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Trophic transfer of microplastics does not affect fish personality
Louise Tosetto
*
, Jane E. Williamson, Culum Brown
Department of Biological Sciences, Macquarie University, North Ryde, NSW, Australia
article info
Article history:
Received 25 May 2016
Initial acceptance 5 August 2016
Final acceptance 6 October 2016
MS. number: 16-00465R
Keywords:
Bathygobius krefftii
beach hopper
behaviour
food web
microplastic
plastic pollution
Microplastics are ubiquitous in the marine environment. Their small size makes them bioavailable to a
range of organisms and studies have reported ingestion across the food chain. Few studies have
demonstrated physical transfer of microplastics between organisms, and no research has assessed the
ecological impacts of transferred microplastics and contaminants over different trophic levels. Con-
taminants associated with plastics can alter animal behaviour; thus, exploring changes in behaviour may
be fundamental in understanding ecosystem effects of microplastics. This study explored the effects of
microplastics and associated contaminants through the food chain in the marine intertidal zone. We
exposed beach hoppers, Platorchestia smithi, to environmentally relevant concentrations of microplastics
and then fed them to Krefft's frillgobies, Bathygobius krefftii,ray-finned fish that inhabit shallow coastal
ecosystems. We tested fish personality to see whether there were any changes that could be attributed to
trophic transfer of microplastics, as even subtle changes in behaviour can have cascading effects on other
organisms and the wider ecosystem. Exploring behavioural changes in response to contaminant expo-
sure is a developing area in ecotoxicology due to its increased sensitivity compared with the traditional
LD50 approach. While gobies readily ingested contaminated beach hoppers, we detected no effect of
microplastic trophic transfer on fish personality relative to control groups. While chronic exposure
studies assessing a suite of behaviours are required, it is possible that the transfer of microplastics via
trophic interactions does not provide an additional exposure pathway for contaminants through the food
web.
©2016 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Food webs and trophic interactions are essential for the transfer
of energy and nutrients throughout ecosystems. Urbanization,
development and industry have influenced pollutants in aquatic
systems, with chemicals, metals, pesticides and pharmaceuticals all
presenting risks to ecosystems and associated biota (von Glasow
et al., 2013; Islam &Tanaka, 2004). Contaminants can accumulate
and concentrate in biota (bioaccumulation) and then subsequently
transfer through the food web (Borgå, Fisk, Hoekstra, &Muir, 2004;
Nfon, Cousins, &Broman, 2008). These pollutants can impact ani-
mal physiology (Hontela, 1998) and can also alter behaviours
important for foraging, reproduction, social interactions and anti-
predator behaviour (Clotfelter, Bell, &Levering, 2004; S€
offker &
Tyler, 2012).
Plastic is a contemporary pollutant in our marine environment.
While the deleterious effects of large plastic debris on marine life
have received much publicity (Barnes, Galgani, Thompson, &
Barlaz, 2009), microplastics are of increasing concern (Cole,
Lindeque, Halsband, &Galloway, 2011). Their small size and vari-
able densities mean they occur throughout marine (Law &
Thompson, 2014) and freshwater (Eerkes-Medrano, Thompson, &
Aldridge, 2015) environments and are bioavailable to a broad
range of organisms (Thompson et al., 2004). Microplastics present a
physical and chemical risk to organisms. (Rochman, 2013). Poten-
tially toxic additives such as phthalates, bisphenol A (BPA) and
flame-retardants are added to many plastics at manufacture to
increase functionality and extend their life (Browne, Galloway, &
Thompson, 2008; Rochman, 2013). Furthermore, plastics are
porous and accumulate and concentrate contaminants including
polychlorinated biphenyls (PCBs), pesticides and fertilizers at high
intensities from the surrounding sea water (Mato et al., 2001).
Many of the additives and the absorbed contaminants are known
endocrine disruptors, carcinogens and mutagens (Lithner,
Damberg, Dave, &Larsson, 2009) and it is thought that contami-
nants may transfer from plastics to organisms following ingestion
(Teuten, Rowland, Galloway, &Thompson, 2007; Teuten et al.,
2009).
Many of the contaminants associated with plastics such as BPA
and PCBs can affect foraging efficiency, alter schooling behaviour or
*Correspondence: L. Tosetto, Department of Biological Sciences, Macquarie
University, North Ryde, NSW 2109, Australia.
E-mail address: louise.tosetto@mq.edu.au (L. Tosetto).
