Environmental Pollution 255 (2019) 113300

Contents lists available at ScienceDirect

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

Acute effects of nanoplastics and microplastics on periphytic biofilms

depending on particle size, concentration and surface modification*
Lingzhan Miao a, b, Jun Hou a, b, *, Guoxiang You a, b, Zhilin Liu a, b, Songqi Liu a, b,
Tengfei Li a, b, Yujuan Mo b, Song Guo b, Hao Qu b
a
Key Laboratory of Integrated Regulation and Resource Development on Shallow Lake of Ministry of Education, College of Environment, Hohai University,
Nanjing, 210098, China
b
College of Environment, Hohai University, Nanjing, China

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

Article history: Microplastics (MPs) can disintegrate into smaller sized microplastics and even nanoplastics (NPs). The
Received 6 May 2019 toxicity of nanoplastics and microplastics on freshwater organisms have been well explored recently,
Received in revised form however, very little is known about the potential impacts of NPs on freshwater biofilms, which are
20 September 2019
essential for primary production and nutrient cycling in aquatic ecosystems. In this study, we studied the
Accepted 22 September 2019
Available online 2 October 2019
acute effects (3 h of exposure) of polystyrene beads (PS, with diameter range from 100 nm to 9 mm) on
five biological endpoints targeting community and ecosystem-level processes in biofilms: chlorophyll a,
photosynthetic yield, and three extracellular enzyme activities. The results showed that the large size PS
Keywords:
Nanoplastics
beads (500 nm, 1 mm, and 9 mm) exhibited negligible effects on the determined biological endpoints in
Microplastics biofilms within the range of concentrations (5e100 mg/L) in this study. However, high concentration of
Biofilm PS beads (100 nm, 100 mg/L) significantly decreased the content of chlorophyll a, and the functional
Extracellular enzymes enzyme activities of b-glucosidase and leucine aminopeptidase, suggesting negative effects on the car-
ROS generation bon and nitrogen cycling of freshwater biofilms. Moreover, the influences of PS NPs (100 nm) on biofilms
strongly depended on the surface modification of PS particles, with the positively charged PS NPs
(amide-modified) exhibiting the highest toxicity to biofilms. The excess generation of reactive oxygen
species (ROS) in this study indicated oxidative stress induced by PS NPs, which might lead to the
observed nano-toxic effects on biofilms. In response, the antioxidant activity of biofilm was enhanced as
indicated by the increased total antioxidant capacity (T-AOC). Overall, our findings highlight nanoplastics
have potential to disrupt the basic ecological functions of biofilms in aquatic environments.
© 2019 Elsevier Ltd. All rights reserved.

1. Introduction essential roles in carbon fixation and nutrient cycling in aquatic

Periphytic biofilms are complex microbial communities present
on submerged surfaces, and are ubiquitous in freshwater environ-
ments (Battin et al., 2016). They are taxonomically diverse and
mainly consist of algae and bacteria, assembled in a polymeric
matrix of extracellular polymeric substances (EPS) (Flemming et al.,
2016). Biofilms not only provide community structure and primary
productivity that support higher trophic levels, but also play
*
This paper has been recommended for acceptance by Lian-Jun Bao.
* Corresponding author. Key Laboratory of Integrated Regulation and Resource
Development on Shallow Lake of Ministry of Education, College of Environment,
Hohai University, Nanjing, 210098, China.
E-mail addresses: hhuhjyhj@126.com, hjy_hj@hhu.edu.cn (J. Hou).
environments (Battin et al., 2016). Further, biofilms have long been
used in ecotoxicological studies because they serve a bioindicator
for the evaluation of pollutant effects on aquatic ecosystems (Battin
et al., 2016; Besemer, 2015).
As biofilms are typically found in streams, river and lakes, they
might represent a first environmental medium that interact with
potential stressors (Tang et al., 2018), such as microplastics (MPs),
commonly defined as plastic particles diameter less then 5 mm
(Thompson et al., 2004). The discharge of MPs into environment
has drawn increasing environmental concerns in the past decade
due to their potential impact on aquatic organisms in freshwater
systems (Li et al., 2018; Su et al., 2016). Polyethylene (PE), poly-
styrene (PS), and polypropylene (PP) were the most common types
of microplastics found in aquatic environments (Koelmans et al.,
2019; Silva et al., 2018). Variable concentrations of microplastics

