Evaluation and optimisation of extraction methods suitable for the analysis of microplastic particles occurring in the edible part of seafood - EFSA
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Evaluation and optimisation of extraction
methods suitable for the analysis of microplastic
particles occurring in the edible part of seafood
Max Rubner-Institut, Federal Research Institute of Nutrition and Food
Department of Safety and Quality of Milk and Fish
Bild Dorade: © Peter Kirchhoff/PIXELIO
Julia Süssmann
Microplastic in seafood: How much do we eat?
Translocation
Pb
Migration Cr
Leaching
Hg
Cd
?
3700 ± 2500100[11] 0 - 24450[14]
150 µm
µm
400 - 8100100[6]µm 3000 ± 900
[3] 980 ± 266010[10]
µm
10 µm
138000 ± 202300[16] [13] 970 ± 261010[2]µm
10 µm 700 - 290010 µm 250 - 3605[4]µm
1600 - 3500 [6]
0 - 680020[9]µm 160 ± 13010[8]
259400 ± 114100[16]
100 µm µm
0 - 35020[5]µm
10 µm
650 - 1330[7] 10 µm
0 - 4280100[17]
µm
[12]
1600 - 270010
5300 ± 500[19] µm
[15]
560 - 1380100 800[1]
10 µm
10 µm
1000 - 4000[18]
µm 10 µm
Figure 1: Microplastic content in mussels (Mytilus spp.) in particle number per kg soft tissue (studies from 2014 – 2020).
No harmonised methods, limitation in comparison. Results are influenced by: resolution of analytical technique, possible polymer loss due to digestion
method ( , ), sub-optimal density separation ( ), incomplete ( ) or no identification ( ).
illustrations: designed by freepik.com
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15.04.2021 2
Microplastic extraction: What do we have to consider?
procedural contamination // Loss due to adsorption on labware surfaces
insufficient digestion filter pore size polymer identification
degradation of plastics filter material
Digestion Filtration Analysis
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15.04.2021 3
Evaluation of sample preparation protocols
Literature research efficiency
Which protocols are regularly applied when
digesting aquatic biota? integrity
time
Evaluation of digestion methods
• Are fish fillets, the soft tissue of mussels steps
and crustaceans digested sufficiently for
filtration with pore size 1 µm? costs
• Are plastic particles not degraded? alkaline (60 °C) alkaline (25 °C) acidic alkaline-acidic
• Is the method suited for routine analysis? oxidative alkaline-oxidative enzymatic enzymatic-alkaline
Figure 2: Performance of digestion methods applied for isolating MP from fish fillet.
Optimisation 100.00
fishes crustaceans molluscs
digestion efficiency [%]
• Which parameters have to be changed 99.50
for minimizing plastic degradation? 99.00
• What measures have to be applied 98.50
regarding different analytical techniques 98.00
97.50
or a broad range of sample matrices?
97.00
In-House-Validation
Is the protocol suited for quantitative isolation
of microplastics from seafood? Figure 3: Digestion efficiency of edible parts from different seafood species.
Fishes are sorted according to their fat content (increasing).
MRI – Institut für Sicherheit und Qualität bei Milch und Fisch 15.04.2021 4
Optimisation: Towards a negligible impact on plastic particles
polymer recovery identification • recovery based on weight
weight [%] area [%] FTIR Raman py-GC/MS might not detect changes
PA6 96 ± 2 104 ± 2 + + + in small surface layer
• loss of small micro- &
40 ºC 95 ± 1 not tested nanoplastics undetected
98 ± 2
PA12 / + + +
• reduction of PET-particle
PAN - - - +/ +/
- - area at 60 ºC alkaline
PC 96 ± 2 97 ± 1 + + + digestion but not at 40 ºC
Figure 4: Photograph of a PET-particle before
40 ºC 95 ±
Optimisation: The importance of filter choice
• improving filtration speed & preventing filter clogging, depending on…
→ pore size: larger pore size = less prone to clogging, but also loss of
cellulose nitrate glass fiber
smaller, probably more abundant, plastic particles
→ filter material: adsorption of matrix residues (e.g. proteins)
• compatibility of filter material and sample preparation, analytical methods
→ e.g. degradation of filter material by digestion solutions cellulose acetate polycarbonate
Figure 6: Photograph of membrane
→ e.g. inorganic filters for thermal analysis filters (pore size ~ 1 µm) after filtering
digested fish fillet.
• filter structure: impact on particle retention & detection[20]
knitted lattice pressed fiber
→ missing fragments with multilayer/fiber-type (hidden between layers)
(e.g. cellulose nitrate, cellulose fiber/paper, glass fiber)
→ loss of fibers with singlelayer-type (passing pores lengthwise)[20]
nylon cotton fiber
(e.g. polycarbonate, Al2O3) multilayer-hole singlelayer-hole
mixed cellulose polycarbonate
Figure 7: SEM-image of surface
morphology-types of membrane filters;
Cai et al. (2020).
