Ingested Micronizing Plastic Particle Compositions and Size

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Ingested Micronizing Plastic Particle Compositions and Size Distributions within Stranded Post-Hatchling Sea Turtles Evan White, Samantha Clark, Charles A. Manire, Benjamin Crawford, Shunli Wang, Jason Locklin, and Branson W. Ritchie Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02776 • Publication Date (Web): 17 Aug 2018 Downloaded from http://pubs.acs.org on August 21, 2018

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Environmental Science & Technology

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Ingested Micronizing Plastic Particle Compositions

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and Size Distributions within Stranded Post-

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Hatchling Sea Turtles

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Evan. M. White1,5*†, Samantha Clark2, Charles. A. Manire2, Benjamin. Crawford4, Shunli.

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Wang1,3 Jason. Locklin1,3,4†, Branson. W. Ritchie1†.

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1

New Materials Institute, University of Georgia, Athens, GA 30602, USA.

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2

Loggerhead Marinelife Center, Juno Beach, FL 33408, USA.

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3

College of Engineering, University of Georgia, Athens, GA 30602, USA.

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Department of Chemistry, University of Georgia, Athens, GA 30602, USA

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Department of Small Animal Medicine & Surgery, Athens, GA, 30602, USA

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*Corresponding Author. Email: [email protected]

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†Present Address: The University of Georgia, 220 Riverbend Road, Riverbend Research South,

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Room 177, Athens, GA 30602, USA.

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KEYWORDS

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Sea turtles, keystone specie, ingestion, micronizing, microplastic, ocean plastic, Raman

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microscopy, size distribution, nanoparticle

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1 ACS Paragon Plus Environment


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ABSTRACT

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From July 2015 to November 2016, 96 post-hatchling sea turtles were collected from 118 km of

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Atlantic coastline in Florida, USA, including loggerhead, green, and hawksbill sea turtle species.

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Forty-five of the recovered turtles were rehabilitated and released, but the remaining 52 died and

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were frozen. At necropsy, the gastrointestinal tracts of most the turtles contained visible plastic,

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and collected particles of 27 individuals were chemically characterized by Raman microscopy as

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polyethylene, polypropylene, polyethylene terephthalate, and polystyrene. Mesoparticle plastic

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fragments 1.0 to 8.7 mm, microparticle fragments 20 to 1000 µm, and nanoparticles 5 to 169 nm

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were identified in the turtles. Polyethylene and polypropylene were the most common plastics

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ingested from specimens representing 54.1% and 23.7% of the total observed mesoparticles and

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11.7% and 21.0% of the total observed microparticles, respectively. A plastic-to-body mass ratio

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of 2.07 mg/g was determined for this group. The authors suggest that ingestion of micronizing

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plastic by post-hatchling sea turtles is likely a substantial risk to survival of these endangered and

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threatened species. This study also provides some of the first evidence for the formation of

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nanoscopic plastic particles that we theorize forms in the post-hatchling and juvenile environment

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and are present post-ingestion.

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INTRODUCTION

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Plastic pollutants were first described in the western Sargasso Sea of the Atlantic Ocean by

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Carpenter and Smith in 1972,1, 2 and ingestion of marine plastic by sea turtles has been documented

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for decades.3 The accumulation of plastic debris in all oceans and seas is well recognized as an

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evolving international problem with reports of entanglement or post-ingestion morbidity or

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mortality in at least 690 marine species of sea turtles, seabirds, seals, sea lions, whales, fish,


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invertebrates, and other groups.4-9 Although some conservation efforts have had a positive impact

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on population growths, like green sea turtles, other regional groups may collapse if breeding

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populations cannot be replaced.10 Life threatening encounters with marine plastics have been

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described in all life stages of all seven species of sea turtles,4,

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(gastrointestinal impaction or injury) and indirect (reduced nutrient intake or abnormal buoyancy)

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impacts on post-hatchling and juvenile turtles, and various factors have been studied in regard to

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determining why sea turtles are ingesting plastics.7, 13, 14

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Plastic is now the most common form of marine debris, and in the decades since it was first

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introduced the total annual production has increased between 1950 and 2015 from 1.5 million to

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between 275 and 299 million metric tons.15,

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mismanagement of non-biodegradable plastics that are ultimately accumulating in the world’s

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oceans and coastal zones continues,17 and production is increasing despite the recognized threats

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this long-lived waste poses for marine life and human health.14, 16, 18 Almost one third of the non-

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biodegradable plastic produced is used to manufacture single-use consumer goods.19 As poorly

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managed plastic waste has accumulated in marine environments,16 there has been a corresponding

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increase in reports describing ingestion of plastic debris by sea turtles in all size classes. Recent

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reviews on microplastic analyses outline the breadth and history of the growing environmental

