Dissertations in Forestry and Natural Sciences
DISSERTATIONS | EMILIA UURASJÄRVI | MICROPLASTICS – A CHALLENGE FOR ENVIRONMENTAL ANALYTICAL CHEMISTRY |
EMILIA UURASJÄRVI
Microplastics – A challenge for environmental analytical chemistry
PUBLICATIONS OF
THE UNIVERSITY OF EASTERN FINLAND
MICROPLASTICS – A CHALLENGE FOR ENVIRONMENTAL ANALYTICAL
CHEMISTRY
Emilia Uurasjärvi
MICROPLASTICS – A CHALLENGE FOR ENVIRONMENTAL ANALYTICAL
CHEMISTRY
Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences
No 433
University of Eastern Finland Kuopio
2021
Academic dissertation
To be presented by permission of the Faculty of Science and Forestry for public examination in the Auditorium MS300 in the Medistudia Building at the University of Eastern Finland, Kuopio, on November 12,
2021, at 12 o’clock
Emilia Uurasjärvi
MICROPLASTICS – A CHALLENGE FOR ENVIRONMENTAL ANALYTICAL
CHEMISTRY
Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences
No 433
University of Eastern Finland Kuopio
2021
Academic dissertation
To be presented by permission of the Faculty of Science and Forestry for public examination in the Auditorium MS300 in the Medistudia Building at the University of Eastern Finland, Kuopio, on November 12,
PunaMusta Oy Joensuu, 2021 Editor: Nina Hakulinen
Distribution: University of Eastern Finland / Sales of publications www.uef.fi/kirjasto
ISBN: 978-952-61-4322-4 (nid.) ISBN: 978-952-61-4323-1 (PDF)
ISSNL: 1798-5668 ISSN: 1798-5668 ISSN: 1798-5676 (PDF)
Author’s address: Emilia Uurasjärvi
University of Eastern Finland SIB Labs
P.O. Box 1627
70211 Kuopio, FINLAND email: emilia.uurasjarvi@uef.fi
Supervisors: Director of Research Infrastructure, Adjunct professor (Docent) Arto Koistinen, Ph.D. University of Eastern Finland
SIB Labs P.O. Box 1627
70211 Kuopio, FINLAND email: arto.koistinen@uef.fi Professor Mika Suvanto, Ph.D. University of Eastern Finland Depart. of Chemistry
P.O. Box 111
80101 Joensuu, FINLAND email: mika.suvanto@uef.fi
Research Director Marko Lehtonen, Ph.D. University of Eastern Finland
School of Pharmacy P.O.Box 1627
70211 Kuopio, FINLAND email: marko.lehtonen@uef.fi
Reviewers: Associate Professor Jesus Javier Ojeda Ledo, Ph.D Swansea University
College of Engineering Bay Campus
Fabian Way Crymlyn Burrows Swansea
SA1 8EN Wales, UK
email: j.j.ojedaledo@swansea.ac.uk
PunaMusta Oy Joensuu, 2021 Editor: Nina Hakulinen
Distribution: University of Eastern Finland / Sales of publications www.uef.fi/kirjasto
ISBN: 978-952-61-4322-4 (nid.) ISBN: 978-952-61-4323-1 (PDF)
ISSNL: 1798-5668 ISSN: 1798-5668 ISSN: 1798-5676 (PDF)
Author’s address: Emilia Uurasjärvi
University of Eastern Finland SIB Labs
P.O. Box 1627
70211 Kuopio, FINLAND email: emilia.uurasjarvi@uef.fi
Supervisors: Director of Research Infrastructure, Adjunct professor (Docent) Arto Koistinen, Ph.D.
University of Eastern Finland SIB Labs
P.O. Box 1627
70211 Kuopio, FINLAND email: arto.koistinen@uef.fi Professor Mika Suvanto, Ph.D.
University of Eastern Finland Depart. of Chemistry
P.O. Box 111
80101 Joensuu, FINLAND email: mika.suvanto@uef.fi
Research Director Marko Lehtonen, Ph.D.
University of Eastern Finland School of Pharmacy
P.O.Box 1627
70211 Kuopio, FINLAND email: marko.lehtonen@uef.fi
Reviewers: Associate Professor Jesus Javier Ojeda Ledo, Ph.D Swansea University
College of Engineering Bay Campus
Fabian Way Crymlyn Burrows Swansea
SA1 8EN Wales, UK
email: j.j.ojedaledo@swansea.ac.uk
Associate Professor Kristian Syberg, Ph.D Roskilde University
Department of Science and Environment Universitetsvej 1, 11.2
DK-4000 Roskilde Denmark
email: ksyberg@ruc.dk Opponent: Professor Denise Mitrano
ETH Zürich
Department of Environmental Systems Science Universitätstrasse 16
8092 Zürich Switzerland
Email: denise.mitrano@usys.ethz.ch
Uurasjärvi, Emilia
Microplastics – A challenge for environmental analytical chemistry Kuopio: University of Eastern Finland, 2021
Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences 2021; 433 ISBN: 978-952-61-4322-4 (print)
ISSNL: 1798-5668 ISSN: 1798-5668
ISBN: 978-952-61-4323-1 (PDF) ISSN: 1798-5676 (PDF)
ABSTRACT
Microplastics, commonly defined as 1 µm – 5 mm plastic particles, are emerging environmental pollutants, which cause risks especially to aquatic organisms.
Microplastics are abundant worldwide in marine and freshwater environments in surface water, water column, sediments, and biota. Administrations, organizations, and scientists have proposed that concentrations of microplastics in the environment should be monitored to assess and reduce the risks they may cause. However, the knowledge about concentrations and types of microplastics is still limited, especially in freshwater environments. Furthermore, no harmonized or standardized methods exist for sampling and analysis of microplastics, and the current commonly used methods have not been validated comprehensively for qualitative and quantitative analysis of microplastics. The aim of this study was to develop and validate methods to analyse particle concentrations, sizes, and polymer types of microplastics from environmental samples to monitor Finnish water environments.
Surface water and fish samples were collected from Lake Kallavesi, and water column samples from the Baltic Sea. Sample pre-treatment methods were developed to separate microplastics from other solid materials. Pre-treatments included decomposition of non-synthetic polymers with hydrogen peroxide and digestion of targeted biomolecules with enzymes. The choice of reagents depended on the composition of samples. Finally, separated microplastics were filtered and measured with single-point or imaging focal plane array (FPA) Fourier transform infrared (FTIR) microspectroscopy. The methods were validated with blanks and spiked recovery samples.
Microplastics were detected from both surface waters and fish of Lake Kallavesi, but the particle concentrations in water were lower than in other lakes located in more densely populated areas. About one fifth of the studied fish contained microplastics,
Associate Professor Kristian Syberg, Ph.D Roskilde University
Department of Science and Environment Universitetsvej 1, 11.2
DK-4000 Roskilde Denmark
email: ksyberg@ruc.dk Opponent: Professor Denise Mitrano
ETH Zürich
Department of Environmental Systems Science Universitätstrasse 16
8092 Zürich Switzerland
Email: denise.mitrano@usys.ethz.ch
Uurasjärvi, Emilia
Microplastics – A challenge for environmental analytical chemistry Kuopio: University of Eastern Finland, 2021
Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences 2021; 433 ISBN: 978-952-61-4322-4 (print)
ISSNL: 1798-5668 ISSN: 1798-5668
ISBN: 978-952-61-4323-1 (PDF) ISSN: 1798-5676 (PDF)
ABSTRACT
Microplastics, commonly defined as 1 µm – 5 mm plastic particles, are emerging environmental pollutants, which cause risks especially to aquatic organisms.
