ISO 4484-2:2023
(Main)Textiles and textile products — Microplastics from textile sources — Part 2: Qualitative and quantitative analysis of microplastics
Textiles and textile products — Microplastics from textile sources — Part 2: Qualitative and quantitative analysis of microplastics
This document establishes a qualitative-quantitative analytical evaluation (i.e. determination) of microplastics to be able to define their: — particle number; — morphology (morphological characteristics); — dimensional distribution; — the type, chemical origin or nature of polymers and their colour, if present. This document is applicable to the determination of microplastics (from the textile sector) collected in various matrices (for example textile process wastewater, clothes washing water, textile process air emissions, textile process solid waste). This document specifies expression of results in terms of estimated surface area and mass of microplastics (MPs) per unit sample. It enables the expression of the results of the quantification of microplastics (MPs) from various sources, including samples related to the production, processing, treatment and use of textiles (raw material, manufacturing process, sample like wastewater from washing clothes, air, and industrial process water). This document applies to textile sector samples of matrices of different physical states (solid, liquid or aeriform), for example: — solid samples from textile production processes; — water samples from the textile production process and/or from the washing of clothing (e.g. garments or other textiles, ISO 4484-1 or ISO 4484-3 can be applied in order to prepare a liquid to be tested); — air samples to test the air quality in the workplace of textile companies. This document, being able to provide information such as size, shape, surface and mass (estimated), enables the transfer of useful information for ecotoxicological assessments to specialists.
Textiles et produits textiles — Microplastiques d'origines textiles — Partie 2: Analyse qualitative et quantitative des microplastiques
General Information
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Standards Content (Sample)
INTERNATIONAL ISO
STANDARD 4484-2
First edition
2023-09
Textiles and textile products —
Microplastics from textile sources —
Part 2:
Qualitative and quantitative analysis
of microplastics
Textiles et produits textiles — Microplastiques d'origines textiles —
Partie 2: Analyse qualitative et quantitative des microplastiques
Reference number
© ISO 2023
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Published in Switzerland
ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 3
5 Reagents . 4
6 Apparatus . 5
7 Cleaning procedure .7
7.1 Cleaning of the materials and the test environment . 7
7.2 Blank test to evaluate environmental contamination . 8
7.3 Cleaning procedure for filters . 8
8 Sample preparation .9
8.1 Solid matrices (fibre, textile or any derivates) . 9
8.2 Liquid matrices (aqueous) . 9
8.3 Air matrices . 9
8.4 Preliminary checking of sample and pre-treatment. 10
8.4.1 Liquid sample property checking and requirements . 10
8.4.2 Pre-treatment if required . . 10
8.4.3 Test sample homogenization/hydration and/or sonication .12
8.4.4 Pre-dilution .12
9 Preparation procedure .13
9.1 General .13
9.2 Preparation of micro-sized fibres sample . 13
9.3 Standard fibre sample preparation (Water based) . 15
9.4 Water based standard fibre sample filtration . 16
9.4.1 General . 16
9.4.2 Filtration procedure . 16
9.5 Requirements for standard fibre samples . 16
10 Test procedure .17
10.1 Addition of internal standard fibre sample . 17
10.2 Test sample filtration . 18
10.3 Recovery procedure . 18
10.4 Image analysis. 18
10.5 Identification of MPs . 19
10.5.1 General . 19
10.5.2 Detection limits . 19
10.5.3 Calculation . 20
10.5.4 Spectra comparison and MP identification . 20
10.5.5 Determination of external surface area and volume . 21
10.5.6 Calculation of total surface and total mass (optional) . 21
10.5.7 Calculation of recovery rate (R ) COUNTING .22
r
11 Test report .22
Annex A (informative) Microtome for standard preparation .24
Annex B (informative) Examples of statistics elaboration .25
Annex C (informative) Example of classification of MPs .32
Bibliography .34
iii
Foreword
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electrotechnical standardization.