Contents lists available at ScienceDirect
Animal Behaviour
journal homepage: www.elsevier.com/locate/anbehav
http://dx.doi.org/10.1016/j.anbehav.2016.10.035
0003-3472/©2016 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Animal Behaviour 123 (2017) 159e167
increase agonistic encounters in fish (Clotfelter et al., 2004; Sloman
&Wilson, 2006). Complex behaviours such as foraging, predator
avoidance and social interactions are all fundamental to individual
fitness and an animal's functioning in an ecosystem (Wolf &
Weissing, 2012). Changes in behaviour may take effect even with
small quantities of pollutants (Bae &Park, 2014), and while changes
in animal health may not be apparent, these behavioural alterations
may affect how the animal performs in an ecological context (Scott
&Sloman, 2004). Behavioural responses provide useful markers of
pollution effects on individuals, potentially performing as reliable
and economical indicators of sublethal effects of pollutants (Weis,
2014). Accordingly, there is growing emphasis in ecotoxicology on
examining changes in behaviour in response to exposure to con-
taminants (Oulton, Taylor, Hose, &Brown, 2014).
Microplastics can be ingested by organisms across the marine
environment (Fig. 1). A range of studies have assessed effects of
plastic consumption at the individual organism level but few have
assessed the capacity for microplastics and associated contami-
nants to move through the food chain. Our understanding of how
pollutants can bioaccumulate (Borgå et al., 2004) suggest micro-
plastics provide an additional exposure pathway for contaminants
to transfer through the food web. The physical transfer of micro-
plastic fragments between organisms has been demonstrated from
mussels, Mytilus edulis, to crabs, Carcinus maenas (Farrell &Nelson,
2013) and mesozooplankton to macrozooplankton (Set€
al€
a,
Fleming-Lehtinen, &Lehtiniemi, 2014;Fig. 1). Recently, the trans-
fer of microplastic fragments as well as contaminants was
demonstrated in an artificial food chain from Artemia nauplii to the
intestinal tract of laboratory-raised zebrafish, Danio rerio. The study
used high concentrations of microplastics spiked with large
amounts of benzo(a)pyrene (BaP) and there was no assessment of
biological consequences for either Artemia or fish (Batel, Linti,
Scherer, Erdinger, &Braunbeck, 2016). No studies have assessed
the biological effects of a microplastic-contaminated diet on higher
trophic levels in an ecologically relevant setting. Exposing animals
to more environmentally relevant concentrations and evaluating
behaviours in relation to an animal's habitat is important in
providing realistic insights to the risks our marine ecosystems face.
We established a model food web to assess the effects of
contaminated microplastics via trophic transfer. As microplastics
accumulate on shorelines (Set€
al€
a, Norkko, &Lehtiniemi, 2016),
coastal biota are exposed to them. Coastal ecosystems are often
dominated by small crustaceans such as talitrid amphipods. In this
study, coastal talitrids (beach hoppers, Platorchestia smithi), pri-
mary consumers that inhabit the sediment, were exposed to
environmentally relevant concentrations of naturally contaminated
microplastics and then fed to Krefft's frillgobies, Bathygobius krefftii,
teleost fish that inhabit shallow coastal ecosystems. Beach hoppers
Seabirds
(Derraik, 2002,
Hammer et al., 2012)
Marine mammals
(Derraik, 2002, Eriksson & Burton, 2003)
Amphipods
(Chua et al., 2004,
Ugolini, 2013)
Estuarine fish
(Possatto et al., 2002)
Turtles
(Hammer et al., 2012,
Lazar & Gracan, 2011)
Sharks & Rays
(Hammer et al., 2012)
Crustaceans
(Farrell & Nelson, 2013,
Murray & Cowie, 2011)
Piscivorous fish
(Jantz et al., 2013)
Zooplankton
(Setala et al., 2014,
Cole et al., 2015)
Marine larvae
(Kaposi et al., 2014)
Mussels
(Farrell & Nelson, 2013,
Browne et al., 2008) Microplankton
(Setala et al., 2014)
Planktivorous fish
(Boerger et al., 2010)
Corals
(Hall et al., 2015)
Bryozoans
(Ward & Shumway 2004)
Holothurians
(Graham & Thompson
2009)
Lugworms
(Thompson et al., 2004)
Isopods
(Hamer et al., 2004)
Artificial food chain (Artemia to Zebrafish)
(Batel et al., 2016)
Figure 1. The range of marine biota that have been shown to ingest microplastics. The green arrows indicate where physical transfer of microplastic fragments has been
demonstrated. The box in the dotted lines illustrates a recent study where transfer of microplastics and associated contaminants was demonstrated via BaP spiked microplastics in
an artificial food chain study Hall et al. 2015.
L. Tosetto et al. / Animal Behaviour 123 (2017) 159e167160
are an important food source for birds (Dugan, Hubbard, McCrary, &
Pierson, 2003), insects (Poore &Gallagher, 2013) and fish (Fanini &
Lowry, 2014) and therefore important in the transfer of energy
between the different trophic levels (Griffiths, Stenton-Dozey, &
Koop, 1983). Gobies are intermediate predators that include am-
phipods in their diet (Souza, Dias, Marques, Antunes, &Martins,
2014), thus making the model suitable to assess trophic transfer
of microplastics. This study used wild-caught fish rather than
laboratory-raised animals to recreate a more realistic setting in
assessing impacts of contamination. Fish came from an urban bay
and so it is likely that some background levels of contaminants
were present in them.
