Environmental Pollution 219 (2016) 166e173

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Effects of nanoplastics and microplastics on toxicity, bioaccumulation,

and environmental fate of phenanthrene in fresh water*
Yini Ma a, Anna Huang a, Siqi Cao a, Feifei Sun a, Lianhong Wang a, Hongyan Guo a,
Rong Ji a, b, *
a
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, 163 Xianlin Avenue, 210023 Nanjing, China
b
Institute for Marine Science, Nanjing University, 163 Xianlin Avenue, 210023 Nanjing, China

a r t i c l e i n f o a b s t r a c t

Article history: Contamination of fine plastic particles (FPs), including micrometer to millimeter plastics (MPs) and
Received 23 June 2016 nanometer plastics (NPs), in the environment has caught great concerns. FPs are strong adsorbents for
Received in revised form hydrophobic toxic pollutants and may affect their fate and toxicity in the environment; however, such
18 October 2016
information is still rare. We studied joint toxicity of FPs with phenanthrene to Daphnia magna and effects
Accepted 21 October 2016
Available online 27 October 2016
of FPs on the environmental fate and bioaccumulation of 14C-phenanthrene in fresh water. Within the
five sizes particles we tested (from 50 nm to 10 mm), 50-nm NPs showed significant toxicity and physical
damage to D. magna. The joint toxicity of 50-nm NPs and phenanthrene to D. magna showed an additive
Keywords:
Microplastics
effect. During a 14-days incubation, the presence of NPs significantly enhanced bioaccumulation of
Nanoplastics phenanthrene-derived residues in daphnid body and inhibited the dissipation and transformation of
Phenanthrene phenanthrene in the medium, while 10-mm MPs did not show significant effects on the bioaccumulation,
Joint toxicity dissipation, and transformation of phenanthrene. The differences may be attributed to higher adsorption
Bioaccumulation of phenanthrene on 50-nm NPs than 10-mm MPs. Our findings underlined the high potential ecological
Environmental fate risks of FPs, and suggested that NPs should be given more concerns, in terms of their interaction with
hydrophobic pollutants in the environment.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction
Environmental contamination caused by fine plastic particles
(FPs), including micrometer to millimeter plastics (microplastics,
MPs) and nanometer-scale plastics (nanoplastics, NPs), has been
catching more and more concerns in recent years, especially in
aquatic environment (Andrady, 2011; Browne et al., 2011; Eerkes-
Medrano et al., 2015; Mattsson et al., 2015b). MPs have been
widely found in water, sediments, and marine animals (Boerger
et al., 2010; Browne et al., 2008; Cole et al., 2013, 2015; Davison
and Asch, 2011; Murray and Cowie, 2011; von Moos et al., 2012).
Environmental concentrations of FPs varied significantly among
different regions. In some protected areas in the Atlantic, as high as
*
This paper has been recommended for acceptance by Dr. Harmon Sarah
Michele.
* Corresponding author. State Key Laboratory of Pollution Control and Resource
Reuse, School of the Environment, Nanjing University, 163 Xianlin Avenue, 210023
Nanjing, China.
E-mail address: ji@nju.edu.cn (R. Ji).
100 g L
1 of MPs was found in sediments (Baztan et al., 2014). Due
to the small sizes, FPs could be easily ingested by zooplankton and
zoobenthos, cause obstruction of feeding appendages, aggregate,
and block the alimentary canal, limit food intake or be translocated
into the circulatory system (Barnes et al., 2009; Browne et al., 2008;
Murray and Cowie, 2011). Moreover, FPs accumulated in small an-
imals could be further translocated into higher trophic level and
cause more damage (Mattsson et al., 2015a). However, most studies
so far were focusing on MPs. NPs, which had potentially much
higher surface area and mobility in aquatic systems, were still
rarely studied.
Hydrophobic organic compounds in the aquatic environment
have very high tendency to be associated with suspended sediment
particles (Eek et al., 2010; Xia et al., 2006). With similar physical
properties and even higher surface hydrophobicity than naturally
occurring suspended organic matter, plastic fragments especially
FPs have even higher sorption capacity and may further influence
the environmental fate of hydrophobic persistent organic pollut-
ants (POPs) (Lee et al., 2014; Mato et al., 2001). Large amount of
hydrophobic POPs, including polycyclic aromatic hydrocarbons

