College of Science and Engineering
College of Science and Engineering
Trophic Transfer of Microplastics
Michaela’s passion for the underwater world began as an undergraduate in New York, USA, only a stone’s throw across the Long Island Sound from her hometown in Connecticut. Six months after graduation she headed across the world to do her Masters at JCU. After her completion of a minor project with Frederieke Kroon, Michaela was offered employment with AIMS where she continued working on microplastic projects. As her love for the field grew, she decided she was ready for the next daunting step: a PhD. Having only just commenced, Michaela hopes to understand how microplastics can travel and accumulate within a food web in the coming years.
Trophic Transfer of Microplastics
2019 to 2023
This project aims to investigate the fate and impact of marine microplastics following entry into the food web of a tropical marine ecosystem. Model organisms from the GBR will be assessed to understand the baseline level of microplastic contamination that is occurring, combined with laboratory experiments to uncover the pathways microplastics take within a multi-level food chain. The laboratory experiments also aim to investigate the physical and chemical (i.e. chemical additives) impact microplastics can have on organisms, both prey and consumers. This will aid in assessing the ecological impact that microplastics may have on the marine environment as a whole.
Plastic pollution has been on the rise since the mass production of plastic materials began in the 1950s. In order to enhance polymer properties and prolong their life, many plastic polymers are altered within the manufacturing process to include chemical additives (i.e. Bis(2-ethylhexyl) phthalate, or DEHP, a priority pollutant with carcinogenic properties known to result in endocrine disruption. However, following mechanical abrasion and exposure to UV light, these additives can leach out and plastics will fragment over time. Plastic litter occurs across the globe within various environments, ranging from the ocean surface and deep sea, within oceanic sediment, and ingested by marine biota. This litter has been generally classified into macro- (> 25 mm), meso- (5 – 20 mm), micro- (< 5 mm) or nano-plastics (< 1 µm) (GESAMP, 2019). Among these categories, microplastics are of particular concern due to their potential and continued uptake by marine organisms.
Uptake of microplastics has been reported for various organisms collected in the field ranging from primary consumers such as zooplankton, and deep sea amphipods (Jamieson et al., 2019) to planktivorous fishes and filter feeders, all the way up to omnivores and carnivores. Despite most species ingesting microplastics, it is still unclear if the recovered plastics are resulting from contaminated prey, mistaking microplastics for prey items, or environmental exposure (i.e. ingestion via gulping water during prey events). In addition, there is limited knowledge of the impact of microplastics once ingested, such as their retention times and chemical leachability during those retention times. From a human health perspective, it is important to understand the bioaccumulation and biomagnification potential of microplastics and their associated toxins into commercially important species (i.e. prawns, reef fishes, bivavles).
If the trophic transfer and accumulation of microplastics is occurring within oceanic environments, examining field-collected organisms would uncover a trend of increased biomagnification correlated with increased trophic level. Based on current literature, there is not enough evidence to support whether microplastics biomagnify up a food web. Similarly, laboratory-based exposure studies have been unrepresentative of the shape, size, and concentration of microplastics found within the natural environment, making comparisons to in-situ ingestion values difficult. Experimentally-based ingestion and retention rates can only be viewed as ‘worst-case’ scenarios due to the tremendous amounts of microplastics being fed to organisms (i.e.10,000 spheres ml-1) relative to contamination levels found in the marine environment. Additionally, there is currently limited research on the impact of chemical additives (i.e. plasticisers like DEHP) resulting from the ingestion of microplastics on marine organisms, both in the field and within laboratory settings.
To understand the extent of microplastic pollution within the Great Barrier Reef system, field samples will be collected and analysed for microplastic contamination. This will be done by investigating contamination within seawater samples, sediment samples and various marine organisms. First, seawater samples taken at the Yongala National Reference Station (NRS) of the Integrated Marine Observing System (IMOS) will be investigated. A long-term monitoring program for microplastic contamination at this location has been implemented by F.Kroon at AIMS. As a result, samples have been collected every month dating back 3 years, allowing temporal trends of contamination to be uncovered, something rarely looked at in the realm of microplastic research. Additionally, seawater, sediment and biota (copepods, shrimp and wrasse) samples will be collected and evaluated for microplastic and toxin contamination at two reef locations off the coast of Townsville.
