Lauren McGrath

Age 19 | Saint John, NB

Canada-Wide Science Fair 2019 Finalist

Introduction

Plastic is an incredibly versatile material. It has revolutionized various industries and helped billions of people live far more convenient lives. From an economic standpoint, the production of plastic was an incredible success. Since the 1950s, it is estimated that over 8.3 billion tonnes of virgin plastics have been produced (UN Environment, n.d.). However, a lack of foresight has left the world with environmental issues. Over 5.25 trillion plastic particles, measuring around 260 000 tonnes, are floating in the world's oceans (Eriksen et al., 2014). Over time, organisms accumulate microplastics through consumption or absorption (Devriese et al., 2017). Much of the microplastics in marine organisms that are consumed by humans are contained in the gut tissue, which is not habitually consumed. However, marine organisms such as bivalves are consumed whole, and the levels of microplastics in bivalves designated for human consumption have not been studied in the Maritimes. According to Fisheries and Oceans Canada, the Maritime region accounted for 757 million dollars of Canada’s total landed fisheries value in 2012 (Fisheries and Oceans, 2019). Considering that fisheries are a vital industry in coastal areas, information on microplastic levels in seafood marketed for consumption is important for developing strategies to limit this pollution (Fisheries and Oceans, 2019). Fisheries and Oceans Canada recommends investigating and comparing the levels of microplastic pollution in two of the most consumed bivalves, blue mussels (Mytilus edulis) and eastern oysters (Crassostrea virginica), to quantify the problem (2019).

Microplastics

Microplastics are generally defined as particles of plastic less than 5 mm in diameter. These particles can be categorized as either primary or secondary. Primary microplastics are those produced at a microplastic size, such as microbeads. Due to their environmental threats, toiletries that contain microbeads cannot be legally imported, produced, or sold in Canada as of July 1st 2018 (Government of Canada, 2018). Canada joins many other countries in their efforts to reduce primary plastic reduction; however, this reduction is only predicted to reduce microplastic pollution in oceans by 1-2% by 2025 (Government of Canada, 2018); this may be because secondary microplastics, microfibers, form over time from various sources of plastic materials like clothing and fishing line. Microfibers are the largest contributor to microplastic pollution (Government of Canada, 2018).

Microplastics’ Ability to Harbour Pollutants. Plastics generally experience surface weathering over time. Researchers (Bond et al., 2018; Da Costa et al., 2018; Weinstein et al., 2016) examined plastic degradation to understand how this affects plastic’s ability to harbour contaminants. With time, a “diffusion-based process” will eventually disintegrate plastic completely (Bond et al., 2018). In a study conducted in saltwater marshes on the types of degradation experienced by plastics in a saltwater environment, it was found that the process of degradation happens extremely quickly in saltwater marshes compared to air and freshwater environments (Weinstein et al., 2016).

Both biotic and abiotic factors lead to the production of secondary microplastics; hydration and dehydration will mechanically degrade the plastics whereas microbes will degrade them enzymatically (Weinstein et al., 2016). In a literature review by Bond et al. (2018), it is suggested that as plastics degrade, their surface area is increased by both mechanical and chemical degradation. Weinstein (2016) estimated that from a 4 g polystyrene plate, more than 46 000 microplastic particles would be produced if allowed to fully degrade. It is commonly hypothesized that this increase in surface area leads to a greater uptake of biological pollutants, and that this uptake will affect the organisms living in the area. Contrarily, an analytical chemistry review from the University of Aveiro claims “contamination, i.e., the presence of alien elements, is not a synonym of pollution, meaning that these alien elements exert biological effects on the biomes of affected habitats” (Silva et al., 2018). This statement is valid considering that these bio-contaminants are already in the water column, and the only added effect the microplastics have is an increase in concentration of these contaminants (Da Costa et al., 2016).

Stress of Microplastics on Marine Organisms. The effects of microplastics on marine organisms have been studied in terms of their presence as foreign objects and the intake of contaminants alongside the microplastics. In one study, Devriese et al. (2017) exposed microbeads to polychlorinated biphenyls (known as PCBs, which are long-lasting chemicals that are difficult to destroy) and then fed them to lobsters to test how this affected the lobsters’ nutritional levels. The authors found that the microplastics did not affect the nutritional levels of the lobster, but recommended using smaller microplastics, since it has been previously shown that microplastics can cross cellular membranes if small enough (Devriese et al., 2017; Kershaw et al., 2015). If these membrane-permeable microplastics were contaminated, it is possible that this contamination could disrupt chemical balances within the cells. While Devriese et al. (2017) only looked at the impact of contaminated microplastics on nutrition levels, Magara et al. (2018) completed a study on how both the physical presence and biological contamination affected the blue mussel (Mytilus edulis). The authors found that the addition of the bio-contaminants did not have any significant effect on any aspect of the blue mussels; however, the presence of the microplastics consistently caused an increase in the oxidative stress levels (Magara et al., 2018). Bour et al. (2018) observed a marked decrease in energy reserves of two bivalve species, Ennucula tenuis and Abra nitida, when exposed to microplastics. They compared their study to a similar one on blue mussels, and this study found that there was no impact on the energy reserves of the mussels (Ribero et al., 2017, as cited in Bour et al., 2018). Bour et al. concluded that the discrepancy stems from the exposure time, as Ribero et al.’s study did not have a very long exposure time (2017, as cited in Bour et al., 2018), whereas their exposure time lasted for four weeks (Bour et al., 2018).

