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Microplastic Surveillance in Central VA
- 1. Surveillance of Microplastic Pollution in Central Virginia Freshwater Ecosystems Alexandra Reddy Department of Biology and Chemistry
- 2. Outline 1. Overview of Plastics 2. Characterization of Plastic Pollution 3. Impacts of Plastic Pollution on Aquatic Organisms 4. Marine vs Freshwater Microplastics 5. Bridging the Gap This Project 6. Methodology 7. Results + Discussion 8. Conclusions 9. Future Directions
- 3. The Origin of Plastic • Synthesized in 1907 • Cheap • Minimal energy • 20 major types (North and Halden, 2013) Retrieved from http://exclusive.multibriefs.com/content/walmart-joins- worldwide-effort-to-reduce-its-plastic-consumption/waste- management-environmental Retrieved from https://www.recycleandrecoverplastics.org/plastic- materials/plastic-bottles/plastic-bottles-istock-926200658-medium-2/ Retrieved from https://www.dailydot.com/irl/straw-ban-ableism- restaurant-thread/
- 4. Plastic Production • 1950 1.5 million tons • 2014 299 million tons • 2019 1.3 billion tons • 8 million tons enter the oceans (Nelms et al., 2015) Retrieved from https://www.sundried.com/blogs/news/how-bad-is-the-global-plastic-pollution-problem-and-what-can-we- do-about-it
- 5. Distribution of Plastic Pollution Retrieved from https://ourworldindata.org/faq-on-plastics
- 6. Categories of Plastic Pollution • Large plastics o Entanglement o Ingestion with visible effects • Small plastics o Ingestion o Gastric obstruction o Physiological effects o Trophic transfer Retrieved from https://inhabitat.com/peanut-the-story- behind-the-poor-sea-turtle-deformed-by-a-six-pack-ring/ Retrieved from https://ocean.si.edu/ocean- life/seabirds/laysan-albatrosses-plastic-problem
- 7. What are microplastics? • Larger pieces quantified into smaller pieces – Wave movements – Ultraviolet light – Physical abrasion Retrieved from https://www.brushmable.com/blogs/mable-lifestyle/microplastics-the-smallest-big-problem
- 8. What is classified as a microplastic? • Classified according to length • <5 mm considered to be microplastics (Oberbeckmann et al., 2015, p. 554)
- 9. How do microplastics impact aquatic organisms? • Respiration • Direct or indirect ingestion • Gastric obstruction • Physiological effects – Liver toxicity – Endocrine disruption – Pesticides – Microorganisms Retrieved from https://www.odt.co.nz/news/national/still-waiting-—-long-road-marine-protection- area Retrieved from https://www.worldwildlife.org/stories/what-do-sea-turtles-eat- unfortunately-plastic-bags
- 10. Primary vs Secondary Microplastics • Primary – Micron size – Industrial production: textiles, pharmaceuticals, cosmetics • Secondary – Fragmentation and degradation – Agriculture, clothing, fishing, automobile, household Retrieved from https://encounteredu.com/multimedia/images/sources-of-microplastics
- 11. How are microplastics transported throughout the food chain? (Medrano et al., 2015, p. 76)
- 12. Outline 1. Overview of Plastics 2. Characterization of Plastic Pollution 3. Impacts of Plastic Pollution on Aquatic Organisms 4. Marine vs Freshwater Microplastics 5. Bridging the Gap This Project 6. Methodology 7. Results + Discussion 8. Conclusions 9. Future Directions
- 13. Marine Microplastics • Well-researched • Documented in a variety of species – Sea turtles – Dolphins – Rays – Seals Retrieved from https://ars.els-cdn.com/content/image/1-s2.0- S0269749117343294-fx1_lrg.jpg
- 14. Freshwater Microplastics • Research is scarce • Particularly for freshwater invertebrates – Macroinvertebrates – Amphipod crustacean – Water flea Retrieved from https://www.curriculumnacional.cl/614/w3-article-25330.html
