key: cord-0910309-vlohjeya
authors: Harris, Lyda S.T.; La Beur, Laura; Olsen, Amy Y.; Smith, Angela; Eggers, Lindsey; Pedersen, Emily; Van Brocklin, Jennifer; Brander, Susanne M.; Larson, Shawn
title: Temporal Variability of Microparticles Under the Seattle Aquarium, WA: Documenting the Global Covid‐19 Pandemic
date: 2021-08-11
journal: Environ Toxicol Chem
DOI: 10.1002/etc.5190
sha: d83a2400eb5c9c3979fbce3a1de5f9b8a847d2c6
doc_id: 910309
cord_uid: vlohjeya

Anthropogenic debris including microparticles (MP; <5mm) are ubiquitous in marine environments. The Salish Sea experiences seasonal fluctuations in precipitation, river discharge, sewage overflow events, and tourism– all variables previously thought to have an impact on MP transport and concentrations. Our goals are two‐fold: 1) Describe long‐term MP contamination data including concentration, type, and size and 2) Determine if seasonal MP concentrations are dependent on environmental or tourism variables in Elliott Bay, Salish Sea. We sampled 100 L of seawater at depth (~9 m) at the Seattle Aquarium approximately every two weeks 2019 – 2020 and used an oil extraction protocol to separate MP. We found MP concentrations ranged from 0 – 0.64 particles L⁻¹ and fibers were the most common type observed. Microparticle concentration exhibited a breakpoint on April 10, 2020, where estimated slope and associated MP concentration significantly declined. Further, when considering both environmental as well as tourism variables, temporal MP concentration was best described by a mixed‐effects model with tourism as the fixed effect and the person counting MP as the random effect. While monitoring efforts presented here set out to identify effects of seasonality and interannual differences in MP concentrations, it instead captured an effect of decreased tourism due to the global Covid‐19 pandemic. Long‐term monitoring is critical to establish temporal MP concentrations and to help researchers understand if there are certain events, both seasonal and sporadic (e.g. rain events, tourism, or global pandemics), when the marine environment is more at risk from anthropogenic pollution. This article is protected by copyright. All rights reserved.

Accepted Article particle type, and particle size and 2) Determine if seasonal MP concentrations are dependent on environmental or tourism variables in Elliott Bay, Puget Sound.

The Seattle Aquarium, a non-profit and non-government organization, operates a small research team through the Conservation Programs and Partnerships Department.

The microplastics research lab and team are unique in that research focuses on volunteer efforts and community engagement while still carrying out rigorous protocols. The marine microplastic team is composed of permanent and temporary staff as well as volunteers. To ensure the use of the most up to date methods, the Aquarium's research capacity, and composition of volunteers and staff, collection and processing protocols were changed during this study. A timeline of changes is described in Table 1 .

Seawater samples were collected at the Seattle Aquarium located in Seattle, WA (47.6073, -122.3439 ). The water collection procedure described here was adopted from methods used by, and communications with, Ocean Wise plastics research team in Vancouver BC, CAN. Water samples were collected approximately once per month January -July 2019 and approximately every two weeks (bi-monthly) from August 2019 through 2020 (some collection dates were missed due to staff and volunteer availability).

Seawater was drawn directly from Elliott Bay into the Aquarium via a pump located approximately 9 m below the Aquarium pier (depth of water fluctuated with tidal changes; tides ranged from -1.04m to +4.01m during this study; NOAA tide predictions).

To capture anthropogenic microparticles (MP), 100 L of seawater was siphoned from the pumps and passed through either a 10-inch diameter (Jan 2019 -Dec 2019) or a 3-inch

Filters were visually inspected under a microscope (Olympus SZX10 stereoscope) with a camera attachment (Olympus SC50). Microparticles were categorized by morphotype and color, and measured using cellSens software (length or area; OLYMPUS cellSens Entry 2.3). Only MP 330 -5,000 m (length; Masura et al. 2015) were categorized and measured due to the resolution of the microscope and camera.

During visual inspection, it is nearly impossible to determine polymer composition; therefore, all particles that appeared to be of anthropogenic origin were counted and categorized by morphotype and color ( Figure 1 ). Morphotypes included fibers, foils, and fragments. Particle color was recorded as the primary color found on the majority of the particle (some particles had multiple colors, but only one color was recorded for the purpose of analyses). Rare colors (<1% of total observations) were recorded and categorized as "other" for analyses.

