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Developing and optimizing a time-efficient method for in-situ observation of microplastics in the marine environment using flow cytometry
Timperman, J. (2022). Developing and optimizing a time-efficient method for in-situ observation of microplastics in the marine environment using flow cytometry . MSc Thesis. Faculty of Bioscience Engineering, Ghent University: Gent. 104 pp.

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  • Timperman, J., more

    Increasing plastic production, along with poor waste management, has led to a growing amount of plastics entering the environment. They can fragment through various mechanisms into microplastics, which accumulate in our oceans. However, there is still no unambiguous conclusion about the risks and effects of exposure to microplastics. Therefore, reliable microplastic assessment is a must. However, current assessment techniques are time-consuming, have issues with the identification of smallermicroplastics (< 20μm), and furthermore, the concentration in a sample has to be estimated by manual counting. Therefore, this thesis aims to develop a time-efficient semi-automated method to observe microplastics in seawater in-situ using flow cytometry in combination with Nile red staining of the microplastics. This work began with the preparation of dilution series to assess the accuracy of the flow cytometer concentration measurement and to quantify the limit of detection. The concentrations in the dilution series were first verified using three counting methods,namely the Sedgewick Rafter counting chamber, the FlowCAM and vacuum filtration combined with image analysis. Here, the latter method gave the best result. The next step was to develop a suitable staining method. Microplastics were stained in a 75% Nile red (10 μm) dilution in Milli-Q water, which resulted in fluorescent microplastics. Several experiments were conducted to investigate the potential of the flow cytometeras a detection technique. First, the flow cytometer was able to detect both autofluorescent microbeads and stained plastic microbeads. Further on, measurements of the dilution series resulted in an underestimation of the concentrations present, so for now, the method is not reliable for estimating polymer concentrations of samples. PET particles smaller than 20 μm were successfully measured, showing the ability of the flow cytometer to solve the size limit issue. Flow cytometer measurement ofthe mixture of PE and PS particles resulted in a cytogram in which the two polymers appeared as two separate lines. Thus, the flow cytometer can distinguish between different types of polymers. The final step in development was to distinguish between organic matter and polymers, since the method must be able to make the measurements in-situ. The detected stained organic material gave a different fluorescence pattern than the PS particles. However, the measurement of a mixture with stained organic material and polymers did not yet result in a cytogram with a sharp distinction between the two, therefore further analysis is needed here.In conclusion, the flow cytometer shows potential to be used as a microplastic detection technique for more time-efficient and semi-automated observation of microplastics in seawater.

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