MRI – Institut für Sicherheit und Qualität bei Milch und Fisch 15.04.2021 6
Optimisation: The importance of filter choice
Figure 8: Fluorescent PA12-particles on
glass fiber filters. Scan on same focal plane
(left) and stacked images of confocal scan
(range 100 µm).
→ consideration of focal plane of particles for imaging/filter scan
• perspective: adsorption of nanoparticles → incomplete separation
Figure 9: SEM-image of polycarbonate filter
→ further research regarding filtration required (pore size 1 µm) with agglomerated Ø100 nm-PS
adhering to the pores & matrix residues.
MRI – Institut für Sicherheit und Qualität bei Milch und Fisch 15.04.2021 7
Optimisation: Preventing procedural plastic contamination
particle number
• small plastic particles are ubiquitous → 2500
2000
monitoring & mitigation of contamination 1500
• investigating probable sources 1000
500
→ insufficiently cleaned glassware 0
→ reagents / solvents
→ exposure of samples to air
Figure 10: Number of MP-suspect particles rinsed off glass
flasks after application of different cleaning procedures.
Figure 11: Photographs of Nile red-stained filters after
filtration of pepsin from different suppliers. Particles with
green, yellow or orange fluorescence are MP-suspect.
MRI – Institut für Sicherheit und Qualität bei Milch und Fisch 15.04.2021 8
Optimisation: Preventing procedural plastic contamination
450
particle number
extraction sedimentation
400
350 2 – 10 µm 11 – 20 µm
300 21 – 50 µm 51 – 100 µm
250 101 – 200 µm
200
150
100
• current protocol for contamination prevention
50
→ cotton clothes, laminar flow workbench 0
heated fume hood laminar laboratory fume hood laminar
→ pre-filtration (pore size < 1 µm) of all glassware flow flow
reagents & solutions Figure 12: Number of fluorescent particles (Nile red staining, FITC-filter) of
heated glassware, a simulated extraction procedure and sedimented particles
→ cleaning of glassware [and filters] from air.
dishwasher, heating (500 ºC), rinsing 30
→ rinsing of filtration apparatus between 20
10
each sample (3x 10 mL filtered water) 0
• monitoring of blank samples still required
Figure 13: Number MP-suspect particles in blank samples.
MRI – Institut für Sicherheit und Qualität bei Milch und Fisch 15.04.2021 9
Validation of the optimised sample preparation protocol
m/z = 122 m/z = 113
digestion
m/z1 = 130 m/z1 = 70 PA-6
m/z2 = 117 m/z2 = 111
PS
m/z1 = 82
m/z2 = 83
PE
fitration & post-filtration treatment
PET
PP
Figure 15: Pyrogram of nine commercially relevant synthetic polymers
spiked to herring fillet and isolated with the optimized protocol. The filter
was silanized with TMCS before pyrolysis. The black chromatogram is
the TIC.
Recovery
n = 10 88 ± 16 %
n = 100 89 ± 12 %
n = 1000 103 ± 13 %
further research for
quantification of plastics
with py-GC/MS needed
Figure 14: Schematic overview of optimised sample preparation protocol.
illustrations: designed by freepik.com
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15.04.2021 10Prospective: Consideration of nanoplastics
Sample • concentration and separation of nano- and microplastics
preparation? • detection limit in field-flow-fractionation
• identification of plastic in the fractions
≥ 1 µm
< 1 µm
Detection limit?
Figure 16: AF4-separation of different amounts of nanoplastics.
MRI – Institut für Sicherheit und Qualität bei Milch und Fisch lab equipment: Landesbildungsserver Baden-Württemberg 15.04.2021 11Summary
• Optimised procedure for isolation of microplastics from edible part of seafood:
→ two-step digestion with pepsin (enzymatic) and KOH (alkaline) at ~ 37 ºC
→ filtration with filters of 1 µm pore size, Ø 47 mm (e.g. glass fiber, polycarbonate)
→ if required: filter bleaching with H2O2 (dark residues), degreasing with alcohol
• Necessity of blank samples even with thorough protocol for preventing microplastic
contamination; important aspects: purity of reagents, cleaning of glassware
• Choice of filter material has a great impact on filtration speed/matrix residues,
microplastic retention[20], and particle detection → more research required
• more research required regarding sample preparation for nanoplastics from seafood
details published in: Süssmann, Julia, et al. "Evaluation and optimisation of sample preparation protocols suitable for the
analysis of plastic particles present in seafood." Food Control 125 (2021): 107969.
