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Brain damage and behavioural disorders in fish induced by plastic nanoparticles delivered through the food chain

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The tremendous increases in production of plastic materials has led to an accumulation of plastic pollution worldwide. Many studies have addressed the physical effects of large-sized plastics on organisms, whereas few have focused on plastic nanoparticles, despite their distinct chemical, physical and mechanical properties. Hence our understanding of their effects on ecosystem function, behaviour and metabolism of organisms remains elusive. Here we demonstrate that plastic nanoparticles reduce survival of aquatic zooplankton and penetrate the blood-to-brain barrier in fish and cause behavioural disorders. Hence, for the first time, we uncover direct interactions between plastic nanoparticles and brain tissue, which is the likely mechanism behind the observed behavioural disorders in the top consumer. In a broader perspective, our findings demonstrate that plastic nanoparticles are transferred up through a food chain, enter the brain of the top consumer and affect its behaviour, thereby severely disrupting the function of natural ecosystems.
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Scientific RepoRts | 7: 11452 | DOI:10.1038/s41598-017-10813-0
www.nature.com/scientificreports
Brain damage and behavioural
disorders in sh induced by plastic
nanoparticles delivered through the
food chain
Karin Mattsson1,2, Elyse V. Johnson3, Anders Malmendal1, Sara Linse
1,2, Lars-Anders
Hansson2,4 & Tommy Cedervall1,2
The tremendous increases in production of plastic materials has led to an accumulation of plastic
pollution worldwide. Many studies have addressed the physical eects of large-sized plastics on
organisms, whereas few have focused on plastic nanoparticles, despite their distinct chemical, physical
and mechanical properties. Hence our understanding of their eects on ecosystem function, behaviour
and metabolism of organisms remains elusive. Here we demonstrate that plastic nanoparticles reduce
survival of aquatic zooplankton and penetrate the blood-to-brain barrier in sh and cause behavioural
disorders. Hence, for the rst time, we uncover direct interactions between plastic nanoparticles
and brain tissue, which is the likely mechanism behind the observed behavioural disorders in the top
consumer. In a broader perspective, our ndings demonstrate that plastic nanoparticles are transferred
up through a food chain, enter the brain of the top consumer and aect its behaviour, thereby severely
disrupting the function of natural ecosystems.
e production of plastic material has increased tremendously during the last decades1, 2, and about 10% of the
plastics produced annually end up in the oceans3 through sewage treatment plants, waste handling or aerial
deposition4, constituting 60–80% of the total marine debris5. Plastic debris has been shown to aect over 660
marine species6 through entanglement and ingestion, and is thus a severe and potent pollutant in aquatic envi-
ronments6. Once in the aquatic environment, plastic material breaks up into smaller pieces through the action
of sunlight, waves, living organisms in the water and by the water itself 7, 8. Eventually plastic material is broken
down to nanoparticles912, which may be an even more potent threat since plastic nanoparticles are able to pass
through biological barriers13, penetrate tissues14 and accumulate in organs15 and aect behaviour and metabolism
of organisms16, 17. ose eects are generally not due to the toxicity of the material per se, but rather a result of the
physical features of the nanoparticles. Hence, the particle size plays a pivotal role for their biological impact18 as
size aects the curvature and provide a large surface area18, 19. Smaller particles are generally more toxic than the
corresponding bulk material at the same mass concentration2022, and the mobility, biological fate and bioavaila-
bility depend on size, shape, charge and other nanoparticle properties23, 24.
e freshwater invertebrate Daphnia magna can ingest nano- and micro-sized (20 nm to 70 μm) particles from
water21, 2527, are commonly used in toxicity studies28 and has a pivotal role in many food chains16, 17, 29. Previous
work has shown that the uptake rate depends on particles size25, 30, 31 and charge32. For example, Daphnia magna
were shown to have a lower uptake rate of 20 nm than 1000 nm polystyrene particles, although when compared at
equivalent surface area, the uptake was equal or higher for the small particles. Furthermore, indirect intake rate
via algal food was higher than direct intake from water30. e precise manner in which particle size, charge and
surface area aect the intake and biological impact of nanoparticles is, however, still unknown.
Here we report novel ndings on how plastic nanoparticles strongly aect an aquatic food chain from the
zooplankter Daphnia magna to the top consumer, the freshwater sh, Crucian carp (Carassius carassius), which is
common in anthropogenically aected waters. We exposed Daphnia magna to a range of polymeric nanoparticles
1Department of Biochemistry and Structural Biology, Lund University, P.O. Box 124, SE-221 00, Lund, Sweden.