Microplastics are abundant worldwide in marine and freshwater environments in surface water, water column, sediments, and biota. Administrations, organizations, and scientists have proposed that concentrations of microplastics in the environment should be monitored to assess and reduce the risks they may cause. However, the knowledge about concentrations and types of microplastics is still limited, especially in freshwater environments. Furthermore, no harmonized or standardized methods exist for sampling and analysis of microplastics, and the current commonly used methods have not been validated comprehensively for qualitative and quantitative analysis of microplastics. The aim of this study was to develop and validate methods to analyse particle concentrations, sizes, and polymer types of microplastics from environmental samples to monitor Finnish water environments.
Surface water and fish samples were collected from Lake Kallavesi, and water column samples from the Baltic Sea. Sample pre-treatment methods were developed to separate microplastics from other solid materials. Pre-treatments included decomposition of non-synthetic polymers with hydrogen peroxide and digestion of targeted biomolecules with enzymes. The choice of reagents depended on the composition of samples. Finally, separated microplastics were filtered and measured with single-point or imaging focal plane array (FPA) Fourier transform infrared (FTIR) microspectroscopy. The methods were validated with blanks and spiked recovery samples.
Microplastics were detected from both surface waters and fish of Lake Kallavesi, but the particle concentrations in water were lower than in other lakes located in more densely populated areas. About one fifth of the studied fish contained microplastics,
and the results indicated that fish had ingested microplastics coincidentally, independently of habitat and feeding behaviour. In the Baltic Sea, microplastics were observed to accumulate in the water column to layers, where density, temperature, and salinity of water change rapidly. Stratification prevents vertical mixing of water and based on the results likely affects the vertical abundance and sinking of microplastics, too.
Method validation indicated that the more rigorously samples are pre-treated, the more prone they are to contamination, which increases the limit of detection.
However, the recovery tests showed that rigorous pre-treatments are necessary to separate microplastics efficiently. Other materials covering microplastics prevent the spectroscopic identification, which lowers the selectivity and the recovery rate of the analysis. Before harmonizing or standardizing analysis methods for microplastics, more comprehensive validation with various suitable validation parameters and reference materials is needed. Moreover, the methods need development especially for separating small microplastics efficiently without contaminating samples.
Keywords: Microplastics, plastic litter, water pollution, environmental monitoring, environmental analytical chemistry, method validation, quality control, infrared spectroscopy, microspectroscopy, lakes, seas, Kallavesi, Baltic Sea
Library of Congress Subject Headings: Microplastics, Plastic scrap, Water – Pollution, Environmental monitoring, Speciation (Chemistry) - Methodology, Water – Analysis, Methodology - Quality control, Infrared spectroscopy, Particles – Spectra, Lakes, Seas, Finland, Kallavesi, Baltic sea
Universal Decimal Classification: 543.39, 543.42
ACKNOWLEDGEMENTS
This study was conducted in University of Eastern Finland, SIB Labs infrastructure unit in 2018–2021. It was done in the Doctoral Program in Science, Technology and Computing (SCITECO) and Department of Chemistry, but in close collaboration with Department of Biological and Environmental sciences, School of Pharmacy and Finnish Environment Institute. Academy of Finland (MIF, no 296384/296169 and Dimpex, no 330181) funded the study. Moreover, Regional Council of Pohjois-Savo funded the instrument, which was essential for this study. I thank the funders.
I thank my supervisors PhD Arto Koistinen, Professor Mika Suvanto, and PhD Marko Lehtonen. Special thanks to Arto, who introduced me to the microplastic research and was the initiator of this dissertation. Moreover, I thank my collaborators and co-authors in Finnish Environment Institute (SYKE): Maiju Lehtiniemi, Outi Setälä, Erika Sainio, Julia Talvitie, Pinja Näkki, and Anna-Riina Mustonen. Maiju and Outi, your help in the writing process of the articles was invaluable! I thank all SYKE people for collaboration and help with the laboratory methods. I always enjoyed visiting your lab! It was not easy to crystallize the ideas to people from varying backgrounds, but finally the outcome was this way better than I would have been able to formulate on my own. It has been a privilege to work with experts of many fields and get new perspective to this subject and the world.
I thank my past and present co-workers in SIB Labs – Virpi Miettinen, Ritva Savolainen, Tuomo Silvast, Kaisa Raninen, Mikko Selenius, Jari T.T. Leskinen, Taru Rahkonen, Tuomo Soininen, Samuel Hartikainen, Jani Tuovinen, Laura Tomppo, and Leila Tiihonen, many thanks for help, good team spirit and warm working atmosphere. Thank you for tolerating my twisted sense of humour and habit to tell opinions even when nobody is interested in them. Moreover, I thank Minna Pääkkönen, Hanna Kolari, Niko Kinnunen, and Patrik Lahtinen for excellent MSc theses and laboratory work. Special thanks to Minna for being co-author in this thesis.
Finally, I thank Mikko Heimonen, my partner in life and wilderness adventures, for patient listening when I was moaning and groaning about this dissertation and other projects.
“Tietä käyden tien on vanki, vapaa on vain umpihanki.” – Aaro Hellaakoski Kuopio, 12.11.2021
Emilia Uurasjärvi
and the results indicated that fish had ingested microplastics coincidentally, independently of habitat and feeding behaviour. In the Baltic Sea, microplastics were observed to accumulate in the water column to layers, where density, temperature, and salinity of water change rapidly. Stratification prevents vertical mixing of water and based on the results likely affects the vertical abundance and sinking of microplastics, too.
Method validation indicated that the more rigorously samples are pre-treated, the more prone they are to contamination, which increases the limit of detection.
However, the recovery tests showed that rigorous pre-treatments are necessary to separate microplastics efficiently. Other materials covering microplastics prevent the spectroscopic identification, which lowers the selectivity and the recovery rate of the analysis. Before harmonizing or standardizing analysis methods for microplastics, more comprehensive validation with various suitable validation parameters and reference materials is needed. Moreover, the methods need development especially for separating small microplastics efficiently without contaminating samples.
Keywords: Microplastics, plastic litter, water pollution, environmental monitoring, environmental analytical chemistry, method validation, quality control, infrared spectroscopy, microspectroscopy, lakes, seas, Kallavesi, Baltic Sea
Library of Congress Subject Headings: Microplastics, Plastic scrap, Water – Pollution, Environmental monitoring, Speciation (Chemistry) - Methodology, Water – Analysis, Methodology - Quality control, Infrared spectroscopy, Particles – Spectra, Lakes, Seas, Finland, Kallavesi, Baltic sea
Universal Decimal Classification: 543.39, 543.42
ACKNOWLEDGEMENTS
This study was conducted in University of Eastern Finland, SIB Labs infrastructure unit in 2018–2021. It was done in the Doctoral Program in Science, Technology and Computing (SCITECO) and Department of Chemistry, but in close collaboration with Department of Biological and Environmental sciences, School of Pharmacy and Finnish Environment Institute. Academy of Finland (MIF, no 296384/296169 and Dimpex, no 330181) funded the study. Moreover, Regional Council of Pohjois-Savo funded the instrument, which was essential for this study. I thank the funders.
