This document presents a novel methodology for separating microplastics (<500μm) from particulate organic matter (POM) in water samples. Current separation methods are inefficient at separating suspended microplastics and POM due to similarities in size and density. The developed method uses a two-phase separation where a non-polar solvent is added to draw microplastics into a separate immiscible layer that can be removed and analyzed. Testing recovered over 90% of fluorescent polyethylene terephthalate and polypropylene microplastics added but only 1% of denser polyethylene terephthalate microbeads. This accurate separation method allows for investigation of microplastic ingestion and effects in marine organisms and ecosystems.

1. A Novel Methodology for the Separation of Known Suspended
Microplastics (<500µm) from Particulate Organic Matter (POM)
THE MICROPLASTIC PROBLEM !
Marine plastic pollution is currently ranked as one of the greatest threats to marine life (Todd et al., 2010; Andrady, 2011).
Plastic pollution consists of both macroplastic (> 5 mm) and microplastic (< 5 mm) particles (Hidalgo-Ruz et al., 2012).
Microplastics have been found in the guts of both pelagic and demersal fish species (Lusher et al. 2012).
Ingested microplastics may serve as a delivery mechanism for Persistent Organic Pollutants (POPs) which have a high affinity for the hydrophobic surfaces of the microplastic particles (Mato,
2001).
CURRENT SEPARATION METHODS
Although useful in separating microplastics from sediment samples, density-based separation methods are inefficient at
separating suspended microplastics from POM. Variations in the density of the water samples alters the relative
buoyancies of both the suspended microplastics as well as the POM (Coote, unpublished).
Previously described methods using nitric acid (Andrady, 2011; Claessens et al., 2013) were inefficient and were found to
degrade the microplastics substantially.
THE NEED FOR A SEPARATION TECHNIQUE
The ability to separate suspended microplastics from POM is essential in order to accurately assess the gut evacuation
rates of microplastics, as well as the effects of their prolonged consumption/retention by marine organisms in
experimental systems (Lusher et al., 2012).
To accurately quantify the amount of microplastics ingested, retained and evacuated, it is necessary to separate them from
any particulate organic matter (POM) in the samples.
Observational studies of natural systems are unable to accurately correlate the extent of microplastic consumption with
any physiological effects on marine organisms due to a high degree of natural variability within these systems (Lusher et
al., 2012; Foekema et al., 2013). Such aspects of the eco-physiology of marine organisms may therefore be better
examined in aquacultural systems (Clements et al., 2009; Lusher et al., 2012).
Literature Cited
Andrady, A. L. 2011. Microplastics in the marine environment. Marine Pollution Bulletin. 62 -8. 1596–1605.
Claessens, M., Van Cauwenberghe, L., Vandegehuchte, M. B. & Janssen, C. R. 2013. New techniques for the detection of microplastics in sediments and field
collected organisms. Marine Pollution Bulletin. 70 -1. 227-233.
Coote, M.W. 2014 (Unpublished). The factors affecting the bio-availability of microplastics and their retention in filter-feeding, herbivorous fish (Mugil cephalus L.).
Hons. Thesis. University of KwaZulu-Natal (Westville campus), R.S.A
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.
Ivar do Sul, J. A. & Costa, M. F. 2014. The present and future of microplastic pollution in the marine environment. Environmental Pollution. 185. 352-364.
Lusher, A.L., McHugh, M. & Thompson, R.C. 2012. Occurrence of microplastic in the gastrointestinal tract of pelagic and demersal fish from the English Channel.
Marine Pollution. 1-6.
Martin, A. 1969. Physical pharmacy: Physical chemical principles in the pharmaceutical sciences. Philadelphia. 2Nd ed. Lea and Febiger.
Thompson, R.C., Olsen, Y., Mitchell, R.P., Davis, A., Rowland, S.J., John, A.W.G., McGonigle, D. & Russell, A.E. 2004. Lost at sea: where is all the plastic? Science
304. 838.
Todd, P.A., Ong, X. and Chou, L.M. 2010. Impacts of pollution on marine life in Southeast Asia. Journal of Biodiversity and Conservation. 19. 1063-1082.
Matthew. Coote, Dr. Deborah Robertson-Andersson, G.K Moodley
University of KwaZulu–Natal – Westville Campus, College of Agriculture, Engineering and Science
School of Life Sciences, Private Bag X 54001, Durban, 4000, South Africa
ASSESSING THE PROBLEM
Although microplastics are considered to be virtually ubiquitous throughout the marine environment, most studies are still
thought to be a gross underestimation of the true extent of microplastic pollution in the oceans (Hidalgo-Ruz et al, 2012).
The majority of observational studies have focused specifically on visible microplastics (Andrady, 2011).
According to Hidalgo-Ruz et al. (2012) current techniques are unable to adequately separate suspended microplastic
particles (< 500 μm) from particulate organic matter (POM) due to the similar sizes and densities of the microplastics
and the POM.
AIM OF THIS STUDY
A technique was therefore sought to separate and quantify specific microplastics (< 500 μm) for this purpose.
Polyethylene terephthalate (PET) and Polypropylene (PP) were tested as they were available in the appropriate form.
Additionally, UV fluorescent plastics were chosen as they were easily visible in small quantities in order to ensure the
accuracy of the separation method during the various stages of separation.
RESULTS AND FINDINGS
METHODOLOGY
The two-phase separation method described by Figure 1 was developed for the separation and quantification of
selected microplastics. The addition of a non-polar solvent with a high affinity for the surface of the microplastics
serves to draw them into a separate layer, immiscible with the rest of the sample. This layer may then be removed
and the microplastics within it can be dried and weighed.
RESULTS AND FINDINGS
METHODOLOGY
Treatment 1 (PET microfibres) recovered 93.95 ± 1.79 % (n=10) of the microplastics
Treatment 3 (PP microbeads) recovered 97.23 ± 1.38 % (n=10).
Whilst the control group indicated that no POM was recovered by the xylene layer during the treatments
In contrast Treatment 2 (PET microbeads) recovered only 1.17 ± 1.13 % (n=10) of the microplastics, most likely
the result of the particle density (1.38 g.cm-3).
Figure 1. Microplastic recovery (mean ± S.D) For
PET microfibres and microbeads and PP microbeads.
CONCLUSIONS
The proposed method for the separation of microplastics (20 - 500 μm) from POM and subsequent
quantification thereof, may be applied using specific plastics where appropriate, for experimental
purposes with the use of appropriate correction factors.
This novel methodology has been successfully applied using PET and PP for microplastic gut
evacuation experiments in mullet (M. cephalus L.) (Coote, unpublished) and is currently being
employed in further investigations using other marine organisms (e.g. fish and marine
invertebrates) as models.
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