SUURJ: Seattle University Undergraduate Research Journal Volume Article 2020 Anthropogenic Debris in the Puget Sound: Review, Methods, and Analysis Koryna Boudinot Seattle University Diana DiMarco Seattle University Follow this and additional works at: https://scholarworks.seattleu.edu/suurj Recommended Citation Boudinot, Koryna and DiMarco, Diana (2020) "Anthropogenic Debris in the Puget Sound: Review, Methods, and Analysis.," SUURJ: Seattle University Undergraduate Research Journal: Vol , Article Available at: https://scholarworks.seattleu.edu/suurj/vol4/iss1/8 This Short Communications is brought to you for free and open access by the Seattle University Journals at ScholarWorks @ SeattleU It has been accepted for inclusion in SUURJ: Seattle University Undergraduate Research Journal by an authorized editor of ScholarWorks @ SeattleU Anthropogenic Debris in the Puget Sound: Review, Methods, and Analysis Koryna Boudinot, Biology Diana DiMarco, Marine and Conservation Biology Faculty Mentors: Peter J Alaimo, PhD and W Lindsay Whitlow, PhD Faculty Content Editor: Mark Jordan, PhD Student Editor: Ciara Murphy 18 Abstract Microplastics are widespread aquatic pollutants that contaminate waterways and originate from human sources, such as packaging or cosmetics. They are smaller than 5.0 mm and include both manufactured plastics (primary), or environmentally degraded plastics (secondary) Concerns about the potential impacts of microplastics through chemical pollutant adsorption, marine organism ingestion, and biomagnification indicate the importance of further investigation into the quantity and types of microplastics in the Puget Sound. Our initial visual analysis using light microscopy identified plastics in samples of both sediment and surface water An improved plastic extraction method and chemical analysis procedure was developed In this improved process, samples were filtered through a series of sieves and vacuum filters followed by an adapted enzymatic digestion using proteinase-K in SDS, then density separated using zinc chloride prior to ATR FT-IR analysis Spectra from various locations were then analyzed to identify microplastic type This purification method paired with ATR FT-IR analysis is an efficient method that avoids plastic degradation and bias of manual sorting, two known problems of previously published methods, and thus aids in the identification of microplastic contaminants This allows for increased confidence when making comparisons between identified microplastics 19 Anthropogenic debris, specifically microplastics (MPs), are ubiquitous in the environment and are found in aquatic, terrestrial, and atmospheric environments (Masura et al 2015; Prata 2018) MPs consist of manufactured plastics (primary), or environmentally degraded plastics (secondary), and are defined as plastics that are smaller than mm (Masura et al 2015). Examples of primary MPs include the microbeads found in cosmetic products, such as facial soaps, while secondary plastics are often the result of degraded materials including single-use plastics, such as plastic bags, bottles, straws, and packaging materials (Masura et al 2015). Microfibers shed from synthetic clothing are an especially common form of anthropogenic debris These particles typically enter the marine environment from nonpoint sources, including runoff into rivers, water treatment plants, and industrial factories, and are spread throughout the world’s marine environments by ocean currents (Masura et al 2015). In the marine environment they can accumulate in seafloor sediment, while larger plastic debris ends up in floating garbage patches and washing up on shorelines around the world (Cole et al 2011) MP research has examined the widespread effects of MP ingestion and toxicity on various forms of life Early research showed that Albatross chicks on Sand Island, Midway Atoll, were being fed plastic pieces by their parents, leading to starvation (Auman and Ludwig 1997) Ingestion of macroplastics, larger than 5mm, are harmful for birds since they often clog the gastrointestinal tract, leading to starvation MPs are known to be ingested by numerous organisms and recent studies have shown the transfer of hydrophobic toxins from MP-adsorbed particles in the environment to organisms across trophic levels (Masura et al 2015) Polyethylene microplastics have been shown to adsorb pesticides that have shown to lead to increase toxicity when ingested by marine plankton (Bellas and Gil 2020) Once ingested, these toxins can wreak physiological havoc on bodily tissues, such as the liver, and they have been shown to be especially harmful to the endocrine system (Rochman et al 2013) Biomagnification of toxins can also have similar health consequences in humans and other animals at higher trophic levels (Cox et al 2019) The effects of MP ingestion are continuing to be researched and are helping scientists understand the physiological consequences of microplastics Due to the increasing global abundance