I thank my supervisors PhD Arto Koistinen, Professor Mika Suvanto, and PhD Marko Lehtonen. Special thanks to Arto, who introduced me to the microplastic research and was the initiator of this dissertation. Moreover, I thank my collaborators and co-authors in Finnish Environment Institute (SYKE): Maiju Lehtiniemi, Outi Setälä, Erika Sainio, Julia Talvitie, Pinja Näkki, and Anna-Riina Mustonen. Maiju and Outi, your help in the writing process of the articles was invaluable! I thank all SYKE people for collaboration and help with the laboratory methods. I always enjoyed visiting your lab! It was not easy to crystallize the ideas to people from varying backgrounds, but finally the outcome was this way better than I would have been able to formulate on my own. It has been a privilege to work with experts of many fields and get new perspective to this subject and the world.
I thank my past and present co-workers in SIB Labs – Virpi Miettinen, Ritva Savolainen, Tuomo Silvast, Kaisa Raninen, Mikko Selenius, Jari T.T. Leskinen, Taru Rahkonen, Tuomo Soininen, Samuel Hartikainen, Jani Tuovinen, Laura Tomppo, and Leila Tiihonen, many thanks for help, good team spirit and warm working atmosphere. Thank you for tolerating my twisted sense of humour and habit to tell opinions even when nobody is interested in them. Moreover, I thank Minna Pääkkönen, Hanna Kolari, Niko Kinnunen, and Patrik Lahtinen for excellent MSc theses and laboratory work. Special thanks to Minna for being co-author in this thesis.
Finally, I thank Mikko Heimonen, my partner in life and wilderness adventures, for patient listening when I was moaning and groaning about this dissertation and other projects.
“Tietä käyden tien on vanki, vapaa on vain umpihanki.” – Aaro Hellaakoski Kuopio, 12.11.2021
Emilia Uurasjärvi
LIST OF ABBREVIATIONS
ABS acrylonitrile butadiene styrene ATR attenuated total reflection CCD charge-coupled device ECHA European Chemical Agency
EDS energy-dispersive X-ray spectroscopy FTIR Fourier transform infrared spectroscopy FPA focal plane array
GC-MS gas chromatography mass spectrometry
GESAMP Joint Group of Experts on the Scientific Aspects on Marine Environmental Protection
GIT gastrointestinal tract
IR infrared
IUPAC International Union of Pure and Applied Chemistry LOD limit of detection
LOQ limit of quantitation MP microplastic
MSFD European Marine Strategy Framework Directive NIST National Institute of Standards and Technology
PA polyamide
PAN polyacrylonitrile
PE polyethylene
PET polyethylene terephthalate PMMA polymethyl methacrylate
PP polypropylene
PS polystyrene
PU polyurethane
PVC polyvinyl chloride Pyr pyrolysis
QA quality assurance QC quality control
QCL quantum cascade laser QMS quality management system RSD relative standard deviation SD standard deviation
SDS sodium dodecyl sulphate SEM scanning electron microscopy
SERS surface enhanced Raman spectroscopy SPT sodium polytungstate
TED thermal extraction and desorption TGA thermogravimetric analysis
UEPP Universal enzymatic purification protocol
UV ultraviolet
LIST OF ABBREVIATIONS
ABS acrylonitrile butadiene styrene ATR attenuated total reflection CCD charge-coupled device ECHA European Chemical Agency
EDS energy-dispersive X-ray spectroscopy FTIR Fourier transform infrared spectroscopy FPA focal plane array
GC-MS gas chromatography mass spectrometry
GESAMP Joint Group of Experts on the Scientific Aspects on Marine Environmental Protection
GIT gastrointestinal tract
IR infrared
IUPAC International Union of Pure and Applied Chemistry LOD limit of detection
LOQ limit of quantitation MP microplastic
MSFD European Marine Strategy Framework Directive NIST National Institute of Standards and Technology
PA polyamide
PAN polyacrylonitrile
PE polyethylene
PET polyethylene terephthalate PMMA polymethyl methacrylate
PP polypropylene
PS polystyrene
PU polyurethane
PVC polyvinyl chloride
Pyr pyrolysis
QA quality assurance QC quality control
QCL quantum cascade laser QMS quality management system RSD relative standard deviation SD standard deviation
SDS sodium dodecyl sulphate SEM scanning electron microscopy
SERS surface enhanced Raman spectroscopy SPT sodium polytungstate
TED thermal extraction and desorption TGA thermogravimetric analysis
UEPP Universal enzymatic purification protocol UV ultraviolet
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on data presented in the following articles, referred to by the Roman numerals I-III.
I Uurasjärvi E., Hartikainen S., Setälä O., Lehtiniemi M., Koistinen A.
Microplastic concentrations, size distribution, and polymer types in the surface waters of a northern European lake. Water Environment research, 2020, 92, 149 – 156, https://doi.org/10.1002/wer.1229
II Uurasjärvi E., Pääkkönen M., Setälä O., Koistinen A., Lehtiniemi M.
Microplastics accumulate to thin layers in the stratified Baltic Sea.
Environmental Pollution, 2021, 268, 115700, https://doi.org/10.1016/j.envpol.2020.115700
III Uurasjärvi E., Sainio E., Setälä O., Lehtiniemi M., Koistinen A. Validation of an imaging FTIR spectroscopic method for analyzing microplastics ingestion by Finnish lake fish (Perca fluviatilis and Coregonus albula). Environmental Pollution, 2021, 288, 117780, https://doi.org/10.1016/j.envpol.2021.117780
The original publications are referred by these Roman numerals throughout the thesis.
AUTHOR’S CONTRIBUTION
I The author performed microscopy and FTIR analysis of microplastics.
Moreover, the author analysed the data and was the main writer of the paper.
Other authors planned and conducted the sampling. S. Hartikainen pre-treated samples. O. Setälä, M. Lehtiniemi and A. Koistinen supervised the study and edited and revised the manuscript.
II The author planned sample preparation, analysis, and quality control methods. The author performed them and data analysis together with M.
Pääkkönen and was the main writer of the paper. O. Setälä and M. Lehtiniemi planned and conducted the sampling. O. Setälä, A. Koistinen and M.
Lehtiniemi supervised the study and edited and revised the manuscript.
III The author performed infrared imaging analysis of samples, data analysis, quality control, and was the main writer of the paper. E. Sainio prepared fish samples for FTIR imaging and participated in the writing and editing of the manuscript. O. Setälä, M. Lehtiniemi and A. Koistinen planned the study, collected samples, supervised the study, and participated in the writing and editing of the manuscript.
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on data presented in the following articles, referred to by the Roman numerals I-III.
I Uurasjärvi E., Hartikainen S., Setälä O., Lehtiniemi M., Koistinen A.
Microplastic concentrations, size distribution, and polymer types in the surface waters of a northern European lake. Water Environment research, 2020, 92, 149 – 156, https://doi.org/10.1002/wer.1229
II Uurasjärvi E., Pääkkönen M., Setälä O., Koistinen A., Lehtiniemi M.
Microplastics accumulate to thin layers in the stratified Baltic Sea.
Environmental Pollution, 2021, 268, 115700, https://doi.org/10.1016/j.envpol.2020.115700
III Uurasjärvi E., Sainio E., Setälä O., Lehtiniemi M., Koistinen A. Validation of an imaging FTIR spectroscopic method for analyzing microplastics ingestion by Finnish lake fish (Perca fluviatilis and Coregonus albula). Environmental Pollution, 2021, 288, 117780, https://doi.org/10.1016/j.envpol.2021.117780
The original publications are referred by these Roman numerals throughout the thesis.