2NanoLund, Lund University, SE-221 00, Lund, Sweden. 3CytoViva, Inc. 570 Devall Drive, Auburn, AL, 36832, USA.
4Department of Biology/Aquatic Ecology, Lund University, SE-223 62, Lund, Sweden. Correspondence and requests
for materials should be addressed to K.M. (email: Karin.mattsson@biochemistry.lu.se)
Received: 9 June 2017
Accepted: 14 August 2017
Published: xx xx xxxx
OPEN
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Scientific RepoRts | 7: 11452 | DOI:10.1038/s41598-017-10813-0
directly in water or via algae (Scendesmus sp.). We show that positively charged amino modied polystyrene nan-
oparticles aect both Daphnia and top consumer (sh) in a size dependent manner. We also show that the nano-
particles were transferred through a three-level food chain from algae through zooplankton to sh, which showed
behavioural disorders. Moreover, those behavioural disorders depended on the size of the nanoparticles and anal-
yses by hyperspectral microscopy showed that the plastic nanoparticles were present in the sh brains. Hence, we
here, for the rst time, demonstrate the mechanistic chain from uptake of nanoplastic particles by algae, through
transport up the food chain and, nally, eects on the brain physiology and behaviour of top consumer (sh). On
a broader scale such eects are likely to considerably aect natural ecosystems, since top predators have a crucial
impact on lower trophic levels and ecosystem functions33.
Results
Eects on Daphnia magna. Out of the tested nanoparticles of dierent size and charge (Table1) only ami-
no-modied positively charged polystyrene nanoparticles with a diameter of 52 nm aected Daphnia, whereas
larger particles of the same material (120–330 nm) had no eect on the animals and we therefore focused on this
particle size in our study. For the 52 nm particles, the toxicity was strongly dependent on the particle concentra-
tion. Up to a concentration of 0.025 g/L all Daphnia were still alive aer 24 hours, and above 0.075 g/L all were
dead within 13 h (Fig.1). Comparing the toxicity of dierently sized amino-modied polystyrene nanoparticles,
where either the mass, surface area or the number of particles were the same, revealed that size was the only
important factor for toxicity. To rule out a potential batch dependent toxicity of 52 nm amino modied polysty-
rene particles, the same type of particles but in a size of 53 nm, 57 nm as well as 58 nm were also tested.
Equipped with the insight obtained from Daphnia magna, we next set out to study the eects of plastic nan-
oparticles (53 nm and 180 nm) on the entire food chain and if the eects of nanoparticles are transferred to the
sh in a food chain starting from algae (Fig.2). Analyses of sh feeding times – the time it took for each group
(aquarium) to consume 50% of the provided Daphnia – showed that the sh receiving 53 nm particles ate more
slowly than the control sh. Contrary to our expectations, sh fed with 180 nm particles were the fastest feeders
(Fig.3A; p < 0.030 ANOVA). Furthermore, detailed analyses of the hunting behaviour showed that the sh fed
with 53 nm particles swam a longer distance to eat 50% of the provided zooplankton (Fig.3B, p < 0.02 ANOVA),
and explored less space within each aquarium (Figs3C and S1). ey also had a signicantly lower activity(px/s),
in contrast to the sh fed 180 nm particles which instead showed a higher activity than controls (Fig.3D; p < 0.001
ANOVA). In natural systems, a slower feeding rate combined with a longer swimming distance before successful
feeding likely leads to suboptimal energy use and a more pronounced exposure to predation. Collectively, these
consequences point to considerable eects on tness and thereby on ecosystem function for sh exposed to plas-
tic nanoparticles of about 50 nm size.
Particle Diameter size (nm) Concentration (g/L) Surface charge
PS-NH2 Amino-modied 52, 53, 57, 58, 120, 180, 330 0.005, 0.010, 0.025, 0.050, 0.075, 0.10, 0.15 positive
PS-COOH Carboxylate 261, 601, 92, 160, 190, 220 0.025, 0.050, 0.075, 0.10, 0.2, 0.4 negative
PS-OSO3H Sulfonated 25, 200 0.025, 0.050, 0.075, 0.10, 0.2, 0.4 negative
PMMA 68, 140 0.025, 0.050, 0.075, 0.10, 0.2, 0.4 negative
Table 1. Characteristics for particles tested for toxicity to Daphnia magna, including particle name, diameter
and concentration. Only the PS-NH2 amino modied particles was found to be toxic to Daphnia and we
therefore performed our sh study using this type of particles. 1Two dierent batches were tested.