Max Rubner-Institut – Bundesforschungsinstitut für Ernährung und Lebensmittel 15/04/2021 12Thank you for your support…
Federal Research Institute
of Nutrition and Food
Safety and Quality of Milk
and Fish
Jan Fritsche University of Hamburg
Torsten Krause Center for Earth System
Dierk Martin Research and Sustainability
Ute Ostermeyer Elke Fischer
Enken Jacobsen Matthias Tamminga
Björn Neumann …
Longina Reimann
Food Chemistry
Food Technology and
Bioprocess Engineering Technical University
Ralf Greiner Berlin
Elke Walz
Sascha Rohn
Birgit Hetzer
Andrea Tauer
Christian Geuter
Max Rubner-Institut – Bundesforschungsinstitut für Ernährung und Lebensmittel 15.04.2021 13Thank you for your
attention!
MRI – Institut für Sicherheit und Qualität bei Milch und Fisch 15.04.2021 14References [1] Abidli, Sami, Youssef Lahbib, and Najoua Trigui El Menif. "Microplastics in commercial molluscs from the lagoon of Bizerte (Northern Tunisia)." Marine pollution bulletin 142 (2019): 243-252. [2] Bråte, Inger Lise N., et al. "Mytilus spp. as sentinels for monitoring microplastic pollution in Norwegian coastal waters: A qualitative and quantitative study." Environmental Pollution 243 (2018): 383-393. [3] Catarino, Ana I., et al. "Low levels of microplastics (MP) in wild mussels indicate that MP ingestion by humans is minimal compared to exposure via household fibres fallout during a meal." Environmental pollution 237 (2018): 675-684. [4] Van Cauwenberghe, Lisbeth, and Colin R. Janssen. "Microplastics in bivalves cultured for human consumption." Environmental pollution 193 (2014): 65-70. [5] Cho, Youna, et al. "Abundance and characteristics of microplastics in market bivalves from South Korea." Environmental pollution 245 (2019): 1107-1116. [6] De Witte, B., et al. "Quality assessment of the blue mussel (Mytilus edulis): Comparison between commercial and wild types." Marine pollution bulletin 85.1 (2014): 146-155. [7] Digka, Nikoletta, et al. "Microplastics in mussels and fish from the Northern Ionian Sea." Marine pollution bulletin 135 (2018): 30-40. [8] Ding, Jinfeng, et al. "Detection of microplastics in local marine organisms using a multi-technology system." Analytical Methods 11.1 (2019): 78-87. [9] Fischer, Elke. "Distribution of microplastics in marine species of the Wadden Sea along the coastline of Schleswig- Holstein, Germany." Final Report University Hamburg (2019). [10] Iversen, Karine Bue. Microplastics in blue mussels (Mytilus edulis) from the marine environment of coastal Norway. MS thesis. Norwegian University of Life Sciences, Ås, 2018. MRI – Institut für Sicherheit und Qualität bei Milch und Fisch 15.04.2021 15
References [11] Karlsson, Therese M., et al. "Screening for microplastics in sediment, water, marine invertebrates and fish: method development and microplastic accumulation." Marine pollution bulletin 122.1-2 (2017): 403-408. [12] Li, Jiana, et al. "Microplastics in mussels along the coastal waters of China." Environmental pollution 214 (2016): 177-184. [13] Li, Jiana, et al. "Microplastics in mussels sampled from coastal waters and supermarkets in the United Kingdom." Environmental pollution 241 (2018): 35-44. [14] Lusher, A. L., et al. "Sampling, isolating and identifying microplastics ingested by fish and invertebrates." Analytical methods 9.9 (2017): 1346-1360. [15] Mankin, Chloe, and Andrea Huvard. "Microfibers in Mytilus species (Mollusca, Bivalvia) from Southern California Harbors, Beaches, and Supermarkets.“ [16] Murphy, Fionn, et al. "The uptake of macroplastic & microplastic by demersal & pelagic fish in the Northeast Atlantic around Scotland." Marine pollution bulletin 122.1-2 (2017): 353-359. [17] Reguera, Pablo, Lucía Viñas, and Jesús Gago. "Microplastics in wild mussels (Mytilus spp.) from the north coast of Spain." Scientia Marina 83.4 (2019): 337-347. [18] Li, Jiana, et al. "Microplastics in commercial bivalves from China." Environmental pollution 207 (2015): 190 -195. [19] Gomiero, Alessio, et al. "First occurrence and composition assessment of microplastics in native mussels collected from coastal and offshore areas of the northern and central Adriatic Sea." Environmental Science and Pollution Research 26.24 (2019): 24407-24416. [20] Cai, Huiwen, et al. "Microplastic quantification affected by structure and pore size of filters." Chemosphere 257 (2020): 127198. MRI – Institut für Sicherheit und Qualität bei Milch und Fisch 15.04.2021 16
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