https://doi.org/10.1016/j.envpol.2019.113300
0269-7491/© 2019 Elsevier Ltd. All rights reserved.
2 L. Miao et al. / Environmental Pollution 255 (2019) 113300
in surface water have been reported from different freshwater
environments, and the highest abundance of microplastics in
plankton net samples was observed in Taihu Lake with 3.4e25.8
items/L (Su et al., 2016). The fate and potential impacts of MPs on
aquatic ecosystems have widely investigated (Silva et al., 2018).
MPs can undergo further degradation or fragmentation, eventually
disintegrating into nanoplastics (Gigault et al., 2016; Lambert and
Wagner, 2016; Song et al., 2017), which exhibit larger surface area
than their bulk particles and also interact with living organisms
differently (Gonza
lez-Pleiter et al., 2019; Triebskorn et al., 2019).
Although there is no suitable monitoring method to directly
determine the amounts of nanoplastics in aquatic environments
(Gigault et al., 2016), the environmental concentrations of nano-
plastics are predicted to be much higher than microplastics (Chae
and An, 2017). The inherent nano-specific properties of NPs can
enhance the risk of their uptake into cell and tissues, and induce
generation of reactive oxygen species (ROS), affecting their physi-
ological processes (Tallec et al., 2018). Therefore, with the particle
size shrinking, the potential toxicity might increase, even if the
same material is relatively inert in bulkier form (Silva et al., 2018;
Triebskorn et al., 2019).
Adverse effects of NPs have been observed on the aquatic en-
vironments, and most of ecotoxicological studies were conducted
using freshwater organisms, including daphnia, zebrafish, shrimp,
macroalgae, cyanobacteria, and phytoplankton (Besseling et al.,
2014; Gonza
lez-Pleiter et al., 2019; Prata et al., 2019; Triebskorn
et al., 2019). Periphytic biofilms are suggested to encounter NPs
after their releases into freshwater, yet, to our knowledge, there is
little information of the potential impacts of NPs on natural biofilm
communities. Numerous studies have investigated the toxicity of
nanomaterials (such as Ag, TiO2, and Fe2O3) to microbial commu-
nities in aquatic environments (Chen et al., 2019; Gil-Allue et al.,
2015; Tang et al., 2018; Tang et al., 2017). Given the nano-
particulate nature of both NPs and nanomaterials, could there be
similar toxic potential of these two particles to microbes in aquatic
environments? (Bundschuh et al., 2018; Huffer et al., 2017). A very
recent study demonstrated that PS NPs (50 nm) increased the
generation of reactive oxygen species (ROS) and negatively affected
the inorganic nitrogen conversion efficiencies of marine bacterium
Halomonas alkaliphlia (Sun et al., 2018). Thus, when exposed to
biofilms, NPs may interact closely with microbial cell and enzymes,
affecting their ecosystem functioning.
In order to address this issue, we explored the potential impacts
of MPs and NPs on periphytic biofilms through a 3 h exposure ex-
periments. As one of the most detected plastics in freshwater, PS
with various sizes (with diameter range from nm to mm) frequently
serve as model particles to evaluate the environmental behaviors
and potential impacts of plastic particles (Nolte et al., 2017; Sun
et al., 2018; Triebskorn et al., 2019). Therefore, PS plastics (with
diameter range from 100 nm to 9 mm) at a dosage ranging from 5 to
100 mg/L (reaching or exceeding the upper limit of environmen-
tally relevant concentrations) were used in our exposure experi-
ments. A series of biological endpoints targeting community and
ecosystem-level processes in biofilms were selected to evaluate
the impacts on biofilms, including chlorophyll a, photosynthetic
yield and three extracellular enzymes activities. The extracellular
enzymes, including b-glucosidase (GLU), leucine aminopeptidase
(LAP), and alkaline phosphatase (AKP) were measured to assess the
effects on carbon, nitrogen and phosphorus acquisition by
periphyton. In addition, the generation of ROS and the level of T-
AOC were measured to assess the effects on oxidative stress in
biofilms. This is the first study exploring the impacts of nano-
plastics and microplastics on periphytic biofilms, and the results are
expected to deepen our understanding of the potential impacts of
plastics debris on aquatic ecosystems.
2. Materials and methods
2.1. Nano- and microplastics preparation and characterization
Polystyrene beads (milk white suspension, 25 mg/mL) were
purchased from BaseLine Chromtech Research Centre (Tianjin,
China). The polystyrene beads were monodisperse and supplied in
10-mL aqueous suspensions. The PS beads of different sizes and
surface modifications were applied: carboxyl PS (100 nm, PS-
COOH), amino PS (100 nm, PS-NH2), and unibead PS in four
different sizes (100 nm, 500 nm, 1 mm and 9 mm). These particles
were chosen due to that they are widely used in ecotoxicity tests of
micro/nano plastics, and therefore the results obtained here could
