Conclusions about the methods and future recommendations

6 AIMS OF THE STUDY

8.3 Evaluation of the performance of the methods

8.3.4 Conclusions about the methods and future recommendations

Publication I: The disadvantages of simple pre-treatment and manual selection of particles for FTIR point measurements were high subjectivity, missing procedural blanks and recovery tests, low MP isolation efficiency, slowness, laboriousness and need for high expertise. The method would be suitable for fast and approximate screening of MPs of larger size fractions 500–5000 µm. Manual selection of smaller particles is not practical, as faster methods exist. However, for example Primpke et al. (2020a) state that both light microscopy with manual selection and instrumental techniques, such as imaging (FPA-) FTIR and thermal degradation methods are suitable for monitoring, routine risk assessment, and research applications.

Publications II and III: UEPP and FPA-FTIR spectroscopy have many advantages, including objectivity, automatization, representative measured sample volumes, and possibility to validate the method easily with controls and recovery tests. However, this method still has the same disadvantages listed for the method of publication I: it is time-consuming and requires special expertise. The method is suitable for

comparing similar samples between each other, but it was not accurate enough for providing absolute mass values.

8.3.3 FPA-FTIR and data analysis

The major advantage of measuring FPA-FTIR from reflective filters is typically no need for sub-sampling. The coverage of the methods in publications II and III was therefore 100%, which is a great advantage compared to many other studies, which have analysed only small or subjectively chosen proportion of particles with spectroscopy (Löder et al., 2015). Gold-coated filters in publication II provided good quality spectra, while silver membranes in publication III slightly reduced the spectral quality, mainly because baseline had more noise. On the other hand, gold-coated filters had 0.8 µm pore size, while silver membranes are commercially available in pore sizes up to 5 µm. Because the pore size affects filtration speed remarkably, the larger pore size was practical and feasible for a large set of samples.

Moreover, as FPA-FTIR can detect >10 µm particles, retention of smaller particles is not necessary. Silver membranes are also more rigid and easier to handle than gold-coated membranes.

FPA-FTIR and siMPle are very accurate for determining particle sizes (reported as the longest dimensions in these publications). The mean (± standard deviation) measured size of PS particles of the recovery rate tests in publication III was 90 ± 7.8 µm, which was close to the reported reference value 90 µm. However, there is always deviation in the sizes, because FPA-FTIR had pixel size 5.5 µm. Hence, the pixel resolution of the particle size measurement is 5.5 µm, and the standard deviation 7.8 µm was rather close to the pixel size. However, long fibers which can be curvy or partially detached from the filter surface were not very accurately measured with FPA-FTIR and siMPle. Instead of being detected as one long fiber, they can be split in analysis to multiple smaller particles, or their dimensions are not measured correctly. Currently, this is a major disadvantage of the method, if numbers or sizes of fibers are aimed to be analysed.

Despite being quite automatic process, the effective use of siMPle however needs some validation steps before routine analyses. First, the reference spectra have to be chosen to create a representative spectral library. The selection of reference spectra depends on various aspects, such as:

1) What is the sample matrix? In addition to most common plastic polymers, natural polymers such as proteins and polysaccharides should be included to the library to ensure, that they are not misrecognized as plastics. For example, PA has similar chemical structures compared to proteins, but the

mismatches can be avoided to some extent by including protein references as well.

2) How large set of plastics is practical to include in search? Increasing number of references increases the calculation time, too. The aim of the study specifies how inclusive set of references is practical. If the study aims to quickly monitor MP abundance from a wide set of samples, only the most common plastic types should be included in the library to reduce the time of the analysis. But if the study aims to analyse detailed information about MP types from limited sample set, then an inclusive library is the choice. In publications II and III, the first approach was chosen, as the aim was to test the method for monitoring purposes.