Figure 1. Mortality aer exposure. Number of alive Daphnia magna 0–24 h aer exposure to dierent
concentrations (0.025–0.150 g/L) of 52 nm amino modied polystyrene nanoparticles. e gure shows that
Daphnia mortality rates depend on the concentration of the particles, n = 10 for each concentration.
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Figure 2. Food chain. Food chain from algae-zooplankton-sh, nanoparticles (53 nm mass (dark blue), 53 nm
surface area (light blue) and 180 nm (red)).
Figure 3. Top consumer response. Fish exposed to amino modied polystyrene nanoparticles of dierent
sizes delivered through a food chain of algae and zooplankton behave dierently. (A) sh feeding time (s) aer
exposure to dierent sizes of nanoparticles, p < 0.030 with ANOVA post hoc between 53 nm and 180 nm, (B)
swimming distance during feeding time, p < 0.018 with ANOVA post hoc between 53 nm and 180 nm, (C) sh
exploration within each aquarium during the rst 120 s of the feeding time (D) mean activity (px/s) during the
rst 120 s of the feeding time, p < 0.001 with ANOVA post hoc between all groups. Data are shown for control
sh that were not exposed to nanoparticles (n = 6 aquaria), 180 nm nanoparticles (n = 6 aquaria), and 53 nm
particles with the same mass concentration as 180 nm particles (n = 5 aquaria). All graphs show the mean ± SE.
e graphs shown that sh fed with 53 nm nanoparticles had a longer feeding time, that they swam a longer
distance to feed and that they explored less area of the aquaria, as well as that they had a lower activity compared
to sh receiving 180 nm particles and control sh. Moreover, sh fed with 180 nm particles were the fastest
feeders and had the highest activity.
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Scientific RepoRts | 7: 11452 | DOI:10.1038/s41598-017-10813-0
e observed behavioural changes in the sh suggest that their brains were aected by the particles. To conrm
this, we explored the unique spectral features of tracking polystyrene using a CytoViva Hyperspectral Imaging
System to obtain a wavelength spectrum from each pixel of the light scattered from sh brains. Polystyrene was
detected in the brains from all analysed sh fed with polystyrene nanoparticles, whereas no polystyrene was
detected in brains from control sh (Figs4B and S2).
We also found that sh exposed to nanoparticles had a higher weight loss and less water in their brains than
control sh (Fig.S3). Moreover, the microscope imaging shows that the gyri in the cerebral lobes were larger in
sh exposed to 53 nm particles (Fig.4A; p < 0.025 ANOVA), suggesting also morphological eects from those
particles. Hence, the morphological changes in the brains of the 53-nm-sized nanoparticle-fed sh suggest that
sh brains were directly aected by the plastic nanoparticles and that the eects depended on particle size. Taken
together, our results suggest that some of the plastic nanoparticles fed to sh through a food web end up in thesh
brains. Furthermore, we unravel a mechanistic link between behavioural disorders and the incorporation of nan-
oparticles in brain tissue.
Discussion
e amount of plastics in the world’s water bodies is rapidly increasing and this material degrades in size over
time and will eventually break down into plastic nanoparticles. Due to their small size, they easily enter the basis
of natural food chains, although it is unclear how these particles aect aquatic ecosystems. We show here that
52 nm positively charged amino modied polystyrene nanoparticles are toxic to Daphnia and that sh feeding
on Daphnia containing plastic nanoparticles change their behaviour in terms of activity, feeding time and the
distance they need to swim to consume their provided food. Furthermore, the behavioural changes depend on
the size of the particles. However, sh receiving 180 nm particles were dierently aected as they were the fastest
feeders and had the highest activity. In nature, the particles likely become aggregated with biological or inorganic
material, but we here show that the nano-size eect remains aer passing through the Daphnia digestive system.
For example, Ward et al.34 exposed the blue mussel Mytilus edulis and the oyster Crassostrea virginica to poly-
styrene nanoparticles, aggregated nanoparticles and micro-particles and found a higher ingestion rate for the
aggregated nanoparticles34. Wegner et al.35 exposed the mussel Mytilus edulis to polystyrene nanoparticles both
as nano-sized particles and as aggregated polystyrene nanoparticles. ey found a reduced ltering rate and an
increased production of pseudofeces35. In this context, our results point to an acute need for a deeper understand-
ing of the size-dependent toxicity eects of nanoparticles when released into nature. How these particles aect
organisms higher up in the food web, such as sh, as well as how they aect birds and mammals are unclear. In
Figure 4. Brain eects. (A) Measured gyri size (px2) of sh brains aer exposure to dierent sizes of polystyrene
nanoparticles, including 53 nm (n = 7), 180 nm (n = 11) and control (n = 15) which was not exposed to any
nanoparticles. (B) number of detected pixels corresponding to polystyrene particles in homogenized sh brain
samples, (n = 3 for each treatment). Fish receiving 53 nm nanoparticles had signicantly larger gyri size than
the 180 nm group, p < 0.021 ANOVA with Turkey’s post hoc. Polystyrene was detected in all brains from sh
exposed to nanoparticles, whereas no polystyrene was detected in the control group.