be compared with those in previous studies (Besseling et al., 2014;
Nolte et al., 2017; Sjollema et al., 2016). According to the manu-
facturer’s description, the PS suspension contained up to 0.25%
sodium dodecyl sulfate (SDS; surfactant to prevent particle aggre-
gation). The PS stock suspensions (500 mg/L) were prepared in
Milli-Q water by sonication (20
C, 250 W, 40 kHz) for 10 min
(Zhang et al., 2018). Prior to the exposure experiments, the stock
solution was transferred to a dialysis bag (8000 molecular weight
cut-off) for two days to remove the SDS present within the solution
(Pikuda et al., 2019).
In this study, the hydrodynamic diameter and zeta potential of
micro/nano PS after the remove of SDS were measured in Milli-Q
water (pH 6.9 ± 0.1) and filtered (through 0.22 mm) experimental
solution (used for biofilm cultivation in the hydrodynamic flume,
see section 2.2) by a Zetasizer Nano ZSP instrument (Malvern In-
struments, UK). The measurements were performed in triplicate,
and each containing 12 runs for the Z-average and 11 for the zeta
potential (Miao et al., 2018). The morphology of micro/nano PS used
was determined by a scanning electron microscope (SEM, Hitachi S-
4800 SEM). The PS stock was further ultrasonicated for 10 min
before use.
2.2. Biofilm cultivation
Biofilms were cultivated in an indoor hydrodynamic flume
described in our previous study (Liu et al., 2018). Briefly, the micro-
bial communities colonizing rocks in Xuanwu Lake were scraped and
served as initial inoculum of biofilms. The water quality parameters
in Xuanwu Lake were: pH ¼ 7.7; total nitrogen (TN) ¼ 2.3 mg/L; total
phosphorus (TP) ¼ 0.13 mg/L; ammonia ¼ 0.62 mg/L; and ni-
trate ¼ 0.85 mg/L. The flume was situated in a greenhouse (14/10-h
light/dark cycle) and the flow velocity were initiated and maintained
at 0.12 m s1. Pebble with a diameter of 2 cm was used as solid
substrate for biofilm development. In addition, the nutrient was
maintained by adding WC medium (Tang et al., 2017) (Table S1)
every 7 days. The biofilms were cultivated for two months, and after
that, dense and mature biofilms (Fig. S1) with moisture content ~ 85%
were used in the following exposure experiments.
2.3. Exposure experiment
The pebbles colonized by biofilm were taken out and transferred
to 250 mL Erlenmeyer flasks, and each flask contained 5 pebbles
and 120 mL of water collected from the hydrodynamic flume. After
the biofilms were acclimated for 2 h, they were exposed to micro/
nano plastics. Certain amounts of the PS stock suspensions of
different size (unibead, 100 nm, 500 nm, 1 mm and 9 mm) were
added to obtain the final concentrations of 5, 10, 50, and 100 mg/L.
This experimental setup was intended to investigate the effects of
particle size and concentration on biofilms. The reasons for
selecting these particle concentrations were that: because of the
limitation of the currently monitoring methods, the detectable
 L. Miao et al. / Environmental Pollution 255 (2019) 113300
plastic particles in natural environment were always larger than
330 mm in size, while the environmental concentrations of the sub-
micron or the nano plastics are predicted to be more higher than
microplastics (Chae and An, 2017), and much high putative risk has
to be allocated to these smaller plastics (Triebskorn et al., 2019).
Thus, concentrations higher than those in the natural environ-
ments were selected to in this study to explore the concentration-
response relationships and the related mechanism. Microcosms
were incubated in a rotary shaker at 120 rpm (20
C) to mimic
water flow across the biofilm surface (Gil-Allue et al., 2015). The
exposure treatments and the control were replicated four times.
After 3 h, biofilms from each replicate were scraped off and pooled
together, and used for all biochemical measurements after ho-
mogenization. As extracellular enzymes are located outside the cell,
they might represent a first site of interaction with external
contamination, such as plastic particles. The exposure time of 3 h
was chosen in this study to balance between naturally occurring
enzymatic activity loss in unexposed biofilm samples (Schug et al.,
2014) and a sufficient time to allow for adsorption of plastic par-
ticles by biofilms.
In a second set of experiments performed under identical
exposure conditions, biofilms were exposed to PS nanoplastics
(100 nm) with various surface modification: PS NPs, PS-COOH NPs
and PS-NH2 NPs. These coatings mimic the variety of engineered
and naturally occurring surface modifications of plastics (Nolte
et al., 2017; Triebskorn et al., 2019) and allow us to determine
how surface chemistry influences the effects on freshwater
biofilms.
2.4. Biological endpoints
At the end of the exposure experiments, the effects on chloro-
phyll a (Chl a) and photosynthesis potential of biofilms were
determined. The in vivo chlorophyll a (Chl a) concentration and the
microalgal photosynthesis (indicated by ФPSII) were measured on
fresh samples using Pulse Amplitude Modulation (PAM) fluorom-