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described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO document should be noted. This document was drafted in accordance with the
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This document was prepared by Technical Committee ISO/TC 38, Textiles.
A list of all parts in the ISO 4484 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
Introduction
There is significant evidence that the textile sector releases microplastics (MPs) into the environment.
These particles, when present in the environment, can affect the biota, and so, their number, shape and
size can be relevant parameters for the assessment of their potential impact and, consequently, the
development of a counting technique can be a helpful approach.
Moreover, many of the microparticles analysed are not of synthetic origin and therefore it is necessary
to identify and distinguish them from microplastics (MPs).
This document is designed to provide the nature, numerical concentration, surface area and (estimated)
mass of the microplastics produced or released by the textile sector and collected in a solid, aqueous or
aeriform matrices.
Depending on the matrice, pre-treatment of the sample is necessary to concentrate the microplastics
and eliminate inorganic and organic (for example biological) components that can interfere with
their identification. This document involves a preliminary observation of the sample by an optical
microscope (OM) and then identification of the microplastics (MPs) by molecular spectroscopy. This
document provides the possibility of using two different techniques of molecular spectroscopy, Micro-
FTIR and Micro-Raman to identify and count plastic particles down to submicron size.
This document is designed to allow the re-evaluation of microplastic counting data when toxicological
and environmental impact indications become available.
This document describes the method of analysis for a single filter. However, errors in the qualitative
and quantitative determination of microplastics that can result from the variability between different
filters imply that replicates should be performed to establish precision.
This document provides useful information (e.g. dimensional classes, shape, composition, etc.) that can
be taken into account for a possible eco-toxicological assessment of health and environmental impacts.
It is well known that some microplastics (MPs) are lipophile and can be vehicles for toxic compounds
(e.g. PCBs, PAHs, dioxins) or vehicles of pathogenic microorganisms adhered to their surface and can be
assimilated (with their dose of toxicity) and permeate into organisms and cells.
The sources of microplastics are numerous. Their shapes and sizes are also variable. In the case of those
released by textiles, the typical (but not the only) morphology is fibrous and their diameter and length
can vary depending on the construction parameters of yarns and fabrics or cleaning conditions.
v
INTERNATIONAL STANDARD ISO 4484-2:2023(E)
Textiles and textile products — Microplastics from textile
sources —
Part 2:
Qualitative and quantitative analysis of microplastics
1 Scope
This document establishes a qualitative-quantitative analytical evaluation (i.e. determination) of
microplastics to be able to define their:
— particle number;
— morphology (morphological characteristics);
— dimensional distribution;
— the type, chemical origin or nature of polymers and their colour, if present.
This document is applicable to the determination of microplastics (from the textile sector) collected in
various matrices (for example textile process wastewater, clothes washing water, textile process air
emissions, textile process solid waste).
This document specifies expression of results in terms of estimated surface area and mass of
microplastics (MPs) per unit sample. It enables the expression of the results of the quantification of
microplastics (MPs) from various sources, including samples related to the production, processing,
treatment and use of textiles (raw material, manufacturing process, sample like wastewater from
washing clothes, air, and industrial process water).
This document applies to textile sector samples of matrices of different physical states (solid, liquid or
aeriform), for example:
— solid samples from textile production processes;
— water samples from the textile production process and/or from the washing of clothing (e.g.
garments or other textiles, ISO 4484-1 or ISO 4484-3 can be applied in order to prepare a liquid to
be tested);
— air samples to test the air quality in the workplace of textile companies.
