| Processing Steps |
- Parameter or Variable: microplastic concentration (measured); Units: pieces/m3; Observation Category: in situ; Sampling Instrument: Manta net; Sampling and Analyzing Method: In this study, the selected geographical areas for the monitoring of microplastics (MPs) are located along the southern coast of Apulia in southern Italy (Salento peninsula). This peninsula lies in between the southern Adriatic Sea and the northern Ionian Sea - mainly exposed to the Western Adriatic current and to the anticyclonic Northern Ionian Gyre, whose intersection may lead to high accumulation of MPs along the Salento coastal waters. The collection of MPs was performed from October to December 2020 and from April to June 2021. A total of 18 transects were carried out at different distances from the shoreline between 1.5, 3 and 6 Nautical Miles (NM). The sea surface was sifted using a neuston Manta net of 100 × 50 cm rectangular frame with a 300 μm mesh size. The neuston Manta net was dragged along transects, parallel to the shoreline, by the 14 m research boat “Pelagia” of the University of Salento, with a cruise speed ranging between 2.1 and 2.5 kt. All deployments were made from the boat’s port side beyond the bow wave, to avoid the wave turbulence. During sampling the volume in liters of water filtered by the net was calculated using a flowmeter model 438-110 (Hydro-Bios) by multiplying the number of revolutions of flowmeter by a factor deriving from the Manta net opening. After the collection, and directly on the vessel, the Manta net was rinsed externally with seawater and the content of the cod end was emptied over a 50-μm metal sieve. All retained material was transferred to glass bottles, and ethanol 80% in 1:10 vol ratio was added. The bottles were then capped, and transported to the laboratory for analysis. Ethanol-added samples stored in refrigerated conditions, were filtered using a 300 μm mesh size steel sieve and residues were quantitatively transferred into glass bottles using deionized filtered water. Then, 10% potassium hydroxide (KOH) solution was added at an approximately ratio 1:10 by volume. Samples were heated at 50◦C for 48 h until complete hydrolysis. After discarding KOH by filtering at 300 μm, and rinsing with deionized water, in some cases, residues were added with sulfuric acid (10%) at 1:10 vol ratio and hydrolyzed for 24 h at 50 ◦C. This last step was performed only in plankton-rich samples and was aimed at decomposing crustacean exoskeletons. The acid hydrolysis option was applied considering that the exoskeleton of many organisms present in the zooplankton is made of calcium salts and chitin, and the reported negative effects on MPs attributed to the acid hydrolysis were tested only using highly concentrated acid, 40% HF or 37% HCl. The effects of 10% diluted sulfuric acid solution were evaluated for: 1) its effectiveness in removing the residual organic matter, after alkaline hydrolysis, 2) its effects on the different polymer types. In terms of effectiveness in removing the organic matter resistant to the alkaline hydrolysis, the diluted acid solution result was found to be extremely useful, as it significantly increased the readability of filtered residues for MPs detection under stereoscope and reduced the time of analysis to about one third. Secondly, no effects were observed on polyethylene, polypropylene, polystyrene and polyethylene terephthalate particles, exposed to the acid solution for 24 h at 50 ◦C. Once the digestion was completed, the hydrolyzed sample was sieved using 1- and then 0.3-mm mesh size steel sieves, obtaining two fractions sizes, i.e., from 5 to 1 mm and below 1 mm. Each fraction was rinsed using water milliQ grade and poured on a GFC filtering disk (47 mm of diameter, pore size 1.2 μm). The GFC disk with the retained particles, was placed in a glass Petri dish and left to partially dry at room temperature, facilitating the image analysis by providing a flat surface for the assessment. After drying, samples were analyzed using a stereoscope SMZ745T (Nikon, Tokyo, Japan) equipped with a digital cam TP5 100 A CMOS and controlled by Alexasoft X-plus v. 20.04.01 software (Alexasof, Frorence, Italy). Digital images were captured and analyzed. For each sample fraction, plastic particles were classified by shape and color, measured for their perimeter, max diameter, and area. Particles with suitable dimensions (>500 μm for fragment, films and pellets or >1 mm for fibers) underwent Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR FTIR) using a FTIR Nicolet iS50 (Thermo Fisher Scientific, Waltham, MA, USA) equipped with Quest ATR diamond (Specac, London, UK) for recognition of polymeric nature. Spectra were acquired in the 400-4000 cm-1 wave number range, resolution 2 cm-1, and number of scans 32. The acquired spectra were saved in .SPC file format, uploaded on www.openspecy.org, smoothed, baseline corrected and searched against the database for identification. The identification was mainly based on the presence in the spectra of the characteristic signals for each polymer, the level of score identification ranged between 0.4 and 0.98 (expressed as Pearson’s r correlation index between the searched spectra and that present in database.; Data Quality Method: The most important problem for the analysis of MPs is the accidental contamination of samples with microfibers derived from synthetic clothes, floating in the air and almost ubiquitous. To reduce the risk of microfibers contamination, a dedicated room of the laboratory was used for this analysis. All room surfaces were cleaned using paper towel and alcohol, air turbulence was strictly avoided, and all working team members dressed in white 100% cotton laboratory coat. For each section of analysis, a blank sample was performed following all analytical steps and the number of microfibers eventually found was subtracted from the total number found in all samples analyzed in the same section. In case of clean sample, without fibers on positive blank, subtraction was not performed. During the experimentation, fragments or pellets were never detected in blank samples, whereas Fibers (as microfibers with diameter of 20 μm) were observed with a mean value of 2 items. To evaluate the effects of alkaline and acid hydrolysis used in sequence on the microplastics integrity and IR spectra, 30 microplastics with dimension ranging from 0.1 to 0.3 mm (as maximum diameter), constituted by polyethylene (fragments, filaments or pellets), polypropylene (fragments or films), polystyrene and polyethylene terephthalate (fragments and filaments) were suspended in milliQ water and analyzed as real samples. The test was replicated five times and the mean recovery resulted from 95 to 100% for fragments and pellets, from 90 to 100% for fibers. Neglectable effects were observed on the IR spectra of the three plastic polymers examined..
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