Environment 2270

Term Paper

Tylo Chadney
7789401
December 2016
1. Introduction
Marine debris is a growing concern around the world, threatening the health and well-

being of marine organisms in a variety of ways (Setälä et al., 2013). Plastics in particular are an

issue, and 10% of the plastic materials being produced are finding their way to the oceans (Cole

et at., 2013; Avio et al., 2015). Plastic makes up somewhere between 60-80% of all marine

debris, and accounts for 90% of the floating particles, making it the most common type of

marine litter (Setälä et al., 2014; Green, 2016). Large pieces of plastic are harmful to marine

life, and wildlife in general, with risks of being ingested and therefore causing gut blockages and

possibly obstructing food intake. They can also trap, tangle or drown wildlife (Cole et al., 2013;

Setälä et al., 2014). However, only recently have the effects of small plastic particles –

microplastics – been studied. Microplastics are defined as plastic particles with a diameter <5

mm (Cole et al., 2013; Mazurais et al., 2015; Avio et al., 2015). As Cole et al. (2013) explains,

microplastic debris either consists of small synthetic polymer products, such as those found in

cosmetics and exfoliates, or are the result of fragmentation of larger plastic materials. Once

ingested by an organism, microplastics have the potential to inhibit feeding activity, alter

growth and development, translocate into the circulatory system or become endocrine and

energy disrupters (Cole et al., 2013; Mazurais et al., 2015). Because of their small size, these

microplastics are accessible to a plethora of marine organisms and have the possibility of being

transferred up the trophic levels of the marine food web (Setälä et al., 2014). Due to their large

surface area to volume ratio and composition, microplastics also run the risk of accumulating

toxic hydrophobic chemicals from the water (Cole et al., 2013; Setälä et al., 2014; Green, 2016).

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 Studies have shown that microplastics can in fact be ingested by marine organisms such

as zooplankton (Cole et al., 2013; Setälä et al., 2014), sea bass and other species (Mazurais et

al., 2015) world-wide. As mentioned earlier, the ingestion of these particles run the risk of

clogging an organism’s digestive system, therefore affecting its ability to feed, or perhaps

translocating to the circulatory system. The chemicals which adhere to the particles may also

cause harm by being absorbed into the tissues of the organism (Cole et al., 2013; Green, 2016).

Therefore, microplastics in marine ecosystems have significant adverse effects on marine

organisms.

2. Thesis Supporting Evidence

2.1 External Adherence

Cole et al. (2013) created an experiment to determine the physical effects microplastics

have on zooplankton. To determine the impact microplastics have on the feeding ability of

copepods, the copepod Centropages typicus was exposed to algal concentrations with and

without microplastics. There were 5 concentrations of 7.3 µm fluorescent polystyrene

microbeads the copepods were exposed to; 0, 2.5, 5, 10 or 20 µL. The animals were studied

after being exposed to the solutions for 24 hours.

Cole et al., (2013) not only found microbeads within the experimental plankton species,

they also found evidence of the beads adhering to the external surfaces of the plankton. They

concentrated between the external appendages of the copepods, especially around the

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swimming legs, antennae, furca and feeding apparatus (Cole et al., 2013) (Fig. 1vii). These

findings are significant because they could potentially hinder an organism’s ability to avoid

prey, or sense and capture food themselves. Although the specific outcomes from this adhesion

were not studied in this experiment, it is a cause for concern and should be looked into more

thoroughly. Microbeads aggregating between the swimming legs of copepods could inhibit

their movement, making them more vulnerable to prey. Aggregation around the feeding

apparatus could prevent the organism from ingesting food and acquiring the energy and

nutrients necessary for survival. This could potentially cause a decrease in the copepod

population, which in turn could negatively impact the organisms higher up in the food web.