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2015, the estimated amount of plastics being released into the ocean was between 4.8 and 12.7 million tons, with a
steady increase the coming years36. Eventually this plastic will degrade in size and reach the nanometer size range.
Here we demonstrate how plastic nanoparticles are transported up the food chain and are detected in brain
tissue of the sh top consumer whereas no polystyrene were detected in the control group. Moreover, we also
here report macroscopic changes in the brain structure and water content in sh that have received plastic nano-
particles. By using hyperspectral microscopy, we were able to detect polystyrene particles in sh brain tissue and
thereby we have, for the rst time, demonstrated that the plastics nanoparticles can be transported across the
blood-brain barrier in sh. Moreover, this result suggests a mechanistic link between the observed behavioural
changes and the presence of plastic nanoparticles in the brain tissue. In the present study, we observed changes in
the brain which may have been caused by specic interactions between the plastics and the brain tissue, although
we cannot rule out that other organs may also be aected. Our study lasted for two months, but during the rst
half of the experiment we observed no changes in behaviour of the nanoparticle fed sh, suggesting that sh are
aected by the particles that are accumulated in the sh. In nature, the Daphnia and sh are likely exposed to low
concentrations of plastic nanoparticles during their whole life-time, which allows accumulation processes to act
for a much longer time period than in our study, since sh, such as crucian carp, may live for more than 10 years37.
However, our results also imply that eects on biota from plastic nanoplastics are dependent on both concentra-
tion and size of the particles, which opens up for manufacturers to adjust production of nanoparticles to sizes that
are less hazardous to organism metabolism and thereby ecosystem function.
e main conclusion from our study is that plastic nanoparticles are transferred through three tropic levels,
suggesting that they are likely to be transferred even further up the food web to ultimately reach humans, the
top-level consumer. Hence, in a broader perspective, our results may have implications for human wellbeing,
although such consequences of the accelerating disposal rate of plastics is yet not well recognized or understood.
Materials and Methods
Testing for suitable particles with Daphnia magna. Polystyrene particles with dierent surface mod-
ications, charges, sizes (25 nm to 330 nm) and at a range of concentrations (0.005 g/L to 0.150 g/L) were tested
for toxicity towards Daphnia magna. On day 1, 100 ml algae or water were added to each bottle together with
particles with dierent concentrations except for the control bottle, which received only water (Table1). e
bottles were then shaken for 2 minutes, and the algae were allowed to ingest the particles for 24 hours. On day 2,
900 ml water was added to the algae/water together with 10 adult Daphnia magna with an approximate size of 3
mm. e number of dead Daphnia was counted every hour for 24 hours and the bottles were then gently stirred
to distribute the algae or water evenly. To rule out a potential batch dependent toxicity of 52 nm amino modied
polystyrene particles, the same type of particles but in a size of 53 nm, 57 nm as well as 58 nm were also tested.
Nanoparticle preparation and characterization. Positively charged amino-modified polystyrene
(PAO2N) particles with diameters of 52 nm, 53 nm, 57 nm, 58 nm, 120 nm, 180 nm and 330 nm were purchased
from Bang laboratories (Fisher, IN, USA). e particles were dialyzed with fresh tap water for 24 hours. e
particle size was measured, to ensure that particles size remained constant during the experiment, with Dynamic
Light Scattering (DLS) both before and aer dialysis, as well as one week aer the dialysis. No change in particle
size was recorded during the study. We chose to not use surface labelled particles since this may aect the surface
chemistry. Moreover, it is unknown how passage through the digestive systems of Daphnia and especially sh
might aect the labelling and vice versa.
