Evaluation and optimisation of extraction methods suitable

Bild

Dora

de

: ©

Pe

ter

Kir

ch

ho

ff/P

IXE

LIO

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?


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


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


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


• 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


→ 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


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


• 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

[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."


[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.

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."


[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.

References


Documents

Evaluation and optimisation of extraction methods suitable