2.2 Trophic Transfer

In 2014, Setälä et al. conducted a series of experiments to investigate the ingestion and

transfer of microplastics through the planktonic food web. In order to investigate the trophic

transfer of polystyrene microspheres, mesozooplankton were incubated with 10 µm

microspheres at a concentration of 2000 spheres mL-1 in 500 mL glass bottles for 12 hours. To

determine the number of individuals from the incubation water that actually contained

microspheres, a 20 mL subsample was taken and fixed with glutaraldehyde to be later

examined under an epifluorescence microscope. After the incubation, healthy looking

copepods and Marenzelleria spp. individuals were chosen to be placed in glass 1L jars

containing particle free filtered seawater and mysid shrimp for 3 hours. The concentration of

copepods and Marenzelleria larvae in each bottle was 100 individuals/taxa L-1. Afterwards, the

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mysids were sieved out of the bottles and fixed in 4% buffered formaldehyde solution to be

dissected and examined.

The subsample from the initial incubation water showed that 43% of the copepods and 86% of

the Marenzelleria spp. contained ingested microspheres (Setälä et al., 2014). When the shrimps

from the experiment were studied more closely under the dissecting microscope, it was found

that there was one Mysis mixta and four Mysis relicta individuals. All four of the M. relicta

individuals had fluorescent microspheres inside their intestines (Table 1).

These findings are significant because they demonstrate that marine organisms can acquire

microplastics indirectly through the ingestion of their usual prey. If the particles were to

aggregate and accumulate within each organism this could lead to biomagnification up the food

web as each individual ingests the plastic particles that were previously ingested by their prey.

A biomagnification of the particles could potentially cause increased harm to the top predators.

This means that although organisms at the base of the food web may be the only ones directly

ingesting the plastic particles, the whole food web could be negatively impacted, especially if

the particles contain hazardous chemicals or toxins.

2.3 Increased Larval Mortality Rate

In 2015, Mazurais et al. conducted an experiment to study the mortality rate of larval

sea bass (Dicentrarchus labrax) when exposed to polyethylene microbeads. Larval groups were

separated into 35L tanks to be fed specific microparticulate diets with concentrations of 0

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beads (control, C), 104 (group 1X) or 105 (group 10X) beads. There were 6 replicates of each

concentration. The effect of microbead exposure on larval mortality was determined by

analyzing the trend of the number of living larvae with time, with the slope representing

instantaneous mortality rate. The effect of microbead concentration on larval mortality rate

was determined by taking into account both the concentration group and the number of

individual beads ingested per larva measured at 14, 20 or 34 days post-hatch (dph).

The results show average cumulative mortality rates at 45 dph to be 29%, 31% and 44%

in groups 1X, Control, and 10X, respectively (Fig. 2) (Mazurais et al., 2015). The instantaneous

mortality rate of group 10X was significantly higher than those of groups 1X and Control. It was

also found that an increase in the number of beads scored per larvae was related to an

increased mortality rate at 14 dph and 20 dph. The instantaneous mortality rates per bead were

increased by 21.9% and 21.2% compared to the control group at 14 dph and 20 dph,

respectively (Mazurais et al., 2015).

These findings help to highlight the negative impact microplastics have on marine

organisms. An increase in larval mortality rates can cause a variety of issues, such as a

decreased population size of the species due to pre-reproductive mortality. If larval sea bass is

a primary source of food for other species in the food web, those species could also be

negatively impacted as the sea bass population falls. However, it is highly likely that sea bass

are not the only species experiencing increased larval mortality rates.

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2.4 Increased Stress

In order to study the effects microplastics have on the respiration, filtration and growth

rates in European flat oysters (Ostrea edulis), Green (2016) conducted an experiment involving

10 L outdoor mesocosms. The mesocosms were made using intact cores collected off the coast

of Ireland, and two adult O. edulis of similar size were placed on each mesocosms. Two types of

microbeads were used; the biodegradable polylactic acid (PLA) and the common high density

polyethylene (HDPE), both at concentrations of 80 µg L-1. There were six replicates for each

type of bead, as well as one control that contained no plastic particles. After 60 days, one

random oyster was chosen from each mesocosm and their respiration rates were measured.

The results indicated that respiration rates of the oysters exposed to PLA were higher

than both the control and those exposed to HDPE (Fig. 3). Those exposed to PLA had respiration

rates ̴2.6 times greater than those exposed to HDPE (Green, 2016). This is a significant increase

and is a possible indication of stress in the oysters (Green, 2016). Any changes to the ecosystem

balance in the mesocosms due to microplastic exposure could have lead to the increased stress

in the oysters. The introduction of the microplastics have the potential to alter the behaviour or

abundance of the other organisms in the mesocosms, therefore altering the interactions

between the various species (Green, 2016). These altered interactions are a likely source of the

stress seen in the oysters.

