Quantitative analysis of selected plastics in high commercial value Australian seafood by Pyrolysis Gas Chromatography Mass Spectrometry

Microplastic contamination of the marine environment is widespread, but the extent to which the marine food web is contaminated is not yet known. The aims of this study were to go beyond visual identification techniques and develop and apply a simple seafood sample clean-up, extraction and analysis method using Pyrolysis Gas Chromatography Mass Spectrometry to improve the detection of plastic contamination. The method allows the identification and quantification of polystyrene, polyethylene, polyvinyl chloride, polypropylene and poly (methyl methacrylate) in the edible portion of five different seafood organisms: oysters, prawns, squid, crabs and sardines. Polyvinyl chloride was detected in all samples and polyethylene at the highest total concentration of between 0.04 - 2.4 mg g-1 of tissue. Sardines contained the highest total plastic mass concentration (0.3 mg g-1 tissue) and squid the lowest (0.04 mg g-1 tissue). Our findings show that the total concentration of plastic is highly variable among species and that microplastic concentration differs between organisms of the same species. The sources of microplastic exposure, such as packaging and handling with consequent transference and adherence to the tissues are discussed. This method is a major development in the standardization of plastic quantification techniques in seafood.


List of tables and figures
Table S 1

Chemicals and reference materials
Polystyrene, poly (methyl methacrylate) and polyvinylchloride were purchased from Sigma-Aldrich and low density polyethylene (LDPE) from Thermo Fischer Scientific.
Polyethylene terephthalate (PET) was kindly provided by the Norwegian Institute for Water Research (NIVA) in Oslo, Norway and polypropylene (PP) was donated by a plastic manufacturer from Melbourne, Australia.

Extraction of plastic standards using an Accelerated Solvent Extraction (ASE) method
All samples were extracted by a Dionex ASE-350 system.High temperature Viton-Orings (Dionex 056325) were used in the end-caps, glass fibre filters (Dionex 056781) were used in the cell base and Hydromatrix sorbent was used to cover the sample, reduce solvent and prevent floatation.
To assess extraction efficiency, between 20 and 50 mg of each selected plastic was weighed and placed inside of an ASE cell (34 mL) previously covered with a filter.Five replicates were performed for each type of plastic (PS, PE, PET, PMMA, PP and PVC) in addition to three controls (cells filled with only Hydromatrix).Following extraction the recovered mass of each polymer was determined gravimetrically by evaporating the liquid in the collection bottles to dryness in a fume hood under a gentle stream of nitrogen (40 °C).
Analysis was performed on the dried residues to quantify the mass of polymer recovered by weight (0.01 g of precision).

Pyrolysis Gas Chromatography/ Mass Spectrometry (Py-GC/MS) analysis
Py-GC/MS was performed using a Multi-shot Pyrolyzer (EGA/PY-3030D) equipped with an auto-shot sampler (AS-1020E) (Frontier Laboratories Ltd., Fukushima Japan) coupled to a Shimadzu GC/MS-QP2010 plus.The MS was operated in electron ionisation (EI) mode and compounds separated on a Frontier Laboratories Ultra ALLOY -5 capillary column (30 m x 0.25 mm x 0.25 µm) with helium as the carrier gas.
The initial oven program was set at 40 °C for 2 min, then increased to 320 °C at 20 °C/min and held for 14 min.Data was acquired in full scan mode (mass range 40 to 600 m/z) with a scanning rate of 2000 Hz.Library search of Shimadzu was used for peak identification of the pyrolyzates together with Kovats retention index (RI) data.

Multivariate calibration curve for PE
To generate a multivariate calibration curve for quantification of PE in different matrices, we used a least square approach, where the algorithm minimizes the sum of squares of the distance between each point and the line.The final calibration curve with an adjusted R 2 of 0.99 included four coefficients and the intercept term.These four coefficients consisted of three associated to each independent variable (i.e.peak intensity) and the interaction between 1-decene and 1-dodecene.For the validation of this multivariate calibration curve, we employed leave one out approach, where during each iteration one of the points in the curve will be removed while generating a new curve using the remaining points 1, 2 .This process was repeated in order to have each point the calibration curve removed at least once.

Testing the dissolution of plastics in DCM after ASE extraction
After extraction, the samples were immediately placed in the pyrolysis cups (Eco-Cups 80LF, Frontier Labs, Japan) and evaporated to dryness in a fume hood to avoid airborne contamination of microplastics.The solubility of the selected plastics in DCM after ASE was tested by placing the plastic standard solutions in the sample cups at different times after the solvent extraction procedure (every 15 minutes for a total period of 2.5 hours).

Relation between peak area of specific indicator compounds and split ratio
For all the indicator ions of selected plastics, areas significantly decreased with the increase of the split ratio (p < 0.05, One Way ANOVA) -Figure S4.Assuming linearity, the split ratio was set at 5 ensuring that the selected plastics in our samples were efficiently quantified and saturation of the mass spectrometer was avoided.

Table S 1
Plastic standards used for data base library matching and mass calibration.NS -

Table S 2
ASE conditions for the selected plastics

Table S 5
Recovery of plastic standards after ASE with DCM at 180 ºC Replicate (Rep) 1, 2, 3, 4 and 5 represent the number of experiments for each polymer standard; blue numbers represent the percentage of recovery of each trial; average recovery represents the mean recovery from the five replicates; STD: standard deviation RSD: relative standard deviation (%); poly-(methyl methacrylate) (PMMA), polystyrene (PS), polyethylene terephthalate (PET), polypropylene (PP), polyethylene (PE) and polyvinyl chloride (PVC)

Table S 6
Recovery of spiked oyster samples after ASE with DCM at 180 ºC

Table S 8
Calibration functions for selected plastics.aresultsare based on ions for the plastic as presented in TableS4; Figure S 1 Experimental design for the extraction of oyster's.Filters were spiked with 6 different plastics: PS, PE, PET, PMMA, PP and PVC.Six glass fibre filters were used as a "blank" with no plastic added and the other 36 were spiked with selected plastics (as schematized) PVC Benzene 2.88 min Figure S 2 Total Ion Chromatogram (TIC) pyrograms of extracted plastics by ASE.Detailed decomposition products and retention times are summarized in TableS4.Polystyrene (PS), polypropylene (PP), poly-(methyl methacrylate) (PMMA), polyethylene (PE) and polyvinyl chloride (PVC)