- 15. Bridging the Gap • Keystone species Crayfish • Contribute to freshwater microplastic knowledge • Surveillance of local microplastic pollution Retrieved from https://bugguide.net/node/view/1157618/bgpage
- 16. Outline 1. Overview of Plastics 2. Characterization of Plastic Pollution 3. Impacts of Plastic Pollution on Aquatic Organisms 4. Marine vs Freshwater Microplastics 5. Bridging the Gap This Project 6. Methodology 7. Results + Discussion 8. Conclusions 9. Future Directions
- 17. Sample Collection • Two stream locations: 1. Opossum Creek 2. Rock Castle Creek
- 18. Sample Measurements and Dissection • Total length (mm) • Blotted wet mass (g) • Gender • Dissection – Dissecting microscope – Scanning Electron Microscope – Glass jars Retrieved from https://microscopecentral.com/products/asbestos-stereo-microscope
- 19. Sample Preparation for Chemical Analysis • Ultrasonication • Membrane filtration – 25 mm stainless steel – Glass microsyringe – Glass jars Retrieved from https://www.thomassci.com/scientific- supplies/Filter-Holder Retrieved from https://laboratory.pall.com/en/sterile- filtration-clarification/filter-holders- membranes/products.html Retrieved from https://www.grainger.com/product/AIR-TITE-Reusable-Glass-Syringe- 19G355
- 20. FTIR-ATR • Chemical fingerprints Functional groups • Most reliable technique (Ory et al., 2018, Supplementary Material Handout p. 6) Retrieved from https://www.slideshare.net/savvysahana/atr-ftir-sahana
- 21. Classification (Cozar et al., 2017, Supplementary Material Handout Figure 4)
- 22. Outline 1. Overview of Plastics 2. Characterization of Plastic Pollution 3. Impacts of Plastic Pollution on Aquatic Organisms 4. Marine vs Freshwater Microplastics 5. Bridging the Gap This Project 6. Methodology 7. Results + Discussion 8. Conclusions 9. Future Directions
- 23. Preliminary Investigation • Carolina Biological Supply Company crayfish • Two hard particles – Speculated to be foreign contaminants
- 25. Preliminary Investigation • Polyethylene filled with calcium carbonate – Filler for white lawn furniture • Components of the gastroliths • Identities inconclusive, but compelling
- 26. Rock Castle Creek Findings 84 86 88 90 92 94 96 98 100 102 500 1000 1500 2000 2500 3000 3500 %T (scaled) wavenumber (cm-1) Fiber vs Commercial Translucent PP %T RCfiber1 201419 32s %T PP translucent; scale and shift
- 27. Tank Study • Inadvertent vs intentional ingestion • Tank study Feeding behavior • 8 crayfish from Opossum Creek – 8 individual glass aquaria – Randomized to a plastic treatment: plastic bag, synthetic fabric, or control
- 28. Tank Study • Dissected after 5 months • No microplastic particulates • Shredding behavior • Interpretation: 1. Did not target and ingest 2. Did target, ingest, and excrete
- 29. Gill Filament Analysis • Filter water over gills • Entrapment of plastic fibers • Blue fiber identified in an Opossum Creek crayfish
- 30. Gill Filament Analysis • 10 Rock Castle Creek crayfish • 4 fibers identified
- 31. Outline 1. Overview of Plastics 2. Characterization of Plastic Pollution 3. Impacts of Plastic Pollution on Aquatic Organisms 4. Marine vs Freshwater Microplastics 5. Bridging the Gap This Project 6. Methodology 7. Results + Discussion 8. Conclusions 9. Future Directions
- 32. Big Picture This investigation strengthens the assertion that plastic pollution pervades our aquatic ecosystems and has negative implications for aquatic life.
- 33. Bridging the Gap • Preliminary investigation Baseline • Limited knowledge of freshwater pollution – Particularly freshwater invertebrates • Support investigation of microplastics in crayfish
- 34. Future Directions • Investigation of species in higher trophic levels – Biomagnification of microplastic particulates • Investigation of the microbiota of microplastic particulates – Influence on the normal microbiota of crayfish
- 35. Questions?