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2.5 Absorption of Hydrophobic Pollutants

Avio et al. (2015) conducted an experiment to determine whether or not hydrophobic

pollutants in microplastics are absorbed into the tissues of marine organisms after ingestion.

The subject organism was the marine mussel Mytilus galloprovincialis. Polyethylene (PE) and

polystyrene (PS) microbeads with diameters <100 µm were mixed in a solution of pyrene at a

target concentration of 50 µg L-1. The mussels were exposed to the beads for 7 days in fifteen

6L glass-beakers, with three replicates of each treatment; control, virgin polyethylene, virgin

polystyrene, pyrene-treated polyethylene and pyrene-treated polystyrene.

When the experiment was completed, it was found that there was a significant increase

of pyrene in the gills of the organisms, and a more intense bioaccumulation was seen in the

digestive glands, with the pyrene levels in the tissues increased by more than 13 times

compared to the controls (Fig. 4) (Avio et al., 2015). The concentration of pyrene in the

digestive tissue was almost 3 times higher than those found on the contaminated microbeads.

This is an indication of a bioaccumulation of the pyrene in the tissues of the mussels (Avio et al.,

2015). Using a Weight Of Evidence (WOE) approach, Avio et al. were able to rate the hazards

the virgin and contaminated plastics posed to M. galloproviancialis in terms of cellular effects

and bioaccumulation. The virgin PE and PS beads were classified as Slight and Moderate, while

the contaminated PE and PS polymers were Major and Severe, respectively.

Bioaccumulation of hazardous materials in an organism is significant because it

increases the risk of genotoxic effects and cellular perturbations within the organism (Avio et

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al., 2015). Bioaccumulation within organisms could also lead to a biomagnification up the food

web as individuals contaminated with toxic substances are preyed upon by other organisms.

3. Thesis Refuting Evidence

3.1 Selective Feeding

During their experiment concerning microplastic ingestion by zooplankton, Cole et al.,

(2013) studied the uptake of fluorescent polystyrene microbeads by a variety of zooplankton

taxa. 20 µL of microbeads with diameters 7.3, 20.6 or 30.6 µm were added 20 mL of filtered

seawater. The larger species were added directly to the glass vials containing the solutions for

24 hours, while smaller organisms with shorter life spans were exposed to the suspensions in

petri dishes for 1 hour. The organisms were studied by a light microscope with fluorescence

post-exposure to determine the uptake of the microbeads. Specific taxa were also placed in

solutions containing both natural algal prey and microbeads to determine the ways in which the

beads alter the copepods feeding abilities.

The investigation saw signs of selective feeding in a myriad of zooplankton taxa. When

presented with algae and microbeads with a diameter of 20.6 µm, at least three of the

zooplankton taxa in the experiment preferentially fed on the algae over the microbeads. This

suggests that the plankton can tell the difference between the algae and the unnatural plastic

particles (Cole et al., 2013). Some zooplankton can also reject prey after ingestion if the

characteristics or electric charge of the particles do not agree with the preying organism. Taking

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these facts into account, it is reasonable to believe that microplastics in the marine

environment are not harmful to zooplankton, because the plankton are not ingesting them.

This also reduces the possibility of microplastics being transferred through the food web. If the

base of the web – the plankton – can avoid ingesting the beads, the species that prey on those

plankton will not be ingesting beads when they consume the plankton.

3.2 Concentration Dependence

Setälä et al. conducted an experiment in 2014 to study the ingestion of microplastic

particles by various mesozooplankton taxa. The organisms were separated by taxa and placed

into solutions of seawater with 3 different concentrations of 10 µm fluorescent polystyrene

spheres (A = 1000, B = 2000, C = 10 000 particles mL-1). There was also one mixed

mesozooplankton test group. After an incubation time of 3 hours the animals were washed

onto petri dishes to be evaluated. The ones chosen for microscopy analysis were fixed with 25%

glutaraldehyde and placed onto object slides or onto Utermöhl settling chambers to be

investigated with an inverted epifluorescence microscope under blue excitation light.