This document, being able to provide information such as size, shape, surface and mass (estimated),
enables the transfer of useful information for ecotoxicological assessments to specialists.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 1833-4, Textiles — Quantitative chemical analysis — Part 4: Mixtures of certain protein fibres with
certain other fibres (method using hypochlorite)
ISO 3696, Water for analytical laboratory use — Specification and test methods
EN 481, Workplace atmospheres — Size fraction definitions for measurement of airborne particles
EN 13284-1, Stationary source emission — Determination of low range mass concentration of dust- Part 1:
Manual gravimetric method
EN 13284-2, Stationary source emissions — Determination of low range mass concentration of dust —
Part 2: Quality assurance of automated measuring systems
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
microplastic
MP
material consisting of a solid polymer containing particles, to which additives or other substances may
have been added, and where a weight fraction of ≥1% particles have:
a) all sizes 100 nm ≤ × ≤ 5 mm,
b) for fibres, a length of 300 nm ≤ × ≤ 15 mm and a length/diameter ratio >3
Note 1 to entry: Polymers that occur in nature that have not been chemically modified (other than by hydrolysis)
are excluded, as are polymers that are (bio) degradable.
[SOURCE: ECHA, ANNEX XV Restriction Report - Microplastics, 22 August 2019, par 1.2.2.1, modified on
lower size recommended dimensions, by Commission Recommendation C/2022/3689 of 10 June 2022
on the definition of nanomaterial (OJ C 229, 14.6.2022, p. 1), modified — "≥1% w/w" was changed to "a
weight fraction of ≥1 %"; additional information has been given as a note to entry.]
3.2
significant sample volume
amount of filtered volume to be analysed considering the source of the sample and the values of: total
suspended solid (TSS) and chemical oxygen demand (COD)
Note 1 to entry: See Table 1 and Table 2.
3.3
subsample
aliquot (fraction) of the primary sample diluted (as known) with water
3.4
washing solution
solution used to wash equipment to recover any MP which can be left on the equipment
3.5
image analysis
identification and classification of particles/fibres according to their morphology (shape) and size,
providing additional sample information.
Note 1 to entry: The sample information are, for example, distribution percentage, number and size of
microparticles and microparticles with fibre shape.
3.6
molecular micro-spectroscopy
analysis (FTIR or Raman) coupled to optical microscope (OM)
Note 1 to entry: The equipment is used to identify the polymer that composes the particle and to classify the
particles recognized by image analysis, providing information such as distribution, percentage, number and size
of MPs identified.
4 Principle
In order to be able to determine the MPs in a matrice, it is first necessary to transfer a significant
portion of them to a suitable filter (6.3) to allow subsequent microscopic analysis with regards to
quantity (molecular spectroscopy).
Different approaches shall be followed depending on the physical state of the starting matrice:
— powdery solid, or a mixture of solid materials;
— aqueous (liquid) suspension;
— aeriform.
In the case of solid powdery samples, the preliminary dispersion of a representative aliquot of the
samples is carried out in a known volume of water (5.1), or in a dispersing solution consisting of a
non-ionic surfactant (5.4.2) in filtered water (5.1). If the sample is solid (non-powdery) an appropriate
disintegration treatment (e.g. ultrasonic treatment) shall be carried out.
For the analysis of liquid suspensions, the sample shall be filtered through a filter (6.3) of suitable
material, pore size and shape (depending on the spectroscopic technique used). Analysis of the material
on the filter (6.3) according to the following description:
a) identification of the composition of microparticles present by Micro-FTIR (6.1) /Micro-Raman
(6.2) spectroscopy in order to identify any plastic microparticles and related measurement of their
dimensions by image analysis;
b) counting of the number and identification of the size class of particles and fibres observed, and
possible calculation of the total masses of MP present on the filter (6.3) in accordance with
Formula (6).
Aeriform samples shall be considered in accordance with EN 13284-1, and for dust collection in the air
(air emissions, air working environment) shall be considered in accordance with EN 13284-2.
All MP assessments, regardless of the different matrices analysed, shall be compared. The data of the
produced waste, the discharged water, the air of the workplace or the emissions into the atmosphere
can be analysed and compared to obtain a balance of the MPs of:
— a specific textile production process;
— textile products during their life cycle as garments;
— any other textile processes/semi-finished/finished products.
According to the analytical method used, the different sampling, preparation and purification
procedures shall be considered. They shall be chosen according to the characteristics of the sample to
be tested.
The analytical method shall be adequately applicable to all samples prepared in advance.