Microplastics in bivalves labelled for consumption

The small size and extensive presence of microplastics allows them to penetrate many different organisms commonly consumed by humans. In the South Pacific, a study was conducted on fish which would be sold for consumption, and it was determined that within the guts of these fish, up to 10 pieces of microplastics were found (Rochman et al., 2015). In a Halifax-based study, farmed mussels were compared to wild mussels, and it was found that the farmed mussels had a higher average of microplastic compared to the wild mussels (Mathalon & Hill, 2014).

Discrepancy and Hypothesis

After reviewing the literature about microplastics and their permeation into marine animals, it is evident that there is a lack of research based in the Southern Gulf of St. Lawrence area on microplastics in bivalves designated for human consumption. This study aimed to investigate and compare the levels of microplastics in blue mussels (Mytilus edulis) and eastern oysters (Crassostrea virginica), two bivalve species, sourced from the Southern Gulf of St. Lawrence which were being sold for consumption. Based on the data from Mathalon and Hill (2014), where they found approximately 100 pieces of microplastics in group samples of five blue mussels, as well as the known similarities between the feeding and filtering systems in mussels and oysters, the alternative hypothesis in the current study is that microplastics will be found in every sample, and that there will not be a significant difference in microplastic numbers between the two bivalve species. Therefore, the null hypothesis is that there will be a statistically significant difference in microplastic numbers between the two species and that not all samples will have microplastics.

Methods

Experimental Approach

A quantitative explanatory approach aligns with the purpose of this experiment, as a quantitative experimental determination is necessary to establish the numeric levels of microplastics in the shellfish. Quantitative data aligns with work done in 2015 and 2014 by researchers such as Rochman et al. (2015) and Mathalon & Hill (2014).

The approach also aligns with a study by Mathalon & Hill (2014), which compared microplastic levels in wild mussels and farmed mussels. The researchers used a 30% H2O2 solution to digest the organic material, leaving the microplastics behind (Mathalon & Hill, 2014). However, the H2O2 was determined to be ineffective in completely digesting the organic material, leading to inaccuracies with their results (Mathalon & Hill, 2014). One aspect of Mathalon & Hill’s (2014) methods that was replicated in this experiment was their avoidance of microplastic contamination; any fibers of the same colour as their lab coats was discounted, and two glass petri dishes were observed for microplastic contamination from the air and the lab water; this helped to prevent overestimations of microplastic counts within the mussels (Mathalon & Hill, 2014).

Similar research was conducted on larger organisms by Rochman et al. (2015), looking at microplastic levels in fish. For this experiment, a solution of KOH was used to dissolve the tougher tissue of the fish (Rochman et al., 2015). The use of KOH instead of H2O2 was replicated in this experiment because of its ability to fully dissolve dense tissue (Rochman et al., 2015). Additionally, Kolandhasamy et al. (2017) inspired our use of an incubator, used to shorten the digestion time, and our use of a nitrocellulose filter for the KOH solution.

Animal Collection

Two species of bivalves were used to see if the microplastic uptake differed between similar bivalves. The microplastic levels in blue mussels (Mytilus edulis) and eastern oysters (Crassostrea virginica) sourced from the Southern Gulf of St. Lawrence and being sold for human consumption were quantified and compared. Shellfish type was varied based on availability for purchase in the area surrounding Saint John New Brunswick, a major urban center at the base of the Southern Gulf of St. Lawrence region, as well as statistics from Fisheries and Oceans Canada on the most commonly consumed shellfish (2019). The distributers of the shellfish were contacted to approximate growing location. The stores were varied to ensure the results in the study were not a result of different care practices at the individual stores. Ten eastern oysters were purchased on January 19th, 2019. Five were bought at Sobeys and came from Neguac, New Brunswick, and the other five were purchased at North Market Seafood and were grown in Prince Edward Island. Ten blue mussels were also purchased the same day, five from Sobeys and five from North Market Seafood. Both were grown by Confederation Cove in Prince Edward Island.

