOH solution with a final concentration of 1:10 (tissue weight/KOH volume) and incubated at 40°C for 3 days in an Innova 40 benchtop incubator. Sample vessels (3 L glass volumetric flasks) were gently swirled by hand after 48 h.

Lipids in the presence of KOH undergo saponification to form a soft weak gel (Kent, 2003) that exhibits no flow when in the steady-state (Ferry, 1980). All samples from Experiment A, B and C were visually inspected at the beginning and midway through the digestion period for the formation of a viscous soft gel at the solution’s surface, signifying saponification. Experiments A, B and C were performed sequentially, with the results and general observations influencing the design of the subsequent experiments. For each of the three experiments, the methods used to redissolve the gel were optimized (Figure 1), then incorporated into a generic workflow applicable to any lipid-rich tissue sample.

The objective of Experiment A was to establish a suitable method to redissolve saponified gel present in lipid-rich samples whilst still achieving high digestion efficiencies (> 95%), typical of 10% KOH digestions where saponification does not occur. For samples presenting with a viscous gel, two methods of gel resuspension were tested sequentially. First, samples were vigorously agitated by stirring with a glass rod in an effort to mechanically breakdown the gel. However, this process proved counterproductive to the desired result and was discontinued. EtOH treatments post-KOH digestion were then examined as a means of redissolving the resultant soap (Table 1). To determine the lowest possible concentration of EtOH needed to achieve complete resuspension of the viscous gel, 5 subsamples (approximately 5 ml) of gel were taken from each sample. Subsamples were diluted with either a 0 (control), 1:1, 1:2, 1:4, or 1:10 volume of EtOH (100% EtOH/gel subsample, v/v), respectively. After adding the EtOH, subsamples were stirred with a glass rod until dispersed or up to 1 min at RT, then visually assessed for the presence of a homogenous liquid, indicating resuspension. Subsamples were then vacuum filtered through a 26 μm stainless steel filter. The control subsamples were not treated with EtOH before filtration. The remainder of each of the three samples was then treated with the lowest EtOH concentration required to achieve redissolution, stirred briefly and filtered, sequentially through a number of pre-weighted stainless steel filters to determine digestion efficiency, as described below.

Experiment B was designed to assess whether a reduction in sample weight, by removal of non-target tissue and solid lipid deposits prior to digestion, prevented or reduced the likelihood of saponification. Therefore, all non-target tissue surrounding the GIT was excised and only the individual stomach and individual intestines were digested separately with KOH as described above. The EtOH concentration, determined from Experiment A, was added to each of the samples 24 h before termination of the reaction. For these samples EtOH was not stirred with a glass rod, but the vessel was swirled gently by hand for <1 min to evenly distribute the EtOH. After 24 h, the suspension was then filtered to determine digestion efficiency, as described below.

Experiment C was designed to examine whether EtOH treatment could successfully redissolve saponified samples generated during a 10% KOH digestion conducted at elevated temperatures. Viscera were digested at 40°C for 3 days. The EtOH concentration determined from Experiment A was added to the digestion solution 1 h before termination of the reaction. The vessels were gently swirled by hand for <1 min to circulate the EtOH, then left stationary for the remainder of digestion period. The resuspended solution was then filtered to determine digestion efficiency, as described below.

All samples treated with 100% EtOH were filtered sequentially onto 547 μm (55 mm Ø), 263 and 26 μm (19 mm Ø) pre-weighed stainless steel filters. Using a Teflon wash bottle, these were then flushed liberally and sequentially with Milli-Q water and 80% EtOH, to reduce the possibility of particulates adhering to the sides of the funnel and to remove any chemical residue. Filters and the filtration system were flushed at least 3x or until there were no particulates adhering to the system. Filters were dried overnight in an oven at 40°C then stored in a desiccator, thereafter weights were recorded to calculate the digestion efficiency (Equation 1).

where W_i = Initial weight of lipid-rich materials (w.w), W_a = Weight of dry filter membrane after filtration (d.w), and W_b = Weight of dry filter membrane before filtration (d.w).

