MICROPLASTICS IN THE SCOTTISH AQUATIC ENVIRONMENT: AN OVERVIEW OF SAMPLE COLLECTION, PROCESSING, IDENTIFICATION & BIOLOGICAL EFFECTS MASTS ANNUAL SCIENCE MEETING 2014 Brian Quinn, Fionn Murphy & Ciaran Ewins Institute of Biomedical & Environmental Health Research (IBEHR), University of the West of Scotland, Paisley Email: [email protected] MICROPLASTICS IN THE SCOTTISH AQUATIC ENVIRONMENT: AN OVERVIEW OF SAMPLE COLLECTION, PROCESSING, IDENTIFICATION & BIOLOGICAL EFFECTS MASTS ANNUAL SCIENCE MEETING 2014 Brian Quinn, Fionn Murphy & Ciaran Ewins Institute of Biomedical & Environmental Health Research (IBEHR), University of the West of Scotland, Paisley Email: [email protected] Microplastics: • MP definition: <5 mm? • Primary V Secondary MP • Sources: • Plastic rubbish, WWTP, Fisheries, Aquaculture • Worldwide issue • Marine V Freshwater environments • Biological effects??? UWS Approach to Microplastics: Sample Collection Sediment Processing Microplastic Identification Biological Effects UWS Approach to Microplastics: Sample Collection Sediment Processing • Sediment - Site selection - Beach location - Protocol development • Effluent - WWTP selection - Treatment stage Microplastic Identification Biological Effects UWS Approach to Microplastics: Sample Collection Sediment Processing • Sediment - Site selection • Protocol development - Beach location • Initial separation (sieves) - Protocol development • Density separation • Effluent - WWTP selection - Brine solution? - Treatment stage • Adaption of Forensic Science techniques Microplastic Identification Biological Effects UWS Approach to Microplastics: Sample Collection Sediment Processing • Sediment - Site selection • Protocol development - Beach location • Initial separation (sieves) - Protocol development • Density separation • Effluent - WWTP selection - Brine solution? - Treatment stage • Adaption of Forensic Science techniques Microplastic Identification Biological Effects • Physical characteristics - microscope (size, colour & shape) • Fourier transform infrared spectroscopy (FT-IR) • Raman Spectroscopy • Polarized light (florescence) UWS Approach to Microplastics: Sample Collection Sediment Processing • Sediment - Site selection • Protocol development - Beach location • Initial separation (sieves) - Protocol development • Density separation • Effluent - WWTP selection - Brine solution? - Treatment stage • Adaption of Forensic Science techniques Microplastic Identification Biological Effects • Physical characteristics • Demersal fish gastrointestional - microscope (size, colour & shape) tract examination • Fourier transform infrared • Freshwater Hydra attenuata spectroscopy (FT-IR) • Marine Actina equina • Raman Spectroscopy • Endpoints: • Polarized light (florescence) - Predator – prey interactions • Vector for contaminant exposure Sample Collection: Site selection Gourock Port Glasgow Erskine bridge Dalmuir WWTP* Erskine WWTP Wemyss bay Dalmarnock WWTP* Daldowie WWTP* Largs Irvine Marine Estuarine / Freshwater * Effluent Sediment Collection • Quadrates (0.25 m2) thrown at random along HWM • Sediment taken from 2 cm depth = ~2 kg sample • Foil trays & labelled • Based on Brown et al., (2010) (Brown et al., 2010. Environ. Sci. Technol. 