WESTPAC Training Workshop on

“Distribution, Source, Fate and Impacts of Marine

Microplastics in Asia and the Pacific”

20–22 September 2017, Phuket, Thailand

Methodological Limitations for Microplastic

Quantification in the Ocean: Recommendations for

Overcoming the Defects

Director, ProfessorPLASTIC MARINE DEBRIS RESEARCH CENTER

EAST CHINA NORMAL UNIVERSITYInvestigate Principal

IOC-WESTPAC Marine MP Program

Daoji Li

Plastic Marine Debris Research Center East China Normal University

1. Microplastic Collection

2. Microplastic Separation

3. Microplastic Identification

4. QA/QC

CONTENTS

1 MICROPLASTIC COLLECTION

1.1 Collection of microplastics from the environment

Influenced by many factors

Water, sediment, soil, air or biota

Determine the abundance, size and shape

No universally accepted methods

The methods available all have potential bias

1.2 Standardization of collection

No standardization for collecting microplastics

The abundance or the concentrations with

differing units of measurement, incomparable

Unit conversion , both numerical and mass

concentrations

Method standardization in microplastic analysis

by the EU Joint Programme Initiative (JPI)

Ocean

Laboratory methods by the National Oceanic

and Atmospheric Administration (NOAA) in the

United States

Selective sampling

Items visible to the naked eye are directly extracted from

the environment

1.3 Sampling method

Volume reduced sampling

The volume of the bulk sample is reduced until only

the specific items of interest for further analysis remains

1.3 Sampling method

Bulk sampling

The entire sample is taken without reducing its volume

Barrows et al. 2017

1.3 Sampling method

Environmental parameters

To consider and record the prevailing weather conditions

wind direction

the time of Sampling

tidal height

rainfall

1.3 Sampling method

wind velocity instrument

Contamination mitigation

Microfibers

From ambient air, but also via the use of sampling or laboratory

equipment, improper storage of samples or even from the

clothing of the researchers themselves

downwind sampling

using non-plastic tools or containers

avoid synthetic clothing and natural fibres

avoid fleece

1.3 Sampling method

Monofilament nylon mesh plankton nets of

various designs

1.4 Surface water sampling

Neuston net

Manta trawl

Catamaran

the typical mesh sizes : 53 μm to 3 mm, most frequent

size being 333 μm (used for plankton studies)

fine mesh will clog quickly, it will need to be towed

slowly.

commonly 350–400 cm long

a flow meter should be fitted to the net

Flow meters is strongly recommended

1.4 Surface water sampling

A neuston catamaran (a) and a manta trawl (b)

Neuston net tow 333 or 335 μm

1.4 Surface water sampling

Vertical sampling of the water column

1.5 Water column sampling

Bongo net can be used for horizontal or vertical sampling of the water column

direct in situ filtration

bulk sampling with subsequent filtration

continuous Plankton Recorder (CPR)

water pumps

Other water column sampling

1.5 Water column sampling

Advantage:

large volume

easy comparison between data sets

Drawback:

size of plastic captured is limited

samples are prone to be contaminated

Advantage and Drawback of net sampling

Advantage and Drawback of bulk sampling

Advantage:

no size limitation

highly efficient

easy to control contamination

Drawback:

small sampling volume

Larger volume of samples by bulk sampling

is highly recommended to assessing MPs in

the waters

Recommendations

Sediments and sands

sediments are considered to be ideal mediums for

environmental pollutants and tend to experience

long-term contamination

1.6 Sediment Sampling

Microplastics (<5–1 mm) \mini-microplastics (<1 mm to 1 μm) in size on coastal sediments

Using visual identification and sorting by hand

Exclusion of mini-microplastics can result in highly underestimated concentrations of microplastics

Mini-microplastics are considered to represent 35–90% of all the microplastics present in the marine environment

As the size of microplastics decrease, they become progressively

more difficult to collect from the environment

The lower size limit of microplastics reported

is highly dependent on the sampling and

separation methods used.

For small irregularly shaped microplastics,

bulk sampling is also the favored method to

obtain a representative sample

1.6 Sediment Sampling

For sediment samples, may also be reported the

units of weight (g or kg) in both wet and dry

weight, as well as volume (mL or L)

Recommended !

1.6 Sediment Sampling

sampling strategy of sedimental samples

TransectsQuadrats

Dekiff et al. 2014

Recommendation:

from the wrack line

to the vegetation line

1.6 Sediment Sampling

Sampling depth

Variant sampling depths reported: from 1 to 200 cm

Recommendation:

The top 5 or 2.5 cm of sand and selectively

sampling variable depth layers

1.6 Sediment Sampling

2 MICROPLASTIC SEPARATION

2.1 Separation of microplastics from samples

Microplastics (<5–1 mm) ,visual detection or binocular

microscopy

Mini-microplastics (<1 mm to 1 μm) , high

magnification fluorescence microscope

Suspected microplastics, based upon their physical

characteristics, such as their size, shape, texture, color

and lack of biological structures

Further identification , using spectroscopic techniques,

such as Raman spectroscopy, to positively confirm the

type of plastic

2.2 Water samples

Filtration

The pore size of the filter paper

used can vary between studies, it

is generally 1–2 μm in size

Sieving

The size in most studies

ranged from 38 μm to 4.75 mm,

mesh size must not exceed 5 mm

A vacuum filtration system

Crawford 2017

Multi-tier sieving

2.2 Water samples

Crawford 2017

2.3 Sediment samples

Density separation

density separation : using a saturated sodium

chloride (NaCl) solution, density of 1.202 g/cm3

The atmosphere should be avoided

The experimental setup of the three-

necked round-bottomed flask

A publication by the

National Oceanic and

Atmospheric Administration

(NOAA) in the United States

recommends the use of 5.4

M lithium metatungstate (1.6

g/cm3) for sediment density

separations

the extraction ,three times to

achieve high efficiencies

2.3 Sediment samples

Crawford 2017

Heavy liquids that have successfully been used to create high

density solutions for the separation of microplastics from

sediment

2.3 Sediment samples

Crawford 2017

A novel technique based on density separation

has been developed, the Munich Plastic

Sediment Separator

Utilizing a solution of zinc chloride, the MPSS

is reported to reliably separate mini-

microplastics from sediments with an

improvement in the recovery rate from 40% to

95.5% , the rate of recovery for microplastics

greater than 1 mm in size is even higher at 100%

2.3 Sediment samples

Elutriation

Separation with a sodium iodide (NaI)