The initial sample weight was measured with a Sartorius TE3102S balance (2 d.p). Filter weights before and after filtration were measured with a Sartorius CP224S balance (4 d.p). The samples were allowed to stabilize on the balance before recording the final weight.

Model MPs were exposed to KOH alone or combined KOH and EtOH to examine whether one or both reagents would significantly degrade polymers, potentially inhibiting recovery and identification. To account for any KOH-induced degradation of the spiked polymers, MPs were exposed to 10% KOH for 14 days at RT or 10% KOH for 3 days at 40°C, in the absence of biological tissue and EtOH. Model MPs comprised of 10 black rayon fibers, 10 green PE fragments, 10 transparent PS fragments and 10 yellow PES fibers.

Lipid-rich tissues from Experiment B and C were spiked with model MPs (Table 1) to investigate whether the additional EtOH treatments would result in MP degradation when combined with KOH. Individual barramundi stomachs (Experiment B) were spiked with 5 black rayon fibers, 5 transparent PS irregular fragments and 5 green PE irregular fragments. Additionally, Experiment C viscera were also spiked with 5 yellow PES fibers per sample as PES fibers are known to degrade following KOH digestion at elevated temperatures, particularly above 60°C (Karami et al., 2017).

Following exposure, all samples were vacuum filtered onto stainless steel filters and retained MPs were assessed for physical and chemical changes by comparing color, shape and infrared spectral profiles with non-treated virgin MPs. MPs were visually identified using a Leica Stereo microscope MZ16A. Polymer spectra of virgin (n = 3) and exposed particles were acquired using ATR-FTIR. Spectral comparisons were conducted using the COMPARE analysis in the PerkinElmer Spectrum IR software (Kroon et al., 2018). Further, carbonyl indices for both non-treated virgin MPs and MPs exposed to KOH and EtOH (Experiment B and C) were compared (A detailed description of the spectral analysis is provided in Supplementary Material).

Results obtained throughout Experiments A, B and C were used to develop a tailored workflow to ameliorate the digestion of lipid-rich tissues. The workflow utilized the following three criteria to establish the complete dissolution of the gel post EtOH treatment: (i) solution appeared homogenous (see Table 2 for guidance in visual assessment), (ii) ability of the treated sample to pass through a 26 μm pore size stainless steel filter in <5 min, and (iii) a digestion efficiency of equal efficacy to KOH digestion without saponification (i.e., >95%). The criteria used to verify the workflow were: (iv) recovery rates of spiked MPs above 90%, and (v) no EtOH-induced changes in physical or chemical characteristics in recovered MPs.

The tailored workflow, based on the results of Experiment A, B, and C, was validated using wild fish (Figure 2). Wild fish were hypothesized to be less fatty than farmed fish and were used to determine if the workflow was suitable for analyzing fish with variable and unknown lipid content. The viscera of coral trout (Plectropomus leopardus), barramundi (Lates calcarifer), and mangrove jack (Lutjanus argentimaculatus), were obtained from fish shops or provided by local fishers from Townsville, Australia.

Viscera was digested in 10% KOH as per Experiment C (3 days at 40°C) in glass Schott bottles. One hour before termination of the digestion, the reaction solution was visually assessed and if a solid gel had developed (Table 2), was processed using the tailored workflow. Using this workflow, EtOH was added to samples with a saponified gel in 1:10 EtOH:KOH ratio. After gently swirling by hand, the samples were returned to the incubator for 1 h. If the gel was visually assessed as having completely dissolved, it was filtered as described above, if not, then additional EtOH was added to achieve a concentration of 1:4 EtOH:KOH. The samples were gently swirled, then visually assessed again. If necessary, the process was repeated using a 1:2 or a 1:1 concentration of EtOH, until the solution presented as a homogenous liquid. Only after complete resuspension was achieved, based on visual assessment, was the sample filtered to assess digestion efficiency.

