Session Chairs: Alexandra ter Halle, CNRS Laboaroire des IMRCP; Julien Gigault, CNRS Geoscience Rennes
This session focuses on microplastics, the remaining 99% part of marine litter at the micrometric and nanoscale.
Plastic debris once discarded in the environment undergoes fragmentation. As more and more data are available on microplastics occurrence (1 – 5 mm) in the environment, it has become indispensable to collect data about the abundance of plastic debris at the micrometric scale and at the nanometric scale. The ecotoxicological evaluation of these very small plastic debris has already started but with very little knowledge about their concentrations in natural waters. There is a need to develop reliable methods for counting and measuring plastic particles at the micrometric and nanometric scale. Could they be automated or semi-automated? How can the fate of microplastics be understood in this context? What are the parameters (density, surface characteristics, and shape) that need to be known in order to gain a predictive knowledge of the subject?
Raman microspectroscopy for analysis of microplastic in environmental samples
presenting: Natalia Ivleva (Technical University of Munich, Institute of Hydrochemistry, Germany); authors: Natalia Ivleva (Technical University of Munich, Institute of Hydrochemistry, Germany), Philipp Anger (Technical University of Munich, Institute of Hydrochemistry), Alexandra Wiesheu (Technical University of Munich, Institute of Hydrochemistry), Reinhard Niessner (Technical University of Munich, Institute of Hydrochemistry), Martin Elsner (Technical University of Munich, Institute of Hydrochemistry)
The accumulation of plastic and especially microplastic (<5 mm) particles in marine and freshwater ecosystems is of growing scientific and public concern. This has stimulated a great deal of research on the occurrence of MP, uptake of MP by aquatic organisms, and the resulting (negative) impact of MP. The crucial step in MP analysis is a reliable identification. Appropriate methods for the chemical identification of MP include ATR-FTIR and µ-FTIR spectroscopy as well as Raman microspectroscopy (RM). ATR-IR is applied for the detection of MP particles larger than 500 µm, while µ-FTIR enables an automated analysis of particles in the size range of 20 – 500 µm. RM is so far the only available method for the identification and quantification of environmental MP with a spatial resolution down to 1 µm  and thus well suited for analysis of small (50 µm – 500 µm) and very small (1 µm – 50 µm) microplastic particles .
Recently we analyzed MP particles from sediment samples in the Lake Garda, Italy and identified MP with a diameter down to 9 µm by means of RM . Apart from MP, we found a large number of pigmented (non)plastic particles with a diameter down to 4 µm. Their size distribution (Fig. 1), suggesting that even smaller pigment particles might be present (down to the nm range). Therefore, further development of methods is needed to assess MP in sub-µm and nm range. For this purpose RM can be combined with techniques for fractionation and concentration of nanoparticles (e.g. AF4). Furthermore, the automatization of RM measurements will enable an efficient analysis of MP down to (sub)µm size in different environmental samples and, hence, will help to assess the risks arising from MP.
 Ivleva et al., Angewandte Chemie International Edition 2017, 56, 1720-1739.
 Imhof et al., Water Research 2016, 98, 64-74.
Towards (Semi-)automation of Microplastic Analysis by means of Raman Microspectroscopy
presenting: Philipp Anger (Technical University Munich, Germany); authors: Philipp Anger (Technical University Munich, Germany), Leonhard Prechtl (Technical University Munich), Reinhard Niessner (Technical University Munich), Martin Elsner (Technical University Munich), Natalia P. Ivleva (Technical University Munich)
Microplastics (MP) in limnic ecosystems are a hot topic in public media as well as in the scientific community. Currently analysis of sediments is very tiresome and takes a lot of time. Sampling may take only one workday. But sample preparation lasts more than a week . The final analysis by means of RM requires a few days when done manually.
Within the BMBF-project (Bundesministerium für Bildung und Forschung) MiWa (Microplastics in the water cycle) one aim is to perform reliable quantification of MP down to 1 µm in reasonable time. We therefore develop a new protocol for (semi)-automated Raman microspectroscopic analysis of MP. Different hardware parameters (objective, magnification, dark/bright field) are compared. Also, different software features that help to automate certain steps of the identification process by means of RM are under close investigation. We therefore implemented a software feature that automatically finds particles and generates coordinates that can be used by the WITec alpha300 R system for the acquisition of Raman spectra and or Raman images (figure 1a). We also developed a filter holder that smoothens surface of analyzed filter (figure 1b).