We assessed how transfer of a microplastic-contaminated diet
affected fish personality. We used personality tests as a sensitive
assay for assessing the impact of a microplastic-contaminated diet
on fish behaviour. Personality is a major driver in population dy-
namics that can influence foraging success, reproduction and
predator interactions (Budaev &Brown, 2011). Variation in per-
sonality traits such as activity or boldness can affect food acqui-
sition and social encounters (Stamps, 2007), with bolder
individuals benefiting in a foraging and social context (Smith,
Miner, Wiegmann, &Newman, 2009). Some studies have found
alterations in personality traits following exposure to pollutants.
These studies include modifications to boldness in three-spined
sticklebacks, Gasterosteus aculeatus, in response to increased car-
bon dioxide (Jutfelt, de Souza, Vuylsteke, &Sturve, 2013),
increased activity and decreased sociality in European perch, Perca
fluviatilis, following exposure to pharmaceuticals (Brodin, Fick,
Jonsson, &Klaminder, 2013), changes to boldness and activity
levels in female Siamese fighting fish, Betta splendens, after expo-
sure to the hormone 17
a
-ethinylestradiol (Dzieweczynski,
Campbell, Marks, &Logan, 2014) and changes in key male be-
haviours in Siamese fighting fish following treatment with the
contraceptive Ruta graveolens (Forsatkar, Nematollahi, &Brown,
2016).A shift in the personality distribution within a population
can have consequences for the local community; thus, changes to
personality on an individual level may be an indicator of wider
ecosystem effects (Weis, Smith, Zhou, Santiago-Bass, &Weis,
2001). Moreover, it is well established that hormone levels can
influence boldness in fish (Chang, Li, Earley, &Hsu, 2012; King,
Fürtbauer, Mamuneas, James, &Manica, 2013), and contaminants
can alter endocrine systems, with many having a feminizing effect
(Vos et al., 2000); thus, we predicted a shift in the personality of
affected fish in response to ingestion of a microplastic-
contaminated diet.
METHODS
Preparation of Microplastics
Commercial polyethylene (PE) microspheres (Cospheric
UVPMS-BG, 1.004 g/ml density, nominal 38e45
m
m diameter,
colour green, Cospheric, Santa Barbara, CA, U.S.A.) were used as
proxies for microplastics in marine environments (Kaposi, Mos,
Kelaher, &Dworjanyn, 2014). To replicate microplastics in the
marine environment, the microplastics were deployed in an urban
bay in Port Jackson, Australia (33
50
0
24
00
,151
15
0
13
00
) for 2 months.
Microplastics were assessed post deployment in Port Jackson for
polycyclic aromatic hydrocarbons (PAHs) as PAHs are one of the
most widespread organic pollutants in the aquatic environment
(Gonçalves, Scholze, Ferreira, Martins, &Correia, 2008). Following
exposure in sea water, the microplastics contained 0.007
m
g/g of
PAHs, suggesting that the microplastics had absorbed a small
amount of contaminants (Tosetto, Brown, &Williamson, 2016).
Study Animals
Platorchestia smithi were collected from March to July 2015 from
the supralittoral zone of Forrester's Beach, NSW (33
24
0
37.06
00
,
151
28
0
05.39
00
). Details of transport and husbandry are provided in
Tosetto et al. (2016). Beach hoppers in treatment groups were
exposed to sediment that had an addition of contaminated PE
microplastics at 3.8% (dry weight) while the beach hoppers in
control groups were exposed to sediment only. Previous studies
reporting negative effects on biota have used microplastics of over
5% wet weight (Besseling, Wegner, Foekema, van den Heuvel-
Greve, &Koelmans, 2013; Graham &Thompson, 2009; Wright,
Rowe, Thompson, &Galloway, 2013). The average distribution of
microplastics on polluted beaches in Hawaii has been recorded as
3.3% by weight (Carson, Colbert, Kaylor, &McDermid, 2011). As
microplastics continue to accumulate in coastal environments
(Turra et al., 2014), the concentration of 3.8% microplastics in the
current study was deemed an environmentally relevant concen-
tration. Beach hoppers were exposed to treatments for 72 h
(exposure period based on previous studies with amphipods (Chua,
Shimeta, Nugegoda, Morrison, &Clarke, 2014; H€
amer, Gutow,
K€
ohler &Saborowski, 2014)). They readily ingested microplastics,
which were found to accumulate in the beach hoppers. Following
72 h exposure there was a 30% increase in PAHs in the treatment
group (3.09
m
g/g) compared with the control group (2.34
m
g/g;
Tosetto et al., 2016).