AUTHOR’S CONTRIBUTION
I The author performed microscopy and FTIR analysis of microplastics.
Moreover, the author analysed the data and was the main writer of the paper.
Other authors planned and conducted the sampling. S. Hartikainen pre-treated samples. O. Setälä, M. Lehtiniemi and A. Koistinen supervised the study and edited and revised the manuscript.
II The author planned sample preparation, analysis, and quality control methods. The author performed them and data analysis together with M.
Pääkkönen and was the main writer of the paper. O. Setälä and M. Lehtiniemi planned and conducted the sampling. O. Setälä, A. Koistinen and M.
Lehtiniemi supervised the study and edited and revised the manuscript.
III The author performed infrared imaging analysis of samples, data analysis, quality control, and was the main writer of the paper. E. Sainio prepared fish samples for FTIR imaging and participated in the writing and editing of the manuscript. O. Setälä, M. Lehtiniemi and A. Koistinen planned the study, collected samples, supervised the study, and participated in the writing and editing of the manuscript.
CONTENTS
ABSTRACT ... 7
ACKNOWLEDGEMENTS ... 9
1 INTRODUCTION ... 17
2 MICROPLASTICS AS ANALYTICAL SAMPLES ... 19
3 SAMPLE PRE-TREATMENT FOR IMAGING SPECTROSCOPY ... 21
4 INSTRUMENTAL TECHNIQUES FOR ANALYSIS OF MICROPLASTICS ... 27
4.1 Light microscopy ... 28
4.2 Vibrational spectroscopy – theoretical background ... 28
4.3 Fourier transform infrared spectroscopy (FTIR) ... 30
4.3.1 Measurement techniques in FTIR spectroscopy ... 31
4.3.2 FPA-FTIR in practise ... 32
4.3.3 Data analysis methods ... 34
4.4 Raman spectroscopy ... 35
4.5 Other common methods for identification and quantitation of MPs ... 37
5 QUALITY CONTROL AND ASSURANCE ... 38
5.1 Representativeness and stability of MP samples... 39
5.2 Contamination ... 40
5.3 Validation parameters for FPA-FTIR ... 41
5.3.1 Selectivity, specificity, and matrix interference ... 41
5.3.2 Recovery ... 42
5.3.3 Accuracy, trueness, repeatability, precision, and uncertainty ... 43
5.3.4 Working range, sensitivity and limit of detection and quantitation ... 44
5.4 Reporting the results ... 46
6 AIMS OF THE STUDY ... 48
7 METHODS ... 49
7.1 Sampling ... 49
7.2 Pre-treatment and laboratory QC/QA ... 53
7.3 FPA-FTIR and data analysis in Publications II and III ... 54
8 RESULTS AND DISCUSSION ... 58
8.1 Microplastic concentrations in the Baltic Sea and Lake Kallavesi ... 58
8.2 Plastic types and particle sizes ... 61
8.3 Evaluation of the performance of the methods ... 64
CONTENTS
ABSTRACT ... 7
ACKNOWLEDGEMENTS ... 9
1 INTRODUCTION ... 17
2 MICROPLASTICS AS ANALYTICAL SAMPLES ... 19
3 SAMPLE PRE-TREATMENT FOR IMAGING SPECTROSCOPY ... 21
4 INSTRUMENTAL TECHNIQUES FOR ANALYSIS OF MICROPLASTICS ... 27
4.1 Light microscopy ... 28
4.2 Vibrational spectroscopy – theoretical background ... 28
4.3 Fourier transform infrared spectroscopy (FTIR) ... 30
4.3.1 Measurement techniques in FTIR spectroscopy ... 31
4.3.2 FPA-FTIR in practise ... 32
4.3.3 Data analysis methods ... 34
4.4 Raman spectroscopy ... 35
4.5 Other common methods for identification and quantitation of MPs ... 37
5 QUALITY CONTROL AND ASSURANCE ... 38
5.1 Representativeness and stability of MP samples... 39
5.2 Contamination ... 40
5.3 Validation parameters for FPA-FTIR ... 41
5.3.1 Selectivity, specificity, and matrix interference ... 41
5.3.2 Recovery ... 42
5.3.3 Accuracy, trueness, repeatability, precision, and uncertainty ... 43
5.3.4 Working range, sensitivity and limit of detection and quantitation ... 44
5.4 Reporting the results ... 46
6 AIMS OF THE STUDY ... 48
7 METHODS ... 49
7.1 Sampling ... 49
7.2 Pre-treatment and laboratory QC/QA ... 53
7.3 FPA-FTIR and data analysis in Publications II and III ... 54
8 RESULTS AND DISCUSSION ... 58
8.1 Microplastic concentrations in the Baltic Sea and Lake Kallavesi ... 58
8.2 Plastic types and particle sizes ... 61
8.3 Evaluation of the performance of the methods ... 64
8.3.1 Contamination controls and limits of detection ...64
8.3.2 Recovery rate tests ...66
8.3.3 FPA-FTIR and data analysis ...68
8.3.4 Conclusions about the methods and future recommendations ...69
9 CONCLUSIONS ... 71
BIBLIOGRAPHY ... 72
1 INTRODUCTION
The presence of plastic waste in the oceans was first scientifically documented in 1970s (Carpenter and Smith, 1972). Since that, the production of plastic and the amount of plastic waste entering the oceans has grown substantially (Geyer et al., 2017), though the plastic waste problem has gained wide public attention and concern (Barnes et al., 2009). The first scientific article about the presence of microplastics (MPs) in the marine environment was published in early 2000’s (Thompson et al., 2004). During the last two decades, the (micro)plastic pollution has gained wide attention among researchers and the public audience, denominated as one of the major current environmental problems. Monitoring the concentrations of MPs in the environment is considered important, because they can cause environmental risks by various mechanisms (Besseling et al., 2019).
To date, MPs have been found worldwide in marine environments, consisting of water column and sediments (Auta et al., 2017; GESAMP, 2016). Because high amount of plastic waste produced in land finally end up in the oceans, the focus of MP research has been on the marine environments (Jambeck et al., 2015). However, freshwater environments, such as lakes and rivers, can similarly suffer from the (micro)plastic pollution than marine environments (Eerkes-Medrano et al., 2015; Li et al., 2018). Therefore, they are studied both as routes of MPs to the oceans, and environments vulnerable to the plastic pollution themselves.
However, the consented definition of “microplastic” lacks still. Mostly, MPs are defined as small plastic particles, which are insoluble to water and have size between 1 µm – 5 mm (Frias and Nash, 2019; GESAMP, 2016). An alternative suggestion defines MPs as plastic particles on a size range 1 µm – 1 mm (Hartmann et al., 2019).
Moreover, Hartmann et al. (2019) define the chemical and physical properties of MPs in more detail: MPs are particles, which consist of synthetic or semi-synthetic polymer(s), and are solid and insoluble to water at 20 °C. Slightly modified natural polymers, such as viscose, are usually not considered as MPs. MPs can be categorized according to their origin to “primary” MPs, which are manufactured to micro size, or “secondary”, which have been fragmented from larger plastic items. Though MPs are usually categorized as one group, they are chemically and physically very diverse, as plastics are produced from wide selection of reagents and the size and shape of MPs vary remarkably (Hahladakis et al., 2018; Rochman et al., 2019).