3) Which measurement method is used? FPA-FTIR measurements can be performed in transmission of reflection mode. Moreover, single-point FTIR measurements can be done in ATR mode. The measurement mode affects the spectral range and quality of the spectra. The recommendation is to use a library, which is measured with the same instrument, method and parameters than samples, and include weathered microplastics in addition to pristine plastics, because the correlation is better between spectra which are measured similarly (Xu et al., 2020). However, in all cases this is not practical or possible and commercial or open-source spectral libraries (e.g.

Primpke et al., 2018) are the choice.

8.3.4 Conclusions about the methods and future recommendations

monitoring MPs on an approximate size range 20–500 µm, as larger particles can be too thick to be measured with FPA-FTIR. In publication III, >500 µm particles were pre-sieved and would have been manually measured with ATR-FTIR, if there were any. When the whole size range of MPs from 1 µm to 5 mm is targeted, pre-sieving size fractions for the feasible analytical methods enhances the accuracy of the results.

If the FPA-FTIR analysis of MPs would be harmonized or standardized in the future, more comprehensive validation by various validation parameters should be conducted.

The methods for identification and quantitation of MPs are highly needed in many research disciplines, such as environmental, biological, and engineering sciences.

Various types of experiments for studying effects and fate of MPs would benefit from validated harmonized or standardized methods. They would generally help to focus on specific research questions, because the effort to prove the quality of the methods is not needed anymore.

In addition to research, practical applications such as routine monitoring of water bodies and quality control in food industry and other industrial sectors would benefit from harmonized or standardized methods for analysis of MPs. To fulfil those needs, the methods developed in this study can be used as a guideline for other sample types than water and fish as well.

The next steps in the future are likely development of standardized reference materials for validation of MP analysis methods, followed by harmonization and/or standardization of methods, which many researchers have called for (e.g. Provencher et al., 2020; Rochman et al., 2017; Stock et al., 2019). This study highlights that FPA-FTIR is currently the most used method for identification and quantitation of MPs from environmental samples. Moreover, this thesis emphasizes the importance of proper validation procedures in attaining reliable results and in standardization of methods.

9 CONCLUSIONS

The first aim of this study was to investigate microplastic concentrations in Finnish marine and freshwater environments by analysing both water and fish samples. The results indicated that Finnish waters are not heavily polluted by MPs on a global scale. This study presents novel information on MP abundance in northern lake water and fish, but more monitoring is needed to assess the risks MPs may cause to the environment.

The second aim of this study was to development and validate analytical methods to quantify microplastics reliably to achieve the first aim. The method development focused on water and fish samples, including sample pre-treatment methods and determination of microplastic concentrations and types with FPA-FTIR spectroscopy. The third aim was to develop quality control for the analytical methods.

The conclusion was that manual selection of particles with light microscopy to point microscopy or ATR FTIR measurements is suitable for larger MPs on an approximate size range 500 µm – 5 mm. Enzymatic and hydrogen peroxide pre-treatment followed by automatic FPA-FTIR spectroscopic analysis of all MPs was suitable for analysing smaller size fraction, approximately 20–500 µm. The methods developed on this study are promising and developable for further sampling campaigns and routine monitoring. However, pre-treatment and laboratory methods need still development to increase the recovery rate and decrease the possibility for contamination.

Before the methods for MP quantitation can be harmonized or standardized, more comprehensive validation is needed. First, the necessary validation parameters for the MP analysis methods should be consented. Moreover, commercially available standardized reference materials are needed for testing and comparing the analytical methods for qualitative and quantitative analysis of MPs. Different methods should be validated systematically with procedural blanks and more representative recovery/spiking tests to compare their suitability. Researchers should report the limits of detection for the analytical methods used. This would lead to the more comprehensive and reliable information about MP pollution and help to assess the risks and create policies to protect the environment.

If the FPA-FTIR analysis of MPs would be harmonized or standardized in the future, more comprehensive validation by various validation parameters should be conducted.

The methods for identification and quantitation of MPs are highly needed in many research disciplines, such as environmental, biological, and engineering sciences.

9 CONCLUSIONS

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