Krefft's frillgobies are small, ray-finned marine fish that grow to
a maximum length of 90 mm (Kuiter, 1993). Fish were caught using
hand nets from Chowder Bay, Sydney, Australia (33
50
0
24
00
,
151
15
0
13
00
) in March and April 2015. A total of 33 fish were
collected and acclimated in the laboratory for 2 months prior to
testing. Individuals were housed in groups of eight in four flow-
through sea water aquaria (640 420 mm and 260 mm deep: 70
litres), held in the flowthrough sea water facility at the Sydney
Institute of Marine Science (SIMS), Australia. All aquaria were
maintained at the same sea water flow rate (1 litre/min), temper-
ature (16e22
C), with a gravel substrate and aeration from an air
stone. Fish were fed live black worms, Lumbriculus variegatus,
during the 1-week settling period and then a mixture of frozen
commercial ‘Hikari Bio-Pure’Mysis shrimp, brine shrimp and black
worms every other day as per White and Brown (2014). Tanks were
physically enriched with terracotta pots and were lit from overhead
fluorescent tubes for 12 h/day. Four weeks before testing, fish were
lightly anaesthetized using a solution of 50 mg of MS222 buffered
with sodium bicarbonate (fish were placed in a bucket containing
1.5 litres of solution until subdued). While anaesthetized their total
length was measured and individuals were tagged using visible
implant fluorescent elastomer tags (Northwest Marine Technology,
Inc., Shaw Island, WA, U.S.A.) to assist identification. Gobies recover
from tagging almost immediately and no long-term effects on
behaviour are apparent (White &Brown, 2013). Tanks were cleaned
weekly and all excess food removed after feeding.
Fish Behaviour Assays
Fish behaviour was tested using combined emergence (Brown &
Braithwaite, 2004) and open-field trial tests (Archard &
Braithwaite, 2011). These assays quantify a range of boldness and
exploration variables that are used as proxies for overall personality
in individual fish (Budaev &Brown, 2011). To maintain novelty
across behavioural trials two slightly different arenas were estab-
lished in 70-litre aquariums identical in size to the housing tanks
for the boldness-exploration tests: (1) the bottom of the aquarium
was covered with 5 cm of sand from a nearby beach and the sides
lined with sand colour shade cloth (Coolaroo 1.83 m Sandstone 70%
L. Tosetto et al. / Animal Behaviour 123 (2017) 159e167 161
UV Shade Cloth, Gale Pacific Australia, Braeside, VIC, Australia); and
(2) shell grit (3 cm) covered the bottom of the tank and the sides
lined with green turf (1.8 m wide Natural Synthetic Turf, Tuff Turf,
Mentone, VIC, Australia). Fish were tested individually in both
arenas. The aquarium was filled with 150 mm sea water. A start box
was placed at one end of the arena. The start box (140 140 mm
and 190 mm high) was made of dark plastic, had a lid to cover the
top and a doorway (30 mm 60 mm) on one side that could be
covered with a sliding piece of black plastic. An air stone was placed
behind the start box for aeration. After capture with a hand net
from the housing tank, the test fish was placed into the start box,
and the lid placed on top. The fish was left for a 2 min settling
period after which a remote pulley lifted the door, allowing the
individual to emerge from the doorway and explore the test arena.
To eliminate impacts of human observers on fish behaviour, ob-
servers stood behind a screen and fish were observed remotely via
a computer monitor connected by firewire (Belkin FireWire cable
IEEE 1394) to a digital camera mounted over the test arena. At the
end of the test period (600 s) fish were netted and returned to their
housing tank.
Experimental Treatments
Six individuals died prior to microplastic exposure and so were
excluded from the treatment groups; 28 fish remained for analysis
of personality. All fish were put through behavioural trials before
exposure to beach hopper treatments. The test was repeated three
times prior to microplastic exposure: trials 1 and 3 were in arena 1
(sandy) and trial 2 in arena 2 (turf). There was at least a 7-day in-
terval between each trial. Following initial tests, gobies were
randomly assigned to two treatment groups and housed accord-
ingly. We placed around 25 beach hoppers in each tank every 2
days (twoethree beach hoppers per fish). We fed the fish with a
pipette so that we could ensure that they all ate the beach hoppers.
Tanks were observed until all fish had fed. The second round of
testing commenced at the end of the first week of the beach hopper
diet. Three trials were undertaken after microplastic exposure.
Trials 4 and 6 were again undertaken in arena 1 and trial 5 in arena
2. There was at least 7 days between trials 4, 5 and 6 (Fig. 2).
Scoring Behaviour
Videos were scored for emergence, exploration and activity
levels using Etholog (Ottoni, 2000). Emergence time was recorded
when the entire fish (i.e. the last of the caudal fin) had emerged out
of the start box. Fish that failed to emerge were given the maximum
score of 600 s. To quantify exploration, i.e. the amount of time fish
spent in various parts of the arena, a grid was placed over the tank
that divided the arena into 10 sections (Fig. 3). The various be-
haviours scored are outlined in Table 1. The start box and cover
were considered safe areas for the fish, the top and middle rows
were identified as risky, and the bottom row and centre were
deemed dangerous as they were furthest from any cover. The total
time spent in each quadrat was recorded, and a fish was not
deemed to be in a new quadrat until the entire fish had moved into
the area. The number of movements, each time the fish swam
forwards or turned left or right, made by the fish over the test
period was a proxy for activity.
