William Li & Rowan Ross
Team Canada ISEF Candidate
ABSTRACT
Background
Nearly half a century ago, microplastics were not yet discovered. Only 50 years later, microplastics are now one of the world’s most prevalent and forthcoming problems. Microplastics are fragments of plastic that are 5mm or smaller. They are formed from the degradation of larger pieces of plastic. Microplastics in the oceans and great lakes are a devastating problem. Microplastics move through the marine food web as they are ingested by animals and will inevitably reach humans. The health concerns from the ingestion of microplastics are still unknown and to this date are being studied. Although research has now begun to explore the subject of microplastics and their respectful extraction, there still remains a lack of solutions to remove microplastics, especially microplastics in the ocean.
Purpose
A successful solution for the extraction of microplastics from the ocean must be both feasible and bio-friendly. In a search to meet those goals, we explored the potential utility of filter feeders to extract microplastics from the ocean. We investigated the ability of solitary filter-feeders, specifically Ascidians, to remove microplastics from ocean water, as a potential bio-filter to reduce ocean pollution.
Hypothesis
We hypothesized that Ascidians will filter microplastics from water. Specifically, if Ascidians are exposed to microplastics in a body of water, then the concentration of microplastics would decrease after the Ascidians filtered the water.
Method
Microplastics were added to three tanks of saltwater that contained living Ascidians. A similar quantity of microplastics was added to three negative control tanks that did not contain Ascidians. To simulate the microplastics found in the ocean, 10μm diameter microbeads were added to each tank.
Results
The concentration of microplastics in the tanks housing the Ascidians demonstrated a rapid 24.7% decrease within 1 day. By day 4, the concentration of microplastics had decreased by 94.7%. In contrast, there was no change in the microplastic concentration in the negative control group.
Conclusions
Ascidians efficiently filtered microplastics from water through their natural feeding and respiratory process. Our research demonstrates that this method of bio- filtration is a potentially effective and viable option for the extraction of microplastics in the ocean. These findings could lay the path for future research in safe microplastic extraction from the ocean using live organisms. Based on our results, we can extrapolate that a 5m x 5m x 5m cage of Ascidians would filter out almost 50kg of microplastics every day. This newfound knowledge can lead to many practical applications in the field of ocean pollution, such as commercially farming Ascidians for the purpose of microplastic extraction.
BACKGROUND RESEARCH
Ascidians are marine invertebrate suspension filter feeders that are found all over the world. There are over 2,300 species of Ascidians that are categorized into 3 main types: solitary, social, and clumped. All three of these types are immobile creatures as Ascidians remain firmly attached to their substrata, such as rocks and shells. Ascidians feed by filtering water. They take in water through their oral siphon, which then flows down their mucus-covered gill slits, into a water chamber called the atrium. Inside this water chamber, various enzymes will extract the nutrients from the water, which then exits through the atrial siphon of the Ascidian.
In our experiment, we tested to see if the natural feeding process of the Ascidian could be utilized to filter out microplastics from water. Microplastics are small pieces of plastics that pollute the environment. By definition, “microplastics” refers to any type of plastic fragments less than 5mm in diameter and in length. Microplastic pollution is so widespread that microplastic pollution is now regarded as a problem from the Northeast Pacific all the way to the Antarctic oceans. Microplastics enter the ocean through a variety of different sources. The main sources of microplastic include tiny microbeads that wash down our drains from personal care products, fragments of single-use plastic items, such as coffee cup lids and plastic straws, and plastic microfibers that shed from textiles in laundry and other processes. There is still research needed to be done regarding the entire cycle of microplastics in the environment; however, it is well- agreed upon that microplastics end up ingested and incorporated into the bodies and tissues of many organisms due to bioaccumulation and natural marine processes.
While we were designing our experiment, we asked ourselves why ocean plastic pollution was such a prevalent issue. With extensive research, we realized that seafood is an important source of food for the human population. The microplastics that are ingested by fish and other seafood are inevitably consumed by humans. There are many toxic and carcinogenic chemicals that makeup plastic, some of which are extremely harmful to the human body. Consequently, microplastic accumulation in humans has a wide range of health problems attached to it. Even among the marine population itself, microplastics are now a major concern to the marine food chain. Marine wildlife is accidentally ingesting microplastics as food. This is extremely harmful to living organisms because once ingested, microplastics can cause digestive system blockage, physical injury, altered feeding behavior, and changes to their cells. This leads to issues that affect growth and reproduction, which often leads to the disruption of an entire food chain. With all this in mind, we sought to explore novel methods of microplastic extraction from the ocean. An effective method to remove microplastics from the ocean would be meaningful to the scientific community and could potentially provide a global solution to the devastating problem of ocean microplastics.