http://dx.doi.org/10.1016/j.envpol.2016.10.061
0269-7491/© 2016 Elsevier Ltd. All rights reserved.
 Y. Ma et al. / Environmental Pollution 219 (2016) 166e173
(PAHs), polychlorinated biphenyls (PCBs), and polybrominated
diphenyl ethers (PBDEs), can be carried by waste plastic particles
(Hirai et al., 2011; Mato et al., 2001; Rios et al., 2007). Rios et al.
(2007) found that total concentration of PAHs ranged from 39 to
1200 ng g
1, PCBs from 27 to 980 ng g
1, and DDTs from 22 to
7100 ng g
1 on plastic fragments and pellets. Despite of the large
risks of hydrophobic POPs carried by FPs in the environment
especially to biota, most studies are limited to the toxicity effects of
FPs alone on marine organisms (Barnes et al., 2009; Besseling et al.,
2014; Browne et al., 2008; Murray and Cowie, 2011). Researches on
the joint toxicity of FPs with POPs are still very rare (Oliveira et al.,
2013). In addition, several studies have shown evidences that POPs,
e.g. PAHs, PBDEs, and PCBs adsorbed on MPs could be transferred
into the body of different marine organisms like mussels and lug-
worms (Avio et al., 2015; Besseling et al., 2013; Browne et al., 2013).
However, the knowledge of the impacts of FPs especially NPs on
bioavailability and fate of hydrophobic organic pollutants in the
aquatic environment is still very limited (Mattsson et al., 2015b).
In this study, we used radioactive tracer and chose phenan-
threne (Phe) as a model compound of PAHs, which have significant
carcinogenic and mutagenic toxicity to organisms (Perera, 1997;
Zhang et al., 2009), to investigate the combined toxicity of FPs
and PAHs, and the effects of MPs and NPs on the transformation and
bioaccumulation of PAHs in fresh water, using the model fresh
water filter feeding zooplankton Daphnia magna. Polystyrene was
selected as a representative model plastic, because of the highest
production volume, making up about 90% of the total plastic de-
mand, and is therefore widely found in the environment (Andrady
and Neal, 2009).
2. Materials and methods
2.1. Chemicals and micro-/nanoplastics
[9-14C]-labelled Phe (14C-Phe) in ethanol with 99% radiochem-
ical purity and 2.02 GBq mmol
1 specific radioactivity was pur-
chased from American Radiolabeled Chemical Inc. (St. Louis, USA).
Phe was purchased from Tokyo Chemical Industry Co. Ltd (Tokyo,
Japan) with >98.5% purity. NPs and MPs suspensions made of
polystyrene with concentrations of 2.5% w/v, with particle size of
50 nm, 500 nm, 5 mm, 10 mm, and 15 mm, were purchased from
BaseLine ChromTech Research Center (Tianjin, China). The size
distributions of the plastic particles in the experiment medium
were measured by transmission electron microscopy (TEM) as
follows: 50 nm (25e75 nm), 500 nm (480e520 nm), 5 mm
(5e7 mm), 10 mm (8e12 mm), and 15 mm (11e16 mm). Images of the
plastic particles and size distributions are shown in Fig. S1.
2.2. Organisms and culturing conditions
The cladoceran D. magna and the green alga Chlamydomonas
reinhardtii on which D. magna feeds were both obtained from the
Institute of Hydrobiology, Chinese Academy of Science. These
daphnids were raised in The Hong Kong University of Science and
Technology (HKUST) laboratory for more than 10 years using GF/C
membrane-filtered pond water, then were raised in Nanjing Uni-
versity laboratory for about five years using aerated tap water at
23.5 ± 1  C on a 14:10 h light-dark cycle with an irradiance of
50 mmol photons m
2 s
1. Daphnids were acclimated in M4 me-
dium (Table S1) (Elendt and Bias, 1990) for one week before ex-
periments. The alga C. reinhardtii was cultivated in WC medium
(Table S2) (Guillard, 1975) under the same environmental condi-
tions as the daphnids.
167
2.3. Acute toxicity test for D. magna
Acute toxicity caused by test compounds (Phe and FPs) after
48 h of exposure was tested according to the United States EPA
guidelines (Agency, U.S.E.P, 1987). Daphnid neonates were exposed
to test compounds at six concentrations (including control) in M4
medium. The tests were performed in three replicates in 50-mL
glass beakers containing 40 mL of M4 medium and five daphnid
neonates (less than 24 h after birth). All beakers were covered with
watch glasses, and randomly distributed in a climate incubator
(20 ± 1  C) with a 16:8 h light-dark cycle (light irradiance of
50 mmol photons m
2 s
1). The temperature and light were
controlled automatically by the incubator and monitored periodi-
cally during the experiments. The daphnids were randomly
distributed over the test vessels and not fed during the experi-
ments. After 48 h of incubation, the daphnids were checked for