Statistical Analysis
To test for the presence of personality, incorporating boldness
and exploration, individual traits were collapsed into principal
component scores using principal components analysis (PCA). PCA
was undertaken using the princomp function in R (R Core Team,
2013). Eigenvectors with a value greater than one were used to
describe the variance in the data set. Each retained principal
component was then used as a composite behavioural variable in
future analyses.
Agreement repeatability (R; the intraclass correlation coeffi-
cient) of component scores was calculated between trials using the
within- and between-variance components in a linear mixed-
effects model (LMM). The restricted maximum likelihood method
(REML) was used in the package rptR, with individual fish identity
as the grouping factor. Adjusted repeatability (R
A
) was assessed
Trial 1 Trial 2 Trial 3
Days
0 8
24
Trial 4 Trial 5 Trial 6
78 86
93
Before exposure After exposure
Figure 2. Schematic diagram of behavioural trials. The dashed line indicates where diet changed from commercial fish food to beach hoppers. Trials with solid lines indicate arena 1
(Sand) and dashed lines indicate arena 2 (Turf).
SB G0 G0
G5
G8
G1
G2
G3
G4
G6
G7
G9
CVR
Figure 3. The test arena used for scoring fish boldness and exploration.
L. Tosetto et al. / Animal Behaviour 123 (2017) 159e167162
across trials for each treatment group before and after exposure
where size and days between trial were included as fixed effects
and individual fish identity as the grouping factor. Confidence in-
tervals and standard errors for both Rand R
A
were calculated from
parametric bootstraps that created the distributions of likelihood
ratios (1000 times). Pvalues for repeatability estimates were
derived from permutation tests (Nakagawa &Schielzeth, 2010). See
the Supplementary Material for the R code for Rand R
A
.
We analysed data in line with their longitudinal nature
(repeated measures over time) using linear mixed models (LMM;
see the Supplementary Material for the R code). The PC1 scores
were not normally distributed but because both positive and
negative values were present in the data set the negative signs were
removed before performing a log transformation on the PC1 values.
The negative sign was replaced following transformation. As the
PC2 scores were normally distributed, no transformation was car-
ried out.
In the analysis, we first assessed whether there was a trend over
time within each exposure (before and after exposure to the beach
hopper diet) for PC1 and PC2. We built a model using days since the
first observation as the principal variable of interest to quantify any
changes in behaviour over time within each of the exposure pe-
riods. The variables treatment (control/treatment) and fish size
were also included as fixed effects. To account for individual fish
variability we included individual fish identity as a random effect.
There was no trend over time before exposure to the contaminated
diet or either PC1 (P¼0.727) or PC2 (P¼0.621). There was also no
effect of fish size for either PC1 (P¼0.738) or PC2 (P¼0.141).
Similarly, when we assessed any effects after exposure to a
contaminated diet there was no effect of time for PC1 (P¼0.812) or
PC2 (P¼0.359), nor was there an effect of fish size in either PC1
(P¼0.999) or PC2 (P¼0.479; Fig. 4).
As there was no effect of days or size within each exposure
period we ran a final model that assessed changes in PC scores
before and after exposure. Fixed effects were treatment and
exposure with an interaction term between them. Random effects
were individual fish identity.
Ethical Note
Our experimental methods conformed to the standards set by
Macquarie University Animal Ethics committee (ARA 2014/003-7).
Fish and beach hopper collections were conducted under NSW
Fisheries Scientific Collection Permit numbers P08/0010-4.2 and
P14/0032-1.1, respectively. Fish were observed at least every second
day to ensure their health and wellbeing and environmental
housing conditions (water temperature, salinity and pH) were also
regularly monitored. At completion of the study the fish were
released to their original environment.
RESULTS
PCA condensed the variables to just two components that
accounted for 56% of the total variance. Component score co-
efficients showed that boldness variables, such as emergence time,
total start box time and time spent in more risky parts of the arena,
Table 1
The 10 variables used in the fish personality testing
Code Variable Description
FE Emergence The time for fish to emerge in full from the start box, recorded when the caudal fin was out of the box
SB Start box The total time (initial time þreturn time) is indicated here. The total time spent in the start box. The start box was a safe
area and fish would return to the start box after emergence
TR Top row Total time fish spent in grids 1, 2 and 3
MR Middle row Total time fish spent in grids 4, 5 and 6
G5 Grid 5 Although related to MR, the centre of the arena was deemed one of the most dangerous areas for the fish to move into
CV Cover The total time spent exploring the area around the start box. Cover was defined as 2 cm around the start box
BR Bottom row Total time fish spent exploring the bottom of the arena, a combination of time spent in grids 7, 8 and 9
G0 Grid 0 Total time spent the top area of the arena once outside the cover of the start box
G8 Grid 8 Related to BR but like G5, a little riskier given it was in the centre of the arena
AC Activity The total movements made by the fish over the course of the test
–2.5
–2.5
0
2.5
5
123
PC1
456
Treatment
Control
Treatment
0
2.5
5
123
PC2
456
Trial
(a)
(b)
Figure 4. Effect of microplastic exposure on PC scores of gobies monitored in the six
behavioural trials. The dotted line between trials 3 and 4 indicates where the diet
changed to beach hoppers. (a) PC1 (boldness) scores. (b) PC2 (exploration) scores.
