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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
340
350
360
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
200
400
600
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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