of MPs, it is imperative to define baseline pollution levels and to determine chemical compositions of MPs Our research at Seattle University has focused on developing a method of MP purification from saltwater, freshwater, and sediment that can be used to determine chemical compositions We conducted a broad review of the MP extraction-identification methods in the literature to understand current practices in the field As leaders in the field, the National Oceanic and Atmospheric Administration (NOAA) has published recommendations for quantifying synthetic particles from waters and sediments that are based on procedures developed in 2015 (Masura et 20 al 2015) Many research institutions in the Pacific Northwest have adopted the methods published by NOAA; however, there is substantial room for improvement For example, the NOAA method requires numerous steps, including sieving, peroxide oxidation, gravimetric analysis using density separation, and microscope examination The NOAA procedure only requires gravimetric analysis, using density separation, and visual analysis, using a dissecting microscope, for MP validation, thus simplifying the procedure Unfortunately, visual identification of MPs using a dissecting microscope is tedious and suffers from observer bias (Hidalgo-Ruz et al 2012) The NOAA method also lacks a systematic chemical verification method, which is required to determine the composition of MPs Other studies have shown that using Fourier-transform infrared spectroscopy (FTIR), which obtains an infrared spectrum of absorption of a solid, liquid, or gas, enables reliable identification of MPs with high resolution over a wide spectral range and is preferable to visual sorting as a method of analysis (Löder et al 2017) Variation in methods used in different research institutions shows that there is a need for standardization of field methods and purification procedures The protocol we developed for enzymatic purification and chemical analysis of MPs is outlined below and can be used to determine baseline levels of anthropogenic pollution MPs were isolated and purified from sediment and water as follows: about five liters of surface water including suspended particles or sediment were collected from various locations (Figure 1) and were filtered through a series of metal sieves to remove particles larger than mm (Figure 2) The filtrate was dried under a vacuum and the mass of the dried solids were recorded Samples were transferred to glass containers and a homogenization buffer was added (400mM Tris-HCl, 0.5% SDS, pH 8) This mixture was incubated at 60º C for 60 minutes to induce protein denaturation, which is more effective at purifying MPs than the NOAA peroxide oxidation method (Karlsson et al 2017) Proteinase-K was added, followed by CaCl then incubated for >2 hours at 50°C, shaken at room temperature for 20 minutes, and incubated at 60°C for another 20 minutes (Löder et al 2017) After enzymatic digestion, samples were suspended in a solution of ZnCl₂ at a density of 1.7 g/cm to separate the plastics by density The top fraction, containing the MPs, was collected throughout a period of several hours The isolated material was then rinsed with purified milli-Q water and left to air dry in a clean environment over the course of several days Using this method, we were able to remove all identifiable organic matter while preserving the chemical composition of the MPs Subsequent analysis was performed by ATR-FTIR which generated clear spectra of the polymers Chemical composition was then determined from the fingerprint region of the FTIR spectra, along with diagnostic C-H stretching bands in the spectral region. This extraction-identification method avoids plastic degradation and the bias of manual sorting, aiding in the identification of microplastic polymers and allowing for increased 2, 21 certainty in analysis This method and subsequent research results can be used or adopted by community science groups to continue MP research in the Puget Sound We plan to continue this project through a long-term collaboration with scientists at the Seattle Aquarium to measure MP concentrations and to track changes over time in the Puget Sound. Continued monitoring of MPs in the Puget Sound is essential in creating environmental policy changes that are required to regulate and limit anthropogenic waste When thinking about MPs as a global environmental and social justice issue, it is especially important to bridge the gap between research establishments and community science organizations. It is imperative to acknowledge our position of power as students of an academic institution with access to funding and laboratory resources As students, we can facilitate collaboration with community science organizations to create positive change around environmental issues There are multiple organizations recognized as community science partners by the Seattle Aquarium, including the Puget Soundkeeper Alliance, Discuren Foundation, City