Counting and sizing microplastic fribres, the accurate and easy way.
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This document describes a method for counting and sizing microplastic fibers using image analysis software. The key points are: - Microplastics are defined as plastic particles less than 5mm in size from primary sources like cleansers or secondary sources like degradation of larger plastics. - A method was developed using fluorescent PET fibers and image analysis software to automatically count, measure length and width, and calculate fiber properties. - The automated method was found to produce similar counts and length measurements as manual counting, but was much faster taking on average 1 minute versus 24 minutes for manual counting. - The automated method provides an accurate and efficient way to analyze microplastic fibers that can be applied to research on micropl
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Counting and sizing microplastic fribres, the accurate and easy way.
- 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
- 8. Introduction contd... “ Experiments sampling wastewater from domestic washing machines demonstrated that a single garment can produce > 1900 fibres per wash.” www17 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) www11 www12 www13 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 www16
- 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
Editor's Notes
- The microplastics content was 550–681 particles/kg in sea salts, 43–364 particles/kg in lake salts, and 7–204 particles/kg in rock/well salts. In sea salts, fragments and fibers were the prevalent types of particles compared with pellets and sheets. Microplastics measuring less than 200 μm represented the majority of the particles, accounting for 55% of the total microplastics,
- The microplastics content was 550–681 particles/kg in sea salts, 43–364 particles/kg in lake salts, and 7–204 particles/kg in rock/well salts. In sea salts, fragments and fibers were the prevalent types of particles compared with pellets and sheets. Microplastics measuring less than 200 μm represented the majority of the particles, accounting for 55% of the total microplastics,