Fish experiment. Two sizes of particles were chosen for the sh experiment, one with the size of 53 nm that was
shown to aect the Daphnia and one larger, 180 nm, that did not show any toxicity towards Daphnia. e particle
size were conrmed with DLS and measured 56 nm (PI: 27%) and 174 nm (PI: 18%) in the water used during the
experiment. Twenty-four aquaria with three sh in each were divided into four groups. e rst group (the 180 nm
group) received 180 nm particles at a concentration of 0.1 g/L. e second group received the same mass concentra-
tion (0.1 g/L) of 53 nm particles (the 53 nm mass group). e third group also received 53 nm particles, but at a lower
concentration corresponding to the same surface area as the group receiving 180 nm particles (the 53 nm surface
area group, concentration 0.029 g/L). e results from this treatment are, for clarity, presented in Supplementary
material, (TableS1). e fourth group, the control group, did not receive any nanoparticles. All sh were measured
and weight before the experiment started. e study was performed under the permission from the Malmö/Lund
Ethical committee (D nr 14 13–12) and was performed according to the current laws in Sweden.
Food chain. Algae (Scenedesmus sp.) with a diameter of approximately 25 μm were cultivated in aquaria. On
day 1, 500 mL algae with a concentration of 450
µgL/
were mixed with water and particles to a total volume of 1 L
in four dierent test bottles (except for the control bottle, which received only water). Aer 24 hours Daphnia
magna (20 Daphnia/sh) were added to the algae medium. Aer 2 hours, the Daphnia were collected on a net
with a mesh size of 150
µm
and washed two times with 150 mL water. Each sh (Crucian carp, Carassius carassius)
was then served 20 Daphnia, i.e. 60 Daphnia per aquarium.
We replicated this natural food chain such that the sh eventually ingested, via algae and Daphnia, the same
type of amino-modied polystyrene nanoparticles as used for the Daphnia toxicity with diameters of 53 nm and
180 nm (Fig.2). To distinguish between size and mass eects, two concentrations of the 53 nm particles were
used, one that corresponded to the same surface area and one that corresponded to the same mass as the 180 nm
particles. e three groups: 180 nm, 53 nm surface area (TableS1) and 53 nm mass were studied together with the
control group, that did not receive any nanoparticles. Sixty Daphnia individuals were introduced as food to each
sh aquarium every third day for a period of 67 days.
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Video analysis. On day 62, we monitored the hunting behaviour of the sh by video recording each aquar-
ium separately during 2 minutes before the sh received food and 10 minutes aer. Since the smaller particles
were toxic to the Daphnia and thereby possibly aected their interaction with the sh, all groups of sh were on
the 62nd day fed with Daphnia that had not received any nanoparticles. Each sh position was registered each sec-
ond during the whole tracking period using the soware ImageJ. e feeding time – the time it took for the sh to
consume 50% of the provided food (Daphnia) – was registered. An ANOVA post hoc was used to test dierences
between treatments.
Brain analysis. On day 64, all sh were collected and anaesthetized using benzocaine. ey were measured
and weighed before the neck was cut and the brain was extracted. All samples were stored at 80 °C. e brain
was weighed and an image was recorded with Olympus SZX7 microscope with an Innity 1 camera and then
freeze-dried and weighed again before it was homogenized in PBS buer. e area of two gyri in all brain images
was measured in pixels2 using ImageJ and further calculated with Matlab. Finally, three brains from each group
were analysed with CytoViva hyperspectral microscope. is microscope was equipped with an enhanced dark-
eld illuminator and visible-near infrared (400–1000 nm) hyperspectral imaging components. e homogenized
brain samples were imaged under 60x magnication. Each image captured one pixel line at a time using an
automated stage. ese pixel lines were compiled to form hyperspectral images, also known as datacubes, which
contain spatial and spectral data for each pixel. For each image of exposed brain that was acquired, a spectral
library corresponding to polystyrene was created. is was accomplished by gathering several regions of interest
from each exposed brain image and ltering the spectra associated with those regions against 3 negative control
images (homogenized brain with no polystyrene). Any spectra that matched spectra in the negative controls were
eliminated from the polystyrene spectral libraries. en, the polystyrene was spectrally mapped and identied in
the exposed brain images using the Spectral Angle Mapper algorithm.
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Acknowledgements
We would like to thank the Centre for Environmental and Climate Research (CEC), the NanoLund at Lund
University, e Swedish Research Council and Mistra.
Author Contributions
K.M., A.M., S.L., L.A.H. and T.C. designed the study. K.M. and E.V.J. performed the experiments. K.M., E.V.J. and
T.C. preformed the analysis, and all authors wrote the manuscript.
Additional Information
Supplementary information accompanies this paper at doi:10.1038/s41598-017-10813-0
Competing Interests: e authors declare that they have no competing interests.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional aliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International
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Supplementary resource (1)

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