of Seattle, City of Burien, King County, University of Washington, Washington Deptartment of Fish and Wildlife, Environmental Science Center, Highline Schools, Seattle Schools, Seattle Rotary, WSU/Island County Cooperative Extension Beach Watchers, National Oceanic and Atmospheric Administration (NOAA) and Fisheries, and Oceans Canada Shorekeepers Collaboration between academic and community organizations removes barriers to science and increases accessibility to contribute to high level research We have found that community science organizations are invaluable due to their ability to collect water samples at a much larger capacity than students at an undergraduate university These samples can be sent to laboratories where the MPs can be isolated and analyzed with methods such as those we have developed It is vital for us to work together with community science groups through creative methods of collaboration in order to monitor MPs on a broader level, which is necessary for an understanding of their roles as pollutants Therefore, we hope our work can be utilized to generate reliable data to be used by the community with the goal to ultimately advocate awareness with policy makers and regulatory groups 22 Figure Sediment and water sample locations denoted by white arrows Initial sediment and water samples were collected from Alki Beach, Gas Works Park, Elliot Bay, and Elliot Bay Combined Sewer Overflow Outlet 23 Figure Purification and analysis methods developed for water and sediment samples 24 References Auman, H, Ludwig, J 1998 Plastic ingestion by Laysan Albatross chicks on Sand Island, Midway Atoll, in 1994 and 1995 Albatross Biol Conserv 239-244 https://www researchgate.net/publication/237813671 Bellas, J, Gil I 2020 Polyethylene microplastics increase the toxicity of chlorpyrifos to the marine copepod Acartia tonsa Environ Pollut 260 doi:10.1016/j.envpol.2020 114059 Cole M, Lindeque P, Fileman E, Halsband C, Goodhead R, Moger J, Galloway TS 2013 Microplastic ingestion by zooplankton Environ Sci Tech 47(12):6646-6655 doi:10.1021/ es400663f Cox KD, Covernton GA, Davies HL, Dower JH, Juanes F, Dudas SE 2019 Human consumption of microplastics Environ Sci Tech 53(12): 7068-7074 doi:10.1021/ acs.est.9b01517 Hidalgo-Ruz V, Gutow L, Thompson RC, Thiel M 2012 Microplastics in the marine environment: a review of the methods used for identification and quantification Environ Sci Tech 46(6): 3060-3075 doi:10.1021/es2031505 Karlsson TM, Vethaak AD, Almroth BC, Ariese F, van Velzen M, Hassellöv M, Leslie H 2017 Screening for microplastics in sediment, water, marine invertebrates and fish: method development and microplastic accumulation Mar Pollut Bull 122(1-2): 403-408 doi:10.1016/j.marpolbul.2017.06.081 Löder MGJ, Imhof HK, Ladehoff M, Löschel LA, Lorenz C, Mintenig S, Piehl S, Primpke S, Schrank I, Laforsch C, Gerdts G 2017 Enzymatic purification of microplastics in environmental samples Environ Sci Tech 51(24): 14283-14292 doi:10.1021/ acs.est.7b03055 Masura J, Baker J, Foster G, Arthur C 2015 Laboratory methods for the analysis of microplastics in the marine environment: recommendations for quantifying synthetic particles in waters and sediments NOAA Technical Memorandum NOS-OR&R-48 Prata JC 2018 Airborne microplastics: consequences to human health? Environ Pollut 234: 115-126 doi:10.1016/j.envpol.2017.11.043 25 Rochman CM, Hoh E, Kurobe T, Teh SJ 2013 Ingested plastic transfers hazardous chemicals to fish and induces hepatic stress Sci Rep 3: 1-7 doi:10.1038/ srep03263 Supplemental References Andrady, AL 2011 Microplastics in the marine environment Mar Pollut Bull 62(8):1596-1605 doi:10.1016/j.marpolbul.2011.05.030 National Ocean Service 2018 What is the biggest source of pollution in the ocean? https://oceanservice.noaa.gov/facts/pollution.html Seattle Aquarium 2020 Community science https://www.seattleaquarium.org/ community-science Van Cauwenberghe L, Janssen CR 2014 Microplastics in bivalves cultured for human consumption Environ Pollut 193: 65-70 doi:10.1016/j.envpol.2014.06.010 Acknowledgments We would like to thank Kaley Duggar, who helped us develop the protocol We would also like to acknowledge Dr Peter Alaimo and Dr Lindsay Whitlow for their generous support that has been invaluable Finally, we would like to thank Julie Masura at UW Tacoma and Shawn Larson at the Seattle Aquarium for sharing their knowledge and providing guidance 26 .. .Anthropogenic Debris in the Puget Sound: Review, Methods, and Analysis Koryna Boudinot, Biology Diana DiMarco, Marine and Conservation Biology Faculty Mentors: Peter J Alaimo, PhD and W Lindsay... plastic degradation and the bias of manual sorting, aiding in the identification of microplastic polymers and allowing for increased 2, 21 certainty in analysis This method and subsequent research... in the Puget Sound. Continued monitoring of MPs in the Puget Sound is essential in creating environmental policy changes that are required to regulate and limit anthropogenic waste When thinking