Preliminary analysis should be performed in accordance with the different sample preparation and
purification procedures and the analytical techniques subsequently applied.
In particular, in the case of textile sector source samples with potential presence of salts and organic
substances, investigative analyses shall first be carried out, for example in the case of liquid samples of
aqueous matrices:
— determination of conductivity;
— determination of COD;
— determination of TSS, with membrane filtration technique;
— pre-screening with OM for estimating microscopic image quality vision.
5 Reagents
All reagents [analytical reagent grade (AR)] or their solutions and the demineralised water (5.1), shall
be filtered through filters (6.3) with a pore size of at least 0,45 µm.
5.1 Demineralised water, Grade 3 quality as specified in ISO 3696.
5.2 Hydrogen peroxide, with a volume fraction of 15 % in demineralised water (5.1).
For the oxidation of organic matter prior to filtration, hydrogen peroxide AR may be used.
5.3 Sodium hypochlorite (NaClO), 1 M or 1 mol/l.
Freshly prepared sodium hypochlorite solution containing (35 ± 2) g/l active chlorine (±1) mol/l, in
accordance with ISO 1833-4 is used for the dissolution of wool fibre during standards preparation.
5.4 Washing solutions
5.4.1 Sodium chloride, with a mass per volume of 1 %.
Dilute 10 g of pure NaCl AR in 1 000 ml with demineralised water (5.1)
The solution shall be prepared by diluting pure NaCl AR salt in demineralised water (5.1).
A sample of this solution shall be analysed to determine the MPs content of the salt to be taken into
account in the results of the analyses.
5.4.2 Non-ionic surfactant, with a mass per volume of 1 %.
Dilute 10 g of pure non-ionic surfactant in 1 000 ml of demineralised water (5.1).
The solution shall be prepared by diluting the surfactant, preferably, not ionic, for example Triton X
(whose composition and/or IR/Raman spectrum shall be known in order to subtract it during analysis),
in demineralised water (5.1).
A sample of this solution shall also be analysed in order to determine the MP content to be taken into
account in the results of the analyses.
5.5 Ethanol solution
Ethanol with a volume fraction of 95 % mixed 1:1 with demineralised water (5.1)
It is possible to use the ethanol solution previously filtered with a filter (6.3) made of mixed esters of
cellulose or cellulose nitrate.
5.6 Acetic acid solution 0,1 mol/l, with a volume fraction of 60 %.
Remove salt or organic material by dosing a solution obtained by diluting 5,7 ml of acetic acid (a volume
fraction of 60 %) in 1 000 ml of demineralised water (5.1).
If necessary, the molarity of the solution can be increased to make the removal of salt or organic
material more efficient.
A sample of this solution shall also be analysed in order to determine the MP content so that it can be
taken into account in the results of the analyses.
6 Apparatus
6.1 Micro-FTIR, for particles greater than 10 μm with the following requirements:
a) transmission/transflectance or reflectance;
-1
b) spectral resolution: minimum 4 cm ;
c) Spectra format: absorbance;
d) Depending on the system an aperture of 150 μm × 150 μm can be used;
-1 -1
e) Detector spectra range 4 000 cm to 675 cm ;
f) Collection time and scans depend on the system and used sources.
6.2 Micro-Raman, for particles greater then 0,2 μm to 0,5 μm with the following requirements:
a) lasers can have a wavelength of 457 nm, 532 nm, 633 nm, and 785 nm;
b) several objectives with different magnifications and numerical apertures;
c) spectroscopic systems;
d) Ultra-High-Throughput spectrometer (UHTS300) in the VIS range Grating: minimum 600 g/mm;
e) Ultra-High-Throughput spectrometer (UHTS400) in the NIR range Grating: minimum 300 g/mm.
6.2.1 CCD camera with one of the following specifications:
a) black illuminated camera for operation in VIS range;
b) low dark current CCD camera in NIR range;
c) dark field microscopy; particle identification and measurement software
6.3 Filters, according to the type of spectroscopy chosen and the size of the particles to be
determined.