Figure 1. Filtration Set-up. Note. Solutions containing digested organic material and remaining microplastics were filtered using a nitrocellulose filter and a Buchner funnel system. — **Figure 1. Filtration Set-up.** *Note*. Solutions containing digested organic material and remaining microplastics were filtered using a nitrocellulose filter and a Buchner funnel system.

Procedure

Before the experiment, a petri dish with a filter soaked in water was placed adjacent to the work area.

To quantify the plastic particles within the shellfish, it was necessary to digest the organic material using a strong alkaline solution. In preparation to make the 10% KOH solution, a lab coat and gloves were worn. Under the fume hood, solid KOH was combined with water in a 500 mL volumetric flask to make the 10% KOH solution. The amount of water or KOH needed was dependent on the size of the trial and was calculated using the following equation:

m = (1.783 M) (V) (56.10 g/mol) (1)

The trials pooled the bivalve tissue based on species and then based on the retailer, leaving four groups: blue mussels from Sobeys, blue mussels from North Market Seafood, eastern oysters from Sobeys, and eastern oysters from North Market Seafood.

One individual from each group was used to test the procedure to ensure the equipment worked effectively. The results from this trial were not included since there were no precautions taken to reduce contamination. The individuals were the largest outliers in size, so that all other shellfish were approximately the same size.

The internal tissues from the bivalves were removed from the shells using a knife and dissection equipment, and the tissue of each organism was placed one at a time in separate beakers. The 10% KOH solution was then poured so it covered all organic material in each beaker. Immediately, to avoid any microplastic contamination from the air, the beakers were placed in an incubator at 65 °C.

After all organic material was digested over approximately 24 h, the beakers were removed from the incubator and placed in a fridge for storage. One at a time, the solutions were removed from the fridge and filtered through a nitrocellulose filter using a Buchner funnel system. For each sample, a filter was placed on the Buchner funnel using forceps and the suction pump was turned on. The filter was sealed to the funnel using water to ensure there were no holes exposed. Then, the KOH solution was poured over the filter. This set-up is demonstrated in figure 1. The filtration part of the procedure was sometimes completed multiple times per sample if the filter took too long, or if the filter was not sealed properly to the funnel. In this case, the filtrate was kept instead of being disposed of and was re-filtered using the same technique. After the solution had filtered through the paper, the filter paper was lifted off the Buchner system using forceps. The filter paper was then put into a sealed petri dish, examined under a dissection microscope at 30x magnification, and the microplastics in the residue were counted, and categorized by approximate size (long, medium, short), possible origin, and colour (see figure 2). All fibers of the colour of the lab coats worn were discounted and the petri dish which was put out at the beginning of the procedure was inspected using dissection equipment and a microscope for microplastics.

Figure 2. Microscopic View of a Microfiber. Note. A secondary microplastic (grey) seen under a dissection microscope at 30x magnification. — **Figure 2. Microscopic View of a Microfiber.** *Note*. A secondary microplastic (grey) seen under a dissection microscope at 30x magnification.

Statistical Analysis

The independent dichotomous variable in this study was shellfish species, and the dependent variable was the count/length of microplastic particles in the organism. The independent variable is categorical data, the relative length is ordinal data, and the count of microplastics is considered ordinal. The microplastic particles were categorized according to colour and relative length.

A power analysis was completed on this study to show the value of the results. Parametric statistics could not be completed due to the small sample size, but all appropriate non-parametric statistical tests were completed. Since the independent variable was categorical and the dependant variable was considered ordinal and on a discrete scale, Spearman rank correlations and two-tailed Mann-Whitney U tests were completed.

The Spearman rank correlation was completed to show the level of association between several variables. It was run between all the individual colours, between the relative lengths, and between each colour and each length. Cohen’s standard was used to evaluate the strength of the relationships, where coefficients between 0.10 and 0.29 represent a small effect size, coefficients between 0.30 and 0.49 represent a moderate effect size, and coefficients above 0.50 indicate a large effect size (Cohen, 1988).

A two-tailed Mann-Whitney U test is a non-parametric test used to assess significant differences in the ordinal dependent variable by a single dichotomous independent variable. The tests use the mean ranks in each group to compute the U statistic, which is used to compute the p value. The p value determines the level of significance. A significant test result would mean that the groups have reliably different scores on the dependant variable.

Results

The average number of microplastics per mussel and oyster sample are presented below, sorted by both colour and relative size. Bar graphs (figures 3 and 4) show exact enumeration of the data for better visualization. Figures 5 and 6 compare the averages between the mussels and oysters. These figures are necessary for comparing the microplastics between the two organisms. Microplastics were found in each shellfish, with a total of 79 microplastics being found in the eight mussels and 70 found in the eight oysters.