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impacts of persistent synthetic plastics,20 describes microparticle ingestion across a number of taxa

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in ocean environments.21-23

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Historical population declines have motivated conservation work globally since the 1950s.24, 25

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These efforts, including beach protection measures, fisheries bycatch regulations, outlawing

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harvest, and creation of marine protected areas, have provided positive trajectories in nesting

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7, 11, 12

including both direct

The production and subsequent waste stream



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females for some species. In the US, conservation groups like Loggerhead Marinelife Center

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(LMC) in Juno Beach, Florida, and over 70 other groups26 work to preserve populations of sea

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turtles. Similar groups dedicated to conserving the sea turtles are found in coastal regions

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worldwide. There is evidence for successful regional population recovery, highlighting the

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importance of continued local conservation work, with often involves community-based

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programs.24, 27 This conservation work requires time, investment and patience, as many species of

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marine turtles can take decades to reach reproductive age.

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Despite these efforts and investments, many of the distinct population segments of sea turtles are

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listed in many regions as vulnerable, endangered, or critically endangered, based on critical

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assessment elements by the National Oceanic and Atmospheric Administration (NOAA) and the

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International Union for Conservation of Nature (IUCN).28-38 Animal consumption of

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anthropogenic plastics may negate conservation efforts (such as in the evaluated population of

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post-hatchlings in Florida) as well as global efforts to conserve keystone species like loggerhead

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(Hoarau et al. 2014) and green sea turtles.39 For the 27 specimens studied by Raman microscopy,

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25 individuals (93%) had some amount of ingested plastic particles, and we theorize that many

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of those individuals had succumbed to either blockage or nutritional deficiencies associated with

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plastic ingestion. These observations were important because data on the potential effects of

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plastic ingestion in free-ranging post-hatchling sea turtles, described as the early portion of the

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“lost years” (the term for the post-hatchling and early juvenile years), is limited.40-42 Studies on

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sea turtle ingestion of plastic materials43-46 and the spectroscopically identified compositions of

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ingested plastics47-49 has increased in the literature in the past decades. Characterizing the

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micronizing particle distributions and compositions will help in understanding the micronizing

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process of ingested plastic. In this study we report the first quantitative microparticle analysis 4 ACS Paragon Plus Environment

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and spectroscopic identification of ingested plastic fragments in post-hatchling sea turtles

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rescued for rehabilitation. The evaluated individuals were washed back to shore where they

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were rescued for rehabilitation. The objective of the study was to estimate the number of

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particles and the chemical composition of particles in the ingested distributions recovered from

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sea turtles using Raman microscopy. The results of this study suggest that the micronizing

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plastic, particles that are continuously fragmenting into smaller and smaller pieces, ingested by

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individuals may eventually result in nanoparticles, making absorption and distribution of plastic

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additives easier (Savoca et al. 2018). This study includes documentation of previously

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uncharacterized nanoparticles in the sub 200 nm range observed by atomic force microscopy

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(AFM).

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MATERIALS AND METHODS

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Post-hatchling Sea Turtle Collection and Recovery

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From July 2015 to November 2016, 96 stranded post-hatchlings (known locally as wash-backs, 5

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– 10cm straight carapace length) and one stranded juvenile sea turtle (11.9 cm) from 118 km of

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beach stretching from Vero Beach to Lake Worth, Florida, were collected and brought to LMC in

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Juno Beach, Florida. The collected sea turtle species were recorded. Upon arrival, a physical

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examination was completed on each turtle including a weight and standard carapace

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measurements, which then determined the medications and treatments to be administered. Many

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of the post-hatchling patients received subcutaneous fluids, enteral nutrition, and an intestinal

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motility stimulant in accordance with institutional guidelines at LMC.50 The post-hatchlings were

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housed in flow-through salt water tanks. Of these, 45 turtles survived and were released offshore

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into floating Sargassum mats 2 – 99 days after entering the rehabilitation facility. The remaining



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52 turtles died and were frozen pending necropsy. Specimens were thawed, weighed, and straight

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carapace length (SCL) measurements were obtained. These post-hatchlings were then necropsied

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using standard procedures at LMC.50 Twenty-seven randomly selected deceased post-hatchling

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samples (13 washed GI tracts and 14 base digested GI tracts) were used in the analysis.

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Recovery and Analysis of Washed Group Ingested Plastic

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The gastrointestinal tract of 12 randomly selected post-hatchlings and one juvenile (13 total) was

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incised continuously from esophagus to cloaca (Figure 1B), and the contents were washed into a

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bowl (washed group). The contents were filtered through a 1 mm mesh colander and materials

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retrieved from the colander were then washed several times with tap water until the food items and

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digesta were removed. The rinsed particles were then floated in tap water, and each piece was

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removed using thumb forceps and then placed into a clean tube for air drying. The stomach

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contents of each individual post-hatchling remained separate and were labeled appropriately. The

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mesoparticle fragments were collected and placed in individual vials labelled by source turtle for

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laboratory analysis (washed group). The particle masses for each individual were weighed prior to

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Raman analysis using an analytical balance (resolution = 0.1 mg).