The pore size that can usually be used are: 0,45 μm, 0,8 μm, 1 μm, 5 μm.
All the filters shall have a suitable shape and fit the instrument of molecular micro-spectroscopy.
Circular or square shapes are preferred.
6.3.1 Micro-FTIR Filters
6.3.1.1 In the case of Micro-FTIR (reflection and transmission) analysis, filters (6.3) with a pore size
area of at least 5 µm shall be used.
The filters shall be one of the following.
-1 -1
a) Aluminium oxide filter, spectral range between 4 000 cm and 1 250 cm , in transmission mode
(pore diameter 0,02 μm to 0,2 μm).
-1 -1
b) Silicon filter, spectral range between 4 000 cm to 600 cm in transmission or reflection mode.
Pore diameter 5 μm, pitch 12 μm, thickness 500 μm.
-1 -1
c) Gold polycarbonate filter, spectral range 4 000 cm to 400 cm in reflection mode (1 μm, 25 mm)
-1 -1
d) Cellulose acetate nitrate filter, spectral range between 4 000 cm to 400 cm in reflection mode or
micro-ATR.
6.3.1.2 In the case of attenuated total reflectance (ATR) analysis, the possible filters (6.3) that can be
used have a pore size area of at least 5 μm, in detail:
a) cellulose acetate nitrate;
b) pore diameter 0,45 μm and/or 0,8 μm; filter diameter 47 mm and/or 25 mm;
c) PVDF (polyvinylidene fluoride);
d) pore diameter 5 μm filter diameter 47 mm.
6.3.2 Micro-Raman filters
In the case of Micro-Raman (6.2), the possible filters (6.3) that may be used are:
a) cellulose acetate nitrate, gold coated polycarbonate membrane, silicon filter;
b) 13 mm diameter, 25 mm diameter, 47 mm diameter, 10 mm side.
All materials used shall be washed with demineralised water (5.1) and subsequently with ethanol (5.5)
beforehand to remove any residual MP, and immediately after washing shall be left to dry in the air
covered with aluminium foil or watch glass.
6.3.3 Reading the filter
To reduce bend upon drying after the filtration process, mount them as flat as possible for measurement.
Silicon filters have two faces (one mirror face and the other darker). Use the filter with the mirror face
facing upwards (face to be used for filtering), in this way the micropores facilitate the adhesion of the
MPs and their maintenance, even when the filter is dry, during the following counting and identification
steps with Micro-FTIR (6.1) or Micro-Raman (6.2).
Silicon filters show strong Raman peaks but no fluorescence. Silicon peaks may be ignored in the
particle identification.
6.4 Light microscope, suitable for fibre identification, involves the use of projection microscopes
and visual microscopic image analysers. Transmitted-light microscopes with direct graduated scale
equipped with an optical lens are also applicable.
6.5 Filtration system, made of steel or glass (see Figure 1), with funnel of 100 ml, 500 ml, 1 000 ml,
2 000 ml complete with sintered septum, clamp and coded Erlenmeyer flask.
Figure 1 — Examples of filtration system, filter holder and filters
6.6 Flasks and bottles, made of glass, with glass cap.
6.7 Tweezers, made of steel.
6.8 Petri dishes, made of glass.
6.9 Microslides, made of glass.
6.10 Filter holder, (see Figure 1).
6.11 Mechanical stirrer.
6.12 Conductivity meter, accurate to a minimum of 5 microSiemens/cm (μS/cm).
6.13 Equipment for COD determination, rapid kits are also allowed.
6.14 Analytical balance, accurate to a minimum of 0,1 mg.
6.15 Ultrasonic bath, (see 7.3).
6.16 Vacuum filtration system.
6.17 Software, for automatic image analysis, morphological identification, dimensional classification,
mapping.
7 Cleaning procedure
7.1 Cleaning of the materials and the test environment
Airborne fibre contamination is possible and using procedures to reduce it is recommended.