White triangles indicate control individuals and black circles indicate treatment in-
dividuals. The solid lines indicate the population mean with the solid line representing
control fish and broken lines representing treatment fish.
L. Tosetto et al. / Animal Behaviour 123 (2017) 159e167 163
were the largest contributors to PC1. Exploratory variables, such as
time spent exploring around the start box (cover) and the more
exposed and furthest areas from the start box (bottom row),
contributed most to PC2. Boldness contributed to almost half of the
variance compared with exploratory variables that explained a
relatively small proportion of the variance, suggesting the experi-
ment was largely a boldness assay (Table 2).
Both repeatability (R) and adjusted repeatability (R
A
) results are
provided in Table 3.Asfish size and number of days were not
included in the final mixed model we used the repeatability (R)
values in all further results. All trials wererepeatable for PC1 before
exposure (R¼0.476 (0.116),95% CI 0.220 to 0.664, P<0.001) and
after exposure (R¼0.684 (0.085),95% CI 0.474 to 0.816, P<0.001)
suggesting that the personality traits were robust. Only trials after
exposure were repeatable for PC2 (R¼0.401 (0.119),95% CI 0.129 to
0.603, P<0.001). When repeatability was assessed for treatment
and control groups separately, there was no significant difference in
repeatability values for PC1 before or after exposure to treatments
suggesting that consumption of prey items contaminated with
microplastics did not affect repeatability of boldness traits. There
was, however, an increase in repeatability values for PC2 in both
treatments following exposure. This implies an effect of familiar-
ization to the test arena rather than the exposure to a contaminated
diet (Table 3).
Exposure to a microplastic-contaminated diet did not affect
boldness or exploration in fish. No significant interactions occurred
between exposure (before and after exposure to beach hoppers)
and treatment (contaminated or not) for either PC1 (boldness:
t
137
¼0.035, P¼0.891) or PC2 (exploration: t
137
¼0.197,
P¼0.557). There was a significant effect of exposure to beach
hoppers on the PC1 scores (t
27
¼0.418, P<0.05) with fish in both
treatment and control groups decreasing in boldness following
exposure to the beach hopper diet (Fig. 4). In scores indicating
exploratory behaviour (PC2), there was a trend for fish in the
control group to show greater exploratory tendencies across both
exposures; however, this was not significant (t
27
¼0.594,
P¼0.058; Fig. 4).
DISCUSSION
This is the first study to assess possible behavioural conse-
quences of trophic transfer of microplastics and associated con-
taminants, in this case through consumption of contaminated prey
by a fish. As toxic contaminants found in plastics have been asso-
ciated with endocrine disruption (S€
offker &Tyler, 2012), we ex-
pected a shift in the behaviour and personality of affected fish. Here
we have shown that, contrary to expectations, transfer of
contaminated microplastics from beach hoppers to fish did not
affect fish personality. There was, however, an overall shift in
boldness with fish becoming shyer after exposure to the beach
hopper diet irrespective of treatment. This was perhaps due to fish
habituating to the experimental tasks, or possibly the alteration in
diet. Exploration remained relatively constant before and after
exposure possibly due to the variation in individuals between
treatments, with control fish being more exploratory throughout.
Repeatability values for boldness were not significantly different
before or after exposure to treatments. Exploration repeatability
values increased across both control and treatment groups after
exposure suggesting acclimatization with the test arena.
Concern surrounding microplastics is largely due to the associ-
ated contaminants and their capacity to desorb into animals (Bakir,
Rowland, &Thompson, 2014), and subsequently accumulate
through the food web in different trophic levels (Teuten et al.,
2009). The assumption that pollutants biomagnify at all levels of
the marine food chain has been disputed (Gray, 2002) but the
biomagnification of organic pollutants from lower trophic levels to
fish has been demonstrated (Kelly, Ikonomou, Blair, Morin, &
Gobas, 2007; Nfon et al., 2008). Whether microplastics are a
viable pathway for contaminant exposure is currently under
debate. Conceptual models assessing the rates of desorption in
organisms suggest that microplastics do not provide a relevant
exposure pathway (Gouin, 2011; Koelmans, 2013, 2014). However,
positive relationships between microplastic consumption and
increased contamination concentration in animal tissue have been
demonstrated in seabirds (Ryan, Connell, &Gardner, 1988), am-
phipods (Chua et al., 2014) and bivalves (Avio et al., 2015).