etry (Heinz Walz GmbH, Effeltrich, Germany) as described in our
previous study (Liu et al., 2018).
To determine the activities of extracellular enzyme, 0.5 g fresh
samples were ground with PBS (pH 7.2e7.4) and centrifuged at
2500 g for 15 min. The liquid supernatant was collected for extra-
cellular enzyme (b-glucosidase (GLU), leucine aminopeptidase
(LAP), and alkaline phosphatase (AKP) quantification, which was
accomplished by double antibody sandwich-enzyme-linked
immunosorbent assays (ELISAs) using a microbiological ELISA kit
(Feiya Biotechnology, Jiangsu, China) (details given in the SI).
2.5. ROS generation and T-AOC measurements
Oxidative stress was commonly determined in the toxicological
studies of nanoparticles, to investigate the disruption of physio-
logical mechanisms or the damage of biochemical integrity in
exposed organisms (Almeida et al., 2017; Tang et al., 2018). In this
study, oxidative stress was represented by the production of ROS
and the level of T-AOC, which are among the most investigated
biomarkers due to their known susceptibility to toxicants (Han
et al., 2016; Jaikumar et al., 2018). Intracellular ROS generation
was determined by a ROS detection kit containing H2DCF-DA ac-
cording to our previous study (Liu et al., 2018). The level of T-AOC in
biofilms was detected using a T-AOC kit (Jiancheng Bioengineering
Co. Ltd., Nanjing, China) in accordance with the manufacturer’s
instructions.
3
2.6. Statistical analysis
All assays were conducted in four replicates, and data were
expressed as the mean ± standard deviation. Data from the
biochemical measurements were converted to percentage of con-
trol (% control) for easy comparison. Significant differences be-
tween treatments were analyzed in Origin version 8.0 using one-
way ANOVA followed by post hoc Tukey test, and p < 0.05 was
taken as significant cut-off. Asterisk (*) represents significant dif-
ferences between the controls and treatments, and different letter
(a, b, c) represent significant differences among the various PS
treatments.
3. Results
3.1. Characterization of PS beads in Milli-Q water and experimental
solution
As seen from the SEM images (Fig. S2), the morphology of the
applied PS beads in this study is basically spherical. As displayed in
Table 1, the particle size distribution of PS particles (100 and
500 nm PS) was significantly different when suspended in Milli-Q
water and experimental solution, however, the particle size dis-
tribution of microplastics (1 and 9 mm PS) maintained changeless in
the two solutions. The hydrodynamic diameters of PS (500 nm), PS
(100 nm), PS-COOH NPs (100 nm) were significantly higher in
experimental solution than those in Milli-Q water (p < 0.05).
Obvious aggregation of PS-NH2 NPs was observed in the two so-
lutions. In addition, the absolute values of zeta potential of micro/
nano PS were significantly lower in experimental solution than
those in Milli-Q water (p < 0.05), suggesting that the electrostatic
repulsion between PS particles were reduced (Xu et al., 2018). Due
to the high ion strength (IS) in the experimental solution, the sur-
face charge of PS particles was decreased through screening effect
(Miao et al., 2016), resulting in obvious aggregation of PS beads,
especially for the nanoplastics. Similar results were obtained by
Sun et al. (2018) who studied the particle behaviors of PS beads in
distilled water and sterile seawater.
3.2. Effects of nano- and micro- PS on freshwater biofilm
3.2.1. Effects on the microalgal growth and photosynthesis
The dose-effects of PS beads with different particle sizes on
biofilms were investigated after 3 h exposure, and the surface of PS
used in these experiments was not modified. As shown in Fig. 1, no
significant change of the concentrations of in vivo Chl a in biofilms
was observed in 5, 10 and 50 mg/L PS treatments, regardless of the
particle size used in this study. Only in 100 mg/L PS (100 nm and
500 nm) treatments, the concentrations of in vivo Chl a were
reduced by 15% and 11%, respectively (p < 0.05). However, large PS
beads (1 mm and 9 mm) didn’t affect the microalgal growth, even at
the highest concentration of 100 mg/L (all p > 0.05). As for the
photosynthesis, there was no significant effect of PS (<10% inhibi-
tion compared to control, p > 0.05) on the algal photosynthesis in
biofilms, regardless of the particle size and concentration used
(Fig. 1) in this study.
3.2.2. Effects on the activities of extracellular enzymes
In this study, three extracellular enzymes (GLU, LAP, and AKP)
were determined to investigate the short-term effects of PS beads
on the metabolic potential of biofilms in terms of carbon, nitrogen,
and phosphorus acquisition and transformations. As shown in
Fig. 2, only PS NPs (100 nm) affected on the activities of GLU, LAP,
and AKP, and the three enzymes exhibited different responses to
the PS beads within the range of concentrations in this study. When
4 L. Miao et al. / Environmental Pollution 255 (2019) 113300