RESEARCH QUESTION
Can Ascidian class Tunicates act as a natural and Bio-Friendly option to filter out microplastics from contaminated water?
HYPOTHESIS
During Ascidians’ respiration and feeding, they take in water through the incurrent (inhalant) siphon and expel the filtered water through the excurrent (exhalant) siphon. With this in mind, it is expected that microplastics may also be filtered out of the water during the natural feeding process of the Ascidians. We hypothesize that due to the filter-feeding attributes of Ascidian class Tunicates, Ascidians placed in microplastic contaminated water will filter the microplastics from the water.
PROCEDURE
High-level overview
Tanks containing equal amounts of fluorescent microplastics (2.5mL solution of microbeads) were set up. Three tanks were used as negative control groups and only contained the microplastics, while the other three tanks contained three Ascidians in each tank. Every day three 1mL samples were collected from each tank. This process was conducted over 4 days. Subsequent to this, the samples were viewed under a fluorescent plate reader and under a fluorescent activating microscope to measure microplastic concentrations.
A. Water tanks set up containing living Ascidians:
The tanks in which the Ascidians were housed were completely sterilized. At first, they were washed with soap and hot water, followed by a rinse and wash with boiling water to rinse off any residual soap and kill off any lasting bacteria.
12L of compliments brand distilled water was poured into each tank. Following that, 343 g of Aquavitro salinity mix was added to each tank to reach a salinity of 31 ppt.
The Aquavitro salinity solution was then completely dissolved into each tank using a stirring rod which had been sterilized with boiling water. Each tank received 2 minutes of stirring to ensure that the salinity was controlled. Following that, a hydrometer was used to ensure that the salinity was accurate and controlled amongst each experimental group.
Before adding the Ascidians to the tanks, the Ascidians were given 30 minutes to acclimate to the new tank temperature so that there would be no sudden temperature shock, which could have resulted in almost immediate death.
The tanks were kept at a controlled temperature of 20-21 degrees C.
A total of 9 Ascidians from “Gulf Marine Specimen” (Florida, USA) were carefully placed
into each of the 3 experimental tanks, labelled Group A, Group B, and Group C.
B. Fluorescent microplastics are added to each tank:
The tanks were kept in a light-controlled environment (dark) to minimize fluorescence degradation over time.
The Ascidians were fed twice a day at 7 am and 7 pm to keep them alive. The Ascidian feeding process consisted of 1mL of MarineSnow per feeding per tank. The food was added to all the tanks to maintain consistency.
The first set of water samples were collected prior to the microplastics being added to be used as a negative control.
A microplastic suspension of 2.5mL (10μm diameter, 2.5% w/v, Polystyrene Fluorescent beads from Magsphere) per 12L of water was prepared by extracting 2.5mL of the aqueous solution. This was done by using a pipette accurate to 1 μL.
The microplastic suspension was then added to the tanks and stirred for 1 minute for each tank repeating the process of sterilizing the stirring rod before and after each tank using boiling water.
C. Water samples were collected from the tanks each day to monitor the levels of microplastics
A set of water samples were then collected from each tank every day at 7 pm. The samples consisted of 1mL of water from its corresponding tank and were placed in glass vials. The samples were collected via a pipette accurate to 1μL and new sterile pipette tips were used for each collection of each tank.
All samples collected during the entirety of the experiment were kept in a climate- controlled (4 degrees Celsius) and light-controlled dark environment to ensure no bacteria grew and that the fluorescence of the microplastics was not affected.
D. The levels of fluorescent microplastics were measured using a Fluorescence Plate Reader
The samples were brought to UBC to the laboratory of Dr. Abby Collier (UBC) and measured with the assistance of Dr. Alexander Smith of the Collier Lab to measure the levels of fluorescence in the samples, as a method to measure the amount of microplastics remaining in the water.
The samples were first shaken for 15 seconds to ensure the microplastics were distributed equally.
Each sample of water was then divided into three separate samples, each having a volume of 250μL. These samples were placed on a 96 well plate to be assayed by the plate reader.
The 96 well plate was then inserted into the plate reader (FlexStation® 3 Multi-Mode Microplate Reader). The settings of which were placed at Emission: 480 nm, Excitation: 538 nm.
E. The levels and localization of fluorescent microplastics were examined using a Fluorescence Microscope
The remaining 250μL from each sample was then used to analyze the water samples under the microscope. The microscope was both used under a Fluorescent and a light setting to observe both abnormalities and the levels of fluorescence microplastics. The microscope was also used to capture photos of the water samples.