Therefore, various analytical methods are needed for identifying chemical composition of MPs and quantifying them from environmental samples.
The legislation and management of plastic waste, single-use plastics and MPs is under development in many countries worldwide, aiming to tackle the plastic
8.3.1 Contamination controls and limits of detection ...64
8.3.2 Recovery rate tests ...66
8.3.3 FPA-FTIR and data analysis ...68
8.3.4 Conclusions about the methods and future recommendations ...69
9 CONCLUSIONS ... 71
BIBLIOGRAPHY ... 72
1 INTRODUCTION
The presence of plastic waste in the oceans was first scientifically documented in 1970s (Carpenter and Smith, 1972). Since that, the production of plastic and the amount of plastic waste entering the oceans has grown substantially (Geyer et al., 2017), though the plastic waste problem has gained wide public attention and concern (Barnes et al., 2009). The first scientific article about the presence of microplastics (MPs) in the marine environment was published in early 2000’s (Thompson et al., 2004). During the last two decades, the (micro)plastic pollution has gained wide attention among researchers and the public audience, denominated as one of the major current environmental problems. Monitoring the concentrations of MPs in the environment is considered important, because they can cause environmental risks by various mechanisms (Besseling et al., 2019).
To date, MPs have been found worldwide in marine environments, consisting of water column and sediments (Auta et al., 2017; GESAMP, 2016). Because high amount of plastic waste produced in land finally end up in the oceans, the focus of MP research has been on the marine environments (Jambeck et al., 2015). However, freshwater environments, such as lakes and rivers, can similarly suffer from the (micro)plastic pollution than marine environments (Eerkes-Medrano et al., 2015; Li et al., 2018). Therefore, they are studied both as routes of MPs to the oceans, and environments vulnerable to the plastic pollution themselves.
However, the consented definition of “microplastic” lacks still. Mostly, MPs are defined as small plastic particles, which are insoluble to water and have size between 1 µm – 5 mm (Frias and Nash, 2019; GESAMP, 2016). An alternative suggestion defines MPs as plastic particles on a size range 1 µm – 1 mm (Hartmann et al., 2019).
Moreover, Hartmann et al. (2019) define the chemical and physical properties of MPs in more detail: MPs are particles, which consist of synthetic or semi-synthetic polymer(s), and are solid and insoluble to water at 20 °C. Slightly modified natural polymers, such as viscose, are usually not considered as MPs. MPs can be categorized according to their origin to “primary” MPs, which are manufactured to micro size, or “secondary”, which have been fragmented from larger plastic items. Though MPs are usually categorized as one group, they are chemically and physically very diverse, as plastics are produced from wide selection of reagents and the size and shape of MPs vary remarkably (Hahladakis et al., 2018; Rochman et al., 2019).
Therefore, various analytical methods are needed for identifying chemical composition of MPs and quantifying them from environmental samples.
The legislation and management of plastic waste, single-use plastics and MPs is under development in many countries worldwide, aiming to tackle the plastic
pollution problem (Lam et al., 2018). For example, the State of California, in the United States of America, has pioneered in developing the legislation for MPs. It has enacted two bills, which require monitoring of MPs from drinking water and aquatic environments (Wyer et al., 2020). However, the Californian statewide MPs strategy acknowledges the need to develop analytical methods before the monitoring is viable. It requires the ocean protection council to “Develop standardized methodologies for sampling, detecting, and characterizing microplastics in the environment”. Moreover, international organizations such as GESAMP (Joint Group of Experts on the Scientific Aspects of Marine Environmental Protection) have created guidelines for monitoring plastic litter and MPs in the oceans (GESAMP, 2019).
In the European Union, the European Chemical Agency (ECHA) has published ANNEX XV restriction report, which aims to restrict intentionally added MPs in consumer products, such as microbeads in cosmetics (ECHA, 2019). However, the EU legislation has not been enacted yet. Moreover, the EU circular economy action plan addresses that important actions to tackle the (micro)plastic pollution would be for example development and harmonization of analysis methods for MPs and assessing the risk and occurrence of MPs in the environment, drinking water and foods (European Commission, 2020). EU-funded project FanpLESStic-sea -
“Initiatives to remove microplastics before they enter the sea” has more comprehensively reviewed the existing policies related to MPs (FanpLESStic-sea, 2019). European Marine Strategy Framework Directive (MSFD) (European commission, 2017) states that littering of the marine environments should be monitored and sources of micro-sized litter, including microplastics, should be resolved to develop action for reducing the amount of litter entering the seas.
Though the needs for monitoring MPs in the environment and develop methods are recognized worldwide, the analytical methods have not been harmonized or standardized yet (Koelmans et al., 2019; Provencher et al., 2020). Instead, researchers use a wide variety of methods, with varying performances. The methods for monitoring should be comparable and repeatable. Analytical chemistry plays a major role in the development of harmonized, standardized, validated, accurate and precise fit-to-purpose methods.
At the beginning of this study, the knowledge about MP concentrations in freshwaters and especially lakes was very limited. The question was, however, locally very important, as Finland is known as “land of the thousand lakes”. Actually, the proverb is a harsh underestimation, because Finland has more than 100 000 lakes, varying from small ponds to large ones. Moreover, Finland has marine environments in the Baltic Sea, which is semi-enclosed, shallow, brackish and suffers from low oxygenation and land-based load of nutrients and pollutants. The already
endangered environment may be vulnerable to the risks caused by MPs. The aim of this study was to determine, how much MPs Finnish water environments contain, and provide information for the risk assessments. Considering the state of the field at the times, this study provided also internationally new data about the concentrations of MPs in dimictic lake and stratified brackish sea, as they are unique environments globally and were not studied in detail previously. To achieve the knowledge about microplastic concentrations in Finnish waters, the fit-to-purpose methods were developed for measuring them. Therefore, the second aim of this dissertation was to develop methods for quantifying MPs from environmental samples. The third aim was to validate the methods by measuring or estimating various validation parameters: accuracy, precision, limit of detection, range, selectivity/specificity, repeatability, matrix interference, recovery, and stability.
2 MICROPLASTICS AS ANALYTICAL SAMPLES
MP research aims to analyse several parameters, such as concentrations of MPs in the environmental samples, and chemical and physical properties of MPs. Not all the parameters can be detected with single analytical method. Instead, the field requires versatile and accurate analytical methods to acquire knowledge for monitoring and risks assessments.
MPs are chemically diverse group of plastic particles, composed of synthetic polymers and additives (Rochman et al., 2019). Therefore, developing analytical methods for detecting them is not as straightforward as for detecting an analyte, which is a single molecule or an element. Analysis and methods can aim to detect all MPs generally, or selected polymer types only. However, if all MPs are aimed to be identified from a sample, the term “MP” must still be defined first. Because there are no consensus about what MP explicitly mean (Hartmann et al., 2019), the range of the analytical method should be defined by polymer types, size, or other applicable features.
MPs have several chemical and physical features, which can have environmental effects and are worth analysing (Andrady, 2017) (Figure 1). Because different analytical methods are suitable for analysing different features of MPs, the choice of methods depends on aim of the study.
pollution problem (Lam et al., 2018). For example, the State of California, in the United States of America, has pioneered in developing the legislation for MPs. It has enacted two bills, which require monitoring of MPs from drinking water and aquatic environments (Wyer et al., 2020). However, the Californian statewide MPs strategy acknowledges the need to develop analytical methods before the monitoring is viable. It requires the ocean protection council to “Develop standardized methodologies for sampling, detecting, and characterizing microplastics in the environment”. Moreover, international organizations such as GESAMP (Joint Group of Experts on the Scientific Aspects of Marine Environmental Protection) have created guidelines for monitoring plastic litter and MPs in the oceans (GESAMP, 2019).