The results of the experiment showed that ingestion of the microspheres varied

between taxa, and that in multiple cases certain taxa did not ingest any spheres (Table 1)

(Setälä et al., 2014). In concentration B, the copepod L. macrurus did not ingest any particles,

and the rotifers Synchaeta spp. did not ingest particles in concentration A. These findings are

significant because if organisms are not ingesting any plastic particles, there is no chance of any

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particles accumulating in their stomach or causing gut blockages. This also limits the possibility

of toxic chemicals adhered to the particles negatively affecting the organism.

3.3 Gene Expression

While performing their experiment regarding larval sea bass mortality rates, Mazurais et

al. (2015) also investigated the effects polyethylene microbeads have on the gene Interleukin-1

beta (IL-1β) and its expression in sea bass larvae. IL-1β is an important mediator of the

inflammatory response, which is believed to be impacted in other species when exposed to

microplastics (Mazurais et al., 2015). In order to assess any potential alteration to the gene,

RNA was extracted from random individuals in each group at 27 and 43 dph and the target gene

transcript was studied.

The results indicate that no significant change in the expression of the gene IL-1β was

detected from any group, at any point during the experiment (Mazurais et al., 2015). These

findings are significant because they suggest that microplastics do not alter or harm individuals

at the molecular level during early development. Alteration at the molecular level could

potentially do irreversible damage to individuals in a population and any offspring they may

later produce. This could eventually lead to a large part of a species or population being unable

to function in a normal, life-sustaining manner. However, if gene alteration is not occurring

during one of the most influential time periods in an organism’s life, it is possible that

microplastics are not as harmful as originally believed.

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3.4 Macrofaunal Abundance

During her experiment relating to the respiration, filtration and growth of oysters when

exposed to microplastics, Green (2016) also studied the effects plastics have on macrofaunal

abundance. The study was held in the same mesocosms at the same time as the oyster

experiment. The mesocosms were exposed to 80 µg L-1 of PLA or HDPE microbeads a day for 60

days. At the end of the experiment, 26 macrofaunal taxa inhabited the mesocosms, but only 15

were abundant enough to run univariate analyses. Of these 15 taxa, 11 were not significantly

affected by exposure to the microplastics. This means that approximately three quarters of the

macrofaunal population was relatively unaffected by the plastics (Fig. 5) (Green, 2016).

These stats are significant because it demonstrates that select taxa and/or species are

more affected by microplastics than others, and that few species are affected in general. One of

the major concerns about microplastics in marine ecosystems is the negative impact they may

have on fish populations. The less impact the plastics have on the organisms lower down in the

food web – such as macrofaunal species – the less impacted the larger fish species will be.

3.5 Unaltered Phagocytosis and Acetylcholinesterase Activity

While determining whether toxic chemicals are absorbed into the tissues of an organism

from ingested microplastic particles, Avio et al. (2015) also studied the effects these chemicals

may have on the organism on a cellular level once absorbed. One of the immunological

responses looked at was phagocytosis activity in haemocytes of the gills and digestive tissues of

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the mussel Mytilus galloprovincialis. Acetylcholinesterase (AChE) in haemocytes and gills was

also studied. The activity of both were examined after exposure to one of the following five

treatments after 7 days; control, virgin polyethylene, virgin polystyrene, pyrene-treated

polyethylene and pyrene-treated polystyrene. The contaminated beads were prepared by

mixing a solution of pyrene (50 µg L-1) and microplastics with a diameter <100 µm for 6 days.

After the exposure, it has been determined that the phagocytosis activity did not vary in

a significant manner, and AChE in the haemolymph stayed relatively stable across all

treatments (Fig. 6) (Avio et al., 2015). Phagocytosis is a process important to immunological

activity, so this is an important discovery. If hazardous chemicals absorbed by the tissues of an

organism from contaminated plastic particles affected the phagocytotic abilities of the

organism, the capabilities of that animal to protect itself from disease and foreign bodies would

be greatly diminished. This could result in an increased mortality rate of that species, which

could then alter the balance of the food web and affect other species as well. Knowing that

phagocytosis and acetylcholinesterase in mussels are relatively unaffected by the introduction

of pyrene through polyethylene and polystyrene microbeads reduces the risk of increased

mortality resulting from microbeads in the marine environment.