Once all the data had been collected, the data was inputted into a spreadsheet.
The Ascidians that were used during the study were frozen for later research and in part
to follow the agreement we had signed when purchasing these Ascidians.
At the lab, the Ascidians were dissected into three sections inside a biosafety cabinet. These sections of the Ascidians were placed into transparent plates and viewed under the
microscope.
Photos were taken at different parts of each section and Ascidian.
F. Disposal of Microplastics
The remaining microplastics were filtered out of the water using a 5 μm filter. The water was then poured out and then the microplastics were recycled.
MATERIALS
9 Polycarpa Circumarata Ascidians
72L of Compliments Distilled water
15 mL of MAGSPHERE® 10 μm Fluorescent Polystyrene Latex Particles
500mL of MarineSnow® Planktonic Food
1 METLERTOLEDO pipette (1 μl-1 mL)
6 32L type 5 plastic bins
96 well plate, transparent & oblique
10 cm transparent plate
FisherbrandTM box of Latex Gloves
FlexStation® 3 Multi-Mode Microplate Reader(Fluorescence Plate Reader)
ZoeTM Fluorescent Cell Imager (Fluorescence activating Microscope
VARIABLES
Independent Variable: The presence of Ascidians
Dependent Variable: The concentration of Microplastics
Controlled Variables:
Sanitization of the tanks
Number of Ascidians in each experimental group
Water temperature
Amount of food given
Light exposure
Water purity (distilled water was used)
Salinity of water
The initial starting concentration of microplastics added to each tank
Time (samples collected pre- and post- at defined intervals)
Negative Control Group: For every experiment, we included a negative control that we treated the same as the rest of the experiment, but without the independent variable. The negative control groups were fed the same amount, and at the same time as the experimental group to ensure that the food’s possible fluorescence did not interfere with the experiment’s results. There was one negative control group for every experimental group.
RESULTS
Table 1: The Mass of Microplastics in Each Water Sample (mg/tank)
Table 1 is comprised of the averages of the three samples collected from each day and each tank. The fluorescent microplastics in the water of each tank were monitored over time by measuring the amount of fluorescence in the water samples. The microbead concentrations were significantly reduced in the three tanks that contained Ascidians compared to the controls (Figure 2). The average microbead concentration in the Ascidian tanks was reduced by 24.7% after 1 day (p = 0.04). By day 4, the average microbead concentration had decreased by 94.7% (p < 0.0001) in the tanks containing the Ascidians. The negative control groups showed no significant change in microbead concentrations throughout the study, varying an average amount of 6% from start to end.
Figure 1: Change in Microplastics Concentration
As seen in the Figure 1, the controls (D, E, F) showed very little decrease in change of microplastic concentration over the course of the experiment, while the experimental group showed a steady decrease in microplastic concentration each day, resulting in a significant 94.7% overall decrease in microplastic concentration by the end of the experiment. The decrease in microplastics is almost seen to be linear. We believe that this is due to the fact that the feeding and respiratory process of the ascidians is constant (they pump water on a schedule similar to how humans eat at specific times during the day) therefore the decrease in plastics would also be constant and result in a linear graph.
Figure 2: A representation of the extraction of the microplastics through the Ascidian
Figure 2 demonstrates the filtration through the Ascidian and where the microplastics are held. This information was gathered through dissecting the Ascidians and viewing them through a Fluorescence activating microscope.
Section A represents the water that is being taken in by the Oral Siphon. This water is contaminated with Microplastics.
Section B represents the water that is ejected from the Ascidian once the extraction process is complete. The microplastics that once inhabited the water are now gone and the water is clean.
Section C represents the various parts in which the microplastics are held following the extraction. Based on our results we hypothesize that the plastic particles are absorbed by the gut into the blood or other tissues of the Ascidian.
DISCUSSION
Live Ascidians can be used as Efficient and Bio-Friendly Filters for Microplastics
Based on the results of our study, we concluded that Ascidians are an viable option to extract microplastics from ocean water. . Ascidians can efficiently filter microplastics from water through their natural feeding process. Our research demonstrates that this method of bio-filtration is a potentially effective and viable option for the extraction of microplastics in the ocean. These findings could lay the path for future research in safe microplastic extraction from the ocean using live organisms. Based on our results, we extrapolate that a 5m x 5m x 5m cage of Ascidians would filter out approximately 50kg of microplastics every day. This newfound knowledge can lead to many practical applications in the field of ocean pollution, for instance, commercially farming Ascidians for the purpose of microplastic extraction.