Copious amounts of prey items and sediment were present in the wild fish GIT, which was visible as a thick layer of sediment in the bottom of the Schott bottle. Thus, these were filtered onto larger 47 mm Ø filters (30 μm pore size). The increased surface area accommodated a larger retentate load before clogging. Digestion efficiency was a calculated for these samples as outlined above.

The objective of this study was to assess recovery of spiked polymers, and not to quantify environmental MP burdens. Therefore, although dissection and analyses followed recommended clean laboratory procedures to prevent laboratory contamination, procedural blanks were not collected. These procedures included wearing a 100% cotton lab coat, cleaning glassware and utensils with detergent (if needed) and Milli-Q water before use and between samples, the use of plastic utensils was avoided, laboratory benches were cleaned with 70% EtOH and samples were kept covered with aluminum foil (Lusher and Hernandez-Milian, 2018). Samples were not processed inside a clean air device (Wesch et al., 2017). To apply this workflow to environmental samples, it is recommended to follow established QA/QC method, such as those described in Karami et al. (2017), Bråte et al. (2018), or Hermsen et al. (2018).

All farmed barramundi tissue subjected to 10% KOH digestion resulted in the formation of a viscous gel, which visually resembled gelatin (jelly) (Table 2 and Figure 3). These saponified samples (Experiment A: viscera; Experiment B: stomach/intestines; Experiment C: viscera) could not be easily poured from the glass vessels in this state.

In Experiment A, agitation in the form of shaking and inverting, resulted in the gel breaking into smaller clumps, however, it could not be redissolved. Further, the gel layer reformed shortly after shaking ceased. Continued vigorous stirring with a clean glass rod induced an extremely rapid saponification reaction, with the physical properties of the entire sample changing from a biphasic mixture (lower liquid layer, and upper floating gel), to a homogenous thick gel (Figure 3 and Supplementary Figure S2), which could not be poured, nor broken apart with vigorous shaking. Attempted vacuum filtration of the control (untreated) subsample through a 26 μm filter was unsuccessful, thus no further analysis of untreated saponified samples was carried out. Treatment of the gel subsamples with EtOH (1:1, 1:2, 1:4, and 1:10) resulted in complete dissolution, with all resulting solutions able to pass through the 26 μm filter, with only minimal gelatinous residue being retained. Thus, the lowest dilution, 1:10, was selected to redissolve the remainder of the samples from Experiment A, and for Experiments B and C.

In Experiment B, the complete removal of the stomach and intestines from lipid-rich organs (e.g., liver) and solid lipid deposits did not prevent saponification. The individual stomachs and intestines all presented with floating soft weak gels when digested with KOH. The samples were treated with 1:10 EtOH and visual assessment determined that gel was redissolved. As dissection was not an effective method of preventing or reducing the level of saponification, complete viscera were used in all following tests to reduce sample preparation time.

The formation of a solid gel following KOH digestion was also observed in Experiment C (viscera 1:10 tissue:KOH (w/v) 40°C) (Figure 4A). Visual assessment revealed this solid gel did not completely dissolve after initially adding EtOH (1:10). Increasing the volume of EtOH (1:4) was successful in redissolving the remaining gel and allowed for filtration without any gel residue (Figure 4B).

The results of Experiment A, B and C demonstrated that an ad hoc visual assessment of the physical appearance of the sample (Table 2) was a simple and effective method to establish the presence of saponified gels. Visual inspection was also effective at verifying dissolution, with all samples that were judged redissolved able to pass through a 26 μm filter after EtOH treatment. Hence, visual assessment combined with EtOH treatment was applied in the wild fish case study (Experiment D).