- 36. References • Cozar, A., Marti, E., Carlos, M., Garcia, J., Sebille, E., Ballatore, T., . . .Irigoien, X. (2017). The Arctic Ocean as a dead end for floating plastics in the North Atlantic branch of the thermohaline circulation. Science Advances, 3,1-8. • Medrano, D., Thompson, R., & Aldridge, D. (2015). Microplastics in freshwater systems: A review of the emerging threats, identification of knowledge gaps and prioritization of research needs. Water Research, 75, 63-82. • Nelms, S.E., Duncan, E.M., Broderick, A.C., Galloway, T.S., Godfrey, M.H., Hamann, M., Lindeque, P.K., & Godley, B.J. (2016). Plastic and marine turtles: a review and call for Research. ICES Journal of Marine Science, 73(2), 165–181. • North, E., & Halden, R. (2013). Plastics and environmental health: the road ahead. Reviews on Environmental Health, 28(1), 1-8. • Oberbeckmann, S., Loder, M. & Labrenz, M. (2015). Marine microplastic-associated biofilms – A review. Environmental Chemistry, (12), 551-562. • Ory, N., Chagnon, C., Felix, F., Fernandez, C., Ferreira, J., Gallardo, C., . . .Thiel, M. (2018). Low prevalence of microplastic contamination in planktivorous fish species from the Southeast Pacific Ocean. Marine Pollution Bulletin, 127, 211-216. • All figures not noted by a “Retrieved from” with a URL link are personal figures that I created.
Editor's Notes
- All figures not noted by a “Retrieved from” with a URL link are personal figures that I created.
- Durable, made to last – hence the persistence in our oceans
- Despite the deleterious implications of trophic transfer of pollutants and the consequent increased awareness about plastics in the environment, the demand for plastics has continued to rise over the past sixty years due to their durability and low cost (Kitamoto et al., 2011). Evidence has demonstrated that all continents are affected by plastic pollution, and consequently the gravity pollution of water sources has dramatically intensified (Gregory, 2009).
- As plastics are persistent contaminants, it is inevitable that marine species will encounter plastic items and be impacted through ingestion and entanglement. Although these effects can be easily observed when organisms become entangled in larger plastic items, there are less obvious repercussions from minuscule pieces of plastics known as microplastics. Ingestion with visible effects example: sea turtle with straw lodged in its nasal and throat passages
- Although larger plastics have negative implications for aquatic organisms through ingestion and entanglement, it is the degradation of larger plastics into smaller pieces known as microplastics that poses the greatest threat to aquatic life. (Oberbeckmann et al., 2015, p. 554).
- These small particulates become readily bioavailable and directly impact aquatic life through respiration, ingestion, gastric obstruction, physiological effects, chemical transfer, or trophic transfer, potentially causing liver toxicity, endocrine disruption, decreased fecundity, and lower survival rates (Lusher et al, 2016).
- https://www.tandfonline.com/doi/full/10.1080/26395940.2019.1580151 The fibers of synthetic clothing are mainly released by washing into sewage treatment plants and eventually into aquatic ecosystems. The heavy use of agricultural plastics allows plastics to enter the soil, degrade into microplastics, and reach the ground or surface water through the action of wind and runoff.
- (Medrano et al., 2015, p. 76). Organisms directly target the microplastics as prey, or inadvertently ingest them through filter feeding processes. These microplastics are then transported through the food chain and the interacting food webs, causing these contaminants to biomagnify through higher trophic levels.
- The inadvertent ingestion of microplastic particulates by marine organisms and consequent movement through the marine biota is thoroughly researched. However, research regarding the impacts of microplastic contamination in freshwater ecosystems is scarce. Despite increasing evidence of microplastic pollution in freshwater ecosystems, there is little research concerning the uptake of microplastic particulates by invertebrates in freshwater ecosystems (Scherer et al, 2017).
- Despite increasing evidence of microplastic pollution in freshwater ecosystems, there is little research concerning the uptake of microplastic particulates by invertebrates in freshwater ecosystems (Scherer et al, 2017). This research project aims to explore the occurrence of local microplastic pollution on an abundant and ecologically important species: crayfish. As research regarding microplastic contamination in freshwater invertebrates is scarce, this project will contribute to the knowledge of freshwater microplastic contamination and the understanding of its ecological implications, as well as the implications for public health. Therefore, this project will contribute to the knowledge of freshwater microplastic contamination and its ecological significance, particularly for crayfish organisms (Lusher et al, 2017).
- The crayfish were collected from each stream using a kick seine technique and were then euthanized either by transferring the crayfish into an Eppendorf tube containing isopropyl alcohol or by immediately sacrificing the crayfish on ice. As the physical stress caused by field sampling can induce gut evacuation in the crayfish organisms, which can decrease the number of microplastics observed prior to examination, the time interval between field sampling and analysis of the animal was minimized (Lusher et al., 2017). Additionally, special care was taken to minimize handling stress of the crayfish in the lab in order to decrease microplastic regurgitation (Lusher et al., 2017). In addition to collection of crayfish, representative water samples and sediment samples were collected using Whirl-Pak sampling bags in order to examine the substrate for the presence of microplastics.
