evaluation and optimisation of extraction methods suitable
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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
Julia Süssmann
Microplastic in seafood: How much do we eat?
2MRI – Institut für Sicherheit und Qualität bei Milch und Fisch
HgPb
Cd
Cr
Translocation
Leaching
Migration
?
0 - 350[5]20 µm
5300 ± 500[19]10 µm
160 ± 130[8]10 µm
700 - 2900[13]10 µm
800[1]10 µm
980 ± 2660[10]10 µm
970 ± 2610[2]10 µm
0 - 24450[14]
150 µm
250 - 360[4]5 µm
650 - 1330[7]10 µm
0 - 6800[9]20 µm
1000 - 4000[18]10 µm
138000 ± 202300[16]10 µm
1600 - 2700[12]
10 µm
259400 ± 114100[16]10 µm
1600 - 3500[6]
100 µm
3700 ± 2500[11]100 µm
560 - 1380[15]100 µm
0 - 4280[17]100 µm
3000 ± 900[3]
10 µm400 - 8100[6]100 µm
illustrations: designed by freepik.com
world map: https://mapswire.com/ 15.04.2021
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 ( ).
Microplastic extraction: What do we have to consider?
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Digestion Filtration Analysis
insufficient digestion
degradation of plastics
procedural contamination // Loss due to adsorption on labware surfaces
filter pore size
filter material
polymer identification
15.04.2021illustrations: designed by freepik.com
lab equipment: Landesbildungsserver Baden-Württemberg
Evaluation of sample preparation protocols
4MRI – Institut für Sicherheit und Qualität bei Milch und Fisch
Figure 2: Performance of digestion methods applied for isolating MP from fish fillet.
costs
steps
time
integrity
efficiency
alkaline (60 °C) alkaline (25 °C) acidic alkaline-acidic
oxidative alkaline-oxidative enzymatic enzymatic-alkaline
In-House-ValidationIs the protocol suited for quantitative isolation
of microplastics from seafood?
Optimisation• Which parameters have to be changed
for minimizing plastic degradation?
• What measures have to be applied
regarding different analytical techniques
or a broad range of sample matrices?
Evaluation of digestion methods• Are fish fillets, the soft tissue of mussels
and crustaceans digested sufficiently for
filtration with pore size 1 µm?
• Are plastic particles not degraded?
• Is the method suited for routine analysis?
Literature researchWhich protocols are regularly applied when
digesting aquatic biota?
97.00
97.50
98.00
98.50
99.00
99.50
100.00
dig
estio
n e
ffic
ien
cy [%
]
Figure 3: Digestion efficiency of edible parts from different seafood species.
Fishes are sorted according to their fat content (increasing).
fishes crustaceans molluscs
15.04.2021
• recovery based on weight
might not detect changes
in small surface layer
• loss of small micro- &
nanoplastics undetected
• reduction of PET-particle
area at 60 ºC alkaline
digestion but not at 40 ºC
Optimisation: Towards a negligible impact on plastic particles
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Figure 4: Photograph of a PET-particle before
(blue) and after digestion with 10% KOH at 60 ºC
for 4 h.
• enzymatic-alkaline treatment might alter composition of
the particle (surface) thus hinder chemical identification
• polymer identity was assessed with FTIR, Raman and
Py-GC/MS before and after digestion with virgin particles
→ significant differences only observed for PAN
Figure 5: Photographs of PAN-particles
before and after digestion. Right: FTIR
spectrum of PAN before (black) and after
(red) digestion.
15.04.2021illustration: designed by freepik.com
polymer recovery identification
weight [%] area [%] FTIR Raman py-GC/MS
PA6
40 ºC
96± 2
95± 1
104 ± 2
not tested
+ + +
PA12 98± 2 / + + +
PAN - - - +/-+/-
PC
40 ºC
96± 2
95± <1
97 ± 1
not tested
+ + +
PE 100± 2 103 ± 5 + + +
PET
40 ºC
91± <1
92± 2
92 ± 6
100 ± 4
+ + +
PP 98± <1 100 ± 5 + + +
PS 99± 2 101 ± 2 + + +
PSu 101± 1 101 ± 3 + + +
PTFE 100 ± 2 100 ± 6 + + /
PU 99 ± 2 100 ± 4 + + +
PVC 99 ± 1 / + + /
Table 1: Polymer integrity after pepsin-KOH-digestion.
Alkaline step conducted at 60 ºC if not noted otherwise.