Seawater samples will be collected via a seawater pump to investigate ingestion of microplastics in copepods as well as microplastics within the water column. Sediment samples will be collected by gathering the top layer of sediment within randomly placed quadrats while scuba diving. Also while diving, small crustaceans will be collected from dead coral rubble substrates, and moon wrasse will be collected using barrier nets, dip nets and clove oil. All sampling techniques have been widely used within the literature (GESAMP, 2019) and within the AIMS’ microplastic research group and allow for the collection of plastic contamination within the micro- size category, including all target organisms. Target organisms were chosen to reflect a basic marine food web, as the purpose of this chapter is to provide baseline contamination of organisms that will be use in subsequent chapters. Seawater and sediment samples will be processed using a hypersaline solution (potassium iodide, KI; density of 1.7 g cm-3) to allowing microplastic particles (densities of 0.02 – 1.5) to float and organic material to sink. Organisms will be chemically digested (potassium hydroxide, 10% KOH for wrasse; nitric acid, 70% HNO3 for copepods and crustaceans) to allow for microplastic recovery. Both NaCl, KOH and HNO3 methodologies have been readily used to allow for microplastic extraction within the literature (Miller et al., 2017), as well as validated within the Microplastics Group at AIMS, contributed to by M. Miller herself. Recovered microplastics will be photographed, measured and recorded via microscopy, and then analysed for polymer identification via Fourier Transform infrared (FTIR) spectroscopy, along with the associated NICODOM© database, to determine their polymer composition. Contamination measures (i.e. laboratory blanks) will be put in place throughout collecting, processing and analysis of samples to account for any airborne contamination that may interfere and falsify results. Statistical analysis for this chapter will include using the total number of microplastics per individual to calculate averages, standard deviations, and pairwise comparisons to determine any statistical difference between species and sites.
This chapter will determine (a) ingestion, retention and depuration rates of biofouled microplastics, (b) food selectivity given the option of a prey item and microplastics, and (c) chemical leachability into organisms via ingestion and surrounding water. This chapter will ultimately answer the question: where do microplastics enter the food web? If this chapter is successful, it will allow for the continuation of experiments in chapter 3.
Copepods (Parvocalanus crassirostris; primary consumers), shrimp (Mysidae spp.; secondary consumers), and wrasse (Thalassoma lunare; tertiary consumers) have been chosen as model organisms. Each organism represents a trophic level (assigned using Froese and Pauly (2010) and Palomares and Pauly (2010)) so assessment on an ecosystem scale can be conducted. Organisms will be obtained via field collection and kept within the National Sea Simulator (SeaSim) facility located at AIMS to allow for acclimation to laboratory conditions. For each organism, a multitude of exposure studies will be conducted with various food regimes:
1. Control feed – determine the ingestion rate of prey items and act as a control for other treatments
2. Biofouled microplastics and prey – determine food selectivity of ‘weathered’ particles
3. Biofouled microplastics dosed with DEHP (no prey) – determine ingestion rates of microplastics and chemical leaching resulting from ingestion of ‘weathered’ particles
4. Biofouled microplastics dosed with DEHP behind a barrier to prevent direct ingestion – determine if chemical toxins in organisms is from direct ingestion or via seawater exposure.
There will be 3 replicates for each species per treatment (n=9 tanks per treatment). In each replicate, species density will vary among species. Copepods will be kept in abundance with >1,000 individuals per replicate. Mysid replicates will contain > 100 individuals, and wrasse replicates will contain 6 wrasse. Because ingestion, depuration and egestion will be quantified per individual, varying sample sizes will not become an issue.