The following is a list of mandatory actions to be taken to reduce contamination and keep equipment
clean:
a) All glassware shall be previously washed with demineralised water (5.1) filtered through filters
(6.3) with a pore size of at least 0,45 µm (nitrate, acetate, mixed cellulose esters) and with washing
solution, then rinsed before each use; then washed with ethanol solution (5.5) filtered through
filters (6.3) with a pore size of 0,45 µm (nitrate, acetate, mixed cellulose esters) and with washing
solution, then rinsed before each use with the same ethanol solution (5.5). Final rinse before each
use shall be done 3 times.
The same actions shall be applied to sample and container for which glass and metals should be
preferred.
Avoid any plastic apparatus or components for filtration and sample conservation as they can
release MPs to the sample.
After washing, the glassware shall be stored and protected with the aid of suitable barriers (for
example aluminium foil closing the inlets) to reduce the possible deposition of MP present in the air.
b) It is recommended to wipe down all surface using paper saturated with ethanol, or acetone before
measuring/testing each sample.
c) Rinse all tweezers (6.7), probes and hands before each filtration procedure.
d) Garments (included white laboratory coat) worn by analysts during sample handling should
preferably be made of natural fibres instead of synthetic ones such as synthetic fleece or similar
materials.
e) Minimize movement in the lab or working space. It is suggested to conduct sensitive work inside a
fume hood or laminar flow cabinet.
7.2 Blank test to evaluate environmental contamination
Environmental contamination of MPs can be considered, by carrying out in parallel, for each lot of
analysis, a blank test where a sample of water (5.1) and any other washing solution used on the test
is subjected to the analysis procedure. The MP value detected shall be defined as MPs environmental
contamination and shall be considered in the various analytical assessments carried out on the various
samples subjected to analysis.
The determination of the blank test shall be carried out for each lot of analysis.
7.3 Cleaning procedure for filters
Store filter in glass petri dishes (6.8) in order to reduce contamination from the dish itself. Keep your
filter (6.3) covered whenever possible before the observation.
All filters (6.3) shall be new or cleaned before using.
Before filtration, observe the whole surface of the filter (6.3) on OM to check that they do not have
any interfering particles on their surface which may have come from the packaging, its handling or the
production process itself. Cleaning depends on the kind of filter (6.3).
It is possible to use either a physical treatment which requires an ultrasonic procedure or a chemical
one (e.g. simple immersion in pure ethanol AR for 10 min), for all types of filters.
However, for silicon filters in order to reduce the overall cost of analysis, the possibility of reusing
the same filter for several analyses has been foreseen with following washing and quality control
procedure:
— minimum of 3 repeated sonications in an ultrasonic bath, each of 10 min in demineralised water
(5.1) (to be replaced at each repetition);
— before using immerse the silicon filter in 10 ml pure ethanol AR.
8 Sample preparation
8.1 Solid matrices (fibre, textile or any derivates)
In the case of solid powdery samples, the preliminary dispersion of a representative aliquot of the
samples is carried out in a known volume of water (5.1), or in a dispersing solution consisting of a
non-ionic surfactant (5.4.2) in filtered water (5.1). If the sample is solid (non-powdery) an appropriate
disintegration treatment (e.g. ultrasonic treatment,) shall be carried out.
8.2 Liquid matrices (aqueous)
8.2.1 Choice of sample volume/mass to be filtered and number of filters (6.3).
Filter through one or more filters (6.3) of different dimensions and pore size depending on sample
volume.
Table 1 shows recommended sample volumes for filtration through a 13 mm filter with 1 µm pore size.
Table 2 shows recommended sample quantity for filtration through a 47 mm filter with 0,45 μm pore
size.
