Microscopic organisms are often overlooked in traditional diversity assessments due to the difficulty of identifying them based on morphology. Metabarcoding is a method for rapidly identifying ...organisms where Environmental DNA (eDNA) is used as a template. However, legacy DNA is problematically detected from organisms no longer in the environment during sampling. Environmental RNA (eRNA), which is only produced by living organisms, can also be collected from environmental samples and used for metabarcoding. The aim of this study was to determine differences in community composition and diversity between eRNA and eDNA templates for metabarcoding. Using mesocosms containing field-collected communities from an estuary, RNA and DNA were co-extracted from sediment, libraries were prepared for two loci (18S and COI), and sequenced using an Illumina MiSeq. Results show a higher number of unique sequences detected from eRNA in both markers and higher α-diversity compared to eDNA. Significant differences between eRNA and eDNA for all β-diversity metrics were also detected. This study is the first to demonstrate community differences detected with eRNA compared to eDNA from an estuarine system and illustrates the broad applications of eRNA as a tool for assessing benthic community diversity, particularly for environmental conservation and management applications.
A common method for characterizing microplastics (MPs) involves capturing the plastic particles on a filter after extraction and isolation from the sediment particles. Microplastics captured on the ...filter are then scanned with Raman spectroscopy for polymer identification and quantification. However, scanning the whole filter manually using Raman analysis is a labor-intensive and time-consuming process. This study investigates a subsampling method for Raman spectroscopic analysis of microplastics (operationally defined here as 45–1000 μm in size) present in sediments and isolated onto laboratory filters. The method was evaluated using spiked MPs in deionized water and two environmentally contaminated sediments. Based on statistical analyses, we found quantification of a sub-fraction of 12.5 % of the filter in a wedge form was optimal, efficient, and accurate for estimating the entire filter count. The extrapolation method was then used to assess microplastic contamination in sediments from different marine regions of the United States.
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•Raman is a labor-intensive and time-consuming method for microplastic (MP) analysis•Investigated subsampling method for Raman analysis of microplastics•Using microplastic amended water and natural sediments performed subsampling study•12.5 % filter sub-fraction optimal for extrapolating MP count to entire sample•Applied subsampling method to sediments from around the United States
Microplastics (MP) are found in marine sediments across the globe, but we are just beginning to understand their spatial distribution and assemblages. In this study, we quantified MP in Gulf of ...Maine, USA sediments. MP were extracted from 20 sediment samples, followed by polymer identification using Raman spectroscopy. We detected 27 polymer types and 1929 MP kg−1 wet sediment, on average. Statistical analyses showed that habitat, hydrodynamics, and station proximity were more important drivers of MP assemblages than land use or sediment characteristics. Stations closer to one another were more similar in their MP assemblages, tidal rivers had higher numbers of unique plastic polymers than open water or embayment stations, and stations closer to shore had higher numbers of MP. There was little evidence of relationships between MP assemblages and land use, sediment texture, total organic carbon, or contaminants.
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•Sediments from the Gulf of Maine were extracted and analyzed for microplastics.•Concomitant site data for each station were analyzed versus MP assemblage.•Habitat, hydrodynamics, and station proximity were important drivers of MP assemblage.•Land use and sediment characteristics had little effect on MP assemblage.•Methods must be less time and labor intensive for feasible largescale monitoring.