All microplastic feeds will consist of environmentally realistic concentrations and sizes of the most prevalent polymer type found during Chapter 1 sampling. Microplastics will be created by grinding virgin plastics in a blender to obtain fragmented pieces. Microplastics will be kept in aquaria containing seawater for > 2 weeks to allow for a microbial film to grow, simulating what happens to microplastics during their time at sea (Reisser et al., 2014). Prior to exposure, a sub-sample of biofouled microplastic feed will be chemically characterised (via FTIR) to help determine any detrimental effects ingestion has on the polymers. For each food regime prior to treatment, a sub-sample of biofouled microplastics be measured and photographed under the microscope to determine any physical and chemical changes that may occur from ingestion. For food regime #3 and #4, biofouled microplastics will also be dosed with a realistic concentration of DEHP (10 – 70 % w/w; (Hahladakis et al., 2018)). A sub-sample of the particles dosed with DEHP will be chemically analysed to obtain a starting toxin concentration (via Liquid chromatography–mass spectrometry, or LC-MS).
During experimentation, microplastics or prey will be dosed once at the beginning of experimentation. Throughout the exposure period, tanks will be kept aerated at multiple locations to ensure water flow and aid in the neutral buoyance of plastics. This will ensure the exposure to prey and/or plastics are uniform throughout.
Following 24 hours exposure, organisms will be transferred into clean tanks to allow for a depuration period of 48 hours. Following exposure, organisms will be fed once daily until experimentation is halted. Since the concentration of microplastics being fed to organisms will be known, ingestion, retention and depuration rates can be determined over time. Sub-sampling immediately following exposure (30 min), 6, 12, 24, 48 and 72 hours will occur to allow this. If microplastics have been retained at 48 hours, depuration will be extended to a maximum of 2 weeks. Organisms will be immediately euthanised during sub-sampling to prevent gut contents becoming expelled due to stress. Organisms will be dissected under microscope to determine how many particles have been ingested. Recovered particles will be measured, photographed and chemically characterised to uncover any impact ingestion may have had on the microplastics. Careful consideration will be taken during dissection of organisms exposed to food regime #3 to keep tissues intact (whole organisms for copepods and shrimp, gills and liver for wrasse) for LC-MS procedures. Organisms exposed to food regime #4 will not be dissected for microplastic ingestion investigation and rather preserved for LC-MS only. This, however, requires dissection to isolate the gills and liver of the fish.
Statistical analysis for this chapter will include using the total number of microplastics per individual to calculate averages, standard deviations, and pairwise comparisons to determine any statistical difference between replicates and treatments. DEHP concentrations on an individual level will also be used to calculate averages, standard deviations and pairwise comparisons for the purpose of comparing replicates and treatment #3 and #4. Species, however, will not be compared to eachother statistically for DEHP concentrations, due to variations in internal anatomy and tissues analysed.
This chapter will provide insight into whether microplastics, and associated chemical additives, can be transferred, accumulate and magnify through a linear tropical food chain.
Following confirmation of ingestion, retention and depuration rates of microplastics and associated DEHP in Chapter 2, the next set of experiments will be to determine the capacity of particles to bioaccumulate and biomagnify through a linear food chain. The purpose of these experiments is to simulate environmentally realistic conditions as much as possible. Microplastic particles will be dosed with DEHP following > 2 weeks in seawater (allowing a microbial biofilm to grow) prior to being fed to organisms. All organisms will be fed live prey to include a variable of prey-predator interaction, i.e. consumption will not be guaranteed.
These experiments will start with exposing the base level of the food chain (copepods; Parvocalanus crassirostris) to environmentally realistic microplastics. A subsample of copepods will be taken to confirm ingestion and concentration of DEHP following 24 hours of exposure to biofouled microplastic particles. This will be done via microscopy work in less time than the retention time of microplastics (determined from Chapter 2 experiments) to ensure depuration of microplastics in the original population is limited. Once ingestion is confirmed, copepods from the original population will be fed live to mysid shrimp (Mysidae spp.) at a concentration matching the prey ingestion rate determined during Chapter 2 experiments. Following 24 hours of feed time, organisms will be subsampled to confirm ingestion as per above. Shrimp from the original population will then be fed live to wrasse. Wrasse will be allowed 24 hours of feeding time and will then be subsampled and processed as per above. To analyse DEHP levels, organisms at each level will be subsampled again, tissues dissected (i.e. whole organism for copepods and mysids, liver and gills for wrasse), dried and analysed via LC-MS.