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Samples of the washed group particles were used as received from necropsy. Sample containers

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were opened in a class 10,000 clean room facility to minimize contamination. Researchers

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handling samples were gowned with cotton-based laboratory coats. The particles from each sample

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were spread evenly over a 180 x 110 mm (177 x 107 mm actual scan area) glass plate mounted on

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a Raman microscope (Malvern, Morphologi G3-ID, version 8.21). Between samples, the glass

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plate was washed with copious amounts of 18 MΩ water, then wiped clean with cellulosic paper

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and acetone and dried with a stream of nitrogen gas. A cleaned, blank glass plate was scanned for 6 ACS Paragon Plus Environment

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contamination; however, not one particle of any type could be found. Optical composite images

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were collected using a 2.5X objective while Raman spectra were collected using a 50X objective

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and a 785 nm laser (3 µm diameter target) with a spectral range of 150 – 1850 cm-1. Raman

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spectroscopy on microparticles with a circular equivalent diameter (CED) below 20 µm were not

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considered in analyses, while particles greater than 20 µm in CED were easily targeted with

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confidence for the analysis. Particle sizes fewer than 100 pixels were discarded from analysis.

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Samples were illuminated with episcopic (top) light with a calibration intensity of 80.0 and

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tolerance of 0.90 using a 10% image stitching overlap. Image threshold values of 0 and 35 were

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used for dark and light images, respectively. The Raman shifts of PE have been studied since the

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1970s.51 Several peaks were considered for verification of polymer despite the fluorescence

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background, and their values are reported in the SI.

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Measurements of all sizes presented herein (mesoparticles, microparticles, and naoparticles) are

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calculated as the CED using the pixel area of each particle according to equation 1,

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CED  2

A

(1)



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where A is the top view area of imaged particles in µm2. For particles larger than the field of view

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of the microscope, a Visual Basic for Applications macro was used to determine the particle area

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from the sample composite image, which is available in the supporting information. All values

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herein denoted by the “±” symbol are standard deviations.

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GI Sample Base Digest and Analysis



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The purpose of the base digestion was to account for smaller particles that the washing protocol

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did not recover. The intestinal tracts of another 14 post-hatchlings that had been necropsied as

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described above and refrozen at -20 °C were evaluated. The whole intestinal tract samples (from

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esophagus to cloaca) had been previously excised and washed to remove visible particles as

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described above. The tissue residues, on average 1.787±0.759 g, of the 14 post-hatchlings were

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thawed and cut with scissors into approximately 10 even-sized pieces and stirred gently (60 rpm)

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in 100 °C 3 M NaOH overnight. The resulting samples were cloudy brown dispersions that were

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transferred to a centrifuge tube using minimal 18MΩ water (~2000 µL) affording a mixture with

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a density of 1.11 g·mL-1. This mixture was centrifuged at 3000 RCF (G) for 10 minutes. Particles

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that were floating after centrifugation were siphoned with a glass pipet into a side arm flask under

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vacuum. The resulting material was retrieved via vacuum filtration on type SS 3.0 µm pore

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nitrocellulose filters using Whatman 40 cellulose filters as a support. The samples were then

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washed with ample water (18 MΩ·cm), dried, and characterized by Raman microscopy.

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AFM Analysis of Nanoparticles

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After Raman spectroscopy experiments, the washed group samples were recovered and washed

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with 2 mL of 0.45 μm nylon filtered isopropyl alcohol. Silicon wafers were cleaned by sequential

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sonication in water, acetone, and isopropanol followed by 5 minutes of argon plasma, and the

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wafers were thereafter stored in sealed petri dishes previously washed with filtered isopropanol.

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The sample in isopropyl alcohol was gently swirled and 35 μL of the solution was spin casted onto

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a cleaned silicon wafer (1 X 2 cm) for 40 seconds at 1,000 rpm with the spin coater lid closed. The

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sample was quickly removed from the spin coater and sealed in a petri dish until AFM imaging.


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Three blank silicon wafers were treated with 35 μL of the filtered isopropanol under the same spin

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coating conditions.

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Both 1 μm and 10 μm AFM topology images were taken using ScanAsyst mode on a Bruker

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NanoScope V AFM, with a piezo frequency of 2,000 Hz. NanoScope Analysis software was

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used to select nanoparticles over 5.0 nm (z threshold), and the CED of each nanoparticle was

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calculated from the pixel area over the z threshold.

