DOI: 10.6084/m9.figshare.21259323.v1
title: Woods IJPR Microplastic_Interactions_with_Wetland_Species_Dataset

description: Microplastic retention (Woods IJPR Panicum_Data_Raw) <br> This mesocosm study employed <em>Panicum hemitomon</em> Schult. (Maidencane), a common constituent of freshwater wetlands in the Gulf South (Godfrey and Wooten 1979), as a representative emergent macrophyte. Individual <em>Panicum hemitomon</em> specimens consisting of 4-5 similarly-sized juvenile stems were collected from the Nicholls University Farm Facility with intact soil material to a depth of 8 cm, rinsed thoroughly, and allowed to acclimate in the experimental vessels prior to initiation of microplastic treatments. The experimental design consisted of a 2 emergent vegetation presence (vegetation present, vegetation absent) x 3 microplastic size class (250-500µm microplastics, 43-250µm microplastics, none) completely randomized factorial design with 5 replicates (30 total experimental units). Mesocosm units consisted of 18.9L white HDPE vessels containing soil to a depth of 8cm from the <em>Panicum hemitomon</em> collection area either with or without vegetation present as appropriate and were flooded with tap water to 2.5 cm below the rim for the duration of the study. Microplastics were generated by grinding bright red HPDE source material with an electric grinder equipped with a fine grade grinding stone. The resulting particles were passed through a series of standard sieves to achieve the desired size classes of 43-250µm or 250-500µm. Microplastic treatments were applied by dosing the surface water of each experimental unit with 20mg of prepared microplastics. A linear air compressor (Sweetwater Aquatic Eco-Systems Incorporated Model No. L29) with individual tubing extending into each experimental unit was employed to ensure continual water mixing throughout the study. The total study duration was three months, beginning June 1, 2020 until complete harvest on September 10, 2020. <br> Microplastic sample processing was based on the laboratory methods for the analysis of microplastics developed by the National Oceanic and Atmospheric Administration (Masura et al. 2015) and modified for this specific research as described below. At the conclusion of the study, mesocosm surface water samples were carefully drained from each experimental unit, poured through a series of sieves, and sieves rinsed into individual 250-mL beakers. Plant tissues exposed to surface waters were carefully harvested and were rinsed while applying a light pressure to the vegetation with fingertips to displace any microplastics sequestered in biofilms or on external tissues, and rinse water collected. The rinse water was passed through a series of sieves so that any displaced microplastics could be collected into individual beakers for quantification. <br> <br> All surface water and vegetation samples then underwent wet peroxide oxidation (WPO) protocol using a highly reactive mixture of aqueous 0.05M Fe (II) solution and 30% hydrogen peroxide to oxidize organic material persisting in the sample after sieve separation. Thereafter, samples underwent a density separation protocol adapted from Coppock et al (2017) by adding NaCl to the sample solution (~ 6g NaCl/20mL sample solution; density = 1.25 g cm-3; Table 1) resulting in aqueous layers of non-plastic inorganic material and HDPE microplastics. The samples were then allowed to settle overnight, facilitating isolation of the experimental microplastics. The isolated microplastics were dried onto a series of pre-weighed microscope slides to a constant weight at 60°C, then mass was determined. The slides were then transferred to a microscope for further examination. <br> Enumeration of isolated microplastics was performed using a Nikon AZ100 fluorescence microscope and photo capture hardware (Nikon Digital Sight DS-U2) and software (NIS- Elements). Captured images of microplastics present in a predetermined field of view were imported into ImageJ image analysis software and particle analysis counts were employed to quantify total microplastic quantity in surface waters and plant tissue/biofilm compartments after the study duration. Simple linear regression was performed to develop a predictive equation relating standard microplastic abundance and mass to compare known dosage to microplastics remaining at the conclusion of mesocosm study. Recovered microplastic (particles/L) abundance was compared to the initial amount administered to determine