Hart, Lisa Denise

Kieserg, Christina

Wichels, Antje

English

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Marine Microplastics - an educational module for the identification of microplastics in environmental samples with Nile redLisa Denise Hart1,2, Christina Kieserg1 and Antje Wichels1* * contact: antje.wichels@awi.de1Alfred-Wegener-Institute Helmholtz Centre for Polar and Marine Research, Biologische Anstalt Helgoland (BAH),OPENSEA, Germany2 Karlsruher Institute of Technology (KIT), Karlsruhe, GermanyPhotographs: AWI school lab OPENSEA Fluorescing eggshellsExperimental set-up: “Fluorescing eggshells”Sample inspection on the dark readerStudents discussing first resultsFirst inspection of plastic particlesUntreated plastic particlesThis experiment serves to convey the principle of auto-fluorescence and shows how thisphenomenon is made visible. We developed an experiment with auto-fluorescentProtoporphyrin IX from brown egg shells. The students' task is to make this substance visiblewith the help of the following materials:• Brown eggshells• Ethyl acetate (Nail polish remover)• Acetic acid (Vinegar 5 %)• Dark reader with an amber screenVisualization of fluorescent samples by using a“dark reader”Environmental samples stained with the fluorescent dye Nile red areexamined. When excited with blue light using the “dark reader”, specificfluorescence can be visualized. To introduce the phenomenon and learnabout fluorescence in general, we extracted Protoporphyrin IX frombrown eggs and visualized auto-fluorescence (emission of redfluorescence) exemplarily. We also can make use of fluorescence forenvironmental science related questions. Using Nile Red, the dye boundto polymers emits yellow-red fluorescence. The Nile red fluorescence ofstained plastic polymers depend on their chemical structure, some ofthem can be distinguished due to their emission colors. Learningmodules and experiments for students were developed in the OPENSEAschool lab, to convey how fluorescent staining can be used to analyzeenvironmental samples (see detection of plastic polymers)contaminated with plastics.The principle of fluorescenceSince the middle of the last century the world production of plastics has increased massively and led to a continuousincrease in the amount of plastic waste. The environmental degradation of plastic waste through physical and chemicalalterations creates continuous fragmentation of large plastic items into small ones known as microplastics (MP). How thewaste gets into the sea, how it is detected and discovered by researchers and what consequences it can have for livingorganisms are topics of an education program on marine litter implemented in the AWI school lab OPENSEA. We focus onhands-on experiments which are framed by several modules. Our aim is to impart up-to-date scientific knowledge andawareness through scientific experimental work for school classes (age 16-19). We offer experiments to sample plastic wasteof different sizes in the environment and characterize the plastic material (microscope, infrared spectrometry). A newmodule is based on the fluorescent dye Nile red aiming to visualize microplastic particles in environmental samples.micro2020:333924Detection of plastic polymers by using fluorescenceThe properties of fluorescence can be used for environmental research. In orderto transfer this to laboratory work, the students receive an environmentalsample to work with. Various materials are used:„Dark reader“• Blue light transilluminator for fluorescent samples (excitation 440-510 nm)• Amber screen emission filter (>570 nm)Nile red• Fluorescent dye• Emission ca. 520-600 nm (in EtOH)• Staining of hydrophobic componentsEggshells contain Protoporphyrin IX, which has fluorescent properties when dissolved inalcohol and excited by a specific wavelength. For an introduction to the topic of fluorescenceand to illustrate the phenomenon, the students will receive additional material:• Video about the principle of fluorescence• Helpsheets with structural formulas of all important substances used in the experiment • Atomic model to illustrate the principle of fluorescence ConclusionThis hands- on experimental module is divided into several small experiments aiming to identify plastic particles based on theirspecific fluorescence. Students work independent, individually or in a team on different scientific questions:1. How to identify different plastic polymers?2. Is the fluorescence of stained plastic polymers affected by color or shape of the MP?3. Can Nile red staining be used to detect and identify different MP’s in an artificial sample?Finally, students will be able to evaluate the applicability of the method of Nile red staining in comparison to other application methods (microscope, infrared spectrometry).MethodsDark reader with an amber screenFluorescent plastic particles stained with Nile redArtificial plastic sample before (A) and after (B)  staining with Nile red By staining with Nile red, plastic polymers can be partially differentiated due to their specific fluorescence. Different plasticpolymers in a mixed sample can be distinguished after staining with Nile red, and excited with blue light.AcknowledgementWe are grateful to the Microplastic Working group of G. Gerdts at the AWI  for scientific support and Marie Fischer for her highly appreciated contribution  to the OPENSEA school lab• Nile red on a basis of Ethanol (20 % w/w)• Different plastic polymers• Artificial sample (mixed)• Dark reader with an amber screen• Compartment dryer (60 °C)

Marine Microplastics- an educational module for the identification of microplastics in environmental samples with Nile red

https://epic.awi.de/id/eprint/53389/1/Poster_Micro_2020_333924.pdf

Marine Microplastics- an educational module for the identification of microplastics in environmental samples with Nile red

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