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RESULTS AND DISCUSSION

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Mesoparticle consumption by sea turtles

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Of the collected stranded turtles, 61 were loggerhead, 33 were green and 3 were hawksbill sea

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turtles, which ranged in weight from 0.020 to 0.614 kg and SCL from 5.0 to 11.9 cm (Tables S1

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and S2). Surviving individuals passed some amount of plastics (Figure 1A). From the 52 deceased

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individuals, plastic fragments were recovered from the gastrointestinal (GI) tracts of the 13

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individuals by repeatedly washing the incised tracts and its contents. Notably, the lower sections

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of excised GI tracts in some turtles showed abnormal distension and hyperemia, indicated by the

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arrow in Figure 1B.

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Work by Parker el al.52 from 1990 – 1992 with juvenile and subadult loggerheads and Mrosovsky

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et al.53 from 1968 – 2007 with subadult and adult leatherbacks observed plastic consumed by about

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one third of evaluated turtles, and later work by Schuyler et al.5 from a literature review and Vélez-

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Rubio et al.54 with juvenile and subadult green sea turtles reported higher frequencies of 52% and

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70% of animals that ingested plastic particles, respectively, which are typically buoyant in sea

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water. Any reduction in the efficiency of nutrient acquisition and absorption would be expected to 9 ACS Paragon Plus Environment


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be of the greatest consequence to post-hatchling and juvenile sea turtles with limited body energy

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reserves to prevent starvation or gut function.12, 55, 56 Work by Camedda et al. in 2014 with juvenile

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and subadult loggerheads described the presence of plastics in the digestive tracts of about 14% of

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sea turtles, yet direct damage to the digestive tract and obstruction were not observed to be the

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cause of mortality in this population.57 However in studies like those by Bugoni et al.58 and

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Bjorndal et al.,12 death by obstruction was determined for a small percentage of turtles. Figure 1C

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shows gastrointestinal tract changes at the point of plastic accumulation that we propose

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contributed to the death of this individual, and Figure 1D shows ingested plastic in the distal

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stomach that likely contributed to starvation. Although we observe instances of mortality that were

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likely associated with blockages, the aim of this work was to identify particle compositions and

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sizes. Future work will address the histopathologic changes that contribute to morbidity and

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mortality in stranded post-hatchling sea turtles that have ingested plastic.

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Figure 1. Pre-mortem findings and necropsy of stranded post-hatchling sea turtle that ingested

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micronizing plastic. A photograph of a living specimen excreting plastic particles from the cloaca

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(A), and a sample GI tract excised from the esophagus to the cloaca with distension, adhesions,

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friability, and hyperemia of the lower GI tract, indicated by the arrow (B). Micronizing plastic

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fragments, indicated by the arrow, that likely induced blockages in the colon and contributed to

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the turtle’s death (C). An example of accumulated plastic indicated by the arrow was found in the

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distal stomach of a green turtle, which may have contributed to starvation (D).

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The ingestion of plastic has been proposed as a threat for morbidity and mortality in sea turtles

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through both direct blockage of the gastrointestinal tract, interference with the consumption and

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absorption of sufficient nutrients through dietary dilution,59 and possible accumulation of plastic

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related toxins.60,

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increases, and we suspect this would further encourage adsorption and absorption of persistent

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organic compounds. A recent review outlines the ecological risk, bioavailability and toxicity of

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such pollutants.62

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Size discrepancies seem to vary in the literature, as well as the methods for measuring particles. In

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this study, we used measurements of particle surface area (as observed by top view microscopy)

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to calculate a circular equivalent diameter (CED). Macroparticles are considered to be 20 – 100

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mm in diameter, of which none were observed in this work, due to gape limitations (small size of

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the mouth in this group). We chose the range of 1 – 20 mm for observed mesoparticles, which

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differs slightly from the range established previously (5 – 20 mm) by Ryan et al,63 Barnes et al,63,

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64

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particles could be observed with higher magnification, we found that the 3 µm laser is too large to

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As particles decrease in size, the surface area-to-mass ratio of particles

and the NOAA recommendation65 suggested in work by Thompson el at.66 Although sub-micron



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confidently measure the spectra of particles smaller than 20 µm in automation modes.

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Microparticles reported here have a CED 1µm – 1 mm. Nanoparticles, defined as 1 nm – 1 µm in

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studies focused on small biota,67 were also found by AFM, discussed later. Particles collected from

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both methods, the washed group particles and particles collected from the base digestion protocol,

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were quantified by microscopy. An average of 48±24 mesoparticles were found in each individual

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from the washed group while the base digested GI tissues, described later, yielded only 2±2

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mesoparticles. Work by K. S. Van Houtan et al. in 2016 describes a similar amount of consumed

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mesoparticles from recovered post-hatchling hawksbill sea turtles.41

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In our spectral identification, we tested for 5 common plastics pollutants, polyethylene (PE),

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isotactic polypropylene (iPP), polystyrene (PS), poly(vinyl chloride) (PVC), and poly(ethylene

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terephthalate) (PET). A total of 135 of the 619 mesoparticles were targeted at random using Raman

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spectroscopy for polymer identification and size measurements (characteristic peaks are described

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in the supplemental materials section, Polymer Raman Spectral Analysis). The mesoparticle size

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distribution is shown in Figure S1.