Table 1 — Recommended sample quantity for filtration through a 13 mm filter with 1 μm pore
size
Sample quality - Source (like a) - TSS content TSS Unpretreated Pretreated
sample sample
mg/l
(minimum (minimum
filterable filterable vol-
volume) ml ume) ml
Wastewater - wwtp Out - low content <100 10 100
Wastewater - wwtp In - medium content <2 500 2 20
Activated sludge - Suspended biomass - high <10 000 1 5
content
Table 2 — Recommended sample volumes for filtration through a 47 mm filter with 0,45 μm
pore size
Sample quality - Source (like a) - TSS content TSS (mg /l) Unpretreated Pretreated
sample sample
(minimum (minimum
volume to be volume to be
filtered) ml filtered) ml
Wastewater - wwtp Out - low content <100 50 200
Wastewater - wwtp In - medium content <2 500 10 100
Activated sludge - Suspended biomass - high content <10 000 5 20
8.3 Air matrices
Use the following techniques and sampling volumes.
— The determination of airborne particles shall be in accordance with EN 13284-1.
— The determination of dust shall be in accordance with EN 481.
8.4 Preliminary checking of sample and pre-treatment
In order to identify the possible needs for pre-treatment of the liquid sample with unknown origin,
the values of conductivity and the COD and TSS of the sample to be analysed shall be measured and, in
addition, an observation in OM at 10 x and/or 50 x magnification shall be carried out.
The liquid samples treated are textile process wastewater, laundry washing effluent, industrial process
water.
8.4.1 Liquid sample property checking and requirements
8.4.1.1 Conductivity
Check the conductivity of liquid sample. If the conductivity is higher than 3 000 µS/cm, it shall be
necessary to wash the filter (6.3) with a solution of acetic acid 0,1 mol/l (5.6) to eliminate any traces of
inorganic substances.
8.4.1.2 Chemical oxygen demand (COD)
Check the COD of liquid sample. If the COD is higher than 100 mg O /l, perform a pre-oxidation with
hydrogen peroxide (5.2) 15 % (for a duration of not less than 7 days to 30 days depending on the
complexity of the matrice) where the volume of hydrogen peroxide (5.2) to be added is calculated using
Formula (1), with the aim of eliminating any trace of non-plastic organic material.
CODS×
()
v
V = (1)
70,06
where
V is the volume of hydrogen peroxide (5.2, a volume fraction of 15 %) to dose in l;
COD is the chemical demand of oxygen expressed in mg O /l;
S is the sample volume to be treated in l.
v
8.4.1.3 OM observation
Depending on the TSS and the COD ratio as well as the OM observation, the recommended minimum
significant sample volume should be defined in order to have acceptable filtration times.
In addition, during the OM pre-screening, if the presence of overlapping microparticles is high, pre-
dilute (8.4.4) the sample with water (5.1), to obtain an optimal microparticle concentration lower than
200 (see Annex B). If the concentration is higher, it may be necessary to use different filters to avoid
overlapping.
8.4.2 Pre-treatment if required
8.4.2.1 General
The acidic and/or oxidant pre-treatment may be carried out in accordance with 5.6 and 5.2, during one
of the following analysis steps in which the sample may be:
a) an untreated raw sample;
b) a subsample before proceeding with the filtration procedure;
c) the filter (6.3) used after the microparticle collection (see Figure 2).
Figure 2 — Examples of nitrate/acetate filters after different pre-treatments with hydrogen
peroxide
8.4.2.2 Salt removal
The acetic acid (5.6) treatment may be carried out in two different ways:
a) on the untreated raw sample by dosing acetic acid (5.6) to attain pH 5 in the sample suspension and
in the case of high conductivity or presence of precipitated inorganic salts;
b) by washing the filter (6.3), after completion of the filtration steps, in case of evidence of precipitation
of salts, with a direct dosing of 1 ml acetic acid (5.6). If necessary, this action can be repeated.
8.4.2.3 Organic removal
8.4.2.3.1 General
To be carried out on the sample prior to filtration.
The oxidation process takes place over time and as a result of the dosage of hydrogen peroxide (5.2).