Counts of total microplastic ingestion per individual and DEHP contamination will be used to obtain averages and standard deviations for each trophic level. These will be used to statistically compare (via pairwise comparison) trophic levels to uncover any magnification of microplastics and DEHP contamination.
In this final chapter, the fate of microplastics within a simulated open-flow tropical coral reef environment will be assessed. Mesocosm experiments will be set-up to simulate a realistic Great Barrier Reef environment, with a plethora of organisms that are representative of all trophic levels (i.e. copepods, wrasse, shrimp, sea cucumbers, sea urchins, and coral). Mesocosm tanks will be exposed to environmentally realistic microplastics (based on chapter 1) and examined over time to determine the fate of microplastics within the ecosystem. Microplastic particles will be dosed into the system on a regular basis over the course of 2 months, to simulate a constant influx of microplastic pollution. The system will be flow-through, with a constant inflow and outflow of seawater. A filter will be placed at the outflow to ensure microplastics stay within the confines of this experiment, and to measure how many particles will “flow through” the system, instead of being ingested or trapped within the ecosystem. Seawater, sediment and biota samples will be subsampled throughout exposure to understand how plastics may accumulate in these environments. Statisical analysis will include taking total microplastics ingested by individual species, total microplastic found in sediment and seawater samples over time to calculate averages and standard devations for each time point. Pairwise comparison will be done between time points to understand how these change over time. The goal of this chapter is to understand how microplastics travel through a tropical marine ecosystem, representative of the reefs along the Great Barrier Reef.
Chapter 1 is the only component of this project with preliminary results to date. A total of 34 months (Sept 2016 to Sept 2019; except Dec 2016, Jan 2019, April 2019) of seawater surface samples with 2 replicates per month (n=68 total) have been put through a density flotation step. Samples have been filtered through two stainless steel mesh filters (547 and 26 µm), resulting in a total of 163 filters to process further. The microscopy of these filters has progressed, with 65 (35%) complete to date, leaving a remaining 119 to be done. The chemical analysis of the potential microplastics is also progressing, with FTIR of putative microplastics on 40 filters (22%) completed.
Overall, 418 putative microplastics have been identified during the microscopy step. Fragments (n=215) are more prevalent than fibres (n=203); however, this is likely a result of the number of larger meshes (n=40) having been processed compared to smaller meshes (n=25). Fibres are usually smaller in width (av. 0.03 ± 0.04 mm) than fragments (av. 1.39 ± 0.89 mm), allowing them to pass through the larger mesh and become stuck on the smaller mesh. Because of this, on average, more microplastics are found on the smaller mesh. Additionally, for the putative microplastics (105 fragments & 7 fibres) that have been FTIRed, the most common polymers are: polyethylene, polypropylene, polyethylene:polypropylene blend, polypropylene:cotton blend, and cotton:polyester:rayon blend. As for temporal trends, none are apparent from the number of samples processed thus far. There is a slight increase in summer (Nov to Feb) months, yet this may change or even become more pronounced once more samples are added to the dataset.
Chapter 2 was successful in completing the fieldwork, with the trial of the collection box for rubble/crustacean sampling considered a success. A total of 209 crustaceans, mainly squat lobsters, crabs and amphipods, were collected for microplastic analysis. Additionally, samples of the environment, namely seawater (n = 6) and sediment (n = 6), and associated organisms including copepods (n= 394), crustaceans (n = 203) and fish (n = 40) were collected. These samples are currently in the process of being prepared for further microscopical identification of potential microplastics.
Lastly, Chapter 3 has begun initial designing steps, with experimental set-up being developed for exposing copepods, crustaceans and coral reef fish to microplastics. Results from Chapter 2 will influence this, and detailed designs are currently limited until Chapter 2 is completed in the coming months. One key feature of the experimental design that has occurred was the addition of a video component to understand the specifics surrounding ingestion vs. intake (via respiration) for organisms within the experiment. This will aid in determining if contamination of microplastics stems from actual ingestion or rather environmental exposure intake via gills.