A method for the characterisation of microplastics in sludge

Graphical abstract


Specification
Environmental Science More specific subject area: Microplastics Method name: Characterisation of microplastics in wastewater sludge Name and reference of original method: NA Resource availability: NA

Contamination control
In order to minimise environmental and cross contamination, all steps were conducted in laminar flows hoods. All equipment and containers for storing and measuring the sewage sludge was either glass or metal made. Before use, all wares were rinsed with filtered deionized water. Work surfaces were cleaned with paper towels and a 70 % isopropanol solution. Natural fiber clothes were used at all the times.

Sample preparation
Wet wastewater sludge grab samples (1 L) were collected from the upper section of the sludge holding tank at Cranfield University wastewater treatment plant. Samples were taken from the top layer of the sludge pile with a metal scoop and transferred into 1-L rinsed glass bottles. The sludge samples were mixed in the bottles prior to analysis to prevent the solids settling. To determine the separation efficiency of plastic fragments, wet sludge subsamples (50 mL) were transferred to glass vials and spiked in triplicates with low-density polyethylene (LDPE) fragments, which is the most detected polymer in UK urban river environments [1]. The following size ranges were tested: 5-1 mm, 1-0.5 mm and 500-150 mm. Two spike levels were selected namely, 10 and 100 fragments which corresponded to sample wet weight values of 0.01 and 0.11 %, respectively. The 0.01 % spike replicated a realistic quantity of microplastics in sludge, whereas the 0.11 % spike was applied for a refined indication of the removal efficiency. Spiked subsamples were stirred for 15 min covered with aluminum foil to prevent airborne contamination from clothing and external plastics. All spiked subsamples were run in triplicate and the plastic fragments were counted twice to reduce error. Fig. 1 shows some of the LDPE fragments used in this study.

Water-sludge separation
For matrix separation subsamples were centrifuged at 700Âg for 10 min. Supernatants were passed through stack comprising 1 mm, 500 mm and 53 mm sieves, while solid pellets were removed from the vial, packaged in aluminum foil for freeze-drying. Both materials retained in the sieve stack and packaged solids were freeze-dried for 24 h to remove moisture content.

Purification
Purification of LDPE fragments separated into the supernatant and sludge pellets was done in 250-mL glass beakers by directly adding 20 mL of Fenton's reagent (0.05 M) and 20 mL of H 2 O 2 (30 %) to each freeze-dried fraction [2]. Beakers were then placed on magnetic stirrers and heated to 60 C and left to react for 15 min. Additional 10-mL aliquots of H 2 O 2 were added after 30 and 90 min and finally the mixture was left to react for 12 h. Once the purification was completed, LDPE fragments from both fractions were recombined by consecutively sieving the respective reaction mixtures.

Microplastics identification
To identify and separate natural polymers (e.g. cotton), Rose-Bengal dye was directly applied to fragments retained on the sieves (Fig. 2) [3]. After a drying stage at room temperature overnight, fragments were isolated under a microscope (Wild Heerbrugg) and tested for structural consistent with tweezers. Subsequently, dyed fragments discharged and microplastics shorted. Further dying of microplastics with Nile red was ruled out since biogenic material (e.g. lipids and chitin) may fluoresce and thus interfere with the identification process [4].
To confirm the polymer was low density polyethylene and establish baseline spectrum data for identification purposes, plastic fragments were analysed by attenuated total reflection FT-IR spectroscopy (Bruker HTS-XT Vertex 70). The instrument was operated at 16 scans with a resolution of 4 cm À1 . Polymer composition of spiked LDPE fragments was confirmed by matching acquired spectra to the those included in instrument library. With this purpose, three fragments randomly chosen from each subsample were analysed by FTIR. Fig. 3 shows the sequential order of the aforementioned analytical steps.

Method validation
As showed in Fig. 4, recoveries based on number of LDPE fragments (blue bars) showed good accuracy regardless of particle number or size fraction. For the 10-particle spike (Fig. 4, panel A), the respective average particle counts for size fractions 5 to 1 and 1 to 0.5 mm were 10 and 9, whereas 7 fragments were recovered for the smallest fraction (500-150 mm). A higher LDPE particle number (i.e. 100-particle spike) translated into consistent results closer to the target value since particle counts consistently attained values of 90 across the three particle sizes (Fig. 4, panel B). When expressed as total LDPE weight (Fig. 4, green bars), recoveries over 100 % were found for both 10-and 100-particle spikes. Materials attached to LDPE fragments contributed to the observed weight gain (see Fig. 5).
Residues have a more significant impact on tests conducted with 10 particles and smaller sizes. For instance, a 330 % overestimate was found in the 500 to 150 mm range (Fig. 4, panel A).
Our method yielded comparable recovery values than those obtained after more extensive sample preparation (see Table 1). For instance, Li et al. (2018) used elutriation for removal of the plastics from  the sludge [4], this step was not used in our methodology owing to the protentional interference of organic matter that could attach to MPs thus preventing their separation for analysis.
The authors whose names are listed immediately below certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.

Declaration of Competing Interest
The authors whose names are listed immediately below certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria;