Guidelines for Harmonizing Ocean Surface Microplastic
Monitoring Methods
Version 1.2, May 2023

Ministry of the Environment, JAPANMay 2023

Guidelines for Harmonizing Ocean Surface
Microplastic Monitoring Methods
MICHIDA Yutaka 1, CHAVANICH Suchana 2, CHIBA Sanae3, CORDOVA Muhammad Reza4,CÓZAR CABAÑAS Andrés 5, GALGANI Francois 6, HAGMANN Pascal 7, HINATA Hirofumi 8,
ISOBE Atsuhiko 9, KERSHAW Peter 10, KOZLOVSKII Nikolai 11, LI Daoji 12, LUSHER Amy L. 13,
MARTÍ Elisa 5, MASON Sherri A. 14, MU Jingli 15, SAITO Hiroaki 1, SHIM Won Joon 16, SYAKTIAgung Dhamar 17, TAKADA Hideshige 18, THOMPSON Richard 19, TOKAI Tadashi 20, UCHIDA
Keiichi 20, VASILENKO Katerina 21, WANG Juying 15,
The University of Tokyo, Japan
Chulalongkorn University, Thailand
3 Japan Agency for Marine-Earth Science and Technology, Japan (Current position: North Paci
fic Marine Science Organization (PICES))
4 The Indonesian National Research and Innovation Agency (BRIN), Indonesia
5 University of Cádiz, Spain
6 IFREMER (French Research Institute for Exploitation of the Sea), France
7 Association Oceaneye, Switzerland
8 Ehime University, Japan
9 Kyushu University, Japan
10 Independent consultant - marine environmental protection, United Kingdom(Chair, GESAMP WG40)
11 Pacific Geographical Institute, Russia
12 East China Normal University, China
13 Norwegian Institute for Water Research, NIVA, Norway
14 The Pennsylvania State University, Behrend College, USA
15 National Marine Environmental Monitoring Center, MEE, China
16 Korea Institute of Ocean Science and Technology, Korea
17 Raja Ali Haji Maritime University (UMRAH), Indonesia
18 Tokyo University of Agriculture and Technology, Japan
19 University of Plymouth, United Kingdom
20 Tokyo University of Marine Science and Technology, Japan
21 Vancouver Aquarium Marine Science Centre, Canada
1
2

The suggested citation for this document is:Michida, Y., et al. (2019). Guidelines for Harmonizing Ocean Surface Microplastic
Monitoring Methods. Ministry of the Environment Japan, 74 pp.
For copies of this document, please contact:
Office of Policies against Marine Plastics Pollution, Water Environment Division,
Environmental Management Bureau, Ministry of the Environment, JAPAN
Kasumigaseki 1-2-2, Chiyoda-ku, Tokyo 100-8975, Japan

Acknowledgements
The contributions and cooperation of the following laboratories were extremely important andessential to the preparation of these Guidelines, both in analyzing standard microplastic samples
prepared for experimental analysis, and reporting the results of their analyses to clarify thedifferences between analytical methods and practices.
Thank you to the participation of Association Oceaneye, Chulalongkorn University, Ehime University,
Korea Institute of Ocean Science and Technology, Kyushu University, Norwegian Institute for Water
Research, National Marine Environmental Monitoring Center, MEE, National Oceanic andAtmospheric Administration, Pacific Institute of Geography, Tokyo University of Marine Science andTechnology, University of Cadiz, and Vancouver Aquarium Marine Science Centre.
Special thanks are noted to DELORENZO Marie, BUENAVENTURA Nina T., ROSS Peter S.,
CHASTAIN Stephen, VIYAKARN Voranop, and ZHANG Weiwei for their contributions to the interlaboratory comparison in 2017 (ILC).

Preface:
This document presents the version (1.2) of “Guidelines for Harmonizing Ocean Surface Microplastic
Monitoring Methods” (herein after referred to as the Guidelines). Its primary goal is to propose waysof harmonizing methodologies for monitoring microplastic densities at the ocean surface to delivercomparable results. Specifically, the Guidelines indicate the rationale for various sample collection
methods, sample handling and processing, analytical procedures, reporting requirements, and othermatters necessary or desirable for harmonization.
Preparation of the Guidelines was based on the output of the international workshop held in 2015 asa follow-up to the “G7 Action Plan to Combat Marine Litter” agreed on in the G7 Elmau Summit 2015,
and a follow-up meeting held in 2019 based on “G20 Implementation Framework for Actions on
marine plastic litter” endorsed in the G20 Osaka Summit 2019. It was indicated that Japan would
lead the harmonization efforts for microplastic monitoring methods in the workshop and the followup meeting held in 2019.
The Guidelines were developed on the basis of opinions and recommendations compiled atinternational meetings of microplastic monitoring experts and the results of dedicated in situ andlaboratory experiments newly conducted toward harmonization, as well as existing findings collected
and summarized from published microplastic monitoring survey reports, guidelines, and manuals.
Estimating the abundance and/or distribution of microplastics in water bodies has become
internationally important. At present, several sets of guidelines and other documents are being
developed by some international organizations including GESAMP. The Guidelines presented here
were designed to supplement and complement such documents, and to propose detailed methodologies
focusing on net sampling and analysis. The outcomes of which are to contribute validated andcomparable data which can be used to produce horizontal distribution maps (two dimensional maps;
2-D maps) of microplastics at the global ocean surface.
Many studies are expected to be carried out to monitor microplastics at the ocean surface. The
application of the harmonized methods proposed in the Guidelines will support these efforts togenerate comparable results. Thus, enabling researchers to analyze, consolidate and integrate theresults on a wider scale. Through such an application, we strongly believe that our understanding of
the abundance of microplastics in the ocean will improve. Shared and integrated monitoring results
will promote higher level analysis of microplastic issues and application to policy development.
These outcome and progress will be share at various international meetings including G20.
The first revision of the Guidelines was made one year after their first publication and this second
revision was made two years after the first revision. The Guidelines will be updated and improved asnecessary.

Contents
1. Introduction............................................................................................................................................. 1
1.1 Background and purpose .................................................................................................................. 1
1.2 Scope of the Guidelines ..................................................................................................................... 5
1.3 Composition ....................................................................................................................................... 7
2. Sampling methods .................................................................................................................................. 8
2.1 Outline ............................................................................................................................................... 8
2.2 Sea conditions.................................................................................................................................. 10
2.3 Sampling equipment ....................................................................................................................... 13
2.3.1 Sampling vessels ...................................................................................................................... 14
2.3.2 Net types ................................................................................................................................... 16
2.3.3 Mesh openings .......................................................................................................................... 19
2.4 Tow parameters ............................................................................................................................... 24
2.4.1 Tow duration ............................................................................................................................. 24
2.4.2 Vessel speed .............................................................................................................................. 26
2.4.3 Sweep area and filtered water volume ................................................................................... 26
2.4.4 Tow distance.............................................................................................................................. 27
2.4.5 Tow position .............................................................................................................................. 31
2.4.6 Net immersion depth ............................................................................................................... 35
2.5 Metadata .......................................................................................................................................... 38
2.6 Field sample blanks ........................................................................................................................ 40
3. Laboratory analysis .............................................................................................................................. 43
3.1 Outline ............................................................................................................................................. 43
3.2 Preprocessing for analysis .............................................................................................................. 45
3.2.1 Biological digestion and chemical treatment ......................................................................... 46
3.2.2 Density separation ................................................................................................................... 49
3.2.3 Sample splitting ....................................................................................................................... 51
3.3 Isolation of microplastic.................................................................................................................. 53
3.4 Counting and size measuring......................................................................................................... 55
3.5 Material identification .................................................................................................................... 58
3.6 Weight measurement ...................................................................................................................... 62
3.7 Accuracy control during analysis ................................................................................................... 64
4. Reporting ............................................................................................................................................... 66
5. Conclusions ........................................................................................................................................... 71

List of Acronyms and Abbreviations
CMSM2018: Comparison of Microplastic Sampling Methods 2018
CMSM2019: Comparison of Microplastic Sampling Methods 2019
CPR: Continuous Plankton Recorder
EC: European Commission
FTIR: Fourier Transform Infrared Spectroscopy
GESAMP: The Joint Group of Experts on the Scientific Aspects of Marine Environmental Protection
ILC: Inter Laboratory Comparison 2017
MOEJ: Ministry of the Environment, JapanNOAA: National Oceanic and Atmospheric Administration
SOP: Standard Operational Procedure1. Introduction
1.1 Background and purpose
Marine litter, including microplastics, is now a global challenge. In particular, pollution of the marine
environment by microplastics has been recognized as a serious international issue. Microplastics arelikely to affect marine ecosystems and are extremely difficult to recover. Determining the current
status of distribution and quantity of microplastics in the ocean is an urgent task. It is important forpolicy making and implementation to be based on concrete scientific knowledge. Early intervention
and mitigation can be facilitated when environmental concentrations are understood. Thus, effective
monitoring tools can support preventive measures against plastic litter in the ocean.
In response to the growing interest surrounding microplastics in the ocean, microplastic monitoring(sampling and laboratory analysis) is carried out by many institutions around the world using various
methods. Accordingly, data are gradually accumulating. It is expected that monitoring will continue,
but as different sampling and analytical methods are used - depending on the purpose of the surveys
of each country and research institution - there is a fundamental lack of comparability among
currently available data. In some instances, research will be carried out under limited resource
availability, technical capacity or institutional arrangements. Alternatively, monitoring will be
conducted using emergent methodologies and is not yet globally common. These factors will further
hamper researchers’ ability to build comparisons.
Methods that are of a high technological readiness level (TRL) for plastic monitoring are essential togenerate harmonised and comparable data (Lusher and Primpke, 2023). Methods can be broken into
different analytical steps and a TRL applied to all steps (Aliani et al., 2023). TRLs can help distinguishwhich methods are at a level which is mature enough to be considered for large-scale monitoring
guidelines or those that need to be redirected into research and development (such as investigating
microplastics in air). The TRL scale classifies technology or methods into basic research (TRLs 1–3),
applied research (TRLs 4–5), in development (TRLs 6–8) and implementation (TRL 9) phases. Using
the scale in Aliani et al., (2023), methods for microplastic sampling and sample processing would fallunder development or applied research phase.
The inability to compare data obtained by different monitoring methods may pose an obstacle todetermining the global distribution and fate of microplastics in the ocean. Hence, harmonization (andwhere possible standardization) of monitoring methods for microplastics are recognized as importanttasks.
At the G7 Elmau Summit in 2015, marine litter, especially plastic, was acknowledged as a global
challenge due to its (1) effects on ocean and coastal ecosystems, (2) direct impacts on ecosystems, and(3) potential impacts on human health. In the annex to the G7 2015 declarations, “supporting theinitiation of a harmonized global marine litter monitoring effort and the standardization of methods,
data and evaluation” is listed as one of the priority actions. Subsequently, the communiqué adopted
at the G7 Toyama Environment Minister's Meeting in 2016 states its commitment to implementing
five priority measures including standardization and harmonization of monitoring methodologies formarine litter. Based on shared recognition of these issues, several activities have been initiatedincluding development of guidelines for monitoring, analysis and evaluation by GESAMP and other1

organizations. At the expert workshop in Berlin, November 2015 following the Elmau Summit, it wasagreed that Japan would play a leading role in standardizing and harmonizing the monitoring
methodologies for ocean microplastics.
At the G20 Osaka Summit in 2019, marine plastic pollution was taken up as one of the priority issues.
The “Osaka Blue Ocean Vision”, which aims to reduce additional pollution by marine plastic litter tozero by 2050, was shared as a common global vision, and the G20 leaders called on other members of
the international community to share the vision. They also endorsed the "G20 Implementation
Framework for Actions on Marine Plastic Litter", which includes sharing scientific information andknowledge. The first follow-up meeting and the G20 Workshop on Scientific Knowledge andInnovative Solutions for Marine Plastic Litter were held in 2019. The meeting identified future
activities anticipated for the G20 Implementation Framework, including joint initiatives of Ministryof the Environment, Japan (MOEJ), EU DG Environment and US Environment Protection Agency tovoluntarily take a lead in further elaborating key issues such as harmonized monitoring and datacompilation by MOEJ.

To remedy the situation, the MOEJ has been advancing efforts to ascertain the actual state of marine
pollution by encouraging to horizontal distribution mapping of microplastic densities at the ocean
surface worldwide. The Guidelines were developed based on the results of three projects, implemented
by scientists and supported by the MOEJ (Fig. 1-1). In addition, a comparative study of the research
being undertaken around the world was conducted. For examining analytical methods, an interlaboratory comparison (ILC) was conducted by 12 laboratories in 10 countries (Canada, China, Korea,
Norway, Russia, Spain, Switzerland, Thailand, USA and Japan) in 2017 to cross-check standard
samples containing a predetermined amount of non-plastic material and a predetermined number
density of plastic particles using various analytical methods (Isobe et al., 2019). For examination ofsampling approaches, a comparison of microplastic sampling methods was conducted in FY2018(hereinafter "CMSM2018"), by sampling microplastics in the sea surface of Tokyo Bay. In FY2019,the comparison of microplastic sampling methods (CMSM2019) was implemented in Sagami Bay tofurther enhance the content of the Guidelines. Based on an analysis of differences in the results
obtained in these projects, recommendations for harmonization, as well as points to be noted wheninterpreting monitoring results were summarized. Furthermore, due to the considerations being
made for a newly developing database for marine plastics (Marine Plastic Litter Mapping Database)and progress in other related activities, some definitions used in the Guidelines needed to be amended
for practical reasons.
The Guidelines were prepared with the view of enabling researchers of ocean surface layer
microplastic monitoring to adopt similar monitoring protocols and therefore interpret their resultswith a level of comparability.

Purpose of the Guidelines:
To focus on determining the actual state of microplastics in the ocean surface layer* rather
than other forms of marine plastic pollution.

2

To provide recommendations for harmonizing sampling and analytical methods which will
enable comparison of the obtained results both of currently ongoing and future studies.
To give consideration to studies carried out under various constraints, such as restrictive
human or financial resources.
* Microplastic monitoring surveys have been carried out for many different purposes (Rochman et al., 2017) suchas to evaluate diverse media or the effects of microplastic emission controls. Among these various research
objectives, the Guidelines aim specifically at developing horizontal distribution maps of microplastics at theocean surface.

3

Review of Research Papers

First(Research on MP monitoring in the Ocean Surface Layer)

ExpertMeeting(Dec.

Two Pilot Projects

Review ofInter-Laboratory Comparison in 2017(ILC2017)
Laboratories from 10 countries
Standard samples with known quantity andweight of plastics were distributed to eachlaboratory for analysis and results were reported.
Effects of different pretreatment, methods ofparticle isolation, and analytical measurement
methods were compared.
Comparison of Microplastic Sampling Methods in2018 (CMSM2018)
Implemented in Tokyo Bay
Plastics in ocean surface layer were sampled
under different sampling conditions (net type,mesh openings, tow position, tow duration, etc.)
at the same time
Effects of different sampling conditions on the
results were investigated2016)

Existing
Guidelines

SecondExpertGESAMP (2013,

Meeting

2015, 2019)(Feb.

EC Guidance

2018)(2013)NOAA (2013,
2015)
UNEP (2016)

Third
ExpertMeeting
(Mar.
2019)

Guidelines for Harmonizing Ocean Surface Microplastic Monitoring Methods
FourthComparison of Microplastic Sampling Methods in 2019 (CMSM2019)
Conducted to verify issues not been fully examined in CMSM2018.
Implemented in Sagami Bay.
Plastics in ocean surface layer were sampled under different sampling
conditions (net type, mesh openings, tow position, tow duration, wind directionetc.) at the same time.
Effects of different sampling conditions on the results were investigatedExpertMeeting
(Feb.
2020)

Guidelines for Harmonizing Ocean Surface Microplastic Monitoring Methods (ver. 1.1)
* Microplastic monitoring surveys have been carried out for many different purposes (Rochman et al.,2017) such as to evaluate diverse media or the effects of microplastic emission controls. Among thesevarious research objectives, the Guidelines aim specifically at developing horizontal distribution mapsof microplastics at the ocean surface.

Fig. 1-1. Guidelines development and updating process.

4

1.2 Scope of the Guidelines

Target readers
・ The main target readers of the Guidelines are researchers who conduct oceanographic surveys of
microplastics, and those who intend to analyze and evaluate the state of pollution by using survey
results of their own and/or others from around the world. Consideration has been given to somestudies carried out in various countries including developing countries under various constraints,
such as restrictive human and financial resources.
・ The Guidelines are not intended to present standards, but rather they have been prepared in the
expectation that they will be helpful in choosing harmonized methods that would derivecomparable results.

Subject and monitoring methods
・ The subject matter of the Guidelines is microplastics at the ocean surface and their aim is toharmonize net sampling in the field and analytical methods in laboratories.
・ Plastic particles with a size of less than 5 mm are treated as microplastics in the Guidelines,
similarly to their definition in GESAMP (2019) and to the definition used in internationalorganizations and many research projects that have been implemented in various countries
around the world.
・ Ascertaining microplastic presence inside living organisms is important to investigate the impactof microplastics on living organisms, but it is beyond the scope of the Guidelines.
・ Although the scope of the Guidelines is microplastics at the ocean surface, as shown in Table 1-1,
the sampling and laboratory analytical methods are considered applicable to surface water in bothmarine and freshwater environments. They can also be partially applicable to water columns andsediments of both seawater and freshwater.

5

Table 1-1. Microplastic sampling and analytical methods within the scope of the Guidelines.
Legend ○: Within the scope △: Partially referable ×: Not within the scope of the Guidelines
The Guidelines have been designed primary for marine surveys. It should be noted there would be
more clogging and vertical mixing in fresh water.

Category
Surface

Field Sampling

Laboratory analysis

○*

○

×

△

Net sampling

water
Other methods(Pump, CPR, etc.)
Water column

×

Sediments

×△△

Why focus on ocean surface net sampling?
Presently, there are numerous microplastics in the ocean surface around the world. They are
impacting invertebrates, fish, birds and other organisms living in or on the ocean surface.
At the ocean surface, it is common to collect samples using nets. Net sampling is thought to havethe following advantages:
・ A large mass of water can be efficiently filtered.
・ Nets can be deployed easily, compared to pumps, CPR (continuous plankton recorders), etc.,
・ Abundant knowledge on surface net use and collection methods is available from plankton
research.
・ Proportionally more surveys using nets to sample microplastics have been conducted, so using
nets facilitates comparison with the accumulated data.

6

1.3 Composition
The Guidelines are divided into five chapters. Table 1-2 gives an outline of these. Each chapter isdivided into sections, and the main content in each section is summarized as keynotes.
Each set of keynotes is highlighted in a box and provides the following information:
Introduction of commonly used methods and parameters.
Related results from projects (ILC, CMSM2018 and CMSM2019) and the results of literature
reviews conducted for preparing the Guidelines.
Recommendations based on the above information.
Further comments pertaining to keynotes are provided as explanatory notes.
Table 1-2. Guidelines chapter outlines.
ChapterContents

1. Introduction

Background, purpose and scope of the Guidelines.

2. Sampling methods

Summary of recommendations for harmonization of ocean surface layer
microplastic sampling methods, specifically for sea conditions during thesurvey, sampling equipment, tow parameters, metadata recording,contamination prevention and accuracy control.

3. Laboratory

Summary of recommendations in view of harmonizing microplastic

analysisanalytical methods in the laboratory, specifically for preprocessing,extracting microplastics, particle counting and size measurement,material identification, weight measurement and accuracy control during
analysis.

4. Reporting

Recommendations on methods of reporting microplastic results andmetadata.

5. Conclusions

Summary of the Guidelines, items that require further consideration, etc.

7

2. Sampling methods
2.1 Outline
Microplastics floating at the ocean surface can be collected by towing a net according to theprocedure illustrated below (Fig.2-1).

Fig.2-1. General flow of microplastic collection using a net.

8

Reviews of previous research identified differences in the net type and size of mesh used acrossstudies. A Manta net or Neuston net is most commonly used, and recommended in samplingguidelines, although differences between net mesh openings and towing methods have been
observed between past studies. For this reason, surveys to collect microplastics from the ocean
surface were conducted (CMSM2018 and CMSM2019) to investigate the effects of the following
factors on sampling results: (1) different towing directions relative to wind direction (Section 2.2),(2) differences between Neuston nets and Manta nets (Section 2.3.2), (3) differences in mesh
openings (Section 2.3.3), (4) differences in tow duration (Section 2.4.1), and (5) differences in tow
position (i.e., towing at the stern) (Section 2.4.5).
CMSM2018 was conducted using a research vessel in Tokyo Bay whereas CMSM2019 was
conducted in Sagami Bay closer to the open ocean. This survey was conducted with a small fishingboat equipped with outfitting that made it possible to tow on the sides of the vessel. The survey
method using a small fishing boat is summarized in the Section 2.3.1.
The average density of microplastics at the ocean surface observed in CMSM2018 in Tokyo Bay
was 2.65 particles /m3 (Range: 0.21 - 6.49 particles /m3, 78 x 103 - 2,432 x 103 particles/km2).
Density has generally been reported in either one of the two unit systems: one is the particle
number per unit volume and the other is per unit area. Comparatively, the average density of
microplastics in CMSM2019 at Sagami Bay was 0.51 particles/m3 (Range: 0.03- 2.57 particles/m3,
13 x 103-956 x 103 particles/km2).
Conditions for harmonization were determined based on a comparison between the results of the
CMSM2018 and CMSM2019 where net types with different mesh openings were towed at thesame time in the same sea area. Specifically, two different nets were simultaneously set at port
and starboard of the same survey vessel.
In order to confirm the validity of this method, test runs were conducted using two Neuston netsof the same design towed at port and starboard positions. There were no statistically significant
differences between the densities of microplastics (Section 2.4.5).
The metadata necessary to enable comparison of the survey results were examined based on theenvironmental data acquired during CMSM2018 and CMSM2019.
Recommendations based on our literature review and field study (CMSM2018 and CMSM2019),
are presented in detail in the following section.

92.2 Sea conditions
Keynotes・ Previous studies and available guidelines have stated that the collection of microplastics at theocean surface should be conducted under calm sea conditions whenever possible. Adverse
weather conditions can affect the results obtained.

Outcomes of the pilot projects
・ It was observed that the density of microplastics in the same survey area changed by about
one order of magnitude within several hours. This occurred as sea conditions, including windspeed and wave height, changed.

Recommendations
・ It is desirable to collect samples when sea conditions are as calm as possible. This might not
be practical in areas prone to elevated wind conditions. In such situations, metadata such as
wind speeds and significant wave heights should be recorded to allow comparisons with othersurvey results (for more details, please refer to Section 2.5, Metadata and Section 4, Reporting).
・ It is desirable to avoid unfavorable timing and conditions for sampling, such as high densities
of natural particles or organisms, i.e. algae and plankton blooms. When conducting a survey
under unfavorable conditions is unavoidable due to characteristics of sea areas, it is desirable
to consider appropriate methods such as shortening the tow duration accompanied withrepeated towing, and frequently washing towing nets (Section 2.4.1).

Explanatory Notes
In general, wind speed and wave heights are known to influence the degree of vertical mixing of
the ocean surface layer and affect the amount of microplastics collected. According to recent
guidelines, microplastic surveys should be conducted in conditions where wave heights are under
0.5 meters and the beaufort wind force scale under 3 (GESAMP, 2019).
Several studies that have been conducted propose a method for estimating the vertical
distribution of microplastics in the water column allowing researchers to correct ocean surface
microplastic density depending on sea conditions (Kukulka et al., 2012; Kooi et al., 2016. etc.).
Recording wind speed and wave height during sampling will allow researchers to estimate thevertical distribution of microplastics in the water column and some studies have adopted thesemethods (Isobe et al., 2015; Suaria et al., 2016. etc.).
In CMSM2018, it was observed that the density of microplastics at the ocean surface decreased
in situations where both wind speed and wave height increased during sampling (Fig.2-2). Thiswas probably due to the enhanced mixing of the ocean surface layer caused by changes in the sea
conditions and the dispersion of microplastics to a certain depth (Reisser et al., 2015).
In CMSM2019, the results of microplastic sampling were compared to examine towing direction
with respect to the wind direction in the open sea, where a net was towed perpendicular to thewind direction, with the starboard side facing upwind and the port side facing downwind undercalm sea conditions (wind speed 2-6 m/s).

10

The results showed no statistically significant difference between the particle densities insamples (1-5mm) collected upwind and downwind (Fig. 2-3). It should be noted that results wereobtained using a small boat of 10 tons under calm sea conditions. The results could not necessarily
be applied to other conditions.
Care should be taken when sampling in sea areas near land following rainfall, as it has beenreported that the density and composition of microplastics at the ocean surface can be
influenced by microplastics input from rivers (Kang et al., 2015., Lima et al., 2015, etc.).
In CMSM2018, surveys conducted in the coastal area of Tokyo Bay showed an increased densityof microplastics. This observation was thought to have been caused by the input from nearby
rivers (Section 2.5, Fig.2-16).
In addition, a significant decrease in the amount of microplastics was observed when a large
amount of jellyfish was caught in the same net in CMSM2018. Clogging of the net by plankton,
algae, jellyfish, floating seaweed, etc., affects survey results. It is preferable to avoid collecting
samples at times when they are expected to be observed in mass.
To obtain mutually comparable results, situations with strong winds and/or waves, or in whichplankton are highly abundant should be avoided. Surveys must be conducted when sea conditions
are as calm as possible.
Tidal currents and/or river inflows should be monitored. Sampling should be conducted undermoderate to average sea conditions to increase comparability.
Additionally, under rough sea conditions, it might be difficult to maintain the immersion depth
as constant, and the flowmeter may not be able to measure the filtered water volume correctly
because the meter may sometimes be outside of the sea surface.
The flowmeter should always be periodically calibrated so that its metering conforms to thestandard.

11

12

1.2

10

1

8

0.8

6

0.6

4

0.4
Wind speed
Wave height

2
0
10:30

0.2

11:00

11:30

12:00
Time

12:30

13:00

0
13:30

11:00

11:30

12:00
Time

12:30

13:00

13:30

Wave height (m)Wind speed (m/s)

(a)

100
Microplastic density(items/m3)

(b)

10

1
10:30

Fig.2-2. Example of temporal changes in wind direction and wave height (a) and density of
microplastics (b) measured in CMSM 2018.
Wind speed, wave height and density of microplastics are plotted at towing commencement times.
10

Upwind side (particles/m3)

1.0 ≤ d < 5.0mm

1Note :

0.1

:
:

0.01
0.01

0.1

1

10

"Average" ± "Standard deviation" of"Proportion""Average" ± 2 x "Standard deviation" of“Proportion

Proportion ＝Downwind side (particles/m3)

Upwind side
Downwind side ＋ Upwind side

Fig. 2-3. Comparisons among different positions of sampling gear relative to wind direction
Table 2-1. Influence of sampling gear positions on microplastic samples. Displayed here are results ofin CMSM2019.
Proportion represents the ratio of between postiioning factors

Proportion
Port vs starboard

Numbers of data
Average of proportions
Standard deviation
T value
T testP value

Upwind side vsdownwind side

Towed in the same wind direction(Towed parallel to the winddirection)(Towed perpendicular to thewind direction)

20
0.53
0.13

9
0.53
0.11
-0.04
0.48

12

2.3 Sampling equipment
・ To collect microplastics floating at the ocean surface, most researchers use nets that canefficiently filter a large mass of water (Neuston, Manta or other nets).
・ Since microplastics are considered to exist in the ocean heterogeneously, it is desirable to sample
large volumes of sea water to obtain representative values in the sea area.
・ Since Neuston nets and Manta nets have been widely used in plankton surveys, knowledge has
accumulated on trawling methods that can facilitate their introduction for anyone wanting tostart or expand investigations of microplastics in the future. Also, their use makes it easier forthe data obtained to be compared with data accumulated in the past.
・ When net sampling, particles smaller than the mesh openings escape through the net. Therefore,
when collecting smaller particles, it would be more effective to sample the water using bottles,
buckets, pumps, etc., and filter the water on the vessel, or collect the ocean surface water usinga mesh screen sampler.
・ It should also be noted that the results obtained using other sampling equipment may not bedirectly compared to results obtained by net sampling because the differences in the sampledlayer and collected water volume are extremely large. To compare the results of such surveys,
further discussion on harmonization is needed.
・ Recently, unique devices for sampling have been proposed, for example, a series of sieves withdifferent mesh openings installed within the cod end of Manta net to fractionate plastic particles
by size whilst towing (Syakti et al., 2018).
・ When research vessels are not available, small fishing boats could be used instead of the research
vessels. Small boats usually had no equipment to tow nets. Therefore, it is necessary to prepare
additional simple equipment for towing. In CMSM2019, microplastic surveys were conductedusing small boats after the installation of an appropriate outfitting. The survey method using
small fishing boats is summarized in Section 2.3.1).
・ The following section highlights points to be noted regarding equipment to be used for surveys
by net towing.

13

2.3.1 Sampling vessels
Keynotes・ Oceanic surveys are usually conducted on research vessels. This may not always be convenientas the number of the research vessels is limited and large vessels cannot navigate in shallow
areas. In addition, using research vessels for a single mission may not be economical.
・ Smaller vessels could be used in place of research vessels. However, equipment such as cranes
to perform towing may need which might present a limiting factor in conducting surveys.

Outcomes of the pilot projects
・ A method of towing from the side of a small boat was investigated. A small fishing boat was
outfitted using several pipes, a clamp for fixing the pipes and a rope for supporting the pipes.
Successful microplastic sampling was conducted from the sides of the small boat accordingly.

Recommendations
・ With proper outfitting, it is possible for small fishing boats to tow sampling nets from the sidesof the boat.
・ The following points should be considered.
(1) To avoid the effects of the bow wave, outfittings (pipes) should be set at an appropriate
length to keep the nets away from the hull. Outfittings should also be installed at thesides and as far forward as possible of the boat to avoid the effect of the wake. This isalso considered to be effective methods from the viewpoint of avoiding contamination.
(2) The outfittings should be fixed to a place on the vessel that is strong, secure and stable,
such as a boat bollard.
(3) To prevent damage to the outfittings, the ends of the pipes should be stabilized withtension using a support rope.
・ Nevertheless, it is vital to pay close attention to safety management to prevent damage to theoutfitting, and also to prevent contamination from paint and cushioning materials on the vessel(see Section 2.6).

Explanatory Notes
・ In CMSM2019, the feasibility of towing with the simple outfitting of small boats was examined.
The boat used was a fishing boat with a gross tonnage of 13 t and a whole length of 11.97 m.
Although the vessel was not equipped cranes or booms, it is desirable to sample from sides of theboat therefore simple installations of outfitting were performed before the surveys commenced.

14

Pipe (L=4m, φ=48.6mm)
11.97 m

Clamp
Port

Boat bollard

Starboard

Suppot ropeNet
3m

Fig. 2-4. Towing was performed by simple outfitting on fishing boat in CMSM2019.
・ In the outfitting shown in Fig. 2-4, it was possible to tow the nets beside the boat. However, due
to the simple outfitting, special attention was required in the following points adding
recommendation noted.
Rigging strength is relatively weak on a simple outfitting. Thus, towing at an appropriate
speed (1-2 knots) is considered necessary (Section 2.4.2).
When a net of 0.10 mm mesh opening is towed, or when high quantities of biological material(e.g. fish eggs) are present, attention should be paid to protecting the outfitting, for example,
by washing the net along the way (Section 2.4.1).
The field blank test clearly identified particles of paint and cushioning material fragments
generated from the fishing boat (Section 2.6).

15

2.3.2 Net types

Neuston net

Manta net

Keynotes・ Neuston nets or Manta nets are most commonly used for sampling at the ocean surface.
・ Each type of net has its own features:(1) Neuston nets can capture the ocean surface layer in wavy conditions. However, it isdifficult to estimate the volume of water filtered accurately because the net's immersion
depth changes constantly.
(2) Manta nets can maintain a constant immersion depth at the sea surface. Filteredwater volume can be estimated fairly accurately providing there are no waves on thesea surface and the net maintains position. If the wave height exceeds a certain level,
the net tends to jump and skip on the water surface.
(3) In addition, the results obtained by a Catamaran net, the shape of which lies
somewhere between Neuston nets and Manta nets, were comparable to the results
obtained by a Neuston net when the particle diameters were 1 mm or larger.

Outcomes of the pilot projects
・ The number of particles per unit of filtered water volume was compared for particles larger
than 1 mm and less than 5 mm in their maximum Feret’s diameter (Section 3.4) sampled bysimultaneous towing using a Manta net and a Neuston net in the same area. The results
showed the number of particles captured by the Manta net tended to be slightly higher thanby the Neuston net, although not statistically different. This tendency was thought to becaused by differences in net immersion depth.

Recommendations
・ Results obtained with different net types are thought to be comparable when the nets have
similar immersion depth or the effects derived from different net immersion depth can becalibrated.
・ Assuming that either a Neuston net or Manta net will be selected based on the respective
advantages and limitations of each (to suit the purpose of the survey and conditions in thetarget sea area optimally). It is necessary to report weather and sea conditions at the time ofsampling along with net immersion depth.

16Explanatory Notes
Neuston nets with a side length of about 45 to 100 cm, or Manta nets with a width of 60 to 100
cm and a height of about 15 to 40 cm are most commonly used to collect microplastics from theocean surface. Both net types were developed and designed to collect plankton in the surface layer.
・ The Neuston net used in CMSM2019 (JMA Neuston net, RIGO Co., Ltd., No.5552）had asquare net mouth width and height of 75 cm each, and a net with 0.35 mm mesh openings.
When towing the Neuston net, immersion depth was set to 1/2 of the height (37.5 cm). The
Manta net (Manta net system, Ocean Instruments, Inc., OI-100) had a rectangular net mouth
100 cm wide and 20 cm in height, and a net with 0.35 mm mesh openings (in place of theoriginally attached net). When towed, the Manta net was submerged to the upper end of the net
mouth.
・ A comparison between a Manta net and a Neuston net was conducted by simultaneously towing
the nets during CMSM2019. The results were compared in terms of the number of collected
plastic particles (1-5 mm) per unit filtered water volume for particles. The Manta net tended tohave densities of microplastics which were slightly higher than those of the Neuston net although
there was no statistical difference between the two (Fig. 2-5).
・ The Manta net is thought to have contained a slightly higher number density as it collects thevery surface of the water, where a high density of plastic particles is likely to occur.
・ The proportion in the value by Manta net to the value by Manta and Neuston nets (Fig. 2-5) was
calculated to be 0.58 for observed microplastic densities collected with Manta and Neuston nets
in CMSM2019. The proportion value is expected to be 0.5 if there were no differences between
two nets.
・ As mentioned in Section 2.2, some studies (Kukulka et al., 2012; Kooi et al., 2016, etc.) haveproposed methods for estimating the vertical distribution of microplastics in a water column byusing wind speeds and wave heights during sampling. With an appropriate estimate of thevertical distribution of microplastics in the water column, it is possible to compare the density of
microplastic from nets at different immersion depths.
・ When the differences in their immersion depths were taken into account, the proportion was
estimated to be 0.59 using the formula proposed by KuKulka et al (2012) using the wind speed
and wave height at the time of sampling. When estimated values are close to the observed values
with the correction, this may verify that the differences in particle the collection between Manta
and Neuston nets were attributable to their different immersion depths.
・ Similarly, Eriksen et al., 2017 reported that although there was no statistical difference in thenumber density of plastic particles collected with a Manta net (net immersion depth: 16 cm) andan AVANI net (elongated rectangular Neuston net with an aspect ratio of about 5: 1 and net
immersion depth of 30 to 60 cm), there was a statistically significant difference in weight ofparticles. This difference is speculated to have arisen from a difference in collection layer and atendency for plastics at relatively high densities to float slightly below the surface layer, suchthat the AVANI net would catch more particles in high density areas than the Manta net.

17

・ For nets used for quantitative collection, the net opening ratio (ratio of the total area of the net’smesh openings to the area of the net’s mouth opening) needs to be 5 or more when using a net
with mesh openings of 0.3 mm or more (Tranter & Smith, 1968), and preferably 9 or more whenusing a net with smaller mesh openings (Saito, 2018).
・ For the net sampling of microplastics at the ocean surface, conducting sampling under conditions
that avoid clogging and inhibition of filtering is recommended, in addition to confirming the net
opening ratio of the net to be used.
Table2-2 Advantages and disadvantages of different nets for collecting floating microplastics
identified by CMSM2019.
Manta net
Neuston net

Advantages
Remains in surface water
except in rough water.
Operates in relatively roughwater.

Disadvantages
Tends to jump and skip onrough water.
Needs some efforts to maintainthe stable net immersion depth.

10(particles/m
Manta net
(items/m33))

1.0 ≤ d < 5.0mm

1

Port

Neuston net

Starboard

Manta net

Note ::

0.1:"Average" ± "Standard deviation" of"Proportion""Average" ± 2 x "Standard deviation" of“Proportion

Proportion ＝

0.01
0.01

0.1

1

Manta net
Manta net ＋ Neuston net

10

3) 3)
(particles/m
Neuston net (items/mFig. 2-5. Comparisons between different net types in CMSM2019.
Table 2-3. T test results of significant difference in the density of microplastics between different net
types (in CMSM2019).
Proportion is the ratio of the 'Manta' value to the sum value of 'Neuston' plus 'Manta.'

Numbers of data
Average of proportions
Standard deviation
T value
T testP value

Proportion
Port (Neuston) vsStarboard (Manta) vsstarboard (Neuston)Port (Neuston)
20
6
0.53
0.58
0.13
0.12
-0.84
0.20

18

2.3.3 Mesh openings
Keynotes・ In general, past surveys generally used mesh openings of about 0.3 mm. Reasons for this choiceinclude the ability to filter large amounts of seawater, suitability for sea conditions andplankton abundance. Nets with mesh openings of 0.20 mm or 0.10 mm have also been used.

Outcomes of the pilot projects
・ Two Neuston nets with the same design but with different mesh openings, 0.35 mm and 0.1
mm, were employed in the pilot projects. Both nets were towed simultaneously. There was no
significant difference in the number of particles > 1 mm in size. However, for particles < 1 mm,
the number of particles collected with a net with mesh openings of 0.10 mm was about fourtimes larger compared to those collected with a net with 0.35 mm mesh openings (Table 2-4).
・ Many sticky fish eggs were observed surface waters when the nets were towed to study
differences between mesh openings. Nets with 0.1 mm mesh openings were clogged with thefish eggs. Therefore, this may have attributed to the collection of fewer particles by nets with
0.1 mm mesh openings.
・ Additionally, a significant decrease in precision was observed in ILC on microplastic analysis
for particles less than 1 mm in maximum Feret's diameter.

Recommendations
・ For the purpose of comparing floating microplastic pollution of various sea areas, or from a
broader, global perspective, the use of the most common mesh opening (0.3 mm) is considereddesirable.
・ On the other hand, monitoring using a net with finer mesh openings would be useful because
data on smaller particles are essential for elucidating the behavior of microplastics in the ocean
as well as the effect of uptake of by organisms.
・ Obtaining data related to smaller mesh openings would be beneficial to obtaining information(providing a coefficient to convert between sizes) on smaller particles, although this can be
influenced by sampling location, size distribution and the accuracy of analysis of smaller
particles.
・ When clogging of the net is inevitable, it is necessary to take measures such as shortening the
tow duration accompanied with repeated sampling to obtain the appropriate tow duration or
tow distance (see Section 2.4.1), and appropriate pretreatment to digest a large amount oforganic matter in the samples before analyses (see Section 3.2.1).
・ Microplastics which are similar in size to the mesh openings may be under-sampled if theirshortest length is smaller than the mesh openings. It is advisable to measure and report
particles <1 mm separately from particles 1 mm - 5 mm.

19

Explanatory Notes
Mesh openings as used in the Guidelines is expressed as the side length of aquadrangle separated by mesh thread and through which sea water passes ((A)
in the figure on right), but in some cases the length of the diagonal line ((B) in
the figure on right) is used as the mesh opening. The researcher should confirm
which mesh opening is meant and record the mesh opening used for the survey.

①
(A)
②
(B)

In CMSM2018 for particles less than 1 mm, the number of particles collectedwith the net with mesh openings of 0.10 mm was about two to five times larger compared to thosecollected with the net with 0.35 mm mesh openings.
・ There were fewer particles with the shortest of length less than 0.5 mm when using a net withmesh openings of 0.35 mm (Fig.2-6). It is conceivable that some particles may pass through a net
with mesh openings of 0.35 mm and not be collected.
・ The net with mesh openings of 0.35 mm used in CMSM2018 had rectangular openings with aside length of 0.35 mm separated by mesh thread through which sea water passed. Assuming theparticles and the mesh did not distort, particles with the shortest length of 0.49 mm or less, whichis the diagonal length of the mesh openings, could pass through the screen.
・ In general, using a net with mesh openings of 0.1 mm or 0.2 mm enables the collection of small
particles which could be under-sampled when using one with larger openings of 0.3 mm, which
was also supported in CMSM2018. Considering the possibility of clogging, however, the samplingtime may have to be limited in case there are high densities of natural particles or organisms,
and problems may arise from the viewpoint of securing the required amount of filtered water.
・ Also, in CMSM2019, similar to CMSM2018, the same two Neuston nets with different mesh
openings of 0.35 mm and 0.10 mm were simultaneously towed at the port and starboard sides,
and their microplastic densities were compared. There were no statistically significant
differences in the numbers and densities of particles larger than 1.0 mm.
・ On the other hand, the number of particles collected with the 0.10 mm mesh opening net was
fewer than those with 0.35 mm mesh openings for particles smaller than 1 mm in CMSM2019.
(Table 2-5, Fig.2-7) At this time, it was observed that a rotation counts of a flowmeter attachedwith 0.10 mm net was lower than the other and Neuston nets with 0.10 mm mesh openings wereclogged with fish eggs. It might be caused by reducing in filtered volume for the 0.10 mm net due
to plenty of sticky fish eggs. This clogging may cause fewer particles collected with nets of 0.10
mm mesh openings.
・ If surveys are to be conducted using a net with finer mesh in sea areas or seasons where clogging
may occur, depending on the purpose of the survey, it is necessary to set appropriate survey
parameters to minimize the effects of clogging as much as possible parameters include the tow
duration, net-washing interval. In addition, these parameters and clogging conditions should berecorded especially when the level of clogging may change during the sampling season.
Obtaining data related to smaller mesh openings would be beneficial to obtaining information(providing a coefficient to convert between sizes) on smaller particles, although this can be
influenced by sampling location, size distribution and the accuracy of analysis of smaller particles.
In the future, it will be necessary to optimize a method for sampling in bloom conditions.
20

Table 2-4. Comparison of mesh openings (0.35 mm vs. 0.10 mm), number of particles obtained insimultaneous sampling cases, and their ratio in CMSM2018.
(1)'d' is maximum Feret's diameter; (2) 'Ratio' refers to the ratio of the number obtained with '0.10 mm' tothat obtained with '0.35 mm'; (3) For particles of < 1 mm, final results are regarded as underestimated forboth nets, due to discrepancies arising during analytical processing in the laboratory.
Numbers of particles (particles/sample)
Mesh
Sampling
Total
openingsd<1.0mm
1.0 – d < 5.0 mm
No.
(d < 5.0 mm)(mm)
Number Ratio Number
Ratio
Number Ratio
0.35
146
159
305
No.1
1.98
1.36
1.66
0.10
289
217
506
0.35
105
154
259
No.2
4.46
1.47
2.68
0.10
468
227
695
0.35
116
92
208
No.3
4.78
2.32
3.69
0.10
555
213
768
0.35
122
135
257
Average
3.57
1.62
2.55
0.10
437
219
656

Fig.2-6. Size distribution of plastic particles collected using nets with mesh openings of 0.35 mm
and 0.10 mm at the same time in the same area in CMSM2018.
The X axis plots the longest length (maximum Feret’s diameter) and the color of the bars indicates theshortest length (minimum Feret’s diameter).

21

Table 2-5. Comparison of mesh openings (0.35 mm vs. 0.10 mm), numbers of particles obtained insimultaneous sampling cases, and their ratio in CMSM2019.
Mesh
Sampling openings
No.
(mm)
No.1
No.2
No.3
Average

0.35
0.10
0.35
0.10
0.35
0.10
0.35
0.10

Numbers of particles (particles/sample)
Total
d < 1.0 mm
1.0 – d < 5.0 mm(d < 5.0 mm)
Number Ratio Number
Ratio
Number Ratio
30
30
60
0.12
0.58
0.43
4
42
46
37
19
56
0.47
0.42
0.46
33
14
47
55
45
100
0.35
0.52
0.44
29
49
78
41
31
72
0.31
0.51
0.44
22
35
57

50

50

Mesh openings: 0.35mm
40

Minimum Feret’s
diameter (d min)Number of particles

Number of particles

40

Mesh openings: 0.10mm

0.5 ≤ d min (mm)30

0.4 ≤ d min < 0.5
0.3 ≤ d min < 0.4

20

0.2 ≤ d min < 0.3
0.1 ≤ d min < 0.2
d min < 0.1

10

30

20

10

0

0

0

1.0
2.0
3.0
4.0
Maximum Feret’s diameter (mm)

5.0

0

1.0
2.0
3.0
4.0
Maximum Feret’s diameter (mm)5.0

Fig.2-7. Size distribution of plastic particles collected using nets with mesh openings of 0.35 mm and0.10 mm at the same time in the same area (in CMSM2019).
A sample at the time of collection. The bottle on
the left is a sample collected by Neuston net with
0.10mm of mesh openings, and the bottle on theright is a sample collected by Neuston net with
0.35mm of mesh openings. The bottle on the left
side contains more fish eggs and other floating
matter.

Fig.2-8. Plastic particles collected using nets with mesh openings of 0.35 mm and 0.10 mm at thesame time in the same area (in CMSM2019).

22

・ Also, in the laboratory analysis, as described in Chapter 3, the accuracy of separating
microplastic of < 1 mm decreases. Thus, in measuring microplastics collected using a net withmesh openings of about 0.3 mm, it is advisable that the results for particles 1-5 mm be reported
separately from those of particles of < 1 mm in size.
・ Therefore, from the viewpoint of harmonizing monitoring methods, using a net with mesh
openings of about 0.3 mm is recommended as it is currently most commonly used. It should be
noted, however, that even if the longest length is sufficiently greater than 1 mm, particles with asufficiently short shortest length (fibrous particles) may pass through the net.
・ Thus, it should be kept in mind that results for particles which are almost the same size as themesh openings, and those with a much shorter shortest length may be underestimated whencomparing the results collected by nets with different mesh openings.

23

2.4 Tow parameters2.4.1 Tow duration
Keynotes
Tow duration used in surveys has been 10 to 30 minutes. This usually depends on theabundance of plankton or floating matter at the ocean surface and the amount of the sampled
particles required for analysis.

Outcomes of the pilot projects
Quantities of microplastics were assessed using simultaneous net sampling in the same
location to assess tow duration. Two different comparisons were conducted 1) 20 minutes
towing at port side and two consecutive runs of 10 minutes towing at starboard; 2) 10 minutes
towing at portside and two consecutive runs of 5 minutes towing at starboard.
There was no significant difference in the number of particles sampled between any tows of 20
minutes, 10 minutes, or 5 minutes in duration (Fig.2-9). However, when particle density was
relatively high in the ocean (~10 pieces/m3) there were discrepancies between the first andsecond run of consecutive tows (both 5 and 10 minutes). There was also a large variation
between port and starboard results. These findings are similar to those of the study by Van del
Hal et al. (2017).

Recommendations
It is recommended that an appropriate volume is sampled to reduce the influence ofheterogenous microplastic distributions, since in some cases microplastics were found floatingin large patches (Van del Hal et al., 2017). For example, a tow duration of approximately 20
minutes, as seen in many of the previous studies, appears to generate sufficient data.
Appropriate volume could be adjusted depending on the microplastic density in the ocean
surface.
Also, it is desirable to sweep at least 1,000 m2 of ocean surface per sample. This is equivalent
to 200-500 m3 of filtered water when towed with a typical net. However, this may not always
be applicable depending on sampling conditions. For example, high densities of floating
material (e,g, fish eggs, plankton, etc.) may hinder tow durations. It is prudent to bring a sparenet. If clogged, one net could be replaced with a second one to sweep the required area of theocean surface in a combined tow.

Explanatory Notes
・ In many earlier studies, net towing was conducted for 10 to 30 minutes. At the 1st and 2nd
International Expert Meetings, 20 minutes was recommended for tow duration to cover the areaof trawling required to obtain representative values in the sea area.
・ Regarding tow duration, many guidelines state that 15 to 30 minutes is appropriate (Lippiatt etal, 2013; EC, 2013; GESAMP, 2015; etc.). Considering the heterogeneous distribution as
mentioned above, trawling for shorter durations may be inappropriate from the viewpoint ofobtaining representative values in the sea area.

24

・ In CMSM2018, the number of microplastics was compared between those collected by towing at
the port side for 20 minutes or 10 minutes, and in the same sea area and at the same time, with
the net at starboard exchanged in the middle of the trip to make two 10 minutes tows or two 5
minutes tows. The results showed no significant difference observed in the number of plastic
particles of larger than 1 mm and less than 5 mm due to tow duration, but dispersion has been
observed when densities of particles at the ocean surface are relatively high in the survey area.
・ Dispersion in the results may have been due to coincidental sampling of high-density water
masses, for example, from prominently heterogeneous distributions of particles that are formed
when microplastic densities at the ocean surface are high.
・ To capture representative values in the sea area, it is necessary to reduce the influence of suchhigh-density water masses and obtain a leveled result.
・ Consequently, it is deemed desirable to set tow duration at about 20 minutes, within a range that
does not cause clogging of the mesh due to plankton or floating matter.

10 minutes (continuous)
5 minutes (1st)(particles/m3)(particles/m3)

5 minutes (2nd)Fig.2-9. Comparison of microplastic densities at different tow durations.

25

2.4.2 Vessel speed
Keynotes
・ Vessel speeds for sampling have been reported between 1 to 3 knots in earlier surveys.

Outcomes of the pilot projects
・ Tows were conducted with a vessel speed of 1 to 3 knots against water current (normallyreferred to as log speed). Towing at 2 knots (about 1 m/sec) for 20 minutes with a net 75 cm
in width resulted in approximately 1,200 m of tow distance, and samples collected from about
1,000 m2 of sea surface area or approximately 350 m3 of sea water volume.
・ When sampling was carried out using a small fishing boat tows had to be conducted with avessel speed of less than 2 knots because the outrigging was relatively weak.

Recommendations
・ Regarding vessel speeds for towing, if the speed is too fast, the inflow at the net mouth
becomes turbulent and the filtering efficiency may decrease (Ogi, 1991; GESAMP, 2016). It isthought that the towing vessel speed should be set at 1 to 3 knots, although this depends on
the type of equipment and vessels used.
When sampling is carried out using a small fishing boat, simple outfitting should be provided.
As the rigging can be relatively weak in simple outfitting, a reduced towing speed (1-2 knots)
is considered necessary.

2.4.3 Sweep area and filtered water volume
Keynotes
Microplastics observed at the ocean surface are often reported as the number of particles orweight per unit area (/m2, /km2) and/or as the number of particles per unit water volume(/m3). Therefore, it is necessary to obtain the swept area of the net tow and/or the amount offiltered water volume, as calculated by the following equations:
Swept area＝net width × tow distance
Filtered water volume＝(net width × net immersion depth) × tow distance* Net width is the horizontal dimension of the net aperture

Recommendations
Refer to Section 2.4.4 for the estimation of tow distance. Method, equations and numeric
figures used when estimating tow sweep area and filtered water volume should be reported.

26

2.4.4 Tow distance

Flowmeter (RIGO.No.5571)

Neuston net with flowmeter

Keynotes
・ Microplastic abundance at the ocean surface is reported as particle number or weight per unit
water volume or unit surface area. Therefore, the filtered water volume or the swept surface
area of each net sampling should be estimated by the chosen reporting units.
・ Generally, filtered water volume or the swept surface area are obtained by multiplying the tow
distance by the net immersion area or the net aperture width respectively. There are threemethods for obtaining tow distances:(1) Calculate from ground speed obtained from position information measured by GPS, etc.
(2) Calculate from the relative speed of the vessel to seawater (log speed), measured with acurrent meter.
(3) Calculate using the rotation count of a flowmeter installed in the net mouth and its
calibration value.

Outcomes of the pilot projects
・ Tow distances were measured using all three methods simultaneously and the results werecompared. Tow distance calculated using method (1) showed large differences depending on thedominant direction of water flow when compared to methods (2) and (3). Results for methods(2) and (3) were similar to each other.

Recommendations
・ Method (3) appeared to generate the most accurate value for estimating the water volume
passing through the net both theoretically and experimentally.
・ It is recommended that Method (3) be used with a flowmeter set at the net mouth to obtain the
tow distance, the density of microplastics per swept area and also density of microplastics per
filtered water volume. This is further supported by other guidelines (i.g. GESAMP, 2019).
Calibration of the flowmeter is important and necessary. Location/vessel position at the startand end of each tow should be recorded.
・ If a flowmeter is not available, it is necessary to estimate sampled water volume using anappropriate tow distance calculation through other alternative methods, such as using speed
relative to sea water.
27

Explanatory Notes
・ Earlier studies have reported large differences between tow distances calculated from ground
speed and tow distances calculated by a flowmeter (Suaria et al., 2016).
・ In CMSM2019, the tow distance obtained from the vessel speed relative to the ground using GPS,the speed of the vessel relative to seawater measured by the flowmeter, and the rotation number
and calibration value using a flowmeter, were compared. When comparing distances calculated
from the relative speed of the vessel to the seawater (log speed) and distances calculated using aflowmeter, both methods produce similar results. CMSM2019 found there to be no statisticallysignificant difference. However, data obtained using a flowmeter is fundamental, so it isrecommended to use the flowmeter as considered by other guidelines (GESAMP, 2019) anddiscussions of expert meeting.
・ On the other hand, a difference was observed when comparing the tow distances obtained fromthe vessel speed relative to the ground using GPS and calculated using a flowmeter, in some cases(Fig.2-11, left side). It is assumed that tow distance calculated from ground speed may not reflect
the actual amount of filtering.
・ For this reason, it is desirable to estimate the amount of filtered water by attaching a flowmeterto the net mouth.
・ In cases with high waves that may cause the flowmeter to pop above the water surface during
towing, it would be desirable to maneuver the vessel to ensure the flowmeter is submerged sothat the tow distance can be measured correctly.
・ When using a flowmeter, it is necessary to select a model that can accurately measure filteredwater distances at different towing speeds. In addition, it is important to ensure thatmeasurements can be conducted accurately at the planned towing speeds before towing.
・ Flowmeters should be calibrated in advance. For example, nets could be towed horizontally in apool equipped with a water flow generator for calibration. Alternatively, a flowmeter could beinstalled in the net frame without a net and sunk to a certain depth. It is then pulled up to thewater surface, and the distance calculated using the flowmeter could be compared with the actual
sunken depth.
・ When filtered water volume needs to be estimated based on distance measured, for example using
GPS and when there is a strong water current in the survey area , the estimation of the sampled
distance by adjustment of the filtered water volume against the current direction and speed
should be considered (Fig.2-11). However, there were no statistically significant differences(Table 2-6).

28

Distance calculated from log speed (m)Lower filtered distances
measured by flowmeter due
to significant net clogging

Note 1

1000: "Average" ± "Standard deviation" of"Proportion": "Average" ± 2 x "Standard deviation" of“Proportion
Distance calculated from log speed"Distance measured by flowmeter” +"Distance calculated from log speed"
Average of proportion: 0.50, Standard deviationof proportion: 0.069

Proportion ＝Note 2;

100
100
1000
Distance measuerd by flowmeter (m)Fig.2-10. Relationship between tow distance obtained using a flowmeter and distance calculated
from log speed in the CMSM2019 survey.

Towed against the water current
Ground dist. > Filtered dist.

Distance calculated from ground speed
and current data (m)Distance calculated from ground speed(m)

For calculating these data, the tow distance obtained while towing a Neuston net with mesh openings of
0.35 mm was used.

1000

1000

Towed against the water current
Ground dist. < Filtered dist.

100

100

Adjustment of grounddistances against current
data produces better
filtered distances
100
1000
Distance measuerd by flowmeter (m)

100
1000
Distance measuerd by flowmeter (m)Opposite direction from water current
Same100
direction as water current
Direction perpendicular
to water current, or no tidal current
100
1000(slack water)

Fig.2-11. Relationship between tow distance obtained using a flowmeter and distance calculated fromground speed using coordinates obtained by GPS in the CMSM2019 survey.
Note 1: "Average" ± "Standard deviation" of "Proportion": "Average" ± 2 x "Standard deviation" of “Proportion""Distance calculated from ground speed"
Proportion ＝"Distance measured by flowmeter" + "Distance calculated from ground speed"Note 2 Averages and standard deviations of proportion are as follows.
Distance calculated fromDistance calculated fromground speed
ground speed and current data
Average
0.52
0.53Standard deviation
0.078
0.064
Note 3 Current data measured for Sagami Bay by Japan Coast Guard with short-wave ocean radar used above

29

Table 2-6. T test results of significant difference in distances between various methods (in CMSM2019).
Proportion' is the ratio of the 'Manta' value to the sum value of 'Neuston' plus 'Manta.
(A) Proportion of "Port" vs "Starboard" of distances"Port" of distances measured by flowmeter=measured by flowmeter"Port" of distance measured by flowmeter + "Starboard" of distance measured by flowmeter(B) Proportion of "Distance calculated from log speed" vs"Distance measured by flowmeter"="Distance calculated from log speed""Distance calculated from log speed" + "Distance measured by flowmeter"(C) Proportion of "Distance calculated from ground speed
and current data" vs "Distance measured by flowmeter"="Distance calculated from ground speed""Distance calculated from ground speed" + "Distance measured by flowmeter"(D) Proportion of "Distance calculated from ground speed
and current data" vs "Distance measured by flowmeter"="Distance calculated from ground speed and current data""Distance calculated from ground speed and current data" + "Distance measured by flowmeter""Distance
calculated
from log
speed" vs"Distancemeasuredbyflowmeter
" (B)

41

47

0.50

"Distance calculated from ground speed" vs"Distance measured by flowmeter" (C)"Distance calculated from ground speed
and current data" vs "Distance measured
by flowmeter" (D)Direction
Opposite Same
Direction
perpendicular
direction direction perpendicular
to waterfromas water to water
current, or nowatercurrent current, or no
tidal current
current
tidal current(slack water)(slack water)Same
direction
as watercurrent

47

20

12

15

47

20

12

15

0.50

0.52

0.49

0.57

0.51

0.53

0.54

0.52

0.52

0.033

0.059

0.078

0.088

0.062

0.053

0.064

0.074

0.058

0.048

T value--0.019-1.1890.607-4.817-0.757-2.355-3.048-1.134-1.222

P value

-

0.49

0.12

0.27 0.0000067

0.23

0.010

0.0017

0.13

0.11

30

Opposite
direction
from
water
current

Numbers of data
Average of
proportion
Standard
deviation
T test"Port" vs"Starboard" ofdistancesmeasuredbyflowmeter(A)2.4.5 Tow position
Keynotes・ In general, a sampling net is towed at the side of a vessel. In some cases, it may be towed at
the stern provided it is positioned to avoid the wake of the vessel.

Outcomes of the pilot projects
・ Effect of tow position on sample collection was tested using nets towed at one-vessel length andtwo-vessel length behind the stern. There was no statistically significant difference in thedensity of microplastic samples collected at one-vessel length behind the stern of the vessel
and those collected at its side. On the other hand, there was a statistically significant difference
between the particles collected at two-vessel length behind the stern of a vessel and thosecollected at its side.
・ Therefore, it is very difficult to correct particle densities obtained at the stern by sampling at
the sides of the vessel.

Recommendations
・ It is desirable to conduct sampling at the side of the vessel with less influence from turbulencecaused by its wake.

Explanatory Notes
・ Nets are generally positioned on either side of the vessel (port/starboard) or at the stern. In
CMSM2018, densities of microplastics collected were compared by towing Neuston nets at theport, starboard and stern simultaneously.
・ With the net set at the stern (vessel size 16 m, rope length 20 m, towed directly behind thehull), the density of microplastics >1 mm was less than that obtained by collecting at the vessel
side, suggesting the influence of vertical mixing of microplastics caused turbulence from thewake, etc. (Fig.2-12)
・ Furthermore, in CMSM2019, particle collection was tested further using nets towed at onevessel length (12 m) and two-vessel length (24 m) behind the stern. There was no statisticallysignificant difference in the density of microplastics collected at 12 m behind the stern of thevessel and those collected at its side. However, there was a statistically significant difference(p value is 0.001) between the particles collected at 24 m behind the stern of a vessel and thosecollected at its side (Table 2-7).
・ Fewer particles captured by the stern tows could be attributed to the size & shape of a boat, its
speed & position of its propeller, meteorological & hydrographic conditions, and distributions ofparticles.
・ Since it would be very difficult to adjust the particle densities obtained by the stern tows to
those for the side tows, particles should be collected by the side tows.

31

(b) 8Tow position
Port side
Starboard side
Stern

0.6

0.4

0.2

Density of particles (particles/m3)

Density of particles (particles/m3)(a) 0.8

0

6

4

2

0
No. 1

No. 2

No. 3

Sampling No.

No. 4
Sampling No.

Fig.2-12. Particle density comparisons depending on tow position (In CMSM2018).
These are divided into two figures according to density of particles due to wide disparities, reflected in thelarger scale of the Y-axis in (b): (a) 0-0.8 particles/m3, and (b) 0-8 particles/m3.

32

(particles/m3)(particles/m3)(particles/m3)(particles/m3)
Note:
:
:

"Average"±"Standard
deviation" of "Proportion""Average" ± 2 x "Standard
deviation" of “Proportion

Fig. 2-13. Particle density comparisons depending on tow position (In CMSM2019).
Proportion is as follow.
Proportion ＝Density of "Stern" +Density of "Stern"
Density of "Port" + Density of "Starboard"
2

Averages and standard deviations of proportion are as follows.

AverageStandard deviation

Distance from boat
to stern net of 12 m
(same as the boat length)
0.55
0.09

33

Distance from boat
to stern net of 24 m
(two boat length)
0.31
0.12

Table 2-7. T test results of significant difference in the density of microplastics between theparticles collected at the stern of the vessel and those collected at its side. (inCMSM2019).
"Prop." is Proportion. Prop. Port vs starboard and Prop. Stern vs side are as follows.
Density of "Port"
Prop. Port vs starboard ＝Density of "Port" + Density of "Starboard"
Prop. Stern vs side ＝Density of "Stern" +Density of "Stern"
Density of "Port" + Density of "Starboard"2

Proportion
Stern vs side
Distance from boat
Distance from boat
to stern net of 12 m
to stern net of 24 m
(same as the boat length) (two boat length)

Port vs starboard
Numbers of data

20

9

8

Average of proportion

0.53

0.55

0.34

Standard deviation

0.13

0.10

0.12

T value

-0.40

-3.52

P value

0.34

0.001

T test34

2.4.6 Net immersion depth
Keynotes・ Net immersion depths have been recorded between 10 cm and 100 cm. Manta net immersion
depth is measured as the height of the net's mouth, whereas a Neuston net is often set at about
1/2 to 3/4 of the height of the net's mouth.
・ Recording immersion depth of the net during sampling is important as the area of the net
mouth under the sea surface is multiplied by the tow distance to estimate the filtered water
volume.

Outcomes of the pilot projects
・ The Manta net tended to jump and skip above the sea surface when the waves were rough.
・ There were cases when the Neuston net sank over time as a large amount of floating matter
was collected and maintaining a constant immersion depth was considered difficult when the
waves were rough or there was an abundance of drifting seaweed, etc.
・ The immersion depth was kept constant by installing the floats with enough buoyancy.

Recommendations
・ It is necessary to tow the net in a way that keeps the immersion depth constant. Measures
such as attaching a moderate weight, adjusting the length of the tow rope and avoiding highwave conditions that may cause the Manta net to jump and skip on the water surface arerecommended.
・ This is particularly important when towing a Neuston net and in conditions when theimmersion depth cannot be controlled effectively due to large amounts of floating matter.
Periodically recording the net immersion depth during each sampling run is consideredeffective for accurately calculating the filtered water volume.

Explanatory Notes
・ Data on net immersion depth at the time of towing is required for accurate calculation of the
net sampling area and filtered water volume. It is necessary to keep the immersion depth asconstant as possible. It is also important to clarify the depth from the surface at samples arecollected.
・ In CMSM2018 and CMSM2019, Manta nets were observed to jump off the sea surface whenwind and waves were present, making them difficult to tow. Although it is possible to makesome adjustments by attaching a heavier weight to the net mouth or by changing the directionof the net relative to the wind and current, accurate sampling is assumed to be difficult if thewind is strong and waves are high.
・ When a Neuston net was used, there was a greater change in net immersion depth when large
pieces of floating matter (seaweed, jellyfish, etc.) were caught in the net, especially when a net
with finer mesh openings was used or large amounts of plankton were caught. In this case, thechange in net immersion depth could not be reversed even if the length of the rope wasadjusted.
35

・ Therefore, in surveys using a Neuston net, attention is required when comparing results.
Significant changes in immersion depth can be expected when many large pieces of floating
matter are also present. This may also reduce filtering efficiency.
・ In CMSM2019, the Neuston net was kept at constant immersion depth using floats attached to
the frame (Fig.2-15). The buoyancy of those floats was about 40 kg when the mouth of the Neuston
net of W:75 cm x H:75 cm x L:3 m was half immersed (immersion depth: 37.5 cm).
・ When towing in a sea area with a lot of floating matter, it is helpful to set a marker at the net
mouth to indicate the immersion depth, and record the immersion depth at the net mouth by thevideo camera or by taking photographs of the net mouth periodically (Fig.2-14).

36

Immediately after tow

13 min. after tow starts

Samplestarts (net immersion(net immersion depth

increased immersion depth

depth normal)

increased).

(a

large

insidenet

amount

of

with
sea

buckthorn (Zostera marina)
has been caught).
(May 18, Tow No. 5, starboard side)
Fig.2-14. Chronological changes in net immersion depth obtained through image analysis.

Fig.2-15. Adjustment of immersion depth of the Neuston net by attaching appropriate floats.
37

2.5 Metadata
Keynotes・ In general, wind speed and wave height have a large effect on the microplastic density in theocean surface layer (e.g., Reisser et al., 2015, Suaria et al., 2016).

Outcomes of the pilot projects
Microplastic particle density at the ocean surface decreased when wind speed and wave heightincreased.
・ High density of microplastics were observed at the ocean surface when salinity decreased(Fig.2-16). Sampling in water bodies that were affected by river water is a possible cause.

Recommendations
・ To ensure comparability, metadata for each sampling event should be recorded through in situ
observations or onboard instruments where possible. Data required include the time of day anddate (to account for seasonality), as well as environmental variables (e.g., weather conditions,wind speed, wind direction, wave height, Beaufort scale index, chlorophyll, fluorescence,salinity etc.) and additional sampling parameters. For more details, refer to Chapter 4 (p. 66).

Explanatory Notes
・ Start position coordinates and specific method information (such as tow time, tow speed, therotation number of the flowmeter, net position and net immersion depth), must be recorded.
・ A nearly ten-fold difference in microplastic density at the ocean surface was observed over a
relatively short time (about 20 to 30 min.) and a small spatial scale (distance of about 100 to
500 m) in CMSM2018. Some correlations were observed when chronological changes in density
were compared with physical environment data (wind and waves) and water quality data
(water temperature and salinity). Specifically, as wind speed and wave height increased, thedensity of microplastics tended to decrease (see Section 2.2, Fig.2-2), and when salinity
decreased the microplastic density tended to increase (Fig.2-16).
・ It is necessary to record wind direction and wave height before and after each sample. Adverse
weather conditions in the days prior to the survey may affect microplastic densities at the ocean
surface.
・ There were cases in which the density of microplastics may have increased due to an influx ofriver water in CMSM2018. When rain is observed shortly before or on the day of the survey,
data on precipitation would be beneficial. River flows are strongly influenced by rainfall which
may influence both freshwater input and salinity and consequently floating microplastics.
・ The influence of tidal current direction and flow rate on microplastic collection results is notclear, but it is beneficial to record these as they are useful in considering the influence of loads
from land areas via rivers. If the vessel does not have a current meter, it is recommended torecord survey conditions using publicly available oceanographic data for the surveyed area.
・ As water temperature and salinity are generally characteristic for each water mass, watertemperature and salinity are considered useful information in confirming whether the38

properties of the water mass have changed between tows, especially in coastal zones that areeasily affected by rivers and tides.
・ In addition, there are indications that it is possible to minimize variance in collection results bytowing in a way that keeps the direction of the net relative to the wind direction constantly
perpendicular. It would be desirable to record the direction of the net relative to the winddirection and ocean current.
・ The presence of floating matter captured in the net can also be recorded.

Tide level (cm)

(a)

150

31

120

30

90

29

60
30
0
9:30

Microplastics density(items/m3)(b)Tide level
Salinity

28
27

10:00

10:30

11:00

11:30
Time

12:00

12:30

13:00

26
13:30

10:00

10:30

11:00

11:30
Time

12:00

12:30

13:00

13:30

10

1

0.1
9:30

Fig.2-16. Tide level and salinity (a) and chronological changes in microplastic density (b).
Microplastic density plotted at the time of tow start.

39

Salinity (-)

・ Variables may differ from sampling cruise to sampling cruise, or even from sample to sample. If
it is possible to average all variables during a sampling event, e.g., 20-minute tow, this ispreferable over only recording the information at the beginning and the end of each tow.

2.6 Field sample blanks
Keynotes
Generally, in the net sampling of microplastics, the net is cleaned thoroughly from its outside
before the start of a sampling run to ensure no plastic particles are left inside the net. The
influence of plastic particles remaining in the net on the survey results can be significant,especially in sea areas where the number of sampled microplastics is relatively small.
Therefore, cleaning just before each sampling run is particularly important to prevent plastic
particles from clothing, equipment, the vessel's paint, etc. from entering the net and affecting
the results.

Outcomes of the pilot projects
Blank tests were carried out for a research vessel eight times in a similar manner to thosereported (GESAMP, 2019) by washing the net before towing, comparable to washing after
towing, by hanging the net with a crane and pouring pumped sea water from the outside thenet, then counting the microplastics in the cod end. Two microplastic particles were observed
on average.
A similar blank test was carried out for a net that had been kept in a natural fiber bag for along time after being thorough cleaning at the end of a survey. This specific net was observed
to contain many particles, indicating that contamination may occur during storage.
Blank tests for the fishing boat was also carried out, and the results clearly identified particles
of paint and cushioning material fragments generated from the fishing boat.

Recommendations
Nets should be washed thoroughly before each sampling run due to the risk of contamination
during storage.
It is necessary to take extra care to avoid contamination with regard to on-board operations
and the storage of nets.
A field blank test is recommended to be conducted for at least one of several nets to be used fora survey, as it can confirm whether sampling procedures such as washing have been carriedout properly without contamination. When towing multiple times, it would be desirable toperiodically conduct blank tests to ensure particle contamination has been sufficiently
controlled.

Explanatory Notes
・ To understand how accurately procedures such as washing are carried out, implementing a blanktest for at least one out of several nets is recommended.
・ Generally, when net towing is completed, the rotation number of the flow meter is firstrecorded, then the net is hung using a crane or pulley and cleaned thoroughly from the outside.
For washing the net, it is common to use sea water pumped up using a pump installed aboard
the vessel. When doing so, care needs to be taken to avoid having the sea water enter the net
via the mouth.
40

・ In CMSM2018, field blank sampling was conducted by washing unused nets in the samemanner as for sampling, and on average two plastic particles (ranging from 0 to 5) wereconfirmed.
・ Confirmed particles were all sufficiently shorter in shortest length than the mesh openings(0.35 mm), and the composition of the material also differed from those obtained in thesurveyed surface layer. Therefore, it is assumed that plastic particles smaller than the mesh
openings do not cause contamination when washing from the outside of the net.
・ Also, when a net that had been stored for a long time was used in a blank test without washing
immediately before use, more particles were confirmed than when the net was washed inadvance. This net had been thoroughly cleaned after the most recent past survey and stored ina natural fiber bag.
・ Before using a net that has been stored for a long time, it would be desirable to wash it again,even if it was thoroughly washed after the previous survey, taking into consideration thepossibility of contamination with plastic particles during storage.
・ The number of particles collected each day in the survey area was around 100 to 2,000
particles, so the several plastic particles collected in the blank test were considered not to havea significant influence on the survey results (above the limit of detection, LOD). However, ahigher level of plastic particle contamination (24 pieces) was confirmed in a net stored for a longtime, so the influence cannot be ignored when sampling in sea areas where the quantities
collected are small. Therefore, it is necessary to pay attention and avoid contamination as thesurvey is conducted.
In addition, a survey was conducted in CMSM2019 using fishing boat (other than vessels
specified to research), and found particles that may be caused by paints or cushioning materials
of a vessel.
・ When particles confirmed as plastic were found in the blank sample, a high proportion ofparticles were vinyl chloride, polystyrene and polyurethane. These materials are rarely found in
the survey samples. Therefore, it is assumed that the net had been contaminated not only fromits washing, but also from tape used to fix equipment and the vessel's buoy and paint. For
surveys, it is advisable to pay attention to plastic products on the vessel and take measures toprevent contamination, such as keeping them as far away as possible from places where
samples are processed.

41

Fig.2-17. (A) Deterioration of boats(B) corresponding fragments detected in field blank42

3. Laboratory analysis
3.1 Outline
・ In general, analysis of samples that include microplastics obtained by trawling a net throughthe ocean surface layer is carried out in the following order: pretreatment (separation of nonplastic material other than microplastics), isolation of microplastics, counting andmeasurement, and material identification. Depending on the purpose of the study, their weight
may also be measured.
・ The order of the pretreatment process, i.e. density separation, chemical treatment, biologicaldigestion and sample splitting, may differ depending on the purpose of the survey and the stateof the sample.
・ Prior to all analytical processes, fractionation of the samples, including non-plastic material, issometimes performed by sifting through various sizes sieves.

Pretreatment[Section 3.2]

Biological Digestion andchemical treatment[Section 3.2.1]
Density Separation[Section 3.2.2]
Sample Splitting[Section 3.2.3]※Pretreatment processes are selected based on purposeof the study

Isolation of Microplastics [Section 3.3]

Quantity and Sizes Measurement [Section 3.4]

※Counting and measurement ofsizes and weights conducted

Identification of Microplastics [Section 3.5]

based on purpose of the study.

Weight Measurement [Section 3.6]
Fig.3-1. General flow of microplastic analysis.

43

・ When comparing the densities of microplastics obtained through laboratory procedures, careshould be taken to note which method was used, as oversight or loss of microplastics may occur
depending on the pretreatment or separation methods. The proficiency level of the analysts
may also be a source of error.
・ Before preparation of these Guidelines, an international collaborative analysis with theparticipation of 12 laboratories from 10 countries (ILC: Inter Laboratory Comparison) in 2017.
The ILC used standard samples to ascertain the extent of variation in results depending on thevarious analytical methods used. Each of the standard samples contained plastic particles ofthe same number, size and weight, and some non-plastic material (plankton, seashells, woodpieces, crustacean shells, etc.).
・ Two samples were sent to each laboratory, one with a large amount of microplastics and nonplastic material simulating a sample from an inner bay, and the other with few microplastics
and non-plastic materials, simulating a sample from the outer ocean.
・ These samples were analyzed according to the analytical methods of each laboratory, and theresults and analytical procedures used were reported to the secretariat. The differences
between the results reported from each laboratory and the design value of the standard
samples were compared and discussed in terms of whether the differences were systematic.
・ With regards to harmonization, recommendations and points to be noted in each analyticalprocess are introduced in this chapter based on the results of the ILC.
・ These Guidelines focus on the microplastics present in sea water and do not cover the analysisof microplastics taken in by lower organisms such as plankton.
・ A part of the results of the ILC has been published in a scientific journal (Isobe et al., 2019).

44

3.2 Preprocessing for analysis
・ Samples obtained by net towing contain various natural particles as well as plastic particles.
Removing the non-plastic particles through pretreatment, improves the accuracy of subsequent
processing for plastic particles such as isolation, material identification, counting and weighing.
・ For that reason, pretreatment may be performed when sampled particles include non-plastic
material.
・ Fractionation of the samples, including non-plastic material, can be performed by sifting
through sieves of various sizes is sometimes performed before pretreatment.
・ Pretreatment methods include density separation, mainly to remove inorganic particles, anddigestion of organism-derived organic substances by oxidation, hydrolysis or enzymatic
reactions.
・ When there are many plastic particles and/or non-plastic particles per sample, the sample may
be sub-sampled to reduce the amount of counting, measuring and other work at the time ofanalysis.
・ During the ILC, nine out of the 12 laboratories conducted pretreatment; three laboratories
conducted only density separation, two conducted only digestion of organic matter, and four
carried out both pretreatments. Sample splitting was not conducted at any of the laboratories.

45

3.2.1 Biological digestion and chemical treatment

This figure illustrates the implementation of wet peroxide oxidation (WPO). To digest the organic
matter, hydrogen peroxide (H2O2) is added to the sample. In this process, Fe (Ⅱ) is also added asa catalyst. The photographs show the addition of these solutions to the sample to obtain thereaction time.

Keynotes・ As described in Section 2.2 of the Guidelines, sampling in sea areas where the density of nonplastic material (including natural-organic matter) is high should be avoided where possible.
・ When there are many non-plastic materials such as plankton (in the sample), pretreatment todigest organic substances with chemicals or enzymes is performed in many cases to remove thenon-plastic material as well as biofilms that have formed on the surface of the sampled plastic
particles. Here the aim is to minimize the possibility of misidentifying plastic particles,
improving the accuracy of the isolation process and overall work efficiency. If improperly
conducted, however, it may lead to deterioration (deformation and/or weight reduction) of
plastic particles from chemicals added or from heating.
・ The purpose of digesting organic substances is not limited to the removal of non-plastic
material to simplify subsequent processing but may also include analyzing microplastics
ingested by organisms in the lower trophic levels such as plankton –although the latter purpose
is not covered by the Guidelines.
・ Digestion of organic substances is effective when biofilms are formed on the surfaces of plastics
and non-plastic material in the sample to the extent that they may interfere with weightmeasurement and material identification using spectral optical instruments.
・ It should be noted that the size of microplastics incorporated into organisms in the lower
trophic levels, such as plankton is often in the order of 10 µm (Botterell et al., 2018).

Outcomes of the pilot projects
・ In the ILC, there was no systematic difference in the measurement results of plastic particles(larger than 1 mm and less than 5 mm) between the laboratories that performed organic matter46

digestion to those laboratories which did not. For particles < 1 mm, the results for the number
and weight of particles were underestimated by all of the laboratories but the values obtainedby laboratories performing digestion treatments tended to be closer to the original value.
・ There was one laboratory conducting digestion that was unable to measure the number ofparticles correctly because the particles aggregated due to biological residue caused byinsufficient digestion.

Recommendations
・ From the viewpoint of harmonizing monitoring methods for particle density of particles larger
than 1 mm and less than 5 mm in size, it is not always necessary to digest organic matter as apretreatment.
・ On the other hand, when analyzing particles less than 1 mm in size, it would be preferable todigest the organic substances to obtain more accurate analytical results.

Explanatory Notes
・ Digestion is an effective method when sampling is performed where there are many organic
substances. Digesting organic matter can be performed through oxidation, hydrolysis or
enzymatic reactions to make separation of plastic particles easier.
・ Removing biofilms formed on the surface of plastic particles or non-plastic material makes
material identification by spectral optical instruments more accurate (see Section 3.5).
・ Plastics can deteriorate when strong acids are used to digest organic substances (e.g., Miller etal., 2017; Hurley et al., 2018; GESAMP 2019).
・ In the ILC, six out of the 12 laboratories conducted organic matter digestion as a pretreatment:
among these, three laboratories conducted digestion using hydrogen peroxide and divalent ironsolvent (H202, Fe2+), one laboratory used only hydrogen peroxide (H202), one laboratory conductedalkaline digestion using potassium hydroxide (KOH), and one laboratory conducted biochemicaldigestion using corolase enzyme. The advantages and disadvantages of various biologicaldigestions and chemical treatments are shown in Table 3-1.
・ When comparing errors in the measurement results between laboratories that conductedorganic substance digestion and laboratories that did not, there was no systematicallysignificant difference in particle quantity measurement results. Consequently, from theviewpoint of harmonization, microplastic measurement results can be compared regardless ofwhether digestion was performed or not.
・ It should be noted that in the ILC, there was a case in which aggregation of particles occurred.
This was observed because of insufficient digestion and remnant biological residue.
・ Among the particle number and weight measurements in the ILC, the results for particles ofless than 1 mm among laboratories that did not conduct digestion tended to be underestimatedcompared to the design value.

47

・ Laboratories using the digestion of organic substances reported more accurate values forparticles <1 mm. It is assumed that digestion makes it easier to isolating plastics.
・ When conducting digestion, depending on the purpose and equipment of the study, as well as thestate of non-plastic material in the sample, care should be taken to select conditions that do notcause deterioration of the plastics and avoid influence from the digested biological residue(appropriate reagents, temperatures, digestion times, etc.).
・ Previous study has suggested paying attention to temperature condition during laboratory
analysis. Alfonso et al., (2021) assessed the microplastics' weight, size, and polymer changes
under different digestion techniques and suggested that using 40 ℃ for 72 hours prevails allpolymer from size changes. It is also mentioned that any method applying high temperaturedamages microplastics.
Table 3-1. Advantages and disadvantages of various biological digestions and chemical treatments(reproduced from GESAMP, 2019).
Purification method
Oxidative digestion

Advantages

Disadvantages

Inexpensive

Temperature needs

Reference
Masura et al. (2015)

to be controlled
Several applications
may be needed
Acid digestion

Rapid (24 h)

Can attack some

Claessens et al. (2013)

polymers
Alkaline digestion

Effective

Damages cellulose

Minimal damage

acetateDehaut et al. (2016)

to most polymers
Enzymatic digestion

Effective

Time-consuming

Minimal damage(several days)

to most polymers

48

Löder et al. (2017)

3.2.2 Density separation

Density separators
Density separation is often performed bypouring the sample and a dense solvent into afunnel or a separating funnel.

Floating plastic particles in a density separator
Plastics with lower specific gravity than thesolvent float on the surface.

Keynotes・ Density separation may be performed as a pretreatment to remove non-plastic material in thesample. This step is more commonly performed for sediment sample analysis.

Outcomes of the pilot projects
・ There was no systematic difference in analytical results among laboratories that performed
density separation on the standard samples and laboratories that did not perform density
separation.

Recommendations
・ In cases where there is a lot of non-plastic material, density separation would be effective as itenables efficient separation of plastic particles, but from the viewpoint of harmonizing themethods of monitoring microplastics at the ocean surface, it is not necessarily required.

Explanatory Note・ Density separation is an effective method of fractionating low-density plastic particles andhigh-density natural particles of inorganic matter.
・ In general, density separation is conducted by mixing the sample into a solution with a higher
specific gravity than that estimated for the collected plastic particles, letting high-density
inorganic substances settle out and recovering and fractionating the floating low-density plastic
particles. Commonly employed solutions for density separation of microplastics are shown in
Table 3-2.
・ Density separation is a particularly effective process for measuring microplastics in bottom andcoastal sediment samples that include heavy materials such as sand, seashells, etc. It is notnecessarily a common practice in analyzing floating microplastics samples collected with nets atthe ocean surface, but may be required when there are lots of plankton.

49

・ In the ILC, density separation was carried out at seven out of the 12 laboratories, using aqueous
solutions of sodium chloride (NaCl) or sodium metatungstate hydrate (Na2WO4) for the
separation.
・ There were no systematically significant differences in the measurement results between
laboratories that did or did not perform density separation. Consequently, from the viewpoint of
harmonization, the results of surface layer microplastic density per filtered water volume can becompared for microplastics that are 1 mm or larger and less than 5 mm regardless of whether
density separation was performed or not.
Table 3-2. Solutions commonly used for the density separation of microplastics (reproduced fromGESAMP, 2019)
Salt

Density (g cm-3)

Reference

Sodium Chloride (NaCl)

1.2

Hidalgo-Ruz et al. 2012

Sodium Polytungstate (PST)

1.4

Hidalgo-Ruz et al. 2012

Sodium Iodide (NaI)

1.6

Claessens et al. 2013

Zinc Chloride (ZnCl2)

1.7

Imhof et al. 2012

1.6

Zobkov & Esiukova, 2017.

50

3.2.3 Sample splitting

Use of Folsom splitter
Splitter is moved back and forth to mix
thoroughly, then the sample is divided.

Folsom splitter

Keynotes・ Sample splitting before counting is often performed in analyses for zooplankton, especiallywhere the quantities sampled are large. It is not common in the analyses of microplastics.

Outcomes of the pilot projects
・ The standard samples and samples obtained in actual sea areas were divided with a splitter(Folsom splitter) and measured. The estimated values of the total number of particles from thedivided samples had about ± 10% error with respect to the total measured number of particles.
When the splitting was repeated, a tendency for the error to increase was observed.

Recommendations
・ Using a splitter may be effective when the number of particles in one sample is large (e.g.,when it exceeds 1,000 or so) or when there are time or personnel constraints, keeping in mind
that a certain level of error is expected.

Explanatory Notes
・ In a small proportion of analyses for zooplankton and microplastics, the samples are divided andonly a part of each sample is analyzed to improve the efficiency of the analysis. The samples weredivided using a Folsom splitter, known for its high splitting accuracy (Guelpen et al., 1982) insome cases (Fossi et al., 2016, Di Mauro et al., 2017).
・ After confirming no loss of particles when using a splitter on the standard samples in ILC, asplitter (Folsom splitter) was used in a trial with samples obtained in an actual sea area.
・ The samples obtained in the actual sea area were divided using the splitter and the number ofparticles was counted. Compared to a sample for which all particles were counted, the error was
about ± 10% for a sample divided into two using the splitter once, and ± 20% for a sample divided
into four using the splitter twice. It is assumed that almost the same level of accuracy wasachieved in the splitting process for plankton (Guelpen et al., 1982).
・ As described above, dividing microplastic samples using a splitter is an effective means of51

improving efficiency when large amounts of samples need to be divided and time is limited, orwhen analyses need to be conducted with limited human resources or time, keeping in mind thatsome error is expected. When dividing the sample with a splitter, it is necessary to stir it
thoroughly and sufficiently wash and collect the sample sticking to the wall of the splitter. It
would also be desirable to verify the degree of error for the sum of measurements obtained fromeach portion of a sample divided by the splitter compared to the measurement of the original
sample.
Table 3-3. Error due to use of splitter.
Note: ' Ntotal' indicates the total number of microplastics in all sub-samples.
' Nsub' indicates the number of microplastics in each of the sub-samples.

'sub-R' indicates one of the sub-samples split into two.
'sub-LR' and 'sub-LL' indicate one of the sub-samples split into four.
'R' indicates the right side of the splitter, 'L' indicates the left side of the splitter.
‘Coefficient of variation: (standard deviation) / (average)
Average
Sample Number(Coefficient of
1
2
3
4
5
6
7
8
9
10
Variation)

Ntotal

873

929

297

537

732

656 1,141 1,505 1,226 1,434933

Nsub×2/Ntotal sub-R

1.03

0.90

0.88

0.86

0.99

0.88

1.01

0.83

1.02

0.85

0.92

sub-LR

1.20

1.15

0.93

1.23

0.90

0.88

0.94

1.41

0.87

1.15

1.07 (17%)

sub-LL

0.75

1.04

1.32

1.06

1.11

1.35

1.04

0.94

1.09

1.16

1.09 (15%)

Nsub×4/Ntotal

(8%)

1.6
sub-R

sub-LR

sub-LL

Nsub×2 / Ntotal or Nsub×4 / Ntotal

1.4

1.2

1

0.8

0.6

0.4
0

200

400

600

800
Ntotal

1,000

1,200

1,400

1,600

Fig. 3-2. Ratios of number of particles estimated from the result of counting divided samples to thenumber of particles when the total quantity was counted.

52

3.3 Isolation of microplastic

Isolation process
To separate microplastics, it is common to pickthem out manually under a stereomicroscope.

Microplastics on a petri dishMicroplastics collected in CMSM 2018.

Keynotes・ Particle isolation is an important process that greatly affects the accuracy of the microplastic
analysis.
・ There are several methods of separating plastic particles from a sample, such as isolation ofplastic particles out after fractionating the sample by size using sieves of various sieve mesh
opening sizes such as 5 mm, 1 mm, and 0.3 mm, and picking the plastic particles from the filter
paper after directly filtering the sample. Stereomicroscopes are commonly used to facilitate
this.

Outcomes of the pilot projects
・ Despite the use of stereomicroscopes by all the laboratories for hand-picking, the quantities ofseparated plastic particles <1 mm from the standard samples were underestimated by 40 to
80%, and the variance in reported results among the laboratories was also large. This wasthought to have resulted from the loss of particles during the pretreatment process as well as
incomplete particle isolation.

Recommendations
・ To obtain fairly accurate results, conducting the picking process carefully is recommended evenwhen a stereomicroscope is used, and exerting caution to avoid losing particles in
pretreatment. Be especially careful not to overlook particles smaller than 1 mm.
・ Reporting should separate particles by size, specifically for those smaller than 1 mm and forparticles larger than 1 mm to maintain comparability based on sampling errors related to mesh
size.

53

Explanatory Notes
・ The accuracy of particle isolation greatly affects microplastic analysis results, as plastic particles
picked out from the sample, whether pretreated or not, are used for subsequent measurement
and analysis.
・ Particle fractionation using sieves with various mesh openings, such as 5 mm, 1 mm, 0.3 mmmay be carried out before pretreatment when the samples include non-plastic materials.
・ In the ILC, many laboratories filtered the samples through sieves with mesh openings of 5 mm
or 0.3 mm for fractionation and then suction filtered using a filter paper of about 0.8 μm. Many
laboratories used glass fiber filters and polycarbonate filters. These laboratories usedstereomicroscopes to isolate particles from the sieves or filter paper. However, the analysis
results for numbers of particles smaller than 1 mm were less than the design value (about 4080% of the design value). This was seen across all laboratories, and the variation in thereported values between these laboratories was also great.
・ This may reflect the difficulty in visually finding small particles that are mixed in with nonplastic material. Also, as glass petri dishes were used in many cases when isolating the particles,
they may have caused difficulty in isolation transparent particles.
・ For improvement, it is advisable to work with a microscope as much as possible, using not only
the backlight but also the incident light when confirming the existence of particles.
・ It is necessary to pay special attention when isolating fibrous particles as they can be mistaken
for other materials (natural fibers, etc.).
・ At the same time, it is also important to build capacity among analysts to improve the accuracyof the picking process. Therefore, recovery tests and duel identification procedures arerecommended.
・ Although it is convenient to use sieves before particle isolation, special care should be taken to
avoid losing particles that have the longest lengths greater than the sieve openings, which may
nonetheless pass through the sieve (see Section 3.4, Fig. 3-3).
・ When separating with a sieve, it is desirable to re-collect the sample passed through the finest
sieve on filter paper.
・ In the ILC, the research institute that conducted re-collection reported a value closer to thedesign value for fine particles smaller than 1 mm.

54

3.4 Counting and size measuring

Measurement of microplastics

Multi-staged sieve with various mesh openings

Measurement using photos of plastic particles
and image processing software. The longest
length is measured from the captured image andthe number of particles are aggregated by size.

The photo shows a multi-staged sieve with 4
mm, 1 mm, 0.3 mm and 0.1 mm openings.
Plastic particles are poured from the top, andthe number of particles remaining in each sieve
is aggregated as the number of particles by size.

Keynotes
・ Microplastic abundance at the ocean surface is most commonly reported in the number ofparticles by size.
・ There are two common methods for counting the number of particles by size; (1) directly
measuring the longest diameter (maximum Feret’s diameter) of separated particles
individually, and, (2) counting the number of particles remaining in the sample after
fractionating by size using sieves of various mesh opening sizes.

Outcomes of the pilot projects
・ Many laboratories measured the longest length using image processing software or calipers bymethod (1) and summed up the number of particles by size. Some laboratories fractionatedwith sieves of different mesh openings as in method (2), counted the number of particles
remaining in each sieve and reported them as the number of particles by size.
・ For particles sampled during CMSM2018, the number of plastic particles that were measured
for their longest length and particles that were sieve fractionated were compared, with the
hypothesis that plastic particles of up to 7 mm, which is around the diagonal length of a 5 mm
square mesh opening, would pass through a 5 mm sieve. It was found that the number ofparticles smaller than 5 mm obtained by the latter method (using sieves) was about 1.25 times
larger than by the former. The above findings indicate that in sieve fractionation, the numberof particles smaller than 5 mm counted using sieves would be overestimated because particles
with the shortest length smaller than the diagonal length of the mesh may pass through thesieve, even if their longest length exceeds 5 mm.
55

Recommendations
・ In terms of harmonization, measuring the longest length of each particle using image
processing software is recommended.
・ When estimating the number of particles and/or weight of particles by size using only sieves,it is necessary to keep in mind that it would be difficult to compare the results with those ofparticles measured directly for the longest length.
・ We also recommend providing classification of plastic particles by morphological traits such as
beads, fragments foams, pellets and fibers, noticing the difficulty of distinguishing between
natural and synthetic fibers.

Explanatory Notes
・ Feret’s diameter is illustrated in Fig. 3-3. The plastic particle size is determined as Feret's
diameter, which is generally defined as the distance between the two parallel planes restricting
the object perpendicular to that direction (Pabst et al., 2017). The shortest Feret's diameter iscalled “minimum Feret’s diameter” and the longest is called “maximum Feret’s diameter”.
・ Size fractionation by sieving is effective from the viewpoint of efficiency. However, in the case ofusing a 5 mm lattice sieve mesh, particles up to 7.0 mm, which is the diagonal length of the
openings, or fibrous particles having a shortest length may pass through the sieve (Fig. 3-4).
・ For one sample collected during CMSM2018, the number of particles by size was compared. The
results showed that the number of particles smaller than 7 mm that could pass through the
diagonal line of a sieve with a 5 mm lattice was 1,974. When maximum diameters were measured
for the same sample, the number of particles obtained smaller than 5 mm was 1,574. When using
a 5 mm mesh sieve, the result is overestimated by about 20%, as particles with the longest lengthof more than 5 mm could also pass through the sieve.
・ These findings suggest the possibility that sieve fractionation may lead to the overestimation ofparticles < 5 mm in size.
・ When only sieving is used, care should be taken to note the fact that the obtained results cannot
be compared to fractionation results obtained by measuring the longest lengths. Consequently, it
would be desirable to measure the longest lengths and aggregate the number of particles by size.
・ Recently, image capturing devices and software/applications capable of image processing can beobtained inexpensively, and particle size measurement by image processing is relatively easy.
Therefore, it would be desirable to measure particle sizes using these devices and software.
・ When measuring the longest lengths with image processing software, it would be preferable torecord the shortest lengths and projected area simultaneously.
・ Depending on the purpose of the study, the shape and color of the plastic particles may need tobe recorded. Guidelines which recommend recording these include EC, (2013) etc..
・ The shape and color of plastic particles are valuable information for identifying sources. Moreinformation is presented in Rochman et al., 2019. Also, as the color of plastics is considered to be56

related to uptake by organisms (e.g., Des forges et al., 2015, Steer et al., 2017.), it is important toacquire these data for future study.
・ In many studies, classification by shape, commonly used shape categories include fragments,
beads, foam, pellets and fibers.
・ If the projected area of each particle is measured after classifying the particles by shape, it may
be possible to convert the projected area to weight with a conversion formula using volume,
weight and plastic density. Accumulations of data both on the projected area of particles andtheir weight would provide a formula to convert the projected area of a particle to its weight orvice versa. The average weight per particle may be also estimated for each sample by dividing
the total weight of particles sampled by their number.

Fig. 3-3. Feret’s diameter

Fig. 3-4. Relationship of the mesh openings of a sieve to the particle sizes that may pass through.

57

3.5 Material identification

Bruker FTIR

Composition analysis by FTIR

Keynotes・ Spectral optical instruments such as IR/Raman spectroscopy are used most commonly toseparate microplastics from non-plastic materials and identify polymers.

Outcomes of the pilot projects
・ Many laboratories who participated in the ILC used Fourier Transform Infrared Spectroscopy(FTIR, including ATR-FTIR) for material identification, while others used Raman spectroscopy.

Recommendations
・ From the viewpoint of harmonization and accuracy, it is essential to confirm the material ofplastic particles using the spectral optical instruments to ensure accuracy of separation byhand-picking.
・ Even when using spectroscopy, knowledge of chemistry is recommended and appropriate
training is required when conducting separation because it may be difficult to determine
whether particles are made of plastic or not.
・ In addition, when confirming materials by visual inspection without using spectral optical
instruments such as FTIR, having an analyst who is skilled at separating plastic particles isrecommended.

Explanatory Notes
・ Visually identifying microplastics from non-plastic material is generally difficult at the handpicking stage, especially with particles <1 mm. Confirming that particles picked out are
plastics and correcting the counting or measurement results is desirable.
・ Even if it is difficult to analyze characteristics of all microplastic by spectroscopy, confirming
the characteristics of some of the particles by spectroscopy is recommended.
・ The EC guideline (EC, 2013) recommends spectroscopic analysis for a subsample of 10% of theidentified particles to verify visual identification, and this method has been applied in several
reports (e.g., Lusher et al., 2018). The European Union’s Marine Strategy Framework Directive
58

(MSFD) also recommends that a proportion (5-10%) of all samples be routinely checked toconfirm the accuracy of visual examination (Gago et al., 2016).
・ At the International Experts Meeting held to examine these guidelines, it was pointed out thata best-case scenario would be testing all particles for chemical composition with FTIR or similar
devices. In cases where time and resources do not allow this, a representative subsample would
be a part of the total sample that reflects the composition of particles, both in shape and color.
For example, if there are both fragments and fibers in a ratio of 1:5, at least one fragment toevery 5 fibers should be assessed to a value which exceeds 20% of the overall total.
・ In the ILC, laboratories that identified the material only by visual confirmation reported larger
quantities than the design value of the standard samples due to errors in mistaking natural
particles for plastic ones.
・ Particularly for small particles with the longest length of less than 1 mm, it would be easy to
misidentify plastics and non-plastic material when conducting visual inspection only, so evenwhen studying plastic particles larger than 1 mm and less than 5 mm, if there are many small
particles of 1 to 2 mm, use of a spectral optical instrument for composition analysis is
recommended.
・ The accuracy of hand-picking relying solely on visual observation with a stereoscopic
microscope was particularly low, although there were only a few laboratories that did not use
spectral optical instruments to isolate microplastics. Meanwhile, the laboratories that applied
other methods such as pushing the particles with a needle, etc., in addition to visualobservation, reported values close to the design value.
・ Understanding the composition of plastics using spectral optical instruments such as FTIR,
ATR-FTIR, Raman spectroscopy, or infrared cameras, is useful not only for separating theparticles from other materials but also for obtaining useful information in regard to the sourcesof the plastics.
・ It is necessary to note that biofilms adhering to particle surfaces may make it difficult to
identify materials or analyze composition using spectral optical instruments and that a certain
amount of experience is required to be able to determine if the obtained spectra reflect thecharacteristics of plastics or not. If uncertain about particle analysis, it would be desirable tocheck the results with an experienced analyst.
・ Advantages and disadvantages of recent microplastic characterization methods, including
identification of polymer types, are shown in Table 3-4.
・ In recent years, equipment such as automated ATR-FTIR that can perform counting, shapemeasurement and material identification simultaneously has started coming into use.

59

Table 3-4.(1) Advantages and disadvantages of microplastic characterization methods, including
identification of polymer types (reproduced from Shim et al. (2017)).
Identification

Advantages

Disadvantages

SimpleNo chemical information for confirming

methodMicroscopy

composition
Low cost

High possibility of false positives

Color and morphologicalHigh possibility of missing small andinformationtransparent particles

Microscopy +Polymer composition of a sub-Possibility of false positives

spectroscopy

set of the sample(sub-set)

Possibility of missing small and transparent
particles
Sub-set may not be representative
Potential bias in sub-set selection

Microscopy +No false positives –Manual selection of particles means some

FTIR

confirmation of allplastic may be missed

spectroscopy

plastic-like particles
Reduction in false negatives

Expensive instrument

Non-destructive

Laborious and time-consuming for
identification of all particles

20 μm particle detection limit

Requires expertise in spectral interpretation
Contact analysis (ATR)
Need to transfer particles from filter paper tometal plate
Removal of organic material a prerequisite

Microscopy +No false positives –Manual selection of particles means someRamanconfirmation of allplastic may be missed

spectroscopy

plastic-like particles
Reduction in false negatives

Expensive instrument

1 μm particle detection limit

Laborious and time-consuming for
identification of all particles

Non-destructive analysis

Requires expertise in spectral interpretation

Non-contact analysis

Interference by pigments
Risk of laser damage to particles
Removal of organic material a prerequisite
Exact focusing required60

Table 3-4 (2). Advantages and disadvantages of microplastic characterization methods, including
identification of polymer types (reproduced from Shim et al. (2017)).
Identification

Advantages

Disadvantages

Semi-

No manual particle selection

No visual image data on single particles

automated

error

spectroscopy

High automation potential

Production of a large volume of data

(mapping

In principle no false negatives

Long post-processing time

methodbased)

Still requires expertise in spectralinterpretation
Efficient removal of interfering particles a
pre-requisite
Still lacks validation for smaller particles
Expensive instrument

Semi-

High automation potential

Production of a large volume of data

automated

Fewer false negatives

Long post-processing time

spectroscopy

Potential for faster sample

Still requires expertise in spectral(imagethroughput

interpretation

analysis

Size and morphology of single

Efficient removal of interfering particles adirected point

particles

pre-requisite

analysis)

Still lacks validation for smaller particles
Expensive instrument

ThermalSimultaneous analysis foranalysis

polymer type and

Destructive analysis

additive chemicals (PyroGC/MS)
Mass-based information

No quantity or size-based information
Limited polymer type identification (DSC)
Complex data (Pyro-GC/MS)

61

3.6 Weight measurement

Sample drying

Weight measurement

Generally, drying prior to weighing is
performed at room temperature.

Weighing of microplastics in a glass vial.

Keynotes・ Weight measurement is carried out because it is important to understand the mass balance. It
is also difficult to estimate the actual abundance of microplastics from the number of theparticles only. This is because even if the same amount of microplastics exists at the ocean
surface by weight, the number of particles may differ depending on fragmentation processes.
Recommendations and guidelines on weight measurement have been issued by the EC andNOAA (EC, 2013, Masura et al., 2015.).

Outcomes of the pilot projects
・ All laboratories participating in the ILC provided weight measurements of the standard samples
irrespective of whether or not they normally carried out weight measurements.
・ In measuring the weight of the standard samples, for particles 1 mm or larger and less than 5mm, there were no significant differences between the results reported from each laboratory
and the design value. On the other hand, for smaller particles less than 1 mm, results from thelaboratories that had conducted digestion of organic matter before measuring the weight werecloser to the design value than those from other laboratories.
・ In response to a questionnaire distributed after the survey in the ILC, many of the laboratories
reporting relatively low accuracy in weight values suggested that in all probability insufficientdrying affected the weight measurement.

Recommendations
・ It is important to wash each sample with distilled water and dry it thoroughly before measuring
its weight. Attention should be paid to humidity and the laboratory atmosphere.
・ Also, to obtain more accurate results, digesting organic matter in the pretreatment process isrecommended (see Section 3.2).
・ Reporting weight for both particles smaller than 1 mm and particles larger than 1 mm
separately is recommended.
62

Explanatory Notes
・ Weight measurement (dry weight) of microplastics may be carried out based on the purpose of
the survey, such as for detailed analysis of plastic particle distribution in sea areas.
・ In the preliminary questionnaire, only four laboratories out of the 12 laboratories participatingin the ILC reported that they measured the weight (or measured the weight and number) in
their routine measurements. In the ILC, all laboratories were requested to measure weight.
・ Out of the 12 laboratories, drying prior to weight measurement was done at room temperature
at 11 laboratories, and at 60°C at one laboratory.
・ Results of testing showed no significant differences among values reported from each laboratory
regarding the weight of microplastics larger than 1 mm and less than 5 mm contained in thestandard samples.
・ Meanwhile, results of weight measurement of microplastics smaller than 1 mm in the ILC
closer to the design value were obtained by laboratories that conducted digestion of organic
substances compared to those that did not. This is thought to have been the result of
improvement in the precision of isolation of small particles and size fractionation through theremoval of non-plastic material by digestion of organic substances.
・ Also, with respect to microplastics larger than 1 mm and smaller than 5 mm, the digestion oforganic substances is considered an effective process for achieving better accuracy in weightmeasurement because samples obtained from actual sea areas may contain particles with sessile
animals or biofilms adhering to the surface.
・ Furthermore, laboratories with large errors in weight measurement results in the ILC reported
insufficient drying as the major factor influencing weight measurement.
・ From the viewpoint of harmonizing monitoring methods, performing adequate digestion oforganic substances and drying the particles thoroughly are thought desirable for achieving
accurate weight measurement.
・ However, measuring the weight of particles smaller than 1 mm is prone to error at the
separation process, even with adequate digestion of organic substances and thorough drying(see Section 3.3). Hence, reporting is not considered essential as the difficulty is expected in
obtaining comparable results based on the analytical methods presented in these Guidelines.

63

3.7 Accuracy control during analysis
Keynotes・ In laboratory analysis, countermeasures, for preventing airborne contamination such as withfibrous matter and contamination from washing water in the fractionation and filtration
processes, are important, such as conducting blank tests in the laboratory or using filteredwater to wash the equipment (EC, 2013, Masura et al, 2015). In recent articles, specific steps
have been proposed to reduce and quantify this kind of contamination for accurate output(see Table 3-5 reproduced from Lusher, 2018).
・ Hermsen et al. (2018) reviewed many cases of quality control for surveys and experiments on
microplastics. This serves as a very useful reference and referring to it before surveys isrecommended.
・ Spiked recovery tests using relevant reference particles (similar properties as sample particles
but still clearly distinguishable, e.g. by distinct colors) are also effective for assessing extraction
efficiency or loss in the digestion protocols or density separation (GESAMP, 2019).
・ Also, the experience and ability of the analysts are thought to be very important in theaccuracy control.

Outcomes of the pilot projects
・ In preliminary questionnaires for participating laboratories, some laboratories responded thatthey used specific facilities such as clean benches and others excluded fibrous materials as
potential contaminants. In addition, there were several laboratories using specified water
filtered through 0.7 to 1μm filters for washing in the fractionation process.

Recommendations
・ It is desirable to include information in the report on measures taken to prevent contaminationthat may affect the accuracy of the analysis. It is also desirable to record the humidity andtemperature of the laboratory atmosphere.

Explanatory Notes
In the discussions at the International Experts' Meetings, reporting of quality assurance/quality
control data was recognized as important.
Examples of quality assurance/quality control data include blank tests in the analytical process,recovery rates, repeatability, etc.
In the preliminary questionnaire given to the laboratories in ILC, eight laboratories reported
using a clean bench, etc., and one laboratory was excluding fibrous particles as a measure.
Examples of contamination risks and the measures against them are shown in Table 3-6.
When ILC was implemented, there were at least two laboratories using water filtered using 0.71 μm filters for washing the mesh prior to fractionation.

64

What has been particularly prominently mentioned in past research has been airborne
contamination in which cloth-derived synthetic fibers adhere to analytical instruments andsamples via the air in the laboratory (Nuelle et al., 2014, Wesch et al., 2017). Careful attention
is necessary when analyzing fibrous microplastics.
Table 3-5. Examples of steps to prevent microplastic contamination. (modified from Lusher et al.
(2018)).
1

All sample containers should be prewashed with filtered distilled water before use.

2

Samples should be kept covered as much as possible using aluminum foil or glass lids.

3

All equipment used in the processing and analysis stages should be rinsed and checked
under a microscope for any microplastics adhering to them. The vacuum filtering
apparatus should be rinsed with filtered water between each sample.

4

All reagents should be vacuum filtered through Whatman GF/D filter papers or othersterile filter papers immediately prior to use.

5

Sample processing should be performed in a sterile cabinet.

6

Several procedural blanks should be performed as negative control samples through thesample processing and analytical stages in order to test for laboratory contamination.

Table 3-6. Examples of contamination risks and preventive measures.
Contamination risks

Preventive measures

Contamination with plastic

Pour purified water into the apparatus used for analysis

particles

tobeforehand and conduct the same analytical process as for

instruments/

sample treatment to confirm the presence or absence ofanalyticaladhering

apparatuses

microplastics.

Contamination with fibrous

Wear clothing that is not plastic-derived and remove any loose

microplastics duringfibers from clothing with a lint roller before sampling andoperations

analysis.
For example, wear clothing of a unique and visible color so thatthe fiber can be distinguished even if it contaminates the
sample.

Contamination with plastics

Use of clean benches and clean rooms.

from air

Implementation of blank tests in the laboratory.

65

4. Reporting・ Observed abundances of ocean surface microplastics are commonly reported in terms of density,or weight of particles per unit area (/m2, /km2) or unit volume of water (/m3).
・ Densities of microplastics per unit area need to be reported together with the sampling depth toallow comparisons to be made between those per unit area and those per unit volume of water.
・ Reports on the distribution of ocean surface microplastics should include not only theirquantities or weight per unit area or per volume of water, but also their particle sizes andmaterials, and metadata at the time of their sampling.
・ For example, collected quantities of microplastics plus the shapes of individual particles make ittheoretically possible to convert them to weight. If data on wind speeds and wave heights areavailable for estimating the intensity of vertical mixing of water, abundances of underwater
microplastics can also be estimated by sampling at the surface layer (Kukulka et al., 2012., Kooi
et al., 2016.).
・ It is also necessary to record and report how each sample was stored and analyzed. Uponcompletion of these analyses, maintaining visual representations (pictures, etc.) obtained at thetime of measurement would be desirable.
・ As for the scale of samplings required to obtain the typical density of microplastics in a certain
sea area, Dr. Cózar (personal communication) suggested 120 tows (one tow usually ranges
between 500 and 2000 m2) for 174,000 m2, while Goldstein et al. (2012) recommended 250 tows
for 165,000 m2. The total area surveyed may be more important than the number of samplingsin studying the typical density of microplastics in certain sea areas, and further consideration isrequired.
・ In the Guidelines, data to be reported to ensure harmonization of ocean surface microplastic
monitoring are summarized in Table 4-1 to Table 4-3. In these tables, the items were selected
for the harmonization of monitoring methods. Among these items, the “fundamental” items are
the minimum requirements to identify the abundance of microplastics, the sampling time andlocation, and microplastic density which can be displayed in any of the following four units:
number of particles/m3 or km2, weight/m3 or km2. The “essential”items are the minimum
requirements to make the survey results comparable.
・ The data-input form summarizing these items were distributed.

66

Table 4-1(1) List of data to be reported when sampling floating microplastics.
Items

Data necessary to ensure comparability

Sampling
Sample name/ ID
date
and Time difference from GMT.
location
Sampling date and time
SeasonSampling Location (Name)
GPS Log

Sampling
equipment

Classification of net frame
Net aperture

Length of net
Mesh

Tow
Parameters

Name of vessel
Tow distance

Trawl sweep area
Filtered water volume
Tow duration
Vessel speed
Tow position
Distance from vessel

CategoryE
Start of trawl
FA, E
End of trawl
E
E
E
Input style (select sexagesimal (base 60) notation FA, E
or decimal notation to input coordinates.)
Coordinates at the start of trawl
FA, E
Coordinates at the end of trawl
E
Type of net frame
E
Model number and manufacturer
E
Shape of net aperture
FC, E
Width of net aperture (m)FC, E
Height of net aperture (m)E
2Area of net aperture (m )E
E
Openings (Size and select one side length or E
diagonal length)
Model number and manufacturer
E
Distance (m)Calculation method (Using flow meter, speed
relative to water or GPS Log.)
Calculation formulas
Sweep area (m2)Calculation formulas
Water volume (m3)Calculation formulas(minutes)(Speed relative to water, knots)(Side or stern of a vessel)
(m)

FC, E
E

FC, E
FC, E
FC, E
FC, E
E
E

・The meanings of the letters listed in the category column are as follows:
FA, FB and FC represent “fundamental” items. The“fundamental”items are the minimum requirement to identify theabundance of microplastics, the sampling time and location, and microplastic density, which can be displayed in any of thefollowing four units: number of particles/m3 or km2, weight/m3 or km2.
FA: All the “FA” are necessary for displaying the density.
FB: At least one (1) item in “FB” is necessary for displaying the density. The symbol * (asterisk) with FB indicates some
additional items among “FC” would be necessary to calculate microplastic density.
・The letter “E” represents “essential”items which are the minimum requirements to make the survey results comparable.
・The items with no letter in the " Category " column are those that may be obtained optionally, depending on the specific purposeof individual surveys or instrument availability.
・Reporting all data obtained is recommended, including metadata sets.

67

Table 4-1(2) List of data to be reported when sampling floating microplastics.
Items

Data necessary to ensure comparability

Tow
Parameters

Net immersion depth

Metadata(weather,sea
conditions,water
quality)Category(m)
FC, E
Percentage of net immersion depth to sizeof net frame (%)
Whether or not there was any change in the E
immersion depth during tow.
Tow direction
E
Blank tests
Whether or not blank tests were conducted E
Results (particles/test)
E
Wind direction and speed(degrees, m/s)
E
Significant wave height (measure using an onboard wave meter.)※When a wave-height meter is not available on the sampling vessel, wave height data from nearby tide
stations or websites can be recorded instead.

Beaufort scale(Visual observation)
Vessel movements(Heave, pitch, roll)Sea surface temperature(degrees Celsius)
Sea surface salinity(-)
Water current direction and speed (degrees, knot)
Other types of water quality data (Chlorophyll, fluorescence, etc.)
State of floating debris on the sea surface. (Large floating debris, drifting algae,
etc.)

E
E
E
E

E

・The meanings of the letters listed in the category column are as follows:
FA, FB and FC represent “fundamental” items. The“fundamental”items are the minimum requirement to identify theabundance of microplastics, the sampling time and location, and microplastic density, which can be displayed in any of thefollowing four units: number of particles/m3 or km2, weight/m3 or km2.
FA: All the “FA” are necessary for displaying the density.
FB: At least one (1) item in “FB” is necessary for displaying the density. The symbol * (asterisk) with FB indicates some
additional items among “FC” would be necessary to calculate microplastic density.
・The letter “E” represents “essential”items which are the minimum requirements to make the survey results comparable.
・The items with no letter in the " Category " column are those that may be obtained optionally, depending on the specific purposeof individual surveys or instrument availability.
・Reporting all data obtained is recommended, including metadata sets.

68

Table 4-2 List of data to be reported for laboratory analysis of microplastics.
Items

Data necessary to ensure comparability

Category

Density
separation

Whether or not density separation was conductedType of solution used for density separation (NaCl, ZnCl2, etc.)
Concentration of solution used for density separation (%)
Processing time (minutes)
Whether or not biological digestion or chemical treatment was conducted
Methods used for digesting organic matter (acid treatment, alkaline
treatment, enzyme treatment, oxidation treatment, etc.)
Temperature during processing (degrees Celsius)
Reaction time (minutes)
Whether or not sample splitting was conducted
Method or equipment of splitting
Estimated relative error range caused by splitting process
Whether or not pretreatment before particle isolation was conductedType of pretreatment (fractionation by size including non-plastic materials by
sieve, etc.)
Whether or not picking was conducted under stereomicroscope.
Method of size fractionation (whether the maximum diameter was measured
or sieves were used)
Diameters of the measured particles (maximum and minimum Feret's
diameters, area)
Whether or not composition analysis was conducted
Method of composition analysis (FTIR, Raman spectroscopy, etc.)
Percentage of the particles subjected to composition analysis (%)
Temperature of sample drying (degrees Celsius)
Humidity of sample drying (%)
Processing time of sample drying (minutes)
Methods of weight measurement
Blank tests
Whether or not blank tests were conducted
Results (particles/test)
Spiked recovery tests Whether or not spiked recovery tests were conducted
Results (%)

E
E
E

Biologicaldigestion andchemicaltreatment
Samplesplitting
Isolationofmicroplastics

Counting andmeasuringsizesofparticles
Identificationofmicroplastics
Weightmeasurement

QA/QC

E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E

・The meanings of the letters listed in the category column are as follows:
FA, FB and FC represent “fundamental” items. The“fundamental”items are the minimum requirement to identify theabundance of microplastics, the sampling time and location, and microplastic density, which can be displayed in any of thefollowing four units: number of particles/m3 or km2, weight/m3 or km2.
FA: All the “FA” are necessary for displaying the density.
FB: At least one (1) item in “FB” is necessary for displaying the density. The symbol * (asterisk) with FB indicates some
additional items among “FC” would be necessary to calculate microplastic density.
・The letter “E” represents “essential”items which are the minimum requirements to make the survey results comparable.
・The items with no letter in the " Category " column are those that may be obtained optionally, depending on the specific purposeof individual surveys or instrument availability.
・Reporting all data obtained is recommended, including metadata sets.

69

Table 4-3 List of data to be reported for results of microplastic survey.
Items

Data necessary to ensure comparability

Weightand Maximum
Feret'snumberof diameterplastic
1.0mm≤d<5.0mm
particles

Number of particles (particles)
Particle number density (per filtered water volume,particles/m3)
Particle number density (per trawl swept area,
particles/km2)
Total weight (g)Particle weight density (per filtered water volume,
g/m3)
Particle weight density (per trawl swept area, g/km2)
Maximum
Feret's Number of particles (particles)
diameterParticle number density (per filtered water volume,d<1.0mmparticles/m3)
Particle number density (per trawl swept area,
particles/km2)
Total weight (g)Particle weight density (per filtered water volume,
g/m3)
Particle weight density (per trawl swept area, g/km2)
Total
Number of particles (particles)(maximum
Feret's Particle density (per filtered water volume,
diameterparticles/m3)
d<5.0mm)
Particle density (per trawl swept area, particles/km2)
Total weight (g)Particle weight density (per filtered water volume,
g/m3)
Particle weight density (per trawl swept area, g/km2)
Properties of 1.0mm≤d<5.0mm
Shapes of microplastics. (Category, percentage)
theplastic
Material of microplastics. (Category, percentage)
particles
Colors of microplastics. (Category, percentage)d<1.0mm
Shapes of microplastics. (Category, percentage)
Material of microplastics. (Category, percentage)
Colors of microplastics. (Category, percentage)

Category

FB*
FB, E
FB, E
FB*
FB, E
FB, E
FB*
FB
FB
FB*
FB
FB
FB*
FB
FB
FB*
FB
FB
E

・The meanings of the letters listed in the category column are as follows:
FA, FB and FC represent “fundamental” items. The“fundamental”items are the minimum requirement to identify theabundance of microplastics, the sampling time and location, and microplastic density, which can be displayed in any of thefollowing four units: number of particles/m3 or km2, weight/m3 or km2.
FA: All the “FA” are necessary for displaying the density.
FB: At least one (1) item in “FB” is necessary for displaying the density. The symbol * (asterisk) with FB indicates some
additional items among “FC” would be necessary to calculate microplastic density.
・The letter “E” represents “essential”items which are the minimum requirements to make the survey results comparable.
・The items with no letter in the " Category " column are those that may be obtained optionally, depending on the specific purposeof individual surveys or instrument availability.
・Reporting all data obtained is recommended, including metadata sets.

70

5. Conclusions
The Guidelines summarize recommendations for harmonization of ocean surface microplastic
survey methods to facilitate the generation of comparative results with the assumption thatvarious sampling and processing methods will be still used in the future. Those recommendations
are also useful for surveys conducted in freshwater systems.
Many studies are expected to be carried out involving microplastic monitoring at the ocean
surface for various purposes. Application of the harmonized methods proposed in the Guidelines
will facilitate result generation in a comparable manner, enabling researchers to analyze,
consolidate and integrate all the available data.
Data gaps are expected to be filled in the future by surveys in various countries and areas where
surveys have yet to be conducted, and at the same time, comparison of the results obtained fromsurveys conducted worldwide to date and accumulation of data measured using harmonizedmethods are expected to facilitate understanding of the global status of microplastic pollution.
Current data on the abundances of microplastics in the ocean suggests the existence of some
unknown mechanism for their removal and identifying the distributions of ocean surface
microplastic densities is expected to elucidate the process of their generation through to theirdisappearance via migration.
It is important to tackle the following technological challenges for improving the efficiency andaccuracy of identifying the status of oceanic microplastic pollution.
Automation of microplastic analysis (size measurements and composition analyses) andocean sampling for efficiency and speed, including faster speedier analysis. The turnaroundtime from sampling to data acquisition could be shortened, allowing prompt confirmation of
the comparability and adequacy of the samplings, so that complementary samplings can be
conducted as required for improving overall accuracy.
Development of techniques to improve the accuracy of measuring tiny microplastics smaller
than 1 mm.
As one of the further actions, a database for creating a two-dimensional map (2-D maps) of
microplastics at the ocean surface will be constructed in order to fill the above mentioned gaps
and to elucidate unknown processes.
The purposes of the 2-D maps are follows:
Facilitate understanding of the actual state of global microplastic pollution.
Contribute to solving issues such as described in the SDGs Target 14.1.
Provide basic data that can be used for efficient environmental monitoring, for use incomputer simulations and for further harmonization of monitoring methods.
The final goal of administrative interest might be for their use as basic data in biological risk
assessments.

71

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