immobilization. Daphnids were considered immobilized when
they were not able to swim after 15 s of gentle stirring, according to
the United States EPA guidelines (Agency, U.S.E.P, 1987). Different
combination of MPs (0, 2.5, 5, 10, and 50 mg L
1) or NPs (0, 2.5, 5,
8.5, 11, and 14.5 mg L
1) and Phe (0, 0.05, 0.1, 0.2, 0.4, 0.8, and
1.2 mg L
1) were chosen, according to their individual EC50 values,
to test the joint toxicity of FPs and Phe to D. Magna. The concen-
trations of FPs were obtained by diluting stock solutions provided
by the company after ultra-sonication and vortexing. In all tests,
Phe was dissolved in ethanol and added to the testing medium at
0.1% volumetric ratio of solvent to medium. Solvent control (0.1%
ethanol) was also set during the tests and no immobilization was
observed for the control.
2.4. Degradation and accumulation of phenanthrene in D. magna
Phe degradation and bioaccumulation with or without the
presence of 5 mg L
1 NPs (50 nm) or MPs (10 mm) were investigated
with <24-h old (small) and 10-days old (large) daphnids in the M4
medium. The tests were performed with ten daphnids each in three
replicates. The experiment lasted for 14 days and daphnids were
fed once everyday with C. reinhardtii at concentrations between 5
and 20 million cells per beaker. 14C-Phe was used to trace the fate of
Phe in the system. Briefly, 12 mL of 14C-Phe (1.48 kBq mL
1,
324 mg L
1) were added to 40 mL of the M4 medium in 50-mL
beaker to give a final Phe concentration of 0.1 mg L
1 and radio-
activity of 17.6 kBq. MPs or NPs were added to each beaker and
ultra-sonicated for 20 min for better dispense of the plastics in the
beaker. Preliminary experiment proved that the sonication process
did not cause degradation of 14C-Phe and no metabolites were
detected after sonication process. On days 2, 7, and 14, three bea-
kers were taken out, and the daphnids were rinsed three times in
deionized water for 20 min each time (Oberdorster et al., 2006) and
then frozen at
20  C before further analysis. After rinsing, no
adhesion of the plastic particles on daphnid carapace was observed
under optical microscope. 14C-Phe and its transformation products
that remained in daphnid gut and adsorbed tightly on daphnid
skinafter rinsing were also taken into account for calculating bio-
accumulation. The remaining medium was extracted three times
with ethyl acetate and the extracts were concentrated on a rotary
evaporation at 40  C for further analysis.
2.5. Adsorption experiments
The applied dialysis technique (Li et al., 2011) in a polytetra-
fluoroethene (Teflon®) chamber consisting of two half-cells (each
9 mL), separated by a dialysis membrane (regenerated cellulose,
cut-off 1000 Da). The detailed description of the apparatus was
published elsewhere (Ho€llrigl-Rosta et al., 2005). At the beginning,
168 Y. Ma et al. / Environmental Pollution 219 (2016) 166e173
one half-cell was filled with 8 mL of the sterilized M4 medium, and
another half-cell with the M4 medium containing 50 mg L
1 NPs or
500 mg L
1 MPs. The concentrations of FPs were selected according
to their adsorption capacity measured in pre-experiments to make
sure that Phe concentration after equilibrium in each half-cell was
in the range of detecting limits for radioactivity. The system was
mixed by rotating the cells at 10 rpm for 16 h and then solutions of
14
C-Phe in ethanol were added to the MPs/NPs-free half-cell using a
glass micro-syringe (Hamilton, Switzerland). The methanol con-
centration of the solutions was kept at <0.1% (v/v) to minimize co-
solvent effects. The experimental concentrations of Phe ranged
from 0.05 to 1.2 mg L
1, respectively. The cells were sealed by Teflon
plug and rotated in dark at 10 rpm. Preliminary experiments
showed that adsorption equilibrium of Phe on the plastics reached
within 7 days. Thereafter aliquots of solution from both sides of the
membrane were sampled to quantify the 14C-Phe concentrations by
liquid scintillation counting (LSC).
2.6. Analytic methods
Radioactivity was quantitatively counted in a liquid scintillation
counter (LS6500, Beckman, USA). For radioactivity in water and
organic extracts, 200 mL of samples were mixed with 2 mL of
scintillation cocktail (Gold Star, Meridian Biotech Ltd., UK). Radio-
activity background was subtracted from the LSC measurement. For
radioactivity in the daphnids, the animals were combusted on an
oxidizer (OX500, Zinsser Analytic, Germany) and the generated CO2
was absorbed by 10 mL of alkaline scintillation cocktail (Oxysolve
C-400, Zinsser Analytic, Germany) and counted by LSC. The re-