In the European Union, the European Chemical Agency (ECHA) has published ANNEX XV restriction report, which aims to restrict intentionally added MPs in consumer products, such as microbeads in cosmetics (ECHA, 2019). However, the EU legislation has not been enacted yet. Moreover, the EU circular economy action plan addresses that important actions to tackle the (micro)plastic pollution would be for example development and harmonization of analysis methods for MPs and assessing the risk and occurrence of MPs in the environment, drinking water and foods (European Commission, 2020). EU-funded project FanpLESStic-sea -
“Initiatives to remove microplastics before they enter the sea” has more comprehensively reviewed the existing policies related to MPs (FanpLESStic-sea, 2019). European Marine Strategy Framework Directive (MSFD) (European commission, 2017) states that littering of the marine environments should be monitored and sources of micro-sized litter, including microplastics, should be resolved to develop action for reducing the amount of litter entering the seas.
Though the needs for monitoring MPs in the environment and develop methods are recognized worldwide, the analytical methods have not been harmonized or standardized yet (Koelmans et al., 2019; Provencher et al., 2020). Instead, researchers use a wide variety of methods, with varying performances. The methods for monitoring should be comparable and repeatable. Analytical chemistry plays a major role in the development of harmonized, standardized, validated, accurate and precise fit-to-purpose methods.
At the beginning of this study, the knowledge about MP concentrations in freshwaters and especially lakes was very limited. The question was, however, locally very important, as Finland is known as “land of the thousand lakes”. Actually, the proverb is a harsh underestimation, because Finland has more than 100 000 lakes, varying from small ponds to large ones. Moreover, Finland has marine environments in the Baltic Sea, which is semi-enclosed, shallow, brackish and suffers from low oxygenation and land-based load of nutrients and pollutants. The already
endangered environment may be vulnerable to the risks caused by MPs. The aim of this study was to determine, how much MPs Finnish water environments contain, and provide information for the risk assessments. Considering the state of the field at the times, this study provided also internationally new data about the concentrations of MPs in dimictic lake and stratified brackish sea, as they are unique environments globally and were not studied in detail previously. To achieve the knowledge about microplastic concentrations in Finnish waters, the fit-to-purpose methods were developed for measuring them. Therefore, the second aim of this dissertation was to develop methods for quantifying MPs from environmental samples. The third aim was to validate the methods by measuring or estimating various validation parameters: accuracy, precision, limit of detection, range, selectivity/specificity, repeatability, matrix interference, recovery, and stability.
2 MICROPLASTICS AS ANALYTICAL SAMPLES
MP research aims to analyse several parameters, such as concentrations of MPs in the environmental samples, and chemical and physical properties of MPs. Not all the parameters can be detected with single analytical method. Instead, the field requires versatile and accurate analytical methods to acquire knowledge for monitoring and risks assessments.
MPs are chemically diverse group of plastic particles, composed of synthetic polymers and additives (Rochman et al., 2019). Therefore, developing analytical methods for detecting them is not as straightforward as for detecting an analyte, which is a single molecule or an element. Analysis and methods can aim to detect all MPs generally, or selected polymer types only. However, if all MPs are aimed to be identified from a sample, the term “MP” must still be defined first. Because there are no consensus about what MP explicitly mean (Hartmann et al., 2019), the range of the analytical method should be defined by polymer types, size, or other applicable features.
MPs have several chemical and physical features, which can have environmental effects and are worth analysing (Andrady, 2017) (Figure 1). Because different analytical methods are suitable for analysing different features of MPs, the choice of methods depends on aim of the study.
Figure 1. Features of MPs to be analysed.
MP concentration can be analysed and reported for environmental samples as particle concentration (particles per sample) or mass concentration (mass of MPs per sample). Both particle and mass concentrations are recommended to analyse and report when possible, because for example numerous small particles can have the same mass than one large, or many large particles have substantially higher mass than the same number of smaller ones. These MPs cause different environmental effects, despite the number or mass being the same.
The choice of analytical method also defines which chemical components of MPs can be determined. Mostly, MPs are categorized according to the main polymer component, and it is identified in majority of the studies. However, chemically very diverse group of plastic additives can cause environmental problems (Hahladakis et al., 2018; Kwon et al., 2017) and quantitation of them requires usually different methods than quantitation of MPs by polymer types. In addition to intentionally added compounds, MPs can sorb hydrophobic organic contaminants (Hartmann et al., 2017) and heavy metals (Brennecke et al., 2016), but the importance of the risk caused by interaction of MPs and other contaminants has been critically discussed to be generally minor (Koelmans et al., 2016; Wang et al., 2019). Compared to clean and new plastics, MPs found from the environment are weathered, oxidized, and covered
by biofilms, which needs to be considered in the analysis and ecotoxicity studies (Vroom et al., 2017).
Particle size distribution is also environmentally relevant parameter to analyse, because the toxicity of MPs (Jeong et al., 2016) and ingestion of MPs by aquatic organisms (Lehtiniemi et al., 2018; Vroom et al., 2017) are size-dependent.
Additionally to chemical composition and size, morphological properties of MPs, such as shape and colour can affect the toxicity and appeal for aquatic organisms (Anbumani and Kakkar, 2018; Foley et al., 2018).
Therefore, MPs have various chemical and physical features, which are environmentally relevant to analyse. The features of environmental MPs are different compared to pristine plastics. Not a single method is suitable for analysing all the features.
3 SAMPLE PRE-TREATMENT FOR IMAGING SPECTROSCOPY
The aim of sample pre-treatment is to separate MPs from other material, which is called sample matrix. To analyse MP counts, masses or sizes, they have to be separated from other solid particles, which may distort imaging spectroscopic measurements (Löder et al., 2017). Imaging spectroscopy is not capable of measuring MPs selectively from the matrix, because when all solid particles are mixed and stacked, then signal comes from the mixture instead of single MPs. Therefore, effective pre-treatment remarkably increases the selectivity of the analysis. The same principle applies for other instrumental analysis methods than imaging spectroscopy.
Environmental samples can be for example freshwater, seawater, wastewater, sludge, sediment, air, or biota. The more the sample contains other insoluble particles than microplastics, the more laborious the separation process is (Lusher et al., 2020).
The same principles apply for the pre-treatments for all kinds of samples: organic non-plastic materials are removed by decomposing and/or dissolving them with suitable chemicals, and inorganic particles are removed by density separation. For very simple matrixes, such as drinking water, only filtration is enough for separating MPs (Koelmans et al., 2019). Moderately simple matrixes, such as lake water, need chemical treatment with for example hydrogen peroxide and filtration. Nevertheless, complex matrixes, such as sediments, biota, and wastewater influent, require
Figure 1. Features of MPs to be analysed.
MP concentration can be analysed and reported for environmental samples as particle concentration (particles per sample) or mass concentration (mass of MPs per sample). Both particle and mass concentrations are recommended to analyse and report when possible, because for example numerous small particles can have the same mass than one large, or many large particles have substantially higher mass than the same number of smaller ones. These MPs cause different environmental effects, despite the number or mass being the same.