/ not suited for testing; - could not be identified; +/- strong
degradation but polymer type indicated; + no significant
differences to virgin polymer
Optimisation: The importance of filter choice
6MRI – Institut für Sicherheit und Qualität bei Milch und Fisch
• improving filtration speed & preventing filter clogging, depending on…
→ pore size: larger pore size = less prone to clogging, but also loss of
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
→ e.g. inorganic filters for thermal analysis
• filter structure: impact on particle retention & detection[20]
→ 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]
(e.g. polycarbonate, Al2O3)
nylon cotton fiber
mixed cellulose polycarbonate
Figure 7: SEM-image of surface
morphology-types of membrane filters;
Cai et al. (2020).
knitted lattice pressed fiber
multilayer-hole singlelayer-hole
Figure 6: Photograph of membrane
filters (pore size ~ 1 µm) after filtering
digested fish fillet.
cellulose nitrate glass fiber
cellulose acetate polycarbonate
15.04.2021
Optimisation: The importance of filter choice
7MRI – Institut für Sicherheit und Qualität bei Milch und Fisch
→ consideration of focal plane of particles for imaging/filter scan
• perspective: adsorption of nanoparticles → incomplete separation
→ further research regarding filtration requiredFigure 9: SEM-image of polycarbonate filter
(pore size 1 µm) with agglomerated Ø100 nm-PS
adhering to the pores & matrix residues.
15.04.2021
Figure 8: Fluorescent PA12-particles on
glass fiber filters. Scan on same focal plane
(left) and stacked images of confocal scan
(range 100 µm).
MRI – Institut für Sicherheit und Qualität bei Milch und Fisch 8
Optimisation: Preventing procedural plastic contamination
• small plastic particles are ubiquitous →
monitoring & mitigation of contamination
• investigating probable sources
→ insufficiently cleaned glassware
→ reagents / solvents
→ exposure of samples to air
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.
0
500
1000
1500
2000
2500
part
icle
num
ber
Figure 10: Number of MP-suspect particles rinsed off glass
flasks after application of different cleaning procedures.
15.04.2021
MRI – Institut für Sicherheit und Qualität bei Milch und Fisch 9
Optimisation: Preventing procedural plastic contamination
• current protocol for contamination prevention
→ cotton clothes, laminar flow workbench
→ pre-filtration (pore size < 1 µm) of all
reagents & solutions
→ cleaning of glassware [and filters]
dishwasher, heating (500 ºC), rinsing
→ rinsing of filtration apparatus between
each sample (3x 10 mL filtered water)
• monitoring of blank samples still required
Figure 12: Number of fluorescent particles (Nile red staining, FITC-filter) of
heated glassware, a simulated extraction procedure and sedimented particles
from air.
0
50
100
150
200
250
300
350
400
450
heatedglassware
fume hood laminarflow
laboratory fume hood laminarflow
part
icle
num
ber
2 – 10 µm 11 – 20 µm
21 – 50 µm 51 – 100 µm
101 – 200 µm
extraction sedimentation
0
10
20
30
Figure 13: Number MP-suspect particles in blank samples.
15.04.2021
Validation of the optimised sample preparation protocol
10MRI – Institut für Sicherheit und Qualität bei Milch und Fisch
dig
estio
nfitr
ation
& p
ost-
filtra
tio
n tre
atm
en
t
Figure 14: Schematic overview of optimised sample preparation protocol.
Recovery
further research for
quantification of plastics
with py-GC/MS needed
n = 10 88 ± 16 %
n = 100 89 ± 12 %
n = 1000 103 ± 13 %
m/z1 = 70
m/z2 = 111
m/z = 122
m/z1 = 82
m/z2 = 83
m/z1 = 130
m/z2 = 117
m/z = 113
PP
PET
PE
PS
PA-6
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.
15.04.2021illustrations: designed by freepik.com
lab equipment: Landesbildungsserver Baden-Württemberg
Prospective: Consideration of nanoplastics
11MRI – Institut für Sicherheit und Qualität bei Milch und Fisch
• concentration and separation of nano- and microplastics
• detection limit in field-flow-fractionation
• identification of plastic in the fractions
Sample
preparation?
Detection limit?< 1 µm
≥ 1 µm
15.04.2021lab equipment: Landesbildungsserver Baden-Württemberg
Figure 16: AF4-separation of different amounts of nanoplastics.
Summary
12Max Rubner-Institut – Bundesforschungsinstitut für Ernährung und Lebensmittel 15/04/2021
• 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.
Thank you for your support…
13Max Rubner-Institut – Bundesforschungsinstitut für Ernährung und Lebensmittel
Food Technology and
Bioprocess Engineering
Ralf Greiner
Elke Walz
Birgit Hetzer
Andrea Tauer
Christian Geuter
Safety and Quality of Milk
and Fish
Jan Fritsche
Torsten Krause
Dierk Martin
Ute Ostermeyer
Enken Jacobsen
Björn Neumann
Longina Reimann
University of Hamburg Center for Earth System
Research and Sustainability
Elke Fischer
Matthias Tamminga
…
Food Chemistry
Technical University
BerlinSascha Rohn
Federal Research Institute
of Nutrition and Food
15.04.2021
Thank you for your
attention!
14MRI – Institut für Sicherheit und Qualität bei Milch und Fisch 15.04.2021
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15MRI – Institut für Sicherheit und Qualität bei Milch und Fisch 15.04.2021
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16MRI – Institut für Sicherheit und Qualität bei Milch und Fisch 15.04.2021