covery of the oxidizer was >96%.
High-performance liquid chromatography (HPLC) equipped
with an online radioactivity flow detector (Ramona Star; Raytest,
Straubenhardt, Germany) was used to measure 14C-Phe and its
metabolites remained in the medium. HPLC was conducted on an
Eclipse XDB-C18 column (250 mm  4.6 mm, 5 mm; Agilent Tech-
nology, USA) at 30  C with an Agilent HPLC 1100 system. The radio-
detector had a detection limit of 1 Bq. The mobile phase started at
60% acetonitrile for 2 min, increased to 95% acetonitrile in 3 min,
then maintained for 4 min, deceased to 60% acetonitrile in 3 min,
then maintained for 2 min. The flow rate of the mobile phase was
1 mL min
1 and the scintillation cocktail (Gold Flow multipurpose;
Meridian Biotechnologies Ltd., Epsom, UK) was 2 mL min
1.
2.7. Data analysis
The Freundlich isotherm model (Eq. (1)) was used to fit the
sorption data for the best fitting results:
1=n
qe ¼ KF  Ce
where qe is the concentration of Phe adsorbed on the solid phase at
equilibrium (mg kg
1), Ce the concentration of Phe in the solution
at equilibrium (mg L
1), KF the Freundlich affinity coefficient
(mg1
1/n L1/n kg
1), and 1/n (unitless) the Freundlich non-linearity
coefficient. The distribution constants Kd (L kg
1) were calculated
by equation (2):
Kd ¼ qe =Ce
Data on Phe dissipation were fitted to the first-order kinetics
(Eq. (3))
Ct ¼ C0  e
kt
where C0 is the initial concentration, Ct is the concentration at time
t, and k is the degradation rate constant. The regression was carried
out using Prism 6 (GraphPad Software, Inc.). The half-life (t1/2) was
calculated using the equation t1/2 ¼ ln2/k.
Statistical analysis to compare differences between controls and
MPs treatments at different times were performed with the soft-
ware IBM SPSS 22.0 (IBM, Inc) by using two-way ANOVA with
Tukey's Post Hoc test. P < 0.05 indicated statistical significance. The
calculation of EC50 and the analysis of EC50 shifts were conducted
by the software Prism 6 (GraphPad Software, Inc.) by using the
dose-response nonlinear fitting model. The Freundlich isotherm
model and first-order kinetics model were fitted by the software
IBM SPSS 22.0 (IBM, Inc) with non-linear regression.
3. Results and discussion
3.1. Acute toxicity of FPs and Phe to D. magna
Acute toxicity of NPs (50 nm, 500 nm) and MPs (5 mm, 10 mm,
15 mm), were assayed, in terms of the immobilization rate of
daphnids. The toxicity of MPs to D. magna depended on the size of
particles; 50-nm NPs showed significant dose effect from 1 to
50 mg L
1 with an EC50 of 15.13 ± 3.34 mg L
1 (predicted
value ± standard error) (Fig. 1), while no immobilization was
observed with the larger size particles at concentrations of up to
100 mg L
1 (Table S3). The EC50 value for 50-nm NPs was similar to
the EC50 values of carbon nanotubes to D. magna (12.7 mg L
1)
(Petersen et al., 2011) while lower than fullerenes and graphene
(Guo et al., 2013; Oliveira et al., 2013; Pakarinen et al., 2013). The
immobilization was in agreement with the physical damage on
daphnid body caused by plastic particles (Fig. 2). For MPs, no
physical damage was observed under microscope (Fig. 2D), which
showed that MP beads were ingested into the intestinal tract and
excreted through the anus without sticking on the body. In contrast,
50-nm NPs at 10 mg L
1 caused already severe damage on the
thoracopods which are essential to swimming and creating water
currents for filtering feeding, probably owing to the accumulation
of NPs on the surface of the thoracopods (Fig. 2E). When the con-
centration of 50-nm NPs increased to 50 mg L
1, such damage was
observed all around the body (Fig. 2F), in agreement with the 100%
immobilization rate in this treatment (Fig. 1). The stronger
attachment of NPs to daphnid body may be due to the stronger
electrostatic interaction between NPs and daphnids. In addition to
the physical damage, the toxicity of NPs may also be due to toxic
impurities from NPs such as surfactant added during the synthesis
process or remaining styrene monomer. Also, it was uncertain
whether the immobilization of daphnids caused by NPs was also
attributed to a diffusion of NPs into cells of the daphnid, which
(1)
100
Immobilization (%)
EC50 = 15.13 ± 3.34 ppm
80 R2 = 0.975
60
40
20
(2) 0
0.1 1 10 100
Concentration of 50-nm NPs (ppm)
(3) Fig. 1. Concentrationeresponse curves of 48-h acute toxicity of 50-nm NPs expressed
as immobilization rate of D. magna (<24 h old) in M4 medium (N ¼ 3). Error bars show
standard deviations of three replicates.
 Y. Ma et al. / Environmental Pollution 219 (2016) 166e173 169