Figure 3. Microplastics by Colour in Oyster Samples. Note. Total number of microplastics enumerated from eight oyster samples sorted by colour. — **Figure 3. Microplastics by Colour in Oyster Samples.** *Note*. Total number of microplastics enumerated from eight oyster samples sorted by colour.

Figure 4. Microplastics by Colour in Mussel Samples. Note. Total number of microplastics enumerated from eight mussel samples sorted by colour. — **Figure 4. Microplastics by Colour in Mussel Samples.** *Note*. Total number of microplastics enumerated from eight mussel samples sorted by colour.

Figure 5. Average Number of Microplastics by Colour. Note. Average number of microplastics per colour in mussels and oysters. — **Figure 5. Average Number of Microplastics by Colour.** *Note*. Average number of microplastics per colour in mussels and oysters.

Figure 6. Average Number of Microplastics by Relative Size. Note. Average number of microplastics sorted by relative size in mussels and oysters. — **Figure 6. Average Number of Microplastics by Relative Size.** *Note*. Average number of microplastics sorted by relative size in mussels and oysters.

A significant positive correlation was observed between black and blue microplastics (rs = 0.58, p = 0.019). Similar results were observed for almost all other Spearman correlations, which were ran between individual colours and colours versus length. The result of the two-tailed Mann-Whitney U test for black was not significant (U = 19.5, p = 0.179). The mean rank for Mussel group was 6.94 and the mean rank for Oyster group was 10.06. All other Mann-Whitney rank sum tests were found to have non-significant results. These tests were run based on colour and based on origin. The Mann-Whitney rank sum tests which were completed based on origin of the sample showed non-significant results, so there were also non-significant differences in microplastic levels between the origins.

Discussion

This study quantified the microplastic amounts in two types of shellfish commonly consumed in the Southern Gulf of St. Lawrence area, the blue mussel (Mytilus edulis) and the eastern oyster (Crassostrea virginica). Microplastics were counted and categorized based on colour and relative size, and these results were compared between the two types of shellfish. Microplastics were found in each individual, with a total of 79 microplastics being found in the eight mussels and 70 found in the eight oysters. This allows the null hypothesis to be rejected and the alternative hypothesis is accepted.

A power analysis included Spearman correlations and two-tailed Mann Whitney U rank sum tests. Microplastics were found in every sample, and the Spearman correlations were positive between every colour and every relative length run. The positive correlation shows that it is likely that there will be multiple microplastics of various colours and lengths within each sample if one is found. Mann-Whitney rank sum tests were also completed. These tests compared the amount of microplastics of each colour per species, and per origin. It was determined that there were no statistically significant differences between mussels and oysters for any colour, and there was also no significant difference between North Seafood Market or Sobeys for any colour. This shows that likely, the mussels and oysters were exposed to similar amounts of microplastics and they will absorb similar amounts of microplastics. This absorption could be through feeding, or as observed in a paper by Kolandhasamy et al. (2017), through the uptake of microplastics directly through the tissues. Additionally, there is likely no contamination based on distributer, showing that the majority of microplastic contamination is likely encountered in the environment where these mussels were cultivated (for this paper, in Prince Edward Island and in Neguac, New Brunswick). All microplastics found in this study were microfibers, meaning they came from various sources that have broken down in the environment over time.

Limitations and Future Directions

A limitation of this study is the small sample size which prevented us in using parametric tests. Additionally, the lack of quality equipment would likely lead to an underestimation of plastic particles rather than an overestimation. Better filtration systems and more powerful microscopes would most likely have yielded more similar results to those in other studies such as in Mathalon & Hill (2014), with microplastic numbers in five mussels upwards of 100.

To investigate the plastic further, an infrared (IR) spectrometer could be used to identify the type of plastic. Information on the type of plastic could be used to estimate the origins of the microplastics. Knowing the origins of the microplastics would help provide specific recommendations for limiting plastic pollution.

Quantifying microplastics in shellfish is an important aspect in learning how to protect their ecosystem, in addition to estimating the risk of consuming microplastics in higher level trophic organisms. A safe level of these particles should be determined for consumption so that the fishing industry can be regulated to maintain the health of those who consume shellfish and so that microplastic producers can be advised on how to limit this pollution. The study by Kolandhasamy et al. (2017) noted that allowing shellfish to sit in clean, plastic free saltwater can reduce the amount of microplastics and contaminants by up to 70%, and some shellfish production companies have adopted this practice (however, it is not well regulated or required in Canada). Since all the microplastics found in this study were microfibers, and common microfiber polluters are from the fishing industry and clothing fibers from wastewater treatment plants (Government of Canada, 2018), this study serves as an incentive for companies and organizations to work on developing more sustainable ways of producing fishing lines as well as improving the filtration systems in wastewater treatment plants.

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