Table 1
Particle size distribution and zeta potential of the different polystyrene beads.

Polystyrene type Primary particle size Particle size distribution (nm) Zeta potential (mV)

Milli-Q water (pH 6.9 ± 0.1) experimental solutionb Milli-Q water experimental solution

Unibead 9 mm a
11240 ± 2130 11480 ± 2340 ND ND
PS 1 mm 1358 ± 245 1495 ± 257 36.2 ± 3.5 23.4 ± 2.8c
500 nm 635 ± 135 985 ± 198c 43.2 ± 6.1 24.3 ± 2.1c
100 nm 129 ± 34 569 ± 124c 39.4 ± 3.9 19.4 ± 3.5c
PS-COOH 100 nm 135 ± 16 685 ± 156c 42.6 ± 4.8 18.6 ± 3.8c
PS-NH2 100 nm 878 ± 198 924 ± 240 10.2 ± 2 5.1 ± 1.8c

The particle size of unibead PS (9 mm) was measured with a Mastersizer 2000 (Malvern Instruments). The zeta potential of unibead PS (9 mm) was not detected.
a

Experimental solution used here was filtered through 0.22 mm (pH 7.6 ± 0.1).
b
c
An asterisk demonstrates significant differences of mean diameter and zeta potential for the PS beads in the experimental solution compared with those in Milli-Q water
(p < 0.05).

study. In short, the PS NPs (100 nm) resulted in significant in-

fluences on the functional enzyme activity of biofilms, depending
on the concentration of PS beads and the types of enzymes.

3.2.3. Potential impacts on the oxidative stress in the biofilms

Oxidative stress is generally identified as the main potential
mechanism of nanoparticle toxicity to exposed organisms, and
various relevant biomarkers have been used in the toxicological
studies (Fan et al., 2012). In this study, the level of T-AOC and the
generation of ROS in biofilms were determined to represent the
response and outcome of oxidative stress induced by PS beads. As
highlighted in Fig. 3, a concentration-dependent increase in ROS
generation was observed, with significant difference observed from
100 mg/L PS beads (100 nm) treatments (p < 0.05). While, other
large size PS beads (500 nm, 1 mm, and 9 mm) did not induce any
oxidative stress to biofilms within the range of the concentrations
in this study (see Fig. 3).
In response to the excess generation of ROS, an effective anti-
oxidant defense system would be developed by organisms to
maintain normal oxidant stress in the cells (Nel et al., 2006). In this
study, a similar variation in the trend of T-AOC was observed from
the nPS beads (100 nm) treatments, with significant differences
observed at concentration of 50 mg/L and 100 mg/L (p < 0.05).
Accordingly, to prevent the harmful effects of ROS, the antioxidative
mechanisms in biofilms might be activated (enhancing the level of
SOD, CAT, and T-AOC) to eliminate ROS (Han et al., 2017).