Fig. 2. Effects of various MPs and NPs on the morphology of D. magna observed under 40 (A, B, C, E, and F) or 160 (D) optical microscope. Daphnids of less than 24 h old were
exposed to MPs with various sizes and concentrations, and were taken out after 48 h of incubation. (A), (B), (C), (E), and (F) are the whole body of daphnid in the medium containing
neither MPs nor NPs (Control), 10 mg L
1 of 500-nm NPs, 50 mg L
1 of 500-nm NPs, 10 mg L
1 of 50-nm NPs, and 50 mg L
1 of 50-nm NPs, respectively. In (F) the daphnid was dead.
(D) shows the hind gut of daphnid in the medium containing 100 mg L
1 of 10-mm MPs. Arrows indicate examples of the area with concentrated NPs in daphnid body (B, C, E, and F)
or with MPs excreted from the hind gut (D).

could probably cause further physiological damage as in the case of

other carbonaceous nano-materials, such as carbon nanotubes
(Petersen et al., 2009).
Our results suggest that FPs may be ingested by fresh water
animals and NPs could cause immobilization and physical damage
to daphnids. This is corresponding to the studies on marine or-
ganisms, showing that polystyrene MPs ranging from 7.3 to 30.6 mm
were taken up by zooplanktons (Cole et al., 2013, 2015). The toxicity
of NPs to D. magna was comparable to that of metallic oxide
nanoparticles (such as TiO2), which had EC50 on D. magna ranging
from 0.622 to 114 mg L
1 (Zhu et al., 2009). The higher toxicity of
NPs to daphnids than MPs observed in this study (Fig. 2) was in
agreement with the phenomena that polystyrene NPs had more
negative effects than MPs on the survival, development, and
fecundity of the marine copepod Tigriopus japonicas (Lee et al.,
2013).
In absence of plastic particles, 48-h EC50 value of Phe was
0.59 ± 0.05 mg L
1 calculated from the dose-response model
(Fig. 3; Table 1), which was comparable to the values (48-h
EC50 ¼ 0.48 and 0.55 mg L
1) reported by Smith et al. (2010) and
Zhang et al. (2014). With the presence of various concentrations of
50-nm NPs, the immobilization rates of D. magna were significantly
enhanced. For example, with NP concentration of 5 mg L
1, the EC50
shifted to almost half of the control value (48-h
EC50 ¼ 0.34 ± 0.02 mg L
1) (Table 1). The results of isobologram
analysis showed that the joint effects of NPs and Phe followed
concentration addition (Fig. 3). In contrast, with the presence of 10-
mm MPs, the EC50 did not shift significantly and even slightly
increased with high concentration of MPs. This indicated that MPs
had a weaker joint acute toxicity with Phe to D. Magna than NPs,
possibly due to the weaker toxicity of MPs alone (Fig. 2).
Significant sedimentation of particles was observed with MPs,
but NPs were relatively stable in the medium based on their zeta-
potential (Table S4). This sedimentation decreased the concentra-
tion of MPs in the medium; however, observation during our study
showed evidence that daphnids ingested the particles on the bot-
tom of the beakers, therefore the sedimentation might not have
strong effects on the acute toxicity of MPs.
Concentration of phenanthrene (ppm) 0.6
0.4
0.2
0.0
0 4 8 12 16
Concentration of 50-nm NPs (ppm)
Fig. 3. The additive isobologram of the 50%-immobilization effect of NPs and Phe to
D. magna in 48-h acute toxicity tests (N ¼ 3). The doted line with intercepts of EC50
values of NPs and Phe (15.13 mg L
1 and 0.59 mg L
1 respectively) predicts the con-
centration pairs of NPs and Phe with addictive effects of 50%-immobilization of
D. magna.
3.2. Effects of FPs on long-term bioaccumulation of Phe on
D. Magna
During the 14 days of incubation, NPs significantly increased the
bioaccumulation of 14C-Phe-derived residues in the daphnid body
(Fig. 4) and the bioconcentration factor (BCF) (Fig. S2). In the case of
the large size daphnids, 50-nm NPs (5 mg L
1) significantly
(P < 0.05) enhanced the bioaccumulation of Phe-derived residues in
the animals over the whole exposure period, and at the end of
exposure, the 14C-Phe-derived residues in the presence of NPs were
three-times higher of that in the plastic-free control. This indicated
that NPs played as vectors of hydrophobic POPs by adsorbing and
concentrating them on the surface. In the case of small daphnids,
the effects of NPs on the bioaccumulation of 14C-Phe-derived resi-
dues were much lower, possibly due to the lower ingestion rate.
170 Y. Ma et al. / Environmental Pollution 219 (2016) 166e173