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In the washed group, the average mass of all collected particles per individual of 198±140 mg was

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determined with the total mass of the particles ranging from 30 – 479 mg for 13 total individuals

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(Table S2). Of the concomitant mesoparticles, 80.7% were identified as either iPP (later referred

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to as simply PP), PE, or PS (Figure 3a), which accounts for a comparable percentage of the total

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mass of collected particles. Jung et al. found comparable results earlier this year in Pacific sea

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turtle bycatches by longline, line or rope whereby 96% of ingested pieces were plastic,47

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comparable to our findings from an Atlantic population of turtles of 80.7% of ingested

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mesoparticles particles being plastic pieces.


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The average calculated consumed plastic mass for the, pc, for the washed group was determined

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to be 167±126 mg of plastics (determined from equations S2 and S3 and Table S2 in the SI), while

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on average 198 mg of GI contents were collected by the washing method. Because mesoparticles

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are significantly larger than microparticles, the masses of the microparticles were not considered

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in the estimation. It is also important to note that much biological material could have been

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digested and assimilated into the organisms prior to death and analysis, so this value is likely biased

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to be higher than the percentage of plastic mass actually consumed.

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The ratio pb is the average ingested plastic mass to the average body mass in a population,

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described otherwise as the body burden average or as a percent of body mass. By comparison,

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Clukey et al. showed a body burden of 0.659 g/kg in Pacific pelagic bycatch sea turtles, 44 and in

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coastal South African turtles, ingested plastic ranged 0.01 – 0.8% of body mass (0.1 – 8 g/kg).68 A

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summary of the physical qualifiers for mesoparticle ingestion by the post-hatchlings is shown in

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Table 1, and calculations for these values are described in the SI.

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Individuals considered 13 Average post-hatchling 80.8±42.9 mass, ω (g) Average straight carapace 7.92±1.47 length (cm) Calculated mean mass of 0.198±0.140 consumed particles, (g) Average calculated plastic 0.167±0.126 mass, pc (g) Plastic:Body, pb (g/kg) 2.07 Table 1. Physical descriptors of post-hatchlings (12) and one juvenile from the washed group

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with standard deviations. The value of pb represents an average calculated mass ratio of

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consumed plastic mesoparticles, pc, to the total body weight at death, ω.



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In an analysis of debris ingestion by Santos el al.69 evaluating plastic ingestion in juvenile green

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sea turtles with a mean curved carapace length of 38 cm along the Brazilian coast, it was found

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that the critical amount of debris that was sufficient to lead the turtles to death through blockage

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was very small, only 0.5 g of mixed debris sources, and 47% of all turtles died due to ingestion of

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less than 2.5 g of material. Debris ingestion varied widely according to the area, and in the study,

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10.7% of the turtles that died did so as a result of debris ingestion. By comparison, the average

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weight of recovered plastic fragments in our study was 0.167 g in much smaller sea turtles (SCL

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average = 7.92 cm) with an average body weight of 80.8 g and a plastic to body mass ratio of 2.07

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g/kg. Future studies into the effects of physical influences (i.e. lesions or nutritional deficiencies)

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or biochemical or chemical influences (metabolic, mutagenic, or toxic consequences) are required

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to determine direct correlations to pb ratios and morbidity or mortality.

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The observed plastic mesoparticles, PE, PP, and PS, are polymers with densities less than sea

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water, about 1.02 g·mL-1. By contrast, PVC and PET have densities near 1.4 g·mL-1, and not a

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single mesoparticle of these types were observed in the samples. There was an average of 5.2±3.3

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meso-sized fibers and 17.4±12.0 microfibers with aspect ratios less than 0.200 ingested by each

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individual. Of the total 67 meso-sized fibers, 21% and 24% were identified as PP and PE,

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respectively. No PS fibers were found in the samples. On average, 44.6% of mesofibers were some

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form of plastic (Table S3), and 18.0±17.0% of microfibers were plastic (Table S4).

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Raman Microscopy of GI Particles

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The standard Raman spectra for the found polymers are shown in Figure S2 and were used to

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chemically identify meso- and microparticles in both the washed group and base digested group.