Alternative methods for microplastic extraction and identification
presenting: Jeff Wagner (Environmental Health Laboratory, California Department of Public Health, United States); authors: Jeff Wagner (Environmental Health Laboratory, California Department of Public Health, United States), Zhong-Min Wang (Environmental Health Laboratory, California Department of Public Health), Sutapa Ghosal (Environmental Health Laboratory, California Department of Public Health), Stephen Wall (Environmental Health Laboratory, California Department of Public Health)
This work presents alternative sample extraction and analysis techniques used to identify microplastics as small as 15 um and address previously noted issues with other methods. The removal of biomass matrices using aggressive chemicals can be problematic for the smallest microplastics, sometimes eroding their surfaces and creating reaction products that interfere with analyses. New methods utilizing ultrapure water and pulsed ultrasonic extraction were developed to separate plastics from biomass without dissolving either, thereby preserving plastic surface characteristics and any adsorbed chemical species. These methods have been used to separate microplastics successfully from marine surface trawls, as well as the stomachs of laboratory, marine, and freshwater fish. Particles were identified and characterized in terms of type, size, and morphology using complementary optical microscopy, scanning electron microscopy plus energy-dispersive x-ray spectroscopy (SEM/EDS), Fourier Transform infrared (FTIR) micro-spectroscopy, and Raman micro-spectroscopy (RMS). After optical and SEM/EDS screening, FTIR and RMS were used to identify specific plastic types. Together, these data can be used to better understand microplastic sources and environmental fates. Shell pieces were identified in many fish stomachs that resembled microplastics, as were brittle, degraded plastics that were shattered like shells. Studies that rely on optical microscopy alone are thus prone to false positives and false negatives. Current progress and challenges for automation and optimization of these extraction and identification methods are discussed, including identification of particles smaller than 15 um.
A rapid-screening approach to detect and quantify microplastics based on fluorescent tagging with Nile Red
presenting: Thomas Maes (Cefas, United Kingdom); authors: Thomas Maes (Cefas, United Kingdom)
A new approach is presented for analysis of microplastics in environmental samples, based on selective fluorescent staining using Nile Red (NR), followed by density-based extraction and filtration. The dye adsorbs onto plastic surfaces and renders them fluorescent when irradiated with blue light. Fluorescence emission is detected using simple photography through an orange filter. Image-analysis allows fluorescent particles to be identified and counted. Magnified images can be recorded and tiled to cover the whole filter area, allowing particles down to a few micrometres to be detected. The solvatochromic nature of Nile Red also offers the possibility of plastic categorisation based on surface polarity characteristics of identified particles. This article details the development of this staining method and its initial cross-validation by comparison with infrared (IR) microscopy. Microplastics of different sizes could be detected and counted in marine sediment samples. The fluorescence staining identified the same particles as those found by scanning a filter area with IR-microscopy.
presenting: Harry Nelson (Fluid Imaging Technologies, United States); authors: Harry Nelson (Fluid Imaging Technologies, United States), Madeyln Woods (Marine and Environmental Research Center), Claudia Lorenz (3Alfred Wegner Institute, Helmholtz Centre for Polar and Marine Research), Gunnar Gerdts (3Alfred Wegner Institute, Helmholtz Centre for Polar and Marine Research), David Fields (Bigelow Laboratory for Oceans Sciences), Patricia Matrai (Bigelow Laboratory for Ocean Sciences), Michelle Devoe (Fluid Imaging Technologies)
Characterizing microplastics(less than 1 mm in size) presents many challenges to the researcher and manager wanting to gain an understanding as to their prevalence, size, composition and ecological effects in the aquatic environment. Responding to the need to automate the characterization of microplastics, researchers have turned to the FlowCam, an imaging particle analyzer that is used worldwide in the research and monitoring of microorganisms in both marine freshwater systems. Here we present an overview of the technology along with data from two research projects that incorporated use of the FlowCam in the study of microplastics. Methods used to identify and count microplastics in aquatic environments, as well as in tissue and feces, along with results and limitations of the technology will be presented. Additionally we will show that the FlowCam can be used for quality assurance of sample preparation as well as to improve the output of spectroscopic analysis.
Are smaller microplastics underestimated? Comparing anthropogenic debris collected in the western English Channel with different mesh sizes.
presenting: Penelope Lindeque (Plymouth Marine Laboratory, United Kingdom); authors: Penelope Lindeque (Plymouth Marine Laboratory, United Kingdom), Alice Wilson McNeal (University of Exeter), Matthew Cole (Plymouth Marine Laboratory), James Clark (Plymouth Marine Laboratory), Tamara Galloway (University of Exeter)
Microplastic debris (<5 mm) is a prolific pollutant, identified in marine ecosystems across the globe, including the ocean depths, polar waters and mid-oceanic islands far from sources of pollution. Despite their ubiquity, sampling, classifying and enumerating microplastics in marine waters and sediments has proven challenging. Based on existing field data ascertained from global sampling efforts, estimated plastic inputs and calculated fragmentation rates, a large proportion of microplastic appears to be “missing”. We hypothesise that some of this “missing plastic” stems from sampling biases. Typically, microplastic sampling is undertaken using nets with a mesh size >335 µm. Therefore, the abundance of smaller (micrometer and nanometer sized) debris remains unclear. Microplastics <335 µm in size are readily consumed by animals at the base of the marine food web (e.g. zooplankton, bivalves, fish), with the potential to bioaccumulate up the food chain. In this study, we investigated whether sampling bias was a cause of the “missing” microplastic by comparing the amounts of microscopic anthropogenic debris collected using 100, 335 and 500 µm aperture nets. Sampling was conducted across 14 sites in the western English Channel, selected with the aid of a hydrodynamic model. Our findings show that as mesh size decreases, the number of anthropogenic microdebris items collected increases. Furthermore, the mean size of items collected was smaller in nets with a smaller mesh size. These results suggest that not only are a significant proportion of microplastics missed using traditional sampling techniques, but that this missing fraction constitutes smaller debris than is usually accounted for.