44(9): 3404-3409) Issues Erskine Bridge Irvine beach • Difficult to distinguish HWM • Sampling opportunistic, due to site variation • Variability? Truly random? Access? Dalmarnock STW Daldowie STW Kelburn park Wemyss bay Density Separation Density Separation • Depends on: • Density (& size) of plastic • Density of brine solution • Composition of sediment Density Separation • Depends on: • Density (& size) of plastic • Density of brine solution • Composition of sediment Plastic Density g/cm-3 Poly propylene 0.85-0.94 Poly ethylene 0.92-0.97 Poly styrene <0.5-1.00 Polyester 1.24-2.3 Nylon 1.02-1.05 PVC 1.16-1.58 Polyethylene terephthalate 1.37-1.43 Brine Solution Code (sigma) Density g/cm-3 Sodium Chloride S7653 1.22 Sodium Bromide S4547 1.36 Sodium Iodide 383112 1.57 Zinc Bromide 02128 2.57 Density Separation Protocol • Based on Claessens et al., 2013 (Claessens et al., 2013. Mar. Pollut. Bull. 70(2): 227-233) • Make saturated brine solution (Sol.) • Ratio of 3:1 for brine sol. to sediment • Stirred 3 min at 300 rpm, settle for 15 min • Floating particles removed under vacuum • Sol. containing particles filtered • Particles dried (70 oC for 10 min) • Triplicate Brine Solution % Recoveries NaCl Brine Solution % Recoveries NaCl Validation • Use density separation protocol: • Different plastic types • Different size classes • Individually & as mixture • n = 9 Plastic Reference Library Plastic Source Colour Weight (g) Density (g/cm-3) High density Polyethelene (HDPE) Air waves base Blue 6.62g 0.941 g/cm3. High density Polyethelene (HDPE) Milk carton colourless 3.54g 0.941 g/cm3. Low density Polyethelene (LDPE) Air waves lid Blue 3.58g 0.915–0.925 g/cm3 Nylon Thread blue 1.13-1.15g/cm3 Polyethelene (PE) Morrisons bag Clear/colourless 0.8g 0.926–0.940 g/cm3. Polyethelene terephthalate (PET) Lucozade bottle Clear/colourless 6.64g 1.38 g/cm³ Polypropylene (PP) Plastic container Clear/colourless 10.45g 0.855 -0.946g/cm3 Polystyrene (PS) Coffee lid White 15.24g 0.946 g/cm3 Polystyrene (PS) Plastic forks White 14.28 0.946 g/cm3 Polyvinyl chloride (PVC) Un Plasticised Window frame 1.35-1.45 g/cm3. Polyvinyl chloride (PVC) Plasticised Wire Black 1.35-1.45 g/cm3. Polyethylene (180µm) Sigma (bought) white n/a 0.926–0.940 g/cm3. Microplastic Identification • Light microscope • Physical characteristics • FT-IR • FT-IR microscope • SEM • Raman Spectroscopy Sediment results • Examined using NaCl brine only • Marine Samples, few MP found • FW similar results to lake Garda (Imhof et al, 2013*) • Size range: 500 µm – 3.2 mm • Total 32 MP in 6 samples *Imhof et al., 2013. Curr. Biol. 23(19): R867-R868) Biological Effects • Separation and ID of MP in gastrointestional tract of demersal fish from around the Scottish coast • Development of bioassay to investigate biological effects • Marine beadlet anemone • Freshwater Hydra (Actina equina) (Hydra attenuata) http://en.wikipedia.org/wiki/Hydra_vulgaris • Endpoints include: morphology, behaviour, feeding, reproduction, regeneration, mortality Future work: • Co-funded UWS/MSS PhD ‘The investigation of microplastics in the marine environment and their ability to bioconcentrate environmental contaminants’ • Dr Marie Russell Co-supervisor • Start Oct 2015 • Validation of MP separation technique • Sources and Sinks (marine & FW) of MP • Biological effects, • Continue bioassay development • Different species • MP accumulation in wild species UWS • Institute of Biomedical & Environmental Health Research (IBEHR) • Centre for Environmental Research (CER) • Infection and Microbiology Group • Areas of interest include: Ecotoxicology • Fish Behaviour • Environmental Geochemistry • Parasitology • Immunology • Aquaculture •Environmental Toxicology • Entomology • Forensic Chemistry • Microbiology • Bioremediation • Analytical Chemistry • Climate Change Vulnerability / Adaptation • Expertise in: Microplastics • Trace Metal contamination • Toxicity testing • Pharmaceuticals • Endocrine disruption • POPs • PCBs • Climate Change impact • Biomarker expression & development • Bivalves in Biomonitoring • Environmental diagnostics • Fish disease pathogenesis & diagnostics • Pathogen control Acknowledgements: • Dr Marie Russell • Dr Craig Robinson • Craig Close • Jordan Kilday • Jamie Malkin • Jordane Elder • Amelia Waugh • Siobhan Boylan • Courtney Cryans • Stacey Grimshaw.