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The fragments that were targeted for Raman spectroscopy displayed a varying degree of 14 ACS Paragon Plus Environment

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background fluorescence due to physisorbed material; however, spectra with high background

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fluorescence could still be identified by characteristic Raman shifts. If a particle displayed a highly

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fluorescent spectrum with no identifiable plastic peaks, it was defined as “other”.

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Work by Minogianni and coworkers examined the crystallinity of isotactic, atactic, and

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syndiotactic PP with Raman spectroscopy, in which the different conformers may be characterized

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by the crystalline stretching mode 809 cm-1 (ρsCH2, skeletal stretching) and the uncoupled modes

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at 841 cm-1 (ρCH2, amorphous stretching).70 Notably, the entirety of identified PP was found to be

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isotactic due to the presence of the strong peak at 809 cm-1. Unsurprisingly, no syndiotactic PP

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particles were identified in this study, as isotactic PP represents the overwhelming majority of PP

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produced globally.

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Inspection of the optical microscope images of larger mesoparticles shows the often-heterogeneous

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nature of the particle surfaces which accounts for the fluorescent background of many spectra.

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Meso- and micro-sized particles were identified with high fidelity down to 20 µm CED. The laser

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diameter is approximately 3 μm, so particles with sizes above 20 μm were easily targeted with the

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use of automation software. Mesoparticles of fibers and irregular shapes and microparticles were

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found within the animals as shown in Figure 2, illustrating the micronizing plastic.



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Figure 2. Washed group specimen sample. Example photograph of particle sizes and colors (A).

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The 2.5X composite image shows a sample with chemically identified mesoparticles, PE (yellow),

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PP (blue), and PS (magenta) (B).

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An example particle distribution is shown in Figure S3. The microparticles were more commonly

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rounded in shape (circularity mean = 0.54) and were similar in size to fish eggs and larval and

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young stages of vertebrates and invertebrates. The post-hatchling sea turtles feed on micronizing

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plastic that appears similar to these food items. Several studies suggest that white and transparent

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plastics are more commonly consumed,57,

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hatchling and small juvenile sea turtles.71,

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microparticle plastic in the ocean is likely a pending ecological threat for the survival of threatened

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and endangered sea turtles, at least in the study area off the coast of Florida.

71

since the particles may resemble prey of post72

The increasing quantity of mesoparticle and


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Mesoparticle and Microparticle Distributions

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PE is the most common form of mesoparticle (54.1%) as shown in Figure 3A; however, in the

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microparticle distribution, PP is the most numerous type (20.97%), Figure 3B. A total of 13,369

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particles were identified as microparticles in the washed group samples (n = 13 turtles) by the

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microscope software, with an average size of 62.1±18.3 µm, and 6,189 spectra were recorded. The

320

particle analysis shows a smaller number of spectrally identified plastic microparticles compared

321

to observed “other” material that are likely foodstuffs or inorganic particles, often gypsum crystals.

322

A total of 253,795 microparticles were observed from the base digestion samples (15,719±11,694

323

microparticles per individual). In the base-digested group, an average of 445±156 microparticles

324

were targeted at random for spectroscopy for each individual and an average size of 34.0±3.1 μm.

325

The microparticle distribution of base digested GI tracts is shown in Figure 3C. The data from

326

each individual is collated in Tables S1 and S2.

327 328

Figure. 3. Comparison of washed and base-digested micro- and mesoparticles by polymer type.

329

The number of recorded spectra for each group, n, is a representative sample of the total observed

330

particles by microscopy, m, for each group. The washed group mesoparticles (n = 135; m = 619)



Page 18 of 35

331

(A) and microparticles (n = 6,189; m = 13,369) (B), and the microparticles collected from the base

332

digested GI tract (n = 6,108; m = 235,795) (C) are shown for comparison.

333

A comparison of SCL to the total GI content and GI plastic content by mass is shown in Figure

334

S4, which indicates that a diverse amount of plastic by mass was found across a variety of animal

335

sizes, yet still the majority of the mesoparticles (80.7%) are plastics. When the data from the micro

336

and mesoparticles are combined, the percent of the plastic types by count may be divided into size

337

domains as shown in Figure 4.

338

339 340

Figure. 4. Plastic type and percent abundance in tested size ranges. Particles from 20 – 10,000 μm

341

were chemically identified, yet smaller particles were found on the samples. The particle percent

342

plastic and size trend (dashed line) suggests that particles in the sub-micron range are expected to

343

have some minor distribution of these plastics. The particle size and percent plastic prediction fit

344

is modeled by the power function y = axb, where a = 13.22 and b = 0.2088.


Page 19 of 35


345

By a simple power function fit of the cumulative percentage of found plastics of all types, we find

346

it reasonable to calculate the theoretical percent of plastic particles sizes down to 1 µm (13.2%

347

total plastic particles). The remaining percentages of the particle size ranges represent “other”

348

particles while PP and PE are the dominant plastic types found in all sizes. A minority of irregular

349

PS and microfiber PET particles were found as well. The trend line shows that submicron sized

350

plastic particles are likely present as a significant part of the particle population.