Furthermore, recent studies suggest that desorption rates of con-
taminants in gut surfactants are 30 times faster than in sea water
for some contaminants, with pH and body temperature also influ-
encing the rate (Bakir et al., 2014). Polyethylene microplastics
deployed in the marine environment have the capacity to absorb
contaminants, which can accumulate in beach hoppers following
consumption, increasing the contaminant load of the organism
(Tosetto et al., 2016). Given our current understanding of micro-
plastics and contaminants we expected to see a change in fish
personality following consumption of contaminated beach
hoppers.
Using personality to assess anthropogenic impacts on in-
dividuals is a relatively new area of research. Personality is a major
driver in population dynamics that can influence competitive in-
teractions and how animals deal with changes in their environment
(Montiglio &Royaut
e, 2014), particularly those attributed to
anthropogenic impacts such as environmental contamination
(Wong &Candolin, 2015). Assessing how contaminants alter per-
sonality traits provides a far subtler way of establishing effects in
comparison to the standard LD50 approach commonly used in
ecotoxicology (Oulton et al., 2014). Recent studies have observed
sublethal effects of pollutants on behaviour (Brodin et al., 2013,
2014; Dzieweczynski et al., 2014; Forsatkar et al., 2016; Jutfelt
et al., 2013); however, when assessing the effects of hormone
17
a
-ethinylestradiol on female Siamese fighting fish
(Dzieweczynski et al., 2014) and increased carbon dioxide on three-
spined sticklebacks (Jutfelt et al., 2013) some habituation to bold-
ness assays over time was reported.
The current study did not observe significant effects of treat-
ment on fish personality or the repeatability of these behaviours.
There was, however, an alteration in fish behaviour following
exposure to the beach hopper diet with all fish becoming shyer. The
longer fish are exposed to a similar task the more inflexible
behaviour comes, with less motivation to emerge and subsequently
less time given to exploring an area (Kieffer &Colgan, 1992).
Moreover, fish will decrease activity as they become familiar with a
Table 2
PCA loadings for each of the behaviour variables in the personality tests
Behaviour Code Boldness (PC1) Exploration (PC2)
Full emergence FE ¡0.372 0.188
Start box SB ¡0.423 0.161
Cover CV 0.280 ¡0.514
Bottom row BR 0.246 0.513
Middle row MR 0.379 0.260
Top row TR 0.365 0.146
Grids adjacent to start box G0 0.213 0.119
Centre grid G5 0.285 0.091
Bottom centre grid G8 0.287 0.523
Activity AC 0.241 0.164
Eigenvalue 4.99 1.44
Percentage variance explained 49.4 6.4
The corresponding code for each behaviour is provided. Variables in bold contribute
the highest amounts to each behavioural variable.
L. Tosetto et al. / Animal Behaviour 123 (2017) 159e167164
tank environment (Brown, 2001; Sharma, Coombs, Patton, &de
Perera, 2009). By varying the test arenas in trials 2 and 5 we
attempted to reduce habituation to the behavioural assays. While
some variation in boldness traits was observed in the trials before
exposure, we did not see corresponding variation after exposure,
suggesting fish had become familiarized to the task in the later
trials. It is possible that the change in behaviour could be due to
increased fear (Carter, Feeney, Marshall, Cowlishaw, &Heinsohn,
2013) but this is unlikely as the open-field trial was designed to
mirror the ecological setting of B. krefftii which seek shelter in rock
pools and crevices (White &Brown, 2015), and there was no novel
object or risk of predation included in the assay. Alternatively, the
alteration in diet may have affected the behaviour of the fish after
exposure. Hunger, or perceived hunger, has been shown to increase
activity and emergence times in fish (Brown &Braithwaite, 2004;
Thomson, Watts, Pottinger, &Sneddon, 2012); thus, it is possible
the gobies became satiated on beach hoppers in this study. Note
that while there was a shift in fish boldness after exposure to the
beach hopper diet, there was no difference across the treatment
and control groups that could indicate an effect of our treatments
on these processes.
It is possible that a longer exposure timeframe is required to
observe an effect of microplastic exposure. When directly exposed
to marine contaminated microplastics, Japanese meduka, Oryzias
latipes, suffered hepatic distress but this was not obvious until 2
months after initial exposure, suggesting that a certain contami-
nation threshold may be needed before a physiological effect is
observed (Rochman, Hoh, Kurobe, &Teh, 2013). It is possible that
the short duration of this study may not have been long enough to
reach the contamination threshold for either P. smithi or B. kreftii.As
these animals live up to 8 months and several years, respectively, it
is possible they are exposed to far greater amounts of plastics and
associated pollutants in their lifetime.