Table 1
EC50 of Phe in the absence (control) and presence of various concentrations of FPs in 48-h acute toxicity test with D. magna (N ¼ 3). Concentrations of FPs are based on the total
FPs in the test vessels. R2 is the coefficient of determination for EC50 calculation. The ratios of EC50's in the presence of FPs (EC50, FPs) to that in the absence of FPs (EC50, control) are
also calculated. Values behind “±” are standard errors.

Treatment FPs Concentration (mg L
1) EC50 (mg L
1) R2 EC50, FPs/EC50, control

a
Control 0 0.59 ± 0.05 0.94 e
NPs 2.5 0.52 ± 0.02 0.92 0.88 ± 0.03
5 0.34 ± 0.02 0.95 0.57 ± 0.04
8.5 0.33 ± 0.05 0.95 0.56 ± 0.09
11 0.21 ± 0.05 0.84 0.35 ± 0.09
14.5 0.03 ± 0.02 0.94 0.05 ± 0.03
0 2.5 0.60 ± 0.02 0.89 1.01 ± 0.04
5 0.51 ± 0.02 0.95 0.87 ± 0.03
10 0.77 ± 0.08 0.94 1.31 ± 0.13
50 0.75 ± 0.07 0.80 1.27 ± 0.12
a
Not available.

Fig. 4. Percentage of initial 14C radioactivity detected in D. magna during a 14-days incubation with 0.1 mg L
1 of 14C-Phe in the presence of 5 mg L
1 of 50-nm NPs and 10-mm MPs
as well as in control (neither MPs nor NPs in the medium). Two different sizes of daphnids, large and small, were used in the experiment. Different letters indicate significant
(P < 0.05) difference within the same group at each sampling time. Data are averages of three individual experiments with error bars indicating standard deviations.

Similar to the acute toxicity results, 10-mm MPs showed no signif- in carp and daphnid body due to the affiliation of metals on the
icant effect on the bioaccumulation of Phe and its residues in the surface of nano-particles (Hartmann et al., 2012; Yang et al., 2014).
daphnid body (Figs. 3 and 4). The bioaccumulated 14C-Phe-derived Although the joint acute toxicity of NPs and Phe did not show a
residues in daphnids included 14C-Phe and its transformation syngeneic effect, during a long-term incubation the Phe on the
products that were translocated into the daphnid tissue, retained in surface of NPs may be able to diffuse into the cells together with
the daphnid gut, and tightly adhered on the carapace. Zooplanktons NPs like other nano-materials, which may result in a direct contact
are one of the most important groups at lower trophic level in the of Phe to the cell membrane - the toxicity target of Phe and its
aquatic food chain and are usually ingested entirely by predators. metabolites (Schirmer et al., 1998) - of D. magna, and thus strongly
Thus, the total amount of pollutants accumulated in the whole body enhance the chronic toxicity of Phe.
including gut and skin is more important than the pollutants only Batch sorption experiments showed the Freundlich sorption
in the tissue when evaluating the impact of zooplanktons on higher isotherm parameters KF of 3.18  105 mg1
1/n L1/n kg
1 with an n of
trophic level organisms. 0.86 for 50-nm NPs (Table 2, Fig. S3), while for 10-mm MPs the KF
Studies on lugworms and amphipods have shown that hydro- was 1.83  104 mg1
1/n L1/n kg
1. This led to the distribution
phobic organic pollutants like nonylphenol, Phe, and PBDEs
adsorbed on micrometer- to millimeter-sized plastics could be
transferred into the body of zoobenthos (Browne et al., 2013; Chua Table 2
et al., 2014). However, those studies showed the presence of plastic Freundlich sorption isotherm parameters (KF, 1/n) and distribution constant (Kd) of
particles lowered the bioavailability of pollutants compared to free Phe with respect to 50-nm NPs and 10-mm MPs. Batch sorption experiment was
conducted in M4 medium using a polytetrafluoroethene chamber consisting of two
pollutants or pollutants preloaded on the surface of sand. Unlike
half-cells separated by a dialysis membrane. R2 values for the goodness of fitting
MPs, which could be easily excreted out through the gut, NPs much were also shown. Values behind “±” are standard errors.
easily accumulated on the thoracopods and in the digestive tract
1/n
Adsorbent KF (105 mg1 L1/n kg
1) 1/n R2 Kd (105 L kg
1)
(Fig. 2B, C, E, F) and increase Phe bioaccumulation. Agreed with our
results, other studies have also shown that, with the presence of 50-nm NPs 3.18 ± 0.27 0.86 ± 0.03 0.999 6.54 ± 1.71
nano-TiO2, the bioaccumulation of heavy metals like Cd increased 10-mm MPs 0.183 ± 0.026 1.02 ± 0.04 0.996 0.169 ± 0.011
 Y. Ma et al. / Environmental Pollution 219 (2016) 166e173 171