The choice of analytical method also defines which chemical components of MPs can be determined. Mostly, MPs are categorized according to the main polymer component, and it is identified in majority of the studies. However, chemically very diverse group of plastic additives can cause environmental problems (Hahladakis et al., 2018; Kwon et al., 2017) and quantitation of them requires usually different methods than quantitation of MPs by polymer types. In addition to intentionally added compounds, MPs can sorb hydrophobic organic contaminants (Hartmann et al., 2017) and heavy metals (Brennecke et al., 2016), but the importance of the risk caused by interaction of MPs and other contaminants has been critically discussed to be generally minor (Koelmans et al., 2016; Wang et al., 2019). Compared to clean and new plastics, MPs found from the environment are weathered, oxidized, and covered
by biofilms, which needs to be considered in the analysis and ecotoxicity studies (Vroom et al., 2017).
Particle size distribution is also environmentally relevant parameter to analyse, because the toxicity of MPs (Jeong et al., 2016) and ingestion of MPs by aquatic organisms (Lehtiniemi et al., 2018; Vroom et al., 2017) are size-dependent.
Additionally to chemical composition and size, morphological properties of MPs, such as shape and colour can affect the toxicity and appeal for aquatic organisms (Anbumani and Kakkar, 2018; Foley et al., 2018).
Therefore, MPs have various chemical and physical features, which are environmentally relevant to analyse. The features of environmental MPs are different compared to pristine plastics. Not a single method is suitable for analysing all the features.
3 SAMPLE PRE-TREATMENT FOR IMAGING SPECTROSCOPY
The aim of sample pre-treatment is to separate MPs from other material, which is called sample matrix. To analyse MP counts, masses or sizes, they have to be separated from other solid particles, which may distort imaging spectroscopic measurements (Löder et al., 2017). Imaging spectroscopy is not capable of measuring MPs selectively from the matrix, because when all solid particles are mixed and stacked, then signal comes from the mixture instead of single MPs. Therefore, effective pre-treatment remarkably increases the selectivity of the analysis. The same principle applies for other instrumental analysis methods than imaging spectroscopy.
Environmental samples can be for example freshwater, seawater, wastewater, sludge, sediment, air, or biota. The more the sample contains other insoluble particles than microplastics, the more laborious the separation process is (Lusher et al., 2020).
The same principles apply for the pre-treatments for all kinds of samples: organic non-plastic materials are removed by decomposing and/or dissolving them with suitable chemicals, and inorganic particles are removed by density separation. For very simple matrixes, such as drinking water, only filtration is enough for separating MPs (Koelmans et al., 2019). Moderately simple matrixes, such as lake water, need chemical treatment with for example hydrogen peroxide and filtration. Nevertheless, complex matrixes, such as sediments, biota, and wastewater influent, require
multiple treatments, including density separation (Hurley et al., 2018; Löder et al., 2017; Renner et al., 2018).
A wide variety of reagents have been tested for pre-treatment of MP samples (Lusher et al., 2020). Optimal reagents do not physically fragment, chemically decompose or dissolve MPs, but help to dissolve other solids. ‘Microplastics’ is an umbrella term for a wide range of synthetic polymers having varied chemical structures. Therefore, unambiguous rules for choosing reagents that do not harm them does not exist.
However, the most commonly produced plastics polyethylene (PE) and polypropylene (PP) (PlasticsEurope, 2019) are chemically rather inert.
Pre-treatment reagents either dissolve the molecules of the matrix directly or decompose them chemically or enzymatically to more soluble compounds.
Environmental matrixes usually contain natural organic polymers and inorganic particles. Natural organic polymers are for example proteins and polysaccharides from biota and vegetation of the sampling environment. Inorganic particles originate from sediment, sand, and soil, and they consist of various minerals. Concentrate acids dissolve common minerals, such as quartz (silicon dioxide), but decompose MPs also. To avoid this problem, microplastics are separated from inorganic matrix components by density separation procedures. Inorganic particles have higher density than plastics, which allows the separation.
The density separation step can be performed in two ways: In the first protocol, sample is mixed with solution that is slightly denser than common plastics and either left to settle down by gravitation in a separation funnel or centrifuged in a plastic tube. Alternatively, apparatuses specially developed for separating microplastics from sediment exist (Coppock et al., 2017; Felsing et al., 2018; Imhof et al., 2012).
Density separation have been conducted with NaCl (Thompson et al., 2004), ZnCl2
(Haave et al., 2019; Imhof et al., 2012), NaI (Claessens et al., 2013), NaBr (Quinn et al., 2017), ZnBr2 (Quinn et al., 2017) and sodium polytungstate (SPT) (Corcoran et al., 2009) (Table 1). Moreover, for example NaCl and NaI treatments have been combined in a two-step density separation process (Nuelle et al., 2014). A comparison study between various solutions remarked that the higher density solutions recovered microplastics better (Quinn et al., 2017). Because for example ZnCl2 is corrosive and toxic, less hazardous NaI and SPT are better choices.
Table 1. Reagents for density separation of inorganic matrix. SPT = sodium polytungstate.
Reagent Density (kg/m3) Reference
NaCl 1.2 Thompson et al., 2004
ZnCl2 1.7 Haave et al., 2019
Imhof et al., 2012
NaI 1.6 Claessens et al., 2013
NaBr 1.37 Quinn et al., 2017
ZnBr2 1.71 Quinn et al., 2017
SPT / Na6[H2W12O40] <3 Corcoran et al., 2009
NaCl + NaI 1.2/1.6 Nuelle et al., 2014
The other step is to decompose and dissolve organic compounds. Again, various reagents have been reported (Table 2). The most commonly used is H2O2 (Renner et al., 2018), which is efficient and affordable for removing biological material from MP samples (Hurley et al., 2018). Hydrogen peroxide inclusively decomposes biological polymers, but not MPs consisting of the common plastic polymers (Nuelle et al., 2014). When samples are mixed and stirred with H2O2, it spontaneously breaks down to hydroxyl radicals (Arts et al., 1997). The radicals are very reactive and capable of oxidizing biological polymers, in other words breaking the molecules to smaller and more soluble ones. However, the spontaneous formation of radicals is not very fast reaction, but it can be accelerated by increasing temperature. MP samples are usually heated to 50–70 °C at maximum, because high temperatures can change the composition of plastics. Moreover, hydrogen peroxide’s boiling point is 150 °C and it is explosive when heated or evaporated, which also reduces the useful temperature range. The other option is to use Fenton’s reaction, in which formation of radicals is catalysed with Fe2+ ions (Fenton and Jackson, 1899). It is reported to reduce the time of oxidation from days to 10 minutes in MP studies (Tagg et al., 2017).
multiple treatments, including density separation (Hurley et al., 2018; Löder et al., 2017; Renner et al., 2018).
A wide variety of reagents have been tested for pre-treatment of MP samples (Lusher et al., 2020). Optimal reagents do not physically fragment, chemically decompose or dissolve MPs, but help to dissolve other solids. ‘Microplastics’ is an umbrella term for a wide range of synthetic polymers having varied chemical structures. Therefore, unambiguous rules for choosing reagents that do not harm them does not exist.
However, the most commonly produced plastics polyethylene (PE) and polypropylene (PP) (PlasticsEurope, 2019) are chemically rather inert.
Pre-treatment reagents either dissolve the molecules of the matrix directly or decompose them chemically or enzymatically to more soluble compounds.
