counting and sizing microplastic fribres, the accurate and easy way

Counting and sizing microplastic fibres, the accurate and easy way

Kunnen, T. H.Gerber, G.

Coote, M. W.Moodley, G. K.

Robertson-Andersson, D. V.

University of KwaZulu-Natal, School of Life Science

Introduction

• Microplastics defined as being < 5 mm (Hidalgo–Ruz et al., 2012)

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Primary microplastics Are produced for a specific purpose (eg. shower gels, cleansers)

Secondary microplastics From environmental degradation (wave action, UV exposure etc) of larger plastics

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• Global demand for plastic production is not decreasing

• Last estimated at over 311 million tonnes per year (PlasticsEurope, 2015)

• Most common source of secondary microplastics is from your own household

Introduction contd...

Polyester Lastex

Spandex

Acetate

Rayon

Nylon

Acrylic

Kevlar

Orlon

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Quick detour...filters

250 – 350 µm 600 µm

Quick detour...filters

400 µm

Quick detour...filters

Introduction contd...

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550–681 part/kg!

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Introduction contd...

“ Experiments sampling wastewater from domestic washing machines demonstrated that a single garment can produce > 1900 fibres per wash.”

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For cleaner lakes and archipelago

“… waste water treatment plant ± 500 000 connected persons is estimated to receive up to 16.9 ton microplastic fibres per year…”

Materials and Methods

3 Weeks later = 0.1276 g fibres 10 – 50/100 µm long

Ultra–violet (UV) fluorescent polyethylene–terephthalate (PET) textile (395 nm)

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White LightUV Light

Materials and Methods contd...

• Epifluorescent microscope

• Low magnification (20 – 40 x)

• 10 – 12 fields of view taken

• Images analysed with Image Pro Plus (IPP)

• Manual counting and sizing

• Automated counting and sizing

• Length and Width data generation

• Time saving

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Width = 29.5 µmLength = 350 µm

Width = 24.3 µmLength = 390 µm

Width = 20.6 µmLength = 868 µm

Fibre 1:Width = 17.5 µmLength = 754 µm

Fibre 2:Width = 20 µmLength = 534 µm

Fibre 3:Width = 21.7 µmLength = 330 µm

Fibre 4:Width = 20 µmLength = 988 µm

Automated image analysis by Binary Segmentation and Histogram Selection

Materials and Methods contd...

• 5 volunteers

• 50 images of microfibres

• Count Manually

• Length, width and time recorded

• Automatically using IPP

• Recorded time taken

This is 39 lines of the 270 (14.4%)

Figure 1: Average microfibre counts per filter analyzed

Manual Automated82

82.5

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83.5

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84.5

85

85.5

Methodology

Mic

rofib

res.

mus

sel-1

p = 0.9378

Results

Manual Automated320

330

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350

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370

380

Methodology

Mic

rofib

re Le

ngth

(µm

)

p = 0.5478

Figure 2: Average mirofibre lengths (µm) measured utilizing manual and automated methodologies

Manual Automated0

10

20

30

40

50

60

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Methodology

Mic

rofib

re W

idth

(µm

)

p = 0.0079

Figure 3: Average microfibre widths measured utilizing manual and automated methodologies

13.7 µm

113 µm

AutoWidth = 50.2189 µm

ManualWidth = 29.5 µm

AutoWidth = 110.345 µm

ManualWidth = 24.3 µm

AutoWidth = 283.866 µm

ManualWidth = 20.6 µm

Width = Area / Length

AutoWidth = 50.2189 µm

ManualWidth = 29.5 µm

Width equationWidth = 26.57 µm

AutoWidth = 110.345 µm

ManualWidth = 24.3 µm

Width equationWidth = 24.46 µm

AutoWidth = 283.866 µm

ManualWidth = 20.6 µm

Width equationWidth = 25.98 µm

Manual Auto Calculated0

5

10

15

20

25

Methodology

Mic

rofib

re W

idth

(µm

)

Figure 4: Average microfibre widths measured utilizing manual and automated methodologies with the equation for new widths

p = 0.0696

23.23 ± 1.45 µm21.63 ± 0.74 µm

Manual Automated0

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Methodology

Tim

e (s

)1434.52 ± 411.97 sec

60.20 ± 7.76 s

23.90 ± 6.86 mins=

Figure 5: Average time (seconds) taken to count and measure microfibres on one filter

Fibre 1:Width = 17.5 µmLength = 754 µm

Fibre 2:Width = 20 µmLength = 534 µm

Fibre 3:Width = 21.7 µmLength = 330 µm

Fibre 4:Width = 20 µmLength = 988 µm

ManualWidth = 19.8 µmLength = 2606 µm

Width equationWidth = 32.30 µmLength = 2402.740 µm

Conclusion

• Using the designed macro within IPP at segmentation of 27:255

• No difference in number of fibres counted

• No difference in measured lengths

• No difference in calculated widths

• Massive time saving when doing automated analysis

M A

Length

M A

Width

M A

Time

Number of fibres

M A

Current and Future Applications• Automated counting sizing and analysis of fluorescent microplastic fibres is ongoing research in our lab

• Mussels (Perna perna)• Sea Urchins (Tripneustes gratilla and Stomopneustes variolaris)• Mullet• Successfully been applied to 6 projects on microplastic research

• Future work to look at

• Broaden the scope and range of analysis to “naturally occurring” environmental microplastics, i.e. non–fluorescent, brown, white etc

• Work out a watershed spit that will enable the differentiation of close contact fibres

Thank you

Any questions?

AcknowledgementsThank you to the MACE lab volunteers and to the NRF for funding this project. Thanks also go to Theo van Zyl.

References

• Hidalgo–Ruz, V., Gutow, L., Thompson, R.C. & Thiel, M. 2012. Microplastics in the Marine Environment: A Review

of the Methods Used for Identification and Quantification. Environmental Science and Technology. 46. 3060– 3075.

• PlasticsEurope 2015. Plastics – the Facts 2015: An analysis of European plastics production, demand and waste data. Konigin Astridlaan 59, 1780 Wemmel, Belgium.

Full list of internet references available upon request