Presented by Travis Kunnen (PhD candidate at MACE Lab) for the KZN Premier's Award and at SCCP 2016.

1. 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

2. Introduction
• Microplastics defined as being < 5 mm (Hidalgo–Ruz et al., 2012)
www1
www2
www4
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
www3

3. • 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
www5 www6 www7 www8 www9

4. Quick detour...filters
250 – 350 µm 600 µm

5. Quick detour...filters
400 µm

6. Quick detour...filters

7. Introduction contd...
www10
550–681 part/kg!
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8. 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…”

9. 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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www14 www15
White LightUV Light

10. 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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13. Width = 29.5 µm
Length = 350 µm

15. Width = 24.3 µm
Length = 390 µm

17. Width = 20.6 µm
Length = 868 µm

19. Fibre 1:
Width = 17.5 µm
Length = 754 µm
Fibre 2:
Width = 20 µm
Length = 534 µm
Fibre 3:
Width = 21.7 µm
Length = 330 µm
Fibre 4:
Width = 20 µm
Length = 988 µm

20. Automated image analysis by
Binary Segmentation and
Histogram Selection

26. 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%)

27. Figure 1: Average microfibre counts per filter analyzed
0
20
40
60
80
100
120
140
Manual Automated
Microfibres.mussel-1
Methodology
p = 0.9378
Results

28. 0
50
100
150
200
250
300
350
400
450
500
Manual Automated
MicrofibreLength(µm)
Methodology
p = 0.5478
Figure 2: Average mirofibre lengths (µm) measured utilizing manual and
automated methodologies

29. 0
20
40
60
80
100
120
Manual Automated
MicrofibreWidth(µm)
Methodology
p = 0.0079
Figure 3: Average microfibre widths measured utilizing manual and automated
methodologies
13.7 µm
113 µm

30. Auto
Width = 50.2189 µm
Manual
Width = 29.5 µm

31. Auto
Width = 110.345 µm
Manual
Width = 24.3 µm

32. Auto
Width = 283.866 µm
Manual
Width = 20.6 µm
Width = Area / Length

33. Auto
Width = 50.2189 µm
Manual
Width = 29.5 µm
Width equation
Width = 26.57 µm
Auto
Width = 110.345 µm
Manual
Width = 24.3 µm
Width equation
Width = 24.46 µm
Auto
Width = 283.866 µm
Manual
Width = 20.6 µm
Width equation
Width = 25.98 µm

34. 0
5
10
15
20
25
30
Manual Auto Calculated
MicrofibreWidth(µm)
Methodology
Figure 4: Average microfibre widths measured utilizing manual and automated
methodologies with the equation for new widths
p = 0.0696
23.23 ± 1.45 µm
21.63 ± 0.74 µm

35. 0
200
400
600
800
1000
1200
1400
1600
1800
2000
Manual Automated
Time(s)
Methodology
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

36. Fibre 1:
Width = 17.5 µm
Length = 754 µm
Fibre 2:
Width = 20 µm
Length = 534 µm
Fibre 3:
Width = 21.7 µm
Length = 330 µm
Fibre 4:
Width = 20 µm
Length = 988 µm

37. Manual
Width = 19.8 µm
Length = 2606 µm
Width equation
Width = 32.30 µm
Length = 2402.740 µm

38. 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

39. 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

40. Thank you
Any questions?
Acknowledgements
Thank you to the MACE lab volunteers and to the NRF for funding
this project. Thanks also go to Theo van Zyl.

41. 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