351

The plastic type and abundance shown in Figure 4 shows that the amount of PE relative to PP

352

changes with particle size, with a higher proportion of PE for mesoparticles and a lower proportion

353

of PE for sub-millimeter particles. This result suggests that oceanic PP mesoparticles, exposed to

354

UV, may be more brittle than PE mesoparticles. Earlier work by Kelly and White in 1997

355

investigated fracture behaviors of PE and PP under UV oxidation conditions using slow strain

356

rates.73 Significant brittle surface cracking was observed for both PE and PP. Stress-strain

357

experiments showed that both PE and PP yield, or fracture, at lower strains after UV exposure,

358

though the more rigid PP samples yield under lower strains than the more flexible PE. We

359

speculate that a combination of PP rigidity and UV-induced degradation may explain the increase

360

in PP microparticle concentrations relative to the mesoparticle distribution.

361

Nanoparticle Distribution in the Washed Group

362

A total of six individual samples were screened by AFM to characterize nanoscopic particle size

363

distributions. Observed nanoparticles were found to be adsorbed to the micro and mesoparticles of

364

the washed group. Over six 100 μm2 areas on spin coated silicon wafers, a total of 4,601

365

nanoparticles were identified with an average of 766±403 nanoparticles with a CED of 51.8±19.7

366

nm (blank samples had 57±8 nanoparticles with a CED of 41.4±41.8 nm). A sample AFM 19 ACS Paragon Plus Environment


Page 20 of 35

367

topography image relative to a control is shown in Figure S5. Raman Spectroscopy has been used

368

for the physiochemical identification of ingested anthropogenic microparticles in crustaceans,

369

filter feeders and fish,22, 74-76 but a complete quantitative analysis of micro and mesoparticles and

370

evidence of nanoparticle formation were not defined in these works.

371

Due to the brittle nature of ocean plastics, minimal sample manipulation coupled with non-

372

destructive particle identification are the best methods to quantify and characterize ingested

373

anthropogenic and residual natural materials. Also, sample characterization by Raman microscopy

374

was performed in a class 10,000 clean room facility to minimize microplastic contamination. It is

375

important to note that the observed nanoparticles could not be chemically identified by AFM;

376

however, we may deduce from data presented in Figure 4 that a smaller minority of the sub-micron

377

nanoparticles are likely plastic. The size of observed particles may have some influence from the

378

action of water washing, the storage and handling of particles, and the base digestion apparatus. In

379

general, the protocols for handling samples were designed to have minimal sample manipulation.

380

Implications from ingested plastics

381

The majority of plastic debris in sea turtle environments has been shown to be disposable consumer

382

plastics from land-based sources.72, 77 We propose that the physical burden of consuming 2.07 mg

383

of plastic per 1 gram of body weight may decrease the chances of survival in post-hatchling sea

384

turtles. We believe stranded post-hatchling sea turtles (most importantly loggerhead and greens)78,

385

79

386

serve as collection systems and reservoirs for assaying plastics across most marine environments,

387

albeit confined to the habitat of a particular group of turtles. Furthermore, the migratory behaviors

388

of sea turtles are actively tracked by conservation groups,80 and their local, transpacific, and

may be used as indicator species for the study of micronizing oceanic plastic, as the animals


Page 21 of 35


389

transatlantic movement across oceans has been well studied,81-83 which may help monitor plastic

390

accumulation trends in animals. Work by Witherington and coworkers outlines the behaviors and

391

diets of sea turtle post-hatchlings and juveniles in pelagic communities.84

392

The studies estimating the accumulation of plastic have largely been based on measurements of

393

collected visible waste from ocean column samples.85 Micronizing plastic can accumulate with

394

plastic concentrations of 100,000 particles per m3 in some ocean locations74 and also may

395

concentrate in coastal sediments.86 However, plastic debris in marine environments fragments into

396

smaller particles due to photochemical transformations from UV exposure and mechanical

397

degradation processes associated with hydraulic forces,87 and other abiotic degradation

398

processes.88 Despite the accumulation of plastic fragments in marine environments and their

399

resulting bioavailability, there has been limited reports of plastic ingestion by free-ranging post-

400

hatchling sea turtles from coastal South Africa.41, 68 While ocean plastic has been described as a

401

threat to all species of sea turtles,11, 12, 69 this work provides evidence for a substantial availability

402

and concentration of micronizing plastic in the environments of stranded post-hatchling sea turtles

403

with corresponding consumption. Raman spectroscopy of particles ingested by sea turtles likely

404

can serve as a predictive model for estimating the bio-available quantity and chemical

405

identification of fragmenting plastics from different ocean zones.