Inspection of wild-caught coral trout viscera found it to be relatively free of fat deposits. Digestion of this tissue under Experiment D conditions yielded a clear solution with no obvious saponified gel observed. As a result, addition of EtOH was deemed unnecessary and the sample was filtered as is. Wild-caught mangrove jack and barramundi viscera were observed to have solid fat deposits (although notably less than farmed barramundi, Supplementary Figure S3). Digestion with 10% KOH formed a viscous gel, therefore both samples were further treated with EtOH (1:10, as per the tailored workflow). Gel-like solids in the mangrove jack sample dissolved completely resulting in a clarified solution that was easily filtered. Treatment of the wild-caught barramundi sample with EtOH (1:10) resulted in only partial dissolution of the gel, as was also noted in Experiment C. Increasing the volume of EtOH to 1:4 redissolved the remaining gel, and the solution was filtered.

The digestion efficiency, notwithstanding the limited sample replication, for all farmed barramundi (Experiment A, B, and C) was greater than 99% (Table 3). Very little prey and sediment residue was present on the filters for these samples (Supplementary Figure S4). Conversely, the wild-caught fish (Experiment D) contained substantial prey items in their GIT, including whole animals as well as bones, crustacean exoskeletons, shells and algae, all of which were retained on the filter. Sediments were also observed in the digested viscera samples obtained from mangrove jack and barramundi. Thus, viscera samples from wild fish required multiple large (47 mm Ø; 30 μm pore size) filters to prevent clogging (Supplementary Figure S5). Although the digestion efficiency was still considered acceptable for fish tissues (>97%), and the gel was able to be resuspended by the EtOH, the abundance of material remaining on the filter would impede visual examination of the digestate for MPs.

Examination of the MPs exposed to 10% KOH for 14 days at RT found there was no change in color or shape of PS or PE. However, black rayon fibers were bleached and changed from opaque to transparent. Chemical analysis using FTIR found no substantial changes in the spectral profiles compared to virgin plastics (PE – 99.0%, PS- 98.7%, Rayon – 94.9.0% match). Despite a slightly lower % match for rayon, it was still above the accepted similarity threshold of > 70% (Lusher et al., 2013; Kroon et al., 2018). Exposure to 10% KOH at 40°C for 3 days also did not result in significant changes in the chemical spectra for the four polymers (PE – 98.5%, PS- 99.6%, Rayon – 97.9%, PES – 97.6% match). There were no changes in color and shape, apart from rayon, which underwent similar color changes as per the RT KOH exposure.

Recovery of spiked MPs from KOH digested and EtOH treated barramundi tissue was 100% for PS particles, 93.3% for PE particles, 93.3% for rayon fibers, and 100% for PES fibers (Table 3). All spiked samples that produced saponified gels were treated with EtOH. Thus, there were no attempts to recover MPs directly from the gel itself. For all MPs, shape was unchanged after the combined KOH and EtOH exposure. Color was also unaffected by the combined exposure for PS, PE and PES. However, rayon fibers became transparent, likely due to KOH exposure prior to the addition of EtOH (Table 3). Spectral analysis revealed no major change in profile compared to MPs exposed to KOH only. All polymer spectra returned correlations > 90% with the virgin MP after the combined KOH and EtOH exposure (PE – 98.6%, PS- 99.3%, Rayon – 97.1%, PES - 98.0% (Supplementary Table S2). Although not all recovered MPs were able to be FTIR analyzed. Carbonyl indices for all polymers treated with KOH and EtOH were not significantly different to untreated virgin MPs (PE: Mann–Whitney U = 15, n₁ = 14, n₂ = 3, p > 0.05, two tailed) (PS: Mann–Whitney U = 16, n₁ = 15, n₂ = 3, p > 0.05, two tailed) (PES: Mann–Whitney U = 10, n₁ = 8, n₂ = 3, p > 0.05, two tailed) (Rayon: Mann–Whitney U = 11, n₁ = 8, n₂ = 3, p > 0.05, two tailed) (Supplementary Table S3).