Plastic particles are ubiquitous in marine systems and fragment into smaller pieces, such as nanoplastics (NPs). The effects of NPs on marine organisms are of growing concern but are not well ...understood. Marine sediments act as a sink for many contaminants, like microplastics, and are rich habitats for benthic micro- and meiofauna which are ecologically-important components of marine food webs; however, little is known about the sensitivities of specific organisms to NPs or the effects on community diversity and composition. Utilizing molecular methods, such as metabarcoding of environmental DNA/RNA, allows for the rapid and comprehensive detection of microscopic organisms via high-throughput sequencing to assess adverse effects at the community level. The objective of this study was to use a metabarcoding approach to investigate the effects of NPs on benthic micro- and meiofaunal community diversity. Mesocosms were created with sediment cores collected from the Narrow River estuary (Rhode Island, USA) and exposed to 900 nm diameter weathered polystyrene beads at concentrations of 0.1, 1, 10, or 100 mg/kg dry weight in sediment for two weeks. Following exposure, RNA and DNA were co-extracted from the sediment, RNA was reverse-transcribed, 18S and COI markers were PCR-amplified, and amplicons were sequenced on an Illumina MiSeq. Using the 18S marker and eRNA template, increases to α-diversity and significant differences to β-diversity were observed in the highest NP exposures relative to the control. Observed differences in community composition were driven by the differential abundance of several types of protists and arthropods. Significant dose-dependent shifts in composition were observed in β-diversity Jaccard and Unweighted-Unifrac metrics with the 18S marker using the RNA template. To our knowledge, this is the first demonstration of a dose-response relationship for NPs at a community level, and it highlights the value of using community-level endpoints to assess environmental impacts of nanoparticles.
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•Nanoplastic alters benthic community composition and diversity.•Arthropods and unicellular eukaryotes are the most sensitive to nanoplastic.•Nanoplastic exposure resulted in dose-dependent effects at the community level.•eRNA metabarcoding is preferential to eDNA for community-level toxicology assessments.
Microplastics are small plastic particles found ubiquitously in marine environments. In this study, a hybridized method was developed for the extraction of microplastics (45–1000 μm) from sediments ...using sodium bromide solution for density separation. Method development was tested using spiked microplastics as internal standards. The method was then used to extract microplastics from sediments in Narragansett Bay, Rhode Island, USA. Suspect microplastics were analyzed with Raman spectroscopy. Microplastic abundance ranged from 40 particles/100 g sediment to 4.6 million particles/100 g sediment (wet weight). Cellulose acetate fibers were the most abundant microplastic. These results are some of the first data for microplastics in Rhode Island sediments.
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•Microplastic in Rhode Island, USA totaled 40–4.6 million pieces per 100 g sediment.•Cellulose acetate fibers were the most abundant microplastic type.•A novel method was used to extract microplastics from sediment.
Microplastics (MP) are distributed throughout ecosystems and settle into sediments where they may threaten benthic communities; however, methods for quantifying MP in sediments have not been ...standardized. This study compares two methods for analyzing MP in sediments, including extraction and identification, and provides recommendations for improvement. Two laboratories processed sediment samples using two methods, referred to as “core” and “augmentation”, and identified particles with visual microscopy and spectroscopy. Using visual microscopy, the augmentation method yielded mean recoveries (78%) significantly greater than the core (47%) (p = 0.03), likely due to the use of separatory funnels in the former. Spectroscopic recovery of particles was lower at 42 and 54% for the core and augmentation methods, respectively. We suspect the visual identification recoveries are overestimations from erroneous identification of non-plastic materials persisting post-extraction, indicating visual identification alone is not an accurate method to identify MP, particularly in complex matrices like sediment. However, both Raman and FTIR proved highly accurate at identifying recovered MP, with 96.7% and 99.8% accuracy, respectively. Low spectroscopic recovery of spiked particles indicates that MP recovery from sediments is lower than previously assumed, and MP may be more abundant in sediments than current analyses suggest. To our knowledge, likely due to the excessive time/labor-intensity associated with MP analyses, this is the first interlaboratory study to quantify complete method performance (extraction, identification) for sediments, with regards to capabilities and limitations. This is essential as regulatory bodies move toward long-term environmental MP monitoring.
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•A novel augmentation method achieved greater recovery, likely due to the use of separatory funnels rather than beakers.•Visual identification likely overestimates MPs due to misidentification of non-plastic materials persisting post-extraction.•Spectroscopic recovery was low, indicating that MP recovery from sediments is lower than previously assumed.•MP may be more abundant in sediments than current MP analyses suggest.•To our knowledge, this is the first interlaboratory study to compare both extraction and identification of MP in sediments.
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