University of Plymouth
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01 University of Plymouth Research Outputs University of Plymouth Research Outputs
2017-10-20
Development and optimization of a
standard method for extraction of
microplastics in mussels by enzyme
digestion of soft tissues
Catarino, AI
http://hdl.handle.net/10026.1/8166
10.1002/etc.3608
Environmental Toxicology and Chemistry
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Running Head: Standard method for microplastics extraction from mussels
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Corresponding Author: Ana I Catarino, a.catarino@hw.ac.uk, catarino.anai@gmail.com, +44 (0) 131 451
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4502
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Address: Institute of Life and Earth Sciences; School of Energy, Geoscience, Infrastructure and
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Society; John Muir Building, Heriot-Watt University, EH14 4AS, Edinburgh, UK
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Title: Development and optimisation of a standard method for extraction of microplastics in mussels
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by enzyme digestion of soft tissues
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Authors:
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Ana I Catarino†, Richard Thompson‡, William Sanderson†, Theodore B. Henry†§
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† Heriot-Watt University, Institute of Life and Earth Sciences, School of Energy, Geoscience,
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Infrastructure and Society, Edinburgh, UK
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‡ Plymouth University, School of Marine Science and Engineering, Plymouth, UK
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§ Department of Forestry, Wildlife and Fisheries, and Center for Environmental Biotechnology, The
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University of Tennessee, Knoxville, Tennessee, USA
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*Address correspondence to catarino.anai@gmail.com
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Abstract: We compared procedures for digestion of mussel soft tissues and extraction of
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microplastics (MPs). Complete tissue digestion was achieved with 1M NaOH, 35% HNO
3
and by 0.1
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UHb/mL protease, but use of HNO
3
caused unacceptable destruction of some MPs. Recovery of MPs
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spiked into mussels was similar (93±10%) for NaOH and enzyme digestions. We recommend use of
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industrial enzymes based on digestion efficiency, MP recovery and avoidance of caustic chemicals.
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Keywords: Enzymatic digestion, FT-IR, Microplastics, Mussels, Emerging pollutants, Environmental
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toxicology
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INTRODUCTION
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The presence and accumulation of plastic debris within marine environments has become an issue
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of high priority for environmental policy [1]. Microplastics [(MPs), particles between 5 mm [2] and 1
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µm [3]], are reported as the most abundant pieces of plastics found in the marine environment [4]
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and these particles have accumulated at the sea surface on shorelines and in sediments [5]. They are
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also present in organisms [6] and are of toxicological concern [3]. Standardised methods for detection
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of MP accumulation in organisms are necessary to establish levels of exposure, facilitate comparison
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among studies, and to enable robust assessments of MPs risks in the environment.
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Mussels are particularly good candidates for assessment of MP exposure, in the same way as they
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are used as indicator species for other environmental contaminants in monitoring programmes such
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as Mussel Watch (The National Oceanic and Atmospheric Administration, NOAA, USA). If standardised
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methods for extraction and quantification of MPs in tissues are established and based on good
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laboratory practice, mussels can act as sentinel species of MP contamination applicable over a wide
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range of geographical scales. For instance, Mytilus spp. are intertidal mussels with a large
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geographical distribution, they filter large volumes of water, are relatively immobile, and are easily
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accessible for collection throughout the year. Laboratory studies have demonstrated ingestion of MPs
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by Mytilus spp [7–9], and gut retention times for MPs can be above 72 h [8]. Microplastics have also
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been found in both wild and cultured Mytilus spp., but different soft tissue digestion and
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quantification methods make comparison of results challenging. For example, particles were found at
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concentrations between 5 - 75 particles per mussel in Nova Scotia [10], but in other studies the
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reported concentrations were 0.36 particles g
-1
wet weight from North Sea coasts [11]) and up to 0.34
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particles g
-1
wet weight from various European specimens [12].
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A standardised and specific method for extraction and quantification of MPs from mussels is
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necessary to provide the data needed to assess levels of exposure of organisms to MPs, and to
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provide support for environmental monitoring programmes and management decisions. Recently,
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Vandermeersch et al. [12] reviewed and compared acid digestion procedures used for soft tissue
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digestion of Mytilus spp., but their evaluation did not consider approaches for tissue digestions that
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use strong bases or enzymes. Some methods used for extraction of MPs from mussels may not enable
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accurate quantification of MP abundance. Techniques used for extraction of MPs can alter the shape
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or destroy of particles present in samples. Extraction of MPs from bivalves in general, and Mytilus spp.
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mussels in particular, has been accomplished by chemical digestion with simple and/or mixtures of
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strong acids (HCl, HNO
3
, HClO
4
) [6,11,13] and bases (NaOH, KOH) [14–16]; however, some of these
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methods can damage and/or destroy pH-sensitive polymers [11,13]. Hydrogen peroxide (H
2
O
2
) has
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also been used to digest tissue prior to extraction of MPs, but limitations including incomplete soft
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tissue digestion and production of foam was indicated to cause lower MP recovery from samples
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[10,13]. Although not previously used to digest mussel tissues, enzyme digestion has been applied to
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extract MPs from plankton-rich seawater samples, with reported high digestion rates (up to 97.7 %)
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and no damage of particles [17]. Similarly, in forensic studies, enzyme digestion (industrial proteases
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and lipases by Novozymes) has been used as a method for soft tissue digestion, and which
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additionally does not cause bone damage [18], indicating the potential use of industrial enzymes
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(used in washing powder and food industry, for instance) to digest soft tissue in other organisms.
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The need for a standardised method to assess MPs from organisms, including mussels, has been
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highlighted by the International Council for the Exploration of the Sea (ICES) advice provided to the
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OSPAR Commission on plastic monitoring in organisms [19] and more recently by Vandermeersch et
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al. [12]. Our goal was to describe a procedure for extraction and quantification of MPs in marine
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mussels. This method was developed specifically for Mytilus species, as the digestion of soft tissue
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from other organisms will differ in methodological requirements (e.g. chitinous tissues in crustaceans
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and gut content analysis in large fish [20]). Our approach was first to optimise the digestion efficiency
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of Mytilus edulis soft tissues by 3 different methods: strong acid, strong base, and a new enzyme
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procedure. This new enzyme procedure uses industrial enzymes that are less expensive than other
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enzymes used for tissue digestion in laboratory research. Rates of soft tissue digestion were
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compared and the effects of each digestion method on polymer integrity were assessed by FT-IR
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analysis of extracted MPs from spiked samples. In addition, spike recovery rates of MPs were
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determined, airborne fibre contamination assessed, and the industrial enzyme digestion procedure
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was applied to quantify MPs in M. edulis exposed in the field to waters containing MPs. To enable
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reproducibility, a more detailed standard operating procedure (SOP) based on Good Laboratory
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Practice (GLP) guidelines [21], is provided in the supplement section.
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