solution and demonstrated an excellent

recovery

Efficiency for PVC particles at 100% and

fibres at 98%

2.3 Sediment samples

2.4 Biological samples

Visual detection and separation from biological

material

Chemical and enzymatic digestion

fish stomachs and shellfish

Other methods ：use of nitric acid, hydrogen peroxide and sodium

hydroxide and have demonstrated effective rates of tissue digestion

(particularly in mussels), with high recovery yields of polystyrene

microbeads at 94–98%, but highly variable results for nylon fibers at

0–98% recovery

The acid destruction method ： a mixture of 65% nitric acid (HNO3)

and 68% perchloric acid (HClO4) in a 4:1 ratio (HNO3:HClO4 4:1 v:v)

3 MICROPLASTIC IDENTIFICATION

3.1 Identification of microplastics

Sample purification (mechanical, chemical and

enzymatic methods)

Any surface exposed to the marine or freshwater environment will

be subject to some form of fouling, particularly biofouling where a

layer of microorganisms (bacteria, algae, fungi or plankton)

accumulates on the surface, resulting in misidentification

FTIR or Raman spectroscopy

may also be negatively impacted

Mechanical removal methods

ultrasonic bath-might artificially introduce secondary

MPs into samples by breaking weathered plastics

Chemical removal methods

H2O2, NaOH, KOH, HNO3, HCl, HClO4, NaClO, HNO3

and HClO4, Sodium hypochlorite and HNO3, KOH and

NaClO

Sample purification

some chemicals alter the physical properties (color, shape

and size, etc.)

Enzymes

MP samples from river water (Nuelle et al. 2014),

mussels tissue (Catarino et al.2016), and digestive

tracts of turtles (Duncan et al. 2017)

Advantages and Recommendation

1. enzymatic purification ensures MPs integrity

2. less harmful to human and environmental health

Enzymatic purification should be considered

Visual identification

Selection criteria to identify microplastics

Microscope-aided visual inspection:

size range 0.5–5 mm

Naked eyes inspection:

size range >1 mm

High-magnification microscopic examination, as well as

fluorescence microscopy, to preclude the possibility of

biological origin

Drawback--misidentification rate:

from 20 % to 70 % (Hidalgo-Ruz et al. 2012;

Eriksen et al. 2013; Song et al. 2015)

Drawback of Visual identification

Complementary techniques are needed (e.g.

FTIR, Raman, GC/MS).

Scanning electron microscope

Pyrolysis–gas chromatography–mass

spectrometry(Pyr-GC–MS)

Nuclear magnetic resonance (NMR) spectroscopy

Fourier-transform infrared (FTIR) spectroscopy

Raman spectroscopy

3.2 Analysis

SEM can offer a clear image of the size and surface

texture of the plastic-like particles

Scanning Electron Movroscopy (SEM) /

Energy-Dispersive X-Ray Spectroscopy (EDX)

X-ray spectroscopy (EDX) can determine the mainatomic composition of the putative plastic particles,which is useful for identifying carbon-dominant plasticsfrom inorganic particles

Drawback: 1. colors cannot be provided by SEM

2. expensive

Methods: Pyro-GC/MS, TDS-GC/MS, TGA-SPE-

TDS-GC-MS

Fries et al. 2013; Fischer M. and Scholz-Böttcher B.M. 2017

Thermo-chemical analysisl

1. destructive to the analyzed particles

2. not routinely applicable in common labs

3. size limitation

Drawback of thermal-chemical analysis:

Advantage: nondestructive andstraight-forward

characterization techniques

Higher resloution

Spectroscopic Methods

FTIR and Raman spectroscopy

BRUKER

Lumos

Fourier-transform infrared

(FTIR) spectroscopy

FTIR - DRAWBACK

Micro-ATR-FTIR are that: 1) aged and brittle MPs can

be destroyed by the pressure of the ATR probe; 2) ATR crystal

is prone to be damaged by hard inorganic particle remnants

The reflectance mode bears the disadvantage that

measurements of irregularly-shaped microplastics may result

in non-interpretable spectra due to refractive error

The transmittance mode needs IR transparent filters (e.g.

aluminium oxide) and is, owing to total absorption patterns,

limited by a certain thickness of the microplastics sample

Raman spectroscopy

RAMAN - DRAWBACK

Undesired fluorescence

Coming from additives (e.g. filler, colorants) or

environmental organic matter (e.g. biofilm, algae)

superposes the Raman spectra of polymer matrix

and hampers identificationfluorescent spectra

FTIR AND RAMAN IMAGING

The analysis of small microplastics without any visual

presorting.

Käppler et al. 2016

4 QA/QC

Minimizing Contamination in labs

1. Filtering all the liquid solvents

2. Glassware

3. Combustion

4. Airclean Hood with HEPA

field sampling

laboratory environments

atmospheric depositions

samples filtration

negative control samples during the digestion and separation

Procedural Blank

At all stages:

Thanks for Your Attention !

Plastic Marine Debris Research Center East China Normal University