Approximately 10% (76 of 726 total particles) of suspected anthropogenic MPs were picked from sample filters for micro-Fourier Transform Infrared (µFTIR) spectroscopy analysis (Thermo Electron iN5 µFTIR; Thermo Fisher Scientific, Waltham, MA, USA). A subset of filters were shipped from the Seattle Aquarium to Oregon State University for micro-µFTIR analysis. Within the subset of filters, chosen MP were representative of the quantities found over time, sample types (water and blanks), colors, and morphologies of particles observed. Subsampling was performed visually with a Leica EZ4 microscope and Motic 3+ camera. Under a LF hood, subsampled MP were picked from filters, stored on glass microscope slides, and secured with a glass coverslip Accepted Article and tape. Samples were first visualized on slides using a dissecting scope (Leica EZ4 E) to get a matching length measurement and positive ID by comparing them to visual and length data recorded at the time of picking from filters. Samples were then placed on a gold-plated slide in a drop of 70% filtered ethanol to prevent movement during transport to the µFTIR. The slide was placed on the stage of the µFTIR and ethanol was allowed to evaporate before analysis. Reflectance was measured using a fixed aperture with 128 -512 scans on the largest, cleanest portion of the sample. A germanium tip probe was inserted and lowered to contact the sample (~1 -2 µm into material surface) for further spectral analysis. A math match of 70 or greater using either or both overall reflectance and attenuated reflectance (µATR) is the standard during sample analysis. The sample was then retrieved, when possible (sometimes samples stuck to or broke upon contact with µATR tip), after data collection and returned to its respective slide. Polymer identifications from Open Specy, open-source software that performs baseline correction and smoothing, as well as matches to microplastic specific databases (Cowger et al. 2021) were used due to higher matching percentages; however, polymer identifications were cross-checked with identifications from Omnic (Thermo Fisher Scientific software) to confirm MP categorizations.

To reduce airborne and ambient anthropogenic MP contamination, all equipment underwent extensive cleaning prior to sampling and OEP. The 10-inch metal sieve was triple rinsed, and the 3-inch metal sieve was cleaned in a sonic cleaner (Cole-Palmer Ultrasonic Cleaner, Model 08895-04); all sieves were covered with aluminum foil prior to and during each sampling event. All glassware received an acid rinse and DIW rinses This article is protected by copyright. All rights reserved.

(3 total) prior to May 2019, and beginning in late May 2019 all glassware was soaked in an acid bath and rinsed thoroughly with DIW. All glassware was then covered with aluminum foil prior to and during use. Samples collected prior to April 2020 were processed and filtered in a clean room, while samples collected in April 2020 and onward were processed and filtered in a LF hood (Security Air Systems, Inc.). Following OEP, filters were placed in plastic petri slides, where the lids remained on throughout drying and visual quantification. Researchers wore white 100% cotton laboratory coats and latex gloves during collection, processing, and polymer analyses. A blank filter paper in an open petri slide was placed in the necropsy room and LF hood during sample processing to collect ambient air MP. Water sampling occurred roughly every two weeks, but environmental and tourism sites did not share the same locations nor the same sampling timepoints. Therefore, the rolling 14-day average of precipitation, Duwamish discharge, WW effluent, and tourism were calculated and used in analyses.

Environmental variables are often linked, where an increase in precipitation causes river flow and WW effluent to increase as well. To take this into account while also identifying effects of each environmental variable, samples were pooled annually into three seasons based on precipitation records and mixing events: Winter (November -February), spring (March -June), and summer (July -October). In the Salish Sea region, winter is characterized by high precipitation (rain and snow), storm events, decreased seawater salinity, and high river flow and WW effluent; spring is characterized by snowpack melt, medium precipitation (rain), higher than average river flow and seawater This article is protected by copyright. All rights reserved.

mixing disrupting stratification; and summer is characterized by low precipitation (rain), increased seawater salinity and temperatures, and seawater stratification.

All data analyses were completed using R (Version 4.0.3, R Core Team, 2020).

The following packages were used: data.table, car, dplyr, emmeans, faraway, ggplot2, lmerTest, MuMIn, nlme, patchwork, plyr, segmented, and zoo. Level of significance was set at α < 0.05. Homogeneity of variance was confirmed with the Bartlett test and normal distribution with Shapiro-Wilks test. Ambient MP concentrations between locations (clean room and LF hood) were compared using a t-test.