406

Of the five common plastic pollutants analyzed by Raman Spectroscopy, plastic fragments that

407

float (PE, PP and PS) and PET fibers were recovered from the post-hatchlings. Moreover, we

408

suspect the morphology of the PET fibers ( aspect ratio < 0.200) may have facilitated adsorption

409

to food items or other plastics prior to consumption, or the PET microfibers may be associated

410

with the sample washing environment or using municipal water supply, which may contain PET



Page 22 of 35

411

microfibers.89 No PS fibers were found, as PS is seldom manufactured as a fiber. Notably, no large

412

mesofibers of PET were found, rather PET fibers were the most abundant form of microfiber.

413

It was beyond the scope of this report to attempt to model what proportion of all post-hatchling

414

sea turtles that ingest plastic are washing back onto beaches, what proportion may be dying at sea,

415

and whether or not there is any population of post-hatchlings that are not consuming micronizing

416

plastic particles. However, if micronized plastic is causing a high level of mortality in post-

417

hatchling and juvenile sea turtles, the accelerated accumulation of over 5.25 trillion particles of

418

plastic waste,90 coupled with increasing production of non-degradable plastics like PE, PP, PS and

419

PET, likely reduces the survival of individuals to maturity.

420

This study of ingested micronizing plastic in stranded post-hatchling sea turtles correlates with the

421

ratio of production levels of plastic for disposable consumer markets. We suggest that we can

422

reduce the morbidity and mortality of post-hatchling sea turtles, as well as other marine fauna for

423

which the post-hatchlings may serve as an indicator species, by changing from an economy of non-

424

biodegradable disposable consumer waste to an economy of only biodegradable disposable

425

consumer products and thereby reduce the quantity of environmentally stable micronizing plastics

426

that accumulates in our oceans.

427

AUTHOR INFORMATION

428

Corresponding Author

429

*Correspondence and requests for materials should be addressed to Evan M. White. Email:

430

[email protected]

431

Present Addresses


Page 23 of 35


432

†Present Address: The University of Georgia, 220 Riverbend Road, Riverbend Research South,

433

Room 177, Athens, GA 30602, USA.

434

Author Contributions

435

E.M.W., B.W.R., S.C. and C.A.M. conceived the study, designed the analyses and led the writing

436

of the manuscript. S.C. and C.A.M. recovered stranded turtles, characterized specimen physical

437

properties, and prepared the washed group and frozen GI tract samples prior to Raman microscopy

438

analysis. E.M.W., B.C., and S.W. performed analysis on samples via Raman microscopy. E.M.W.

439

and B.C. compiled the data sets, carried out the data processing, and prepared the tables and

440

Figures. J. L. analyzed and verified the procedural methodologies.

441

FUNDING SOURCES

442

Funded in part by a contribution from the RWDC Environmental Stewardship Foundation.

443

ACKNOWLEDGMENT

444

We thank Malvern Instruments for providing access to the Morphologi G3-ID Raman

445

microscope for analysis. We also thank Kara K. Huff for work on Raman microscopy samples.

446

In addition, we thank the volunteers of the Sea Turtle Stranding and Salvage Network for

447

rescuing the turtles and the volunteers and staff of the Sea Turtle Hospital at Loggerhead

448

Marinelife Center for assisting with care of the turtles. This study was carried out under permit

449

number MTP-086 issued by the Florida Fish and Wildlife Conservation Commission.

450

SUPPORTING INFORMATION

451

Standard deviation and plastic mass calculations, Visual Basic for applications information,

452

mesoparticles size distribution, sample Raman spectra, example distributions of micro particles

453

in GI tract, SCL and body mass information, example AFM height images, and example



454

microscopy images are in the SI. Individual turtle data and metrics are also available in the SI.

455

This information is available free of charge via the Internet at http://pubs.acs.org.

456

ABBREVIATIONS

457

SCL straight carapace length, LMC Loggerhead Marinelife Center, CED circular equivalent

458

diameter, AFM atomic force microscopy, PE polyethylene, PP polypropylene, PS polystyrene,

459

PVC poly(vinyl chloride), PET poly(ethylene terephthalate).

460

TABLE OF CONTENTS GRAPHIC

Page 24 of 35

461 462

TOC Graphic. Plastic particle passing through the GI of sea turtles. The particle size distribution

463

of the consumed material follows a continuum from mesoparticles to microparticles to

464

nanoparticles.

465

TOC art: I attest that the artwork for the TOC graphic is author generated and that it meets

466

requirements.

467

Supporting Information. Plastic mass calculations, plastic Raman shifts and microscope settings,

468

example microscopy and AFM images, individual sea turtle data in supporting information.

469

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