8.4.2.3.2 Hydrogen peroxide treatment
The volume fraction of 15 % hydrogen peroxide (5.2) to be added to the sample is proportional to the
COD of the sample to be treated. Dose 1,4 ml of 15 % hydrogen peroxide (5.2) volume fraction for each
100 mg O /l (COD) referred to 100 ml of sample. To calculate the dosage for a real sample, refer to
Formula (1).
Satisfactory results are obtained after 7 days, if they are still not adequate (see Figure 3) extend the
reaction time.
Figure 3 — Water samples treated with different hydrogen peroxide volume fractions of 15 %
8.4.2.3.3 Reaction time
Depending on the matrice, different reaction times can occur.
The minimum oxidation time depends on the quality of the non-plastic organic matrice to be oxidized.
The time can vary between 7 days and 30 days. Verify the oxidation effect visually by checking the
clarity of the sample and the reduction of the colour/turbidity.
Consequently, a production of sediment can occur.
Figure 4 highlights this effect.
a) 1 day b) 3 days c) 7 days
Figure 4 — Water sample treated with hydrogen peroxide (5.2) volume fractions of 15 % at
different times
8.4.3 Test sample homogenization/hydration and/or sonication
The sample should be stirred vigorously or alternatively sonicated to produce the subsample; these
operations can be carried out at room temperature or higher, up to a maximum of 50 °C.
In order to minimize the ability of the microparticles to adhere to the walls due to their high surface
tension and hydrophobicity, all samples are subjected, before being handled and filtrated to a process
of ultrasonication designed to significantly increase the hydrophilicity of MPs and consequently
reduce/minimize the wall effect and agglomeration, as that would result in loss of significance and
representativeness of the sample analysed.
The procedure involves treatment with ultrasonic bath (6.15) 20 kHz to 40 kHz at different treatment
times. The wetting effect of the MPs shall make them perfectly dispersible in aqueous solution.
The subsample shall then be diluted (see 8.4.4) by a factor to perform an adequate particle counting
(without particle overlapping) on the final filter (6.3).
In the calculation of the total volume, the value of the volume used for the washing of the equipment
(for example pipettes, cylinders, filtration container) shall be included.
8.4.4 Pre-dilution
In order to produce a subsample to be tested, pre-dilution shall be performed immediately after the
preliminary checking phase (8.4).
In the pre-dilution phase of the sample, its homogenization shall be prepared according to 8.4.3.
9 Preparation procedure
9.1 General
This protocol provides a consistent method for preparing standardised suspensions containing
[4]
microfilaments/microplastic with fibre shape for:
[1][2]
— standard or reference materials currently unavailable for purchase ;
— evaluation and validation of all approaches from sampling to identification of microplastic.
9.2 Preparation of micro-sized fibres sample
This procedure describes how to cut synthetic threads in order to obtain solutions with a number of
filaments per litre between 70 filaments per litre and 850 filaments per litre. Table 3 provides a series
of types of threads used for the preparation of standard samples and their relative concentrations.
Table 3 — Examples of preparation of standard samples (example)
Sample name
Theoretical concentration
Water (5.1) collection
(polymer, colour,
volume (ml)
(Number of filaments/litre)
number of filaments)
300 227
Polyamide 6,6 blue (68 filaments) 500 136
900 76
300 427
Polyamide 6 multicolour (128 fila-
500 256
ments)
900 142
300 600
Polyamide 6 orange (180 filaments) 500 360
900 200
300 833
Polyester beige (256 filaments) 500 512
900 284
300 240
Polypropylene orange (72 fila-
500 144
ments)
900 80
In order to cut sample from 200 µm to 800 µm a manual microtome (see Figure A.1) can be used.
It is necessary to use a yarn of commercial material with a known number of filaments for the
preparation of the standards. Before cutting, check that the number of filaments is equal to the one
declared by the producer by using an OM (see Figure 5 and Figure 6).
Figure 5 — Example of polyamide thread
Figure 6 — Example of polyamide thread with 180 filaments
Proceed with the cutting using a microtome (only one cut for sample), see Annex A. In order to fill the
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