- After investigation of the tissues of interest under the dissecting microscope, the tissues were then transferred to separate glass microscope slides to further search for potential contaminants under a Scannning Electron Microscope (SEM)
- After dissection of the crayfish and visual investigation using the two categories of microscopes, analytical chemistry tools were utilized to determine the identity of any foreign particulates and determine their frequency within the gill filaments and digestive tracts of the crayfish specimens.
- Following these sample preparation and isolation techniques, the analytical chemistry tool Fourier Transform Infrared Spectroscopy (FTIR) with the Attenuated Total Reflection accessory (ATR) was used to identify the chemical “fingerprints,” or functional groups, of the contaminants. The general consensus among the scientific community upholds that FTIR-ATR spectroscopy is the most accurate and effective technique to determine the chemical identities of plastic polymers (Lusher et al, 2017). FTIR-ATR spectroscopy is a useful method for determining the chemical composition of microplastic debris as it is able to analyze both solid and liquid particles and does not require extensive sample preparation. Expert judgement and reference spectra were used in order to identify the chemical constituents according to the specific absorption bands of the functional groups, with polypropylene and polyethylene as the most commonly identified polymers (Ory et al., 2018).
- With the implementation of this analytical chemistry technique, microplastics could be identified in the crayfish organisms. Any isolated microplastics were then classified into categories such as granules and pellets, rigid fragments, or films (Cozar et al., 2017)
- In order to determine the best dissection and tissue preservation techniques, a male crayfish from the Carolina Biological Supply Company was dissected. Upon dissection of this specimen, the digestive tract was extracted, and two hard particles were discovered at the junction of the stomach, intestine and hepatopancreas (Figure 6). These particles were speculated to be foreign contaminants, and thus chemical investigation was performed to determine the identity of the particulates.
- The unknown particle was examined using FTIR-ATR spectroscopy and compared with the spectra produced by a plastic pipette that contains polyethylene. The foreign particle and the plastic pipette displayed similar stretches from 2750-3000 cm1 and 1250-1500 cm1, which was indicative of shared functional groups. Additional resolution of the spectra displayed distinctly comparable stretches from 2900-2920 cm1 and 2840-2860 cm1, which further indicated a similar chemical identity. A final resolution of the spectra displayed comparable stretches from 1400-1500 cm1 and 700-750 cm1 that also demonstrated a similar chemical composition between the plastic pipette constituents and the foreign particle.
- Following the preliminary investigation, with the preliminary data gathered and compared with the available literature, the spectrum produced by the foreign particle resembled polyethylene filled with calcium carbonate, which is a common filler of polyethylene used in the popular white lawn furniture as a substitute to the expensive polymer fillers (Ozen et al., 2013). Therefore, these substantiated results from the preliminary investigation provided compelling evidence for microplastic contaminants in the digestive tracts of crayfish. However, it was also determined that the identity of the two particles identified in the Carolina Biological Supply Company specimen could have been components of the gastroliths. The gastroliths are synthesized as a pair in the stomach wall of crayfish during their pre-molt life stage (Luquet et al., 2016). The purpose of these structures is to store calcium ions that can be readily accessed after molting in order to calcify the exoskeleton. In particular, the calcium is stored as amorphous calcium carbonate (ACC) within the gastrolith pair (Luquet et al., 2016). During phases of molting, crustaceans like crayfish have the ability to synthesize and resorb calcium-containing minerals. As crayfish possess an inelastic exoskeleton, they are required to molt in order for growth to occur. Like most crustaceans, crayfish achieve hardening of the exoskeleton through calcification by precipitation of calcium carbonate. Although calcium ions are generally accessible in the aquatic biotopes, crustaceans like the crayfish most often utilize calcium ions stores through the development of gastroliths in the stomach wall of the gastric stomach (Luquet et al., 2016). Therefore, in light of this information, the identities of the two particles discovered in the Carolina Biological Supply Company specimen were inconclusive.