control 50-nm NPs 10-µm MPs the initial Phe (no mineralization in the system) after 14 days
(Fig. S5). In the presence of daphnids, the transformation products
100
in the medium increased to 83.1% (large daphnids) and 40.5% (small
with large daphnids
daphnids) (Fig. S5), indicating that biodegradation by daphnid or
80 associated microbial activity was much higher than photo-
degradation. Based on the radioactivity recovery from medium and
% of initial phenanthrene remained in medium

60 daphnid body (Table S5), radioactivity loss by either mineralization

40
20
0 The presence of 50-nm NPs significantly inhibited the dissipa-
0 5
100
with small daphnids
80
60
40
20
0 (Table 2) showed that with the presence of 5 mg L
1 of 50-nm NPs
0 5
Incubation time (day)
14
Fig. 5. C-Phe remained in the medium during exposure of large (top) and small
(bottom) daphnids to 14C-Phe (0.1 mg L
1) for 14 days in the presence of 50-nm NPs
(5 mg L
1) or 10-mm MPs (5 mg L
1) and in control (neither MPs nor NPs in the
medium). Data are averages of three individual experiments with error bars indicating
standard deviations.
constant Kd value to be 40 times higher for 50-nm NPs than 10-mm
MPs in the testing concentration of Ce between 0.001 and
0.05 mg L
1. Correspondingly, the BET surface area of 50-nm NPs
(84.96 ± 2.91 m2 g
1) was 20-times higher than that of 10-mm MPs
(3.71 ± 0.49 m2 g
1) (Table S4). Our results suggested that NPs with
higher specific surface area, i.e., higher sorption capacity for hy-
drophobic organic pollutants, might carry more pollutants into
animals.
3.3. Effects of FPs on dissipation and degradation of Phe
The dissipation of Phe in the system could be attributed to three
pathways: biodegradation, photodegradation, and volatilization. In
the absence of daphnids, photodegradation amounted to 11.8% of
or volatilization mostly happened after 7 days for the large daph-
nids and during the first 7 days for the small daphnids. Since
degradation products counted much lower in the small daphnid
groups than large daphnid groups (Fig. S5), it was likely that
volatilization contributed relatively more than mineralization to
the radioactivity loss.
10 15 20 tion thus increased the half-life (t1/2) of 14C-Phe in the medium
(e.g., from 2.17e3.51 days to 4.59e7.61 days in the medium with
large daphnids), while 10-mm MPs had no effects on the dissipation
with the presence of either large or small daphnids (Fig. 5, Table 3).
Correspondingly, the ratio of metabolites to parent compound
significantly decreased in the presence of 50-nm NPs, especially
with the presence of daphnids, indicating a slower degradation of
Phe with the presence of NPs (Fig. 6). It was not able to determine
the chemical structures of the metabolites, due to their trace
amount in the medium. The inhibition of dissipation should also be
attributed to the high adsorption affinity of NPs to Phe, resulting in
lower concentration of freely available Phe in the liquid phase
(Table 3). Calculation using the Freundlich sorption isotherm model
10 15 20 or 10-mm MPs the free Phe concentration in the medium decreased
from the original 0.1 mg L
1 to 0.0028 or 0.092 mg L
1, respectively.
The inhibition of Phe dissipation in the system could increase
the exposure time of daphnids to Phe and further accumulation
through food web. If the metabolites were less toxic than Phe, this
inhibition could increase the toxicity of Phe. Daphnids of different
ages responded differently to the inhibition of Phe dissipation by
NPs. In the case of the large daphnids about 19.7 ± 2.8% of the initial
Phe retained in the medium at the end of incubation (14 days);
however, in the case of the small daphnids the residual Phe
increased to 27.2 ± 2.8% (Fig. 5), indicating that compared to old
daphnids, neonatal daphnids suffered for a longer time higher
negative effects caused by NPs.
3.4. Environmental implications
This study for the first time provided comprehensive informa-
tion on the impact of FPs on the toxicity, bioaccumulation, and
degradation of a model PAH compound Phe in fresh water system
with a model test organism D. magna. Our findings indicated the
possible ecological and environmental risks of plastic particles, and
suggested that nanometer scale NPs should be given more concern,
in terms of their joint toxicity and interaction with organic