3.3. Surface modification effects of polystyrene on freshwater

biofilm

Previously, the surface modification has been demonstrated to

Fig. 1. Effects of PS beads with different particle sizes on microalgal growth and
photosynthesis in biofilms after 3 h exposure (n ¼ 4). * indicates significant difference
between the controls (100%) and treatments (p < 0.05). Different letters represent
significant differences among the PS treatments (p < 0.05).
exposed to PS beads (100 nm), the activity of GLU (123%) was
significantly higher than the control at concentration of 50 mg/L
(p < 0.05), while, it was significantly decreased (85%) when PS
concentration increased to 100 mg/L (p < 0.05). These
concentration-response relationships of biofilms might be attrib-
uted to hormesis effects with low dose stimulation and high dose
inhibition (Calabrese et al., 2012; Samarajeewa et al., 2017). As for
LAP, only PS beads (100 nm) at 100 mg/L induced a significantly
decrease (87%) in the activity compared to the control (p < 0.05).
The activity of AKP remained constant after 3 h exposure of PS
beads, regardless of the particle size and concentration used in this
influence the potential impacts of polystyrene to exposed organism
(Bergami et al., 2017; Sun et al., 2018). Besides, in the present study,
only PS beads (100 nm) at concentration of 100 mg/L induced sig-
nificant changes in most determined biochemical endpoints from
the first exposure experiments (Figs. 1e3). Therefore, 100 mg/L PS
beads (100 nm) with different functional groups (non-functional-
ized, -COOH and -NH2) were applied in the second experiments to
investigate the surface modification effects of polystyrene on
freshwater biofilms.
As shown in Fig. S3, the concentrations of in vivo Chl a in biofilms
were significantly decreased by the three nPS beads, with the
highest inhibition (25%) observed from the nPS-NH2 treatments
(p < 0.05). Besides, no significant change of algal photosynthesis in
biofilms was observed in the three nPS treatments at concentration
of 100 mg/L. These results suggested that PS nanoparticles could
inhibit the microalgal growth, regardless of the surface modifica-
tion in this study, while, the potential for primary productivity
(algal photosynthesis) are not affected within 3 h exposure
 L. Miao et al. / Environmental Pollution 255 (2019) 113300
Fig. 2. Effects of PS beads with different particle sizes on the activities of extracellular enzymes in biofilms after 3 h exposure (n ¼ 4). * indicates significant difference between the
controls (100%) and treatments (p < 0.05). Different letters represent significant differences among the PS treatments (p < 0.05).
experiments.
As for extracellular enzyme (Fig. 4), the activity of GLU was
significantly decreased by the three nPS beads (p < 0.05), with the
highest inhibition observed from the nPS-NH2 treatments (140 ng/
L) compared to the control (193 ng/g). The activity of LAP was
significantly decreased by PS NPs (non-functionalized) and PS-NH2
NPs (p < 0.05), while only a slight decrease of LAP was observed
from the PS-COOH NPs treatment (p > 0.05). The activity of AKP
remained at similar level among the three PS NP treatments within
the experiment period (Fig. S4). These results indicated that
extracellular enzyme activities were affected by nanoplastics to
varying degree, depending on the surface modification of PS beads
(100 nm).
The two representative biomarkers of oxidative stress (T-AOC
and ROS) were significantly affected by the PS NP treatments
(Fig. 4). The ROS generation was significantly increased by PS NPs
(non-functionalized) and PS-NH2 NPs (p < 0.05), especially in the
PS-NH2 NP treated biofilms (increased by 50%) compared to the
control. Meanwhile, obvious increase of T-AOC was observed from
all the three PS NP treated biofilms (p < 0.05). These results indi-
cated that the influences of PS NPs (100 nm) on the oxidative stress
of biofilms depended on the surface modification of PS beads.
4. Discussion
In this study, the acute toxicity of PS beads with different par-
ticle sizes and surface modifications on biofilms were investigated
by determining integrated biological endpoints, including algal
5
growth and photosynthesis, functional enzyme activities and
representative biomarkers for oxidative stress. As shown from the
obtained results, the toxic effects of PS beads on biofilms strongly
depended on the particle size and the surface modification. At the
end of experiments, most of the biological endpoints remained
constant in the large size PS beads (1 mm and 9 mm) treated bio-
films, regardless of the concentration applied. While, the small size
PS beads, especially nPS beads (100 nm) exhibited nano-toxicity to
biofilms through oxidative stress at high concentration of 100 mg/L.
Significant decreases were observed for microalgal growth, along
with reductions of enzymatic activities (with the exception of AKP)
in biofilms when exposed to 100 mg/L PS NPs, and the inhibition
degree was related to the surface modification of PS NPs. All these
observed effects highlighted that PS NPs (100 nm) significantly
affected biofilms and their associated microbial functions, such as
carbon and nitrogen acquisition and cycling in aquatic
environments.
4.1. The potential toxic mechanisms to biofilms
In this study, the introduced large PS MPs did not induce any
oxidative stress to biofilms, while the high concentration of PS NPs
(100 nm) exposure enhanced the intracellular ROS in biofilms,
likely posing threats to microorganisms due to oxidative stress. As
reported, microplastics are not a highly toxic substance, while the
nanoplastics disintegrated from microplastics have been shown to
exhibit toxic effects on freshwater organism of different trophic
levels, including zooplankter, algae, cyanobacteria, and bacteria
6 L. Miao et al. / Environmental Pollution 255 (2019) 113300
Fig. 3. Effects of PS beads with different particle sizes on the level of T-AOC and the