- Based on the findings of this preliminary investigation, as well as practice of the dissection protocol, collection of crayfish specimen from the field was conducted. First, five crayfish specimens were collected from the urbanized Rock Castle Creek study site. The foreign fiber embedded in the digestive tract was of an opaque color and was clearly entwined in the stomach tissue. This fiber was analyzed using FTIR-ATR and its spectrum was compared with that of a commercial translucent polypropylene lid. The spectra exhibited identical resonances in the 2800-3000 cm-1 and 1400-1500 cm-1 regions. Therefore, it was concluded that the identity of this foreign fiber was polypropylene. It was speculated that the resonances in the fingerprint region were due to another component in the fiber such as a mineral or trace organic material from the crayfish. Thus, this substantiated result supported the hypothesis that crayfish organisms are ingesting microplastic particulates in their aquatic environments.
- After the discovery of the polypropylene fiber in the crayfish specimen collected from Rock Castle Creek, it was evident that crayfish organisms can ingest microplastics that they encounter in their environment. However, it was not known whether this particulate was inadvertently ingested through normal feeding behavior, or if the particulate was mistaken as a food item and thus intentionally ingested. Therefore, a tank study was performed to investigate the feeding behaviors of crayfish organisms in relation to microplastics. In order to minimize the potential compounding factor of microplastic contaminants being already present in the digestive tracts, the crayfish specimens used in the tank study were collected from the rural study site, Opossum Creek. This was due to the understanding that as an aquatic ecosystem not situated near a dominant thoroughfare like Rock Castle Creek, Opossum Creek should have little to no plastic pollution and the crayfish should then possess minimal, if any, microplastic particulates in their digestive tracts. Tanks 1, 6 and 10 served as the control tanks and received no plastic treatment. Three, two by two inch squares of a synthetic green fabric were submerged in tanks 2, 4, and 8. Lastly, three, two by two inch squares of polypropylene shopping bags were submerged in tanks 2 and 11.
- However, it was concluded that the former speculation was more likely to have occurred because the plastic items submerged did not most accurately represent the physical nature of plastic pollutants that crayfish might encounter in the field. Nonetheless, performance of the tank study provided important information about the feeding behaviors of crayfish in association with plastic pollutants. As other crayfish studies were simultaneously being conducted in the laboratory by other research teams at the time of this investigation, many crayfish were housed in glass aquaria for extended periods of time. Upon weekly feeding of these crayfish specimens, it was observed that some of the crayfish from the other studies were shredding the sponge filters that were present in the aquaria. As omnivores and scavengers, crayfish often shred any material that they encounter in their environment. For example, crayfish play a critical role in the shredding of leaf litter and decaying material, therefore making vital nutrients available to other trophic levels of their aquatic ecosystem. Therefore, the observation of crayfish shredding the sponge filters in their glass aquaria was consistent with the shredding behavior that is observed in their natural habitats. Consequently, it was hypothesized that crayfish may be exhibiting this same shredding behavior towards plastic contaminants that they encounter in their freshwater ecosystems like the Rock Castle Creek and Opossum Creek study sites.
- Furthermore, as crayfish continually filter water over their gill filaments, it was speculated that microplastic particulates could become trapped within the gill tissue. Therefore, the investigations of this study included examination of the gill tissue for potential microplastic contaminants. During analysis of a crayfish specimen collected from the rural Opossum Creek study site, a blue fiber was identified in the gill filaments under the SEM at a magnification power of 40X. This blue fiber was then transferred to an Eppendorf tube for analysis using FTIR-ATR. However, due to the microscopic size of this fiber and the difficulty of transferring the fiber to the FTIR-ATR instrument, the identity of the fiber was unable to be determined. However, this fiber was characterized by fibril edges that were consistent with characteristics of synthetic fibers like those found in fabric or plastic tarps that are commonly-found pollutants in freshwater ecosystems. Therefore, it was confidently speculated that the identity of this fiber was of synthetic origin.
- Based on the discovery of the potentially synthetic blue fiber in the gill filaments of the Opossum Creek crayfish specimen, ten additional crayfish were collected from the Rock Castle Creek study site and their gill filaments investigated for potential microplastic pollutants.
- With recent observations of plastic pollution in the Rock Castle Creek study site and speculations about uptake by crayfish organisms, the preliminary results of this study support the investigation of microplastics in the digestive tracts and gill filaments of crayfish organisms. Visual observation of the Rock Castle Creek study site indicated the frequency of plastic pollution, while dissection of the digestive tracts and gill filaments of collected specimens indicated the uptake of these contaminants.