retention of microplastics in vegetative and surface water components per mesocosm. <br> In addition to microplastic retention, impacts of microplastics on growth responses were also evaluated. To investigate influence of microplastics on physiological processes, net CO2 assimilation and stomatal conductance rates were measured using a Portable Photosystem (Li-Cor LI-6400 XT), with leaf chamber conditions set to a light intensity of 1,500 μmol m-2 s-1 and atmospheric CO2 concentration of 400 ppm. Integrated growth responses were assessed by determining stem density and cumulative stem height at the study midpoint and conclusion. At the conclusion of the study, biomass was harvested, aboveground tissues processed for microplastic adherence, and partitioned into live and dead aboveground components, as well as belowground biomass, before drying at 60ºC to a constant weight. <br> To assess differences between microplastics in surfaces waters a one-way ANOVA was employed. To understand the partitioning between microplastics on vegetation surfaces and surface water in vegetated mesocosms, a mixed model ANOVA was performed. Specific a priori linear contrasts were employed to assess differences between the different levels of the microplastic presence/size class treatment (43-250µm, 250-500µm, and None) in these isolated components. To ensure comparability between size classes for abundance statistical analyses, a weight:count ratio correction was applied to normalize the small size class abundance metric to the large size class. Uncorrected abundance data are presented in tables to facilitate direct comparison with other published values. The effect of microplastic size class on biomass partitioning (i.e., integrated growth impacts) of <em>Panicum hemitomon</em> (aboveground live, aboveground dead, and belowground) was analyzed using a one-way ANOVA. To evaluate the effect of time and microplastic presence/size class on repeated growth metrics (stem density and cumulative stem height) and photosynthetic responses (net CO2 assimilation and stomatal conductance) a mixed model repeated measures ANOVA was employed. Alpha was set at 0.05 for all significance assessments. <br> Photosynthetic Responses (Woods IJPR Photosystem_Data_Raw) <br> To determine whether microplastics inhibited photosynthetic processes in wetland vegetation via interference with stomata, a second mesocosm study was implemented at the Nicholls State University Farm greenhouse facility. A 4-vegetation species (<em>Juncus effusus</em>, <em>Sagittaria latifolia</em>, <em>Typha latifolia</em>, and <em>Panicum hemitomon</em>) x 4 microplastic concentration (Low- 7,700 particles m-3, Medium- 77,000 particles m-3, High- 770,000 particles m-3, and Control- 0 particles m-3) completely randomized factorial design with 4 replicates was implemented (64 total experimental units). Young plants, with maximum stem heights less than 20cm to ensure complete submergence during microplastic exposure treatments, were collected at the Nicholls State University Farm Facility and potted into nursery pots. After a one-week acclimation period, each nursery pot was randomly assigned to a microplastic concentration treatment and submerged in an 18.9L reservoir. After 24 hours of microplastic exposure, the pots were removed from each reservoir and left to dry for one hour. Net CO2 assimilation and stomatal conductance measurements were performed on tagged leaves of each pot at intervals of approximately one hour, one day, one week, and two weeks post-exposure to assess potential impacts to photosynthetic processes. Photosynthetic characterization was performed using a Portable Photosystem (Licor LI-6400 XT) with leaf chamber conditions set to a light intensity of 1,500 μmol m-2 s-1 and atmospheric CO2 concentration of 400 ppm. Upon completion of the final recovery period measurements (two weeks), 1-cm2 leaf samples were collected from a random unit of each species and were then examined microscopically for microplastic aggregations impacting stomata. <br> For the physiological impact assessment, the effect of vegetation species and microplastic dosage on the photosynthetic responses (net CO2 assimilation and stomatal conductance) was analyzed using a mixed model repeated measures ANOVA framework with alpha set at 0.05. A simple linear regression was performed to potentially expedite quantification of microplastics based on sample mass in future studies. Statistical analyses were completed using R Statistical Software v. 4.0.3.