generation of ROS in biofilms after 3 h exposure (n ¼ 4). * indicates significant dif-
ference between the controls (100%) and treatments (p < 0.05). Different letters
represent significant differences among the PS treatments (p < 0.05).
(Chae and An, 2017; Gonza
lez-Pleiter et al., 2019; Zhang et al.,
2019). The unique characteristics of nanoparticles (small size and
high surface-volume ratio) make them easily pass through bio-
logical barriers and can lead to oxidative damage to living organ-
isms (Besseling et al., 2014; Tang et al., 2018). Numerous studies
revealed that the toxicity of nanoparticles (such as nPS, TiO2, and
Fe2O3) to microorganisms are mainly attributed to the oxidative
damage (Sun et al., 2018; Tang et al., 2017; Zhu et al., 2019).
Accordingly, oxidative stress generally developed from low level
to high level with increasing exposure duration and nanoparticle
concentrations (Nel et al., 2006). Within the duration in this study,
the antioxidative biomarker of T-AOC increased in response to the
enhanced generation of ROS, suggesting a relative low level of
oxidative stress (Fan et al., 2012), and the PS NPs did not destroy the
antioxidant system within the experimental periods. During this
phase, ROS can be alleviated through enzymatic (such as SOD, CAT)
and nonenzymatic actions (such as T-AOC), protecting cells in
biofilms against oxidative stress (Fan et al., 2012). It was probably
the same situation with this study. However, if the concentration
and exposure time of PS NPs keep increased, the continuous
accumulation of ROS might breakdown the antioxidant defense
systems, leading to higher oxidative stress, and consequently cell
death (Fan et al., 2012; Han et al., 2016). This highlighted the need
to perform long-term exposure experiments investigating the
chronic responses of biofilms to PS NPs in aquatic environments. In
addition to the response mechanisms of antioxidant action, EPS,
attached to the outer layer of the cells, could protect microbial
communities from oxidative stress, as proteins and polysaccharose
(two major components of EPS) are reactive with radicals (Coburn
et al., 2016; Zhu et al., 2019).
4.2. Responses of microalgal growth and photosynthesis
In the present study, the influences of PS beads on the micro-
algal growth and photosynthesis in biofilms were strongly depen-
dent on the particle concentration and size applied. Low
concentrations of PS NPs (5 and 10 mg/L) did not induce any in-
fluence on the microalgal growth and photosynthesis in this study,
consistent with previous study reported by Zhang et al. (2018).
High concentration of PS NPs (100 nm) at 100 mg/L induced sig-
nificant decrease of microalgal growth in biofilms, while the value
of ФPSII remained constant in this study. Similar results were ob-
tained in previous study that small sized polystyrene (0.05 mm)
significantly inhibited the growth of microalgae D. tertiolecta at the
concentration of 250 mg/L, and the microalgal photosynthesis
maintained changeless after 72 h exposure (Sjollema et al., 2016).
While, negative effects of mPVC (average diameter 1 mm) on algal
photosynthesis were observed by Zhang et al. (2017) after 1 h
exposure, and the value of ФPSII recovered to a similar level of the
controls after 96 h. This discrepancy among studies might be due to
different microbial communities or plastic types used in the
exposure experiments.
There are two possible reasons for the inconsistent changes of
microalgal growth and photosynthesis. One is that the ROS induced
by PS NPs (100 nm) beads could lead to oxidative damage to the cell
membrane (Liu et al., 2018), and as a result, the growth of micro-
algal community was inhibited. While, the quantum yield repre-
sents the proportion of photons of incident light which are actually
used to drive the photochemistry of photosynthesis (Almeida et al.,
2017). This normalized index can represent the efficiency of the
photochemical conversion of light energy in PSII of whole micro-
algal community or individual microalgal cells. Thus, the inhibition
of microalgal growth might not affected the level of ФPSII in bio-
films. Another reason is that the sensitivity of algae in biofilms to
nanoparticles varies within algal types, resulting in different re-
sponses of algae to nano-toxic effects. In our previous study, we
found that exposure to Ag2S NPs inhibited the growth of cyano-
bacteria and diatoms, however, increased the content of green
algae in freshwater biofilms (Liu et al., 2018). As green algae has
higher potential of photosynthetic activity than cyanobacteria and
diatoms (Chalifour and Juneau, 2011), the enhanced content of
green algae in biofilms could counterbalance the loss of photo-
synthetic activity induced by other types of algae, and thus main-
tained the normal photosynthetic yield in biofilms (Liu et al., 2018).
4.3. Responses of the activities of functional enzymes
In general, the three functional enzymes responded differently
to PS NPs in this study: GLU was negatively affected in more cases
compared to LAP. The activity of AKP remained changeless within
the exposure duration, regardless of the concentration and surface
modification of PS NPs used in this study. These differences in
sensitivity might result from different localization patterns of the
three enzymes. Previous studies showed that a major fraction of
GLU are located in the soluble fraction (outside layer) in the biofilm
matrix, and thus, GLU might be more accessible for PS NPs and ROS
(Schug et al., 2014). Whereas, LAP and AKP were mainly associated
with the insoluble fraction and they might be less affected
compared to GLU. Similar results were obtained in previous study,
in which the activities of GLU and LAP were significantly inhibited
by Ag NPs, while AKP was not affected after 2 h exposure (Gil-Allue
et al., 2015). The decreased activities of GLU and LAP suggested
 L. Miao et al. / Environmental Pollution 255 (2019) 113300 7