Table 3
Dissipation constant k and half-life (t1/2) of Phe calculated with the first-order decay model from the incubation experiment of exposing large and small daphnids to 14C-Phe
(0.1 mg L
1) for 14 days in the presence of 50-nm NPs (5 mg L
1) or 10-mm MPs (5 mg L
1) and in control (neither MPs nor NPs in the medium). Values in parentheses are 95%
confidence limits. R2 values are coefficients of determination of model fitting.

Parameter With large daphnids With small daphnids

Control 50-nm NPs 10-mm MPs Control 50-nm NPs 10-mm MPs

k 0.258 0.121 0.265 0.193 0.110 0.172

(0.197, 0.319) (0.091, 0.151) (0.185, 0.344) (0.151, 0.235) (0.095, 0.125) (0.141, 0.202)
Half Life (t1/2) 2.68 5.73 2.62 3.59 6.28 4.03
(2.17, 3.51) (4.59, 7.61) (2.01, 3.75) (2.95, 4.57) (5.53, 7.27) (3.42, 4.91)
R2 0.927 0.855 0.873 0.956 0.959 0.968
172 Y. Ma et al. / Environmental Pollution 219 (2016) 166e173

control 50-nm NPs 10-µm MPs 1596e1605.

Andrady, A.L., Neal, M.A., 2009. Applications and societal benefits of plastics. Philos.
Trans. Roy. Soc. B Biol. Sci. 364, 1977e1984.
120 Avio, C.G., Gorbi, S., Milan, M., Benedetti, M., Fattorini, D., d'Errico, G., Pauletto, M.,
with large daphnids
Bargelloni, L., Regoli, F., 2015. Pollutants bioavailability and toxicological risk
100 from microplastics to marine mussels. Environ. Pollut. 198, 211e222.
Barnes, D.K.A., Galgani, F., Thompson, R.C., Barlaz, M., 2009. Accumulation and
fragmentation of plastic debris in global environments. Philos. Trans. Roy. Soc. B
80 * Biol. Sci. 364, 1985e1998.
Baztan, J., Carrasco, A., Chouinard, O., Cleaud, M., Gabaldon, J.E., Huck, T., Jaffres, L.,
* Jorgensen, B., Miguelez, A., Paillard, C., Vanderlinden, J.P., 2014. Protected areas
60 in the Atlantic facing the hazards of micro-plastic pollution: first diagnosis of
% of metabolites in medium

40
20
0 12336e12343.
0 5 10
120
with small daphnids
100
80
60
* linked to health and biodiversity. Curr. Biol. 23, 2388e2392.
40 * Chua, E.M., Shimeta, J., Nugegoda, D., Morrison, P.D., Clarke, B.O., 2014. Assimilation
20
0 Cole, M., Lindeque, P., Fileman, E., Halsband, C., Galloway, T.S., 2015. The impact of
0 5 10
Incubation time (day)
Fig. 6. Percentage of 14C-Phe-derived metabolites compared to total radioactivity
remained in the medium during exposure of large (top) and small (bottom) daphnids
to 14C-Phe (0.1 mg L
1) for 14 days in the presence of 50-nm NPs (5 mg L
1) or 10-mm
MPs (5 mg L
1) and in control (neither MPs nor NPs in the medium). Data are averages
of three individual experiments with error bars indicating standard deviations. “*”
indicates significant difference from control.
pollutants. Our study showed that in addition to the additive effects
on acute toxicity and increasing bioaccumulation potential of hy-
drophobic pollutants, plastic particles may inhibit degradation of
organic pollutants and their metabolites in the environment,
resulting in accumulation of both parent compounds and metab-
olites in the environment. Better knowledge of the impacts of FPs,
especially the NPs, on the degradation pathway of hydrophobic
organic pollutants, their chronic toxicity, and transportation in food
web in fresh water system is a research imperative now.
Acknowledgments
This work was funded by the gs1:National Natural Science
Foundation of China (Grant No. 21407075) and China Postdoctoral
Science Foundation (Grant No. 2014M561624).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.envpol.2016.10.061.
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