4. Conclusions

Microplastics in the oceans are a growing concern, and the more scientists learn about

their impacts on marine organisms, the more valid these concerns become. These five studies

highlight a few of the ways microplastics affect marine organisms, such as adhering externally

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to feeding appendages, being transferred up the trophic levels of the food web, and acting as a

pathway for toxic hydrophobic substances to be absorbed into the tissues of the organism.

They also draw attention to the ways in which the microplastics are harmless. In specific species

ingestion can be avoided, and they may not alter gene expression or basic cellular functions as

many people fear.

However, although there is evidence of microplastics having no effect on certain

functions in more than one species, the adverse effects are undeniable. They can impact the

entire food web through direct ingestion, as well as indirectly through trophic transfer up the

food web. The particles also act as carriers for toxic chemicals that may have their own unique

effects on the functioning of the organism.

It has been demonstrated in more than one study that an increase in the concentration

of microplastic particles in an ecosystem has a direct relationship to the amount and rate at

which the particles are ingested. With the majority of microplastics being the result of

fragmentation of larger plastic pieces, it is extremely likely that the microplastic concentration

in the oceans will only increase with time. This will lead to an increase in plastics being ingested

by marine organisms and their effects on the ecosystem will be greater as time goes on.

Microplastics have a significant adverse effect on marine organisms, and these effects will only

get worse as more plastics enter the worlds oceans.

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5. Tables and Figures

Figure 1: Microplastics of different sizes ingested, egested and adhered to different plankton,
visualized using fluorescence microscopy. Cole et al. 2013.

Table 1: Percentage of organisms with ingested microspheres after incubations in 3

microsphere concentrations (A=1000, B=2000, C=10 000 microspheres mL1-)
D = food web transfer with copepods and Marenzelleria spp. larvae. Incubation 3
hours. Setälä et al., 2013.

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Figure 2: Effect of exposure to polyethylene microbeads on the instantaneous mortality rates of European sea
bass larvae. Mazurais et al. 2015.

Figure 3: Mean respiration of oysters exposed to doses of either biodegradable

(PLA) or conventional (HDPE) microplastics or no microplastics (Control).
Green, 2016.

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 Figure 4: Concentrations of pyrene in gills and digestive glands on mussels exposed to various microplastic
treatments; control, virgin polyethylene, virgin polystyrene, pyrene-treated polyethylene and pyrene-treated
polystyrene. Avio et al. 2014

Figure 5: Mean number of taxa in mesocosms exposed to low or high doses of either biodegradable
(PLA) or conventional (HDPE) microplastics or to no microplastics. Green, 2016.

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Figure 6: Immunological and cholinesterasic biomarkers in mussels exposed to various
microplastic treatments; control, virgin polyethylene, virgin polystyrene, pyrene-treated
polyethylene and pyrene-treated polystyrene. Avio et al. 2015

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6. References

1. Cole M, Lindeque P, Fileman E, Halsband C, Goodhead R, Moger J, Galloway TS. 2013.

Microplastic ingestion by zooplankton. Environmental Science and Technology, 47; 6646-6655.

2. Setälä O, Fleming-Lehtinen V, Lehtiniemi M. 2014. Ingestion and transfer of microplastics in

the planktonic food web. Environmental Pollution, 185; 77-83

3. Mazurais D, Ernande B, Quazuguel P, Severe A, Huelvan C, Madec L, Mouchel O, Soudant P,

Robbens J, Huvet A, Zambonino-Infante J. 2015. Evaluation of the impact of polyethylene

microbeads ingestion in European sea bass (Dicentrarchus labrax) larvae. Marine Environmental

Research, 112; 78-85

4. Green DS. 2016. Effects of microplastics on European flat oysters, Ostrea edulis and their

associated benthic communities. Environmental Pollution, 216; 95-103.

5. Avio CG, Gorbi S, Massimo M, Benedetti M, Fattorini D, d’Errico G, Pauletto M, Bargelloni L,

Regoli F. 2015. Pollutants bioavailability and toxicological risk from microplastics to marine

mussels. Environmental Pollution, 198; 211-222.

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