Environmental matrixes usually contain natural organic polymers and inorganic particles. Natural organic polymers are for example proteins and polysaccharides from biota and vegetation of the sampling environment. Inorganic particles originate from sediment, sand, and soil, and they consist of various minerals. Concentrate acids dissolve common minerals, such as quartz (silicon dioxide), but decompose MPs also. To avoid this problem, microplastics are separated from inorganic matrix components by density separation procedures. Inorganic particles have higher density than plastics, which allows the separation.
The density separation step can be performed in two ways: In the first protocol, sample is mixed with solution that is slightly denser than common plastics and either left to settle down by gravitation in a separation funnel or centrifuged in a plastic tube. Alternatively, apparatuses specially developed for separating microplastics from sediment exist (Coppock et al., 2017; Felsing et al., 2018; Imhof et al., 2012).
Density separation have been conducted with NaCl (Thompson et al., 2004), ZnCl2
(Haave et al., 2019; Imhof et al., 2012), NaI (Claessens et al., 2013), NaBr (Quinn et al., 2017), ZnBr2 (Quinn et al., 2017) and sodium polytungstate (SPT) (Corcoran et al., 2009) (Table 1). Moreover, for example NaCl and NaI treatments have been combined in a two-step density separation process (Nuelle et al., 2014). A comparison study between various solutions remarked that the higher density solutions recovered microplastics better (Quinn et al., 2017). Because for example ZnCl2 is corrosive and toxic, less hazardous NaI and SPT are better choices.
Table 1. Reagents for density separation of inorganic matrix. SPT = sodium polytungstate.
Reagent Density (kg/m3) Reference
NaCl 1.2 Thompson et al., 2004
ZnCl2 1.7 Haave et al., 2019
Imhof et al., 2012
NaI 1.6 Claessens et al., 2013
NaBr 1.37 Quinn et al., 2017
ZnBr2 1.71 Quinn et al., 2017
SPT / Na6[H2W12O40] <3 Corcoran et al., 2009
NaCl + NaI 1.2/1.6 Nuelle et al., 2014
The other step is to decompose and dissolve organic compounds. Again, various reagents have been reported (Table 2). The most commonly used is H2O2 (Renner et al., 2018), which is efficient and affordable for removing biological material from MP samples (Hurley et al., 2018). Hydrogen peroxide inclusively decomposes biological polymers, but not MPs consisting of the common plastic polymers (Nuelle et al., 2014). When samples are mixed and stirred with H2O2, it spontaneously breaks down to hydroxyl radicals (Arts et al., 1997). The radicals are very reactive and capable of oxidizing biological polymers, in other words breaking the molecules to smaller and more soluble ones. However, the spontaneous formation of radicals is not very fast reaction, but it can be accelerated by increasing temperature. MP samples are usually heated to 50–70 °C at maximum, because high temperatures can change the composition of plastics. Moreover, hydrogen peroxide’s boiling point is 150 °C and it is explosive when heated or evaporated, which also reduces the useful temperature range. The other option is to use Fenton’s reaction, in which formation of radicals is catalysed with Fe2+ ions (Fenton and Jackson, 1899). It is reported to reduce the time of oxidation from days to 10 minutes in MP studies (Tagg et al., 2017).
Table 2. Overview of the reagents used for digestion (decomposition and dissolution) of organic matrix. SDS = sodium dodecyl sulphate.
Reagent Category References
SDS Surfactant Enders et al., 2015
Löder et al., 2017
H2O2 Oxidizer Nuelle et al., 2014
Hurley et al., 2018 Fenton’s reaction
Fe2+ + H2O2 Oxidizer Tagg et al., 2017
NaClO Oxidizer Collard et al., 2015
HCl Acid Cole et al., 2015
HNO3 Acid Roch and Brinker, 2017
HClO4 Acid Enders et al., 2017
NaOH Base Cole et al., 2015
Budimir et al., 2018
KOH Base Pereira et al., 2020
Karami et al., 2017
Proteinase-K Enzyme Cole et al., 2015
Lipase, protease, amylase Enzyme Mani et al., 2015
Various enzymes Enzyme Löder et al., 2017
However, not all biological polymers are reactive with H2O2, for example chitin.
Because exoskeletons or shells of crustaceans, insects, zooplankton, and fish compose of chitin, it is very commonly present in natural waters or biota samples. Chitin can be degraded and dissolved with for example concentrated NaOH (Pillai et al., 2009), but NaOH also decomposes some plastic types, such as polyethylene terephthalate (PET) (Dehaut et al., 2016). The most effective, but gentle reagent to dissolve chitin is chitinase enzyme (Löder et al., 2017; Mani et al., 2015). Enzymes target specific molecules and leave others intact. Therefore, they are effective and specific. In addition to chitinase, other enzymes can be used to degrade and dissolve for example
cellulose and other polysaccharides, proteins, and lipids (Löder et al., 2017).
Moreover, sodium dodecyl sulphate (SDS) has been used for decomposing and dissolving organic matrixes. SDS is also a surfactant, which prevents MPs from adhering to laboratory glassware and equipment (Enders et al., 2015) and it macerates proteins, which enhances the following enzymatic or oxidative digestion steps (Löder et al., 2017).
On the other hand, acids and bases have been used for decomposing and dissolving organic matrixes, too. For example, pre-treatments including HCl, HNO3 and NaOH have been tested to decompose intestines of fish and plankton samples (Cole et al., 2015; Roch and Brinker, 2017). Moreover, NaOH followed by HCl has been used for fish samples (Budimir et al., 2018). KOH has been used similarly than NaOH for decomposition of fish samples (e.g. Mizraji et al., 2017; Pereira et al., 2020).
Additionally, HClO4 has been used to digest organic materials, but it is not recommendable, because together with HNO3 they can destroy MPs (Enders et al., 2017). Similarly, a mixture of NaClO and HNO3 has been used for fish samples (Collard et al., 2015). However, because NaClO decomposes and produces toxic chlorine gas during the sample treatment, it is not popular or generally recommendable compared to less hazardous reagents.
The effectivity of the reagent depends on the concentration and temperature. The most of the common reagents can damage MPs in high concentrations or temperatures (Hurley et al., 2018; Lusher et al., 2017). Karami et. al. (2017) tested and compared for fish samples KOH, NaClO, H2O2, HCl, HNO3, and NaOH in four different temperatures. As a result, KOH was found to be the most effective to decompose biological material in low temperatures, but it did not decompose MPs.
Moreover, H2O2 in 50 °C was efficient and did not decompose MPs, as HNO3 and HCl did. For sludge and soil samples, similar comparison study included H2O2, Fenton’s reaction, NaOH and KOH in different concentrations and temperatures (Hurley et al., 2018). Fenton’s reaction coupled with density separation was tested to be the most effective method for separating MPs from soil and sludge.
The universal enzymatic purification protocol (UEPP) (Löder et al., 2017) has been tested for marine and freshwater surface water, sediments, beach sand, wastewater, tissue sample of mussels, daphnia, and fish. The protocol includes SDS, H2O2, protease, lipase, cellulase, amylase, chitinase, and density separation. The reagents and steps are selected for samples according to their compositions. Therefore, UEPP is probably the most versatile pre-treatment method published for MP samples.
Moreover, Lusher et al. (2020) recommend 10% KOH or H2O2/Fenton’s reaction for water and biota samples, and NaI for density separation, based on inclusive literature review. Because enzymes are expensive and time-consuming to use, Lusher et al.