Study Limitations
Since we did not have access to a saltwater lab, the experiment had to be done in a household environment. Our results may have been more accurate if we had a larger tank to better simulate ocean conditions, such as varying pressure and current flow. Also, if we had more microplastics to work with, we would have created multiple microplastic suspensions to examine the effect of varying levels of plastic concentrations, which would be more representative of the real world.
Study Extensions
Regarding further extensions of our project, after conducting our experiment and reviewing our results, we realized that the possibility of extracting microplastics from water may not only be limited to Ascidians. Ascidians are part of the tunicate subphylum of filter feeders, which contains a broad range of filter feeders. We chose Ascidians for this project because the anatomy of the Ascidian appeared well-suited to the task of microplastic filtration. Indeed, we observed that the anatomy of Ascidians allowed the microplastics to be absorbed into their tissue and removed from the external environment. We suspect that other filter feeders, such as sea sponges, could also be used to filter microplastics due to the similarity in feeding mechanics. Another path for extending our project would be to expand our procedure to incorporate more Ascidians in our extraction process to see how effective it would be on a larger scale. In our experiment, we added only three solitary Ascidians in each tank because we were limited by the amount of Ascidians. Since we now know that Ascidians can be used to successfully extract microplastics from water, if we were to put a larger number of Ascidians in each tank, it could perhaps more accurately model how this process could be done on a large scale. Following these implications, serious measures should be taken to reduce and outright ban single use plastics across the world.
REAL WORLD RELEVANCE AND APPLICATIONS
Scalability and Feasibility
Ascidians are found all over the world in shallow ocean waters. This means that nearly all regions on Earth would have easy access to Ascidians. In addition to ease of access, Ascidians are able to reproduce both sexually and asexually, which contributes to them having a very fast reproductive cycle. Ascidians can produce a larva within 24 hours under prime temperature, and once in the larva stage it takes Ascidians approximately 2 days to complete metamorphosis into a juvenile Ascidian, complete with incurrent and excurrent siphons to filter out microplastics. This means that there are a lot of options and opportunities in setting up mass scale ascidian farming operations.
Real world relevance
The driving force behind this project was not only to complete an experiment with scientific accuracy, but one with scientific relevance. We decided to pursue a project on microplastic extraction because we realized that water pollution is a major worldwide problem that desperately requires innovative solutions. We felt that this project was extremely worthwhile to do not only because the conclusions would be relevant to our lives, but also because we would finish this project with a better understanding of how detrimental plastic pollution is to our lives. We believe that our findings significantly contribute to the current scientific understanding of microplastic extraction. This is because there is currently little scientific exploration into the extraction of microplastics with live organisms. With our research, there is now evidence that (1) Ascidians rapidly take up and hold onto microplastics from the water, and (2) extracting microplastics from water using an Ascidian as a bio-filter is feasible and highly effective. This new knowledge could lead to many practical applications in the field of ocean pollution. For instance, Ascidians can be commercially farmed for the purpose of microplastic extraction. These Ascidians could be used on mass scales around the oceans to filter out microplastics. Due to the small size of Ascidians, it would be possible to fit many of them in small areas. Once the Ascidians have absorbed all the microplastics, the Ascidian would be decomposed and the plastics safely recycled. This is far more superior and environmentally beneficial than allowing the microplastics to float around our oceans awaiting to be ingested by marine life and eventually humans.
ACKNOWLEDGEMENTS
We would like to express our deep gratitude to Dr. Alexander D. Smith, of the Faculty of Pharmaceutical Sciences at UBC for his assistance in measuring the water samples. We would also like to thank Dr. Michael Hart, Professor in Biological Sciences at SFU for his advice and assistance in keeping our progress on schedule.
We would also like to extend our thanks to the Collier Lab for their help in offering us the resources in running the program.
Finally, we wish to thank our parents for their support and encouragement throughout our project.
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About the authors
An enthusiastic young scientist, William (right) loves learning about biochemistry. He is an active participant in the science fair community because exploring scientific principles to apply to real-world problems is something he is passionate about. William is an active member of his school community, with leadership roles in the school Model UN Team and Newspaper Club. In his free time, he enjoys playing the piano, researching for MUN, and taking walks while listening to TED podcasts. William wishes to pursue his passion for biochemistry, with a focus on developing solutions to environmental problems.
Rowan (left) is an easily excited young scientist. He has worked on many science fairs in the past working on problems and topics that interest him. When Rowan is not working on science fairs he works at McDonald's as an assistant manager and is a very enthusiastic skier. Part of his passions is derived from his love of the environment and the well being of others. In the future, Rowan would like to follow a career in the medical field.