A breakpoint and change in MP concentration over time was evaluated using change point estimation. Piecewise linear regression analysis was used to estimate the date where the breakpoint occurred. Differences in anthropogenic data (WWTP and tourism) were evaluated across the same dates pre-and post-breakpoint using t-tests. To evaluate the effects of environmental and tourism data on MP concentration over time, all possible linear mixed-models were assessed, where precipitation, Duwamish River discharge, tourism, and WW effluent were fixed effects (interactions included), and the person counting MP was the random effect. Correlations between covariates were tested to avoid overfitting. Model selection occurred by calculating Akaike's Information Criterion (AIC) = 2k -2log L, where k is the number of parameters and log L is the log likelihood for that model. However, because AIC tends to select more complex models, Bayesian Information Criterion (BIC) and AICc with correction for small sample sizes were also calculated. After the best model was identified, model fit was evaluated and model diagnostics were checked. To look at potential seasonality within and across years, data were pooled into three seasons: Winter, spring, and summer. Since the breakpoint analysis pointed towards a difference in MP concentrations (April 10, 2020), data were separated into annual seasons and pre-and post-breakpoint groups. Spring 2020 straddled the breakpoint, with the majority occurring post-breakpoint and was categorized as such. Total MP concentration was dependent on annual seasons and the breakpoint (p = 0.0002 and p = 0.03, respectively; 2-way ANOVA; Figure 4 ; Table 2 ). All seasons in 2019 (pre-

were microplastics and included polyethylene terephthalate (PET), polyester phthalate, polyamide, polyethylene and silicate, and polyester; possibly natural/processed included conflicting results of wool and polyamide (Supplemental Table 2 ). No unprocessed plant or biotic material was identified (e.g. algae, zooplankton, hair). Complete spectral matching and categorization data can be found in Supplemental Table 2 .

This study presents the first long-term monitoring of anthropogenic microparticle (MP) concentrations at depth in Elliott Bay, WA in the Salish Sea. Continuous and frequent sampling at the Seattle Aquarium over time allowed a unique opportunity to measure MP concentrations across multiple years and seasons, as well as through the beginning of the Covid-19 pandemic. Due to the duration of this study, changing capacities and funding of our research program, and the evolving nature of MP sampling methodologies, our experimental protocols were updated as the study progressed. While this led to methodological changes, we are confident the changes had no effect on results and interpretations, and we encourage other long-term studies to adjust methodologies as the field grows. This article is protected by copyright. All rights reserved.

Microparticles categorized as possibly natural/processed accounted for 4% of the subsample analyzed and were keratin-based fibers, possibly from wool, cashmere, alpaca, (Granek et al. 2021) or marine mammals. Due to the inconclusive types, sources, and potential processing, we were unable to definitively determine the exact nature of these MPs.

Physical characteristics of microplastics are known to dictate fate and transportation in marine systems (Zhang 2020). Examining the material properties of MP found in pelagic water samples may offer an explanation on the difference in MP concentrations (morphotype, polymer type, and quantities) across the breakpoint as well as compared to other studies. Sea surface samples capture lighter, more buoyant particles that have not necessarily been in the water very long and thus may not interact with pelagic or benthic marine organisms but may be washed up on beaches. Low density synthetic particles found in pelagic waters (e.g. polyethylene and polyamide) suggest particles were biofouled, causing an increase in density, bypassing surface waters and sinking towards the sediment. Morét-Ferguson et al. (2010) found that low-density polymers such as polyethylene were found on beaches with higher densities than pristine counterparts, concluding that the increase in density resulted from biofouling at sea.

There is still little research on the mechanisms of biofouling and how that contributes to the distribution of microplastics in the water column.

Sampling at depth captures particles that sink due to high density, wave action, currents, or biofouling, all on their way to benthic sediments or to be ingested by benthic organisms. This study provides a glance at MP that are available to pelagic and benthic organisms in the Salish Sea. In the Puget Sound, 63% of oysters, which are proficient tourism leads to a smaller urban population, which can have cascading effects on human movement, WWTP effluent, as well as waterfront activity. All of these anthropogenic factors can affect both the concentration as well as composition of MP pollution in Elliott Bay, further supporting an effect Covid-19 and subsequent decline of tourism on MP concentration and source.

Decline in tourism and activity on the waterfront decreased the quantity of MP observed, however, the long-term Covid-19 effects on marine debris remain unknown.

The MP found here were primarily fibers and likely not from single use plastics, but rather from textile washing and shedding. While the quantity of MP found in this study decreased with the onset of Covid-19 and stay-at-home orders, global single use plastic consumption and subsequent pollution increased substantially (Benson et al. 2021; Prata et al. 2021) . When plastic enters waterways, it is degraded by UV rays and broken apart by physical forces such as wave action over time. As single use plastic consumption remains high throughout the pandemic and the foreseeable future, it is possible that as these plastics break apart they will begin to appear in future water samples. While MP contamination is relatively low and mostly consists of fibers as of 2020, future conditions are likely to worsen due to a return of tourism and current consumption and waste of single use plastics.

Seasonality of MP concentrations may exist, however, with the significance of the breakpoint and the lack of supportive evidence from the environmental data, it is too early to definitively say. In the Salish Sea region (and specifically in Seattle) tourism 

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