Alternatively, it is possible that background contamination
levels in the wild-caught fish masked any environmental effects of
microplastic exposure. Studies assessing contaminant transfer
from microplastics to fish have fed microplastics directly to
laboratory-raised fish that have not had prior exposure to envi-
ronmental pollutants (Rochman et al., 2013). Moreover, a recent
laboratory-based study assessing the capacity for microplastics
and associated contaminants to transfer through an artificial food
chain used concentrations of microplastics and contaminants at
much higher levels than environmentally relevant concentrations
and used animals raised in the laboratory environment (Batel
et al., 2016). Theoretical models predict that the consumption of
microplastics does not increase the burden of pollutants in fish
given the background levels already present in the environment
(Gouin, 2011). Recently Koelmans, Bakir, Burton, and Janssen
(2016), following an assessment of the scientific literature,
determined that the uptake of contaminants through natural
pathways possibly exceeds accumulation via microplastics in most
habitats. This implies that ingestion of microplastics is unlikely to
increase an animal's exposure to contaminants in the marine
environment. Using wild-caught fish is more realistic than using
laboratory-raised animals in assessing ecological impacts of pol-
lutants. As we used fish from an urbanized area in Sydney,
Australia and fed a diet of commercial frozen food during accli-
mation, it is plausible that the experimental effects of plastic
consumption were not discernible from the background levels in
our wild-caught fish.
Gobies collected from highly polluted areas have shown differ-
ences in behavioural responses to stress than counterparts
collected from less contaminated sites (Marentette, Tong, &
Balshine, 2013). It is possible that fish with lower levels of back-
ground pollution may elicit a different response when exposed to
contaminated microplastics. As we expect the distribution of
microplastics to increase and expand through our marine ecosys-
tems (I~
niguez, Conesa, &Fullana, 2016), the effects of microplastic
exposure to wildlife from areas of low contamination should be
explored. Studies have demonstrated that assessment of complex
behaviours provides us with effective tools to assess sublethal
levels of pollutants; however, future studies should also incorpo-
rate analysis of animal digestive tracts and tissues to establish
whether microplastics are changing the contaminant load of ani-
mals through the food web.
Overall, this study found that short-term trophic transfer of
microplastics via prey does not affect the personality of fish.
(Brodin et al., 2014; Dzieweczynski et al., 2014; Jutfelt et al., 2013).
We did observe a shift in boldness after exposure to the beach
hoppers that may be attributed to familiarization, or a change in
diet; however, there was no significant effect between the treat-
ment groups after exposure. The problem of animals learning and
habituating to an experiment will persist across any longitudinal
study. To understand the broad impacts of plastic exposure we
need to take an all-inclusive approach to behavioural experiments
that assess the impacts of microplastics on behaviours applicable to
foraging, sexual activity, sociality and learning. Future long-term,
ecologically relevant studies should directly examine, via tissue
analysis, whether microplastics and contaminants are accumu-
lating through the food web. A suite of personalities and behav-
iours should also be assessed to get a more comprehensive picture
of changes in behaviour in response to microplastics through
different trophic levels. It is possible, however, given the back-
ground levels of contamination already present in coastal envi-
ronments, that trophic transfer of microplastics does not provide
an additional exposure pathway for contaminants to move through
the food web.
Acknowledgments
This research was funded by the Department of Biological Sci-
ences at Macquarie University. We thank members and interns of
Table 3
Repeatability estimates across trials for both Control and Treatment groups
Treatment Exposure PC R(SE) CI PR
A
(SE) CI P
Control Before PC1 0.462 (0.163) [0.089, 0.729] 0.005 0.493 (0.162) [0.124, 0.735] 0.003
PC2 0.072 (0.128) [0.000, 0.047] 0.294 0.155 (0.146) [0.000, 0.495] 0.184
After PC1 0.690 (0.126) [0.361, 0.860] 0.001 0.686 (0.133) [0.353, 0.855] 0.001
PC2 0.328 (0.171) [0.000, 0.642] 0.017 0.343 (0.175) [0.000, 0.649] 0.024
Treatment Before PC1 0.498 (0.161) [0.138, 0.746] 0.004 0.620 (0.145) [0.254, 0.818] 0.002
PC2 0.116 (0.131) [0.000, 0.430] 0.210 0.293 (0.165) [0.000, 0.590] 0.041
After PC1 0.688 (0.129) [0.344, 0.853] 0.001 0.691 (0.146) [0.282, 0.856] 0.001
PC2 0.488 (0.166) [0.096, 0.732] 0.001 0.470 (0.171) [0.066, 0.731] 0.008
The table shows repeatability (R) and adjusted repeatability (R
A
), standard errors (SE), the 95% confidence interval (CI) and associated Pvalue of each estimate (N¼14 in-
dividual fish per treatment). Significant repeatability estimates are shown in bold.
L. Tosetto et al. / Animal Behaviour 123 (2017) 159e167 165
the MEG and BEEF labs for field and laboratory assistance, Sarah
Houlahan for assistance with GCMS and analysis of beach hopper
contamination, Petra Graham for guidance on statistical analysis,
and Alistair Poore for advice on beach hopper husbandry. This
paper is contribution number 193 from the Sydney Institute of
Marine Science.
Supplementary Material
Supplementary material associated with this article is available,
in the online version, at http://dx.doi.org/10.1016/j.anbehav.2016.
10.035.
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