Fig. 4. Effects of PS beads with different surface modifications on enzyme activities and oxidative stress in biofilms after 3 h exposure (n ¼ 4). Different letters represent significant
differences among the PS treatments (p < 0.05).

negative effects of nPS beads on the carbon and nitrogen cycling of better with long term exposure and low concentration of plastic
freshwater biofilms. particles, and the obtained results could be useful to evaluate the
potential impacts in the aquatic ecosystem. Environmental con-
centrations of the applied plastic particles are not well-known but
4.4. Responses to different surface modifications of polystyrene
are expected to be lower than in the present study (Chae and An,
2017), suggesting that the extrapolation of our results requires
As observed from the second experiments, the nano-toxic ef-
caution. Nevertheless, there is little knowledge about the effects on
fects of PS NPs on biofilms was highly dependent on the surface
freshwater biofilms so far, and thus detailed and comprehensive
modification of PS NPs in this study, and the positively charged PS
studies are needed to evaluate the ecotoxicological effects of these
NPs (PS-NH2) exhibited the highest toxicity to biofilms. These dif-
plastic particles. The main purpose of the present study was to
ferences might be possibly attributed to the binding affinity be-
explore the acute toxic effects of PS beads to freshwater biofilms
tween PS NPs and the cell (Nolte et al., 2017). Previously,
after short term exposure and the concentration-response re-
Bhattacharya et al. (2010) found that the positively charged nano-
lationships and the related mechanisms were discussed. Indeed,
plastics exhibited a high binding affinity to algae than negatively
the ecological functions of biofilms were negatively affected in
charged particles, and resulted in more significant effects on algal
terms of carbon and nitrogen cycling. Complementary studies
photosynthesis. Similarly, non-functionalized and positively
aimed to investigate long-term effects would be useful to evaluate
charged PS nanoparticles showed a higher adsorption to the cell
the impacts of PS NPs in aquatic environments, integrating the
wall of Pseudokirchneriella subcapitata than negatively charged PS
responses of community structure and functional properties of
beads (Nolte et al., 2017). In addition, these binding affinity be-
biofilms. Also, because that periphytic biofilms are important re-
tween particle and cell might also be affected by medium condi-
sources for consumers in aquatic environments (Gil-Allue et al.,
tions and material properties, which should be further investigated.
2015), the trophic transfer of PS NPs and their effects on aquatic
food webs should receive more attention.
4.5. Environmental implications

Generally, the estimated exposures in the environment might be

8 L. Miao et al. / Environmental Pollution 255 (2019) 113300
5. Conclusion
In the present study, we demonstrated that the potential im-
pacts of PS beads on freshwater biofilms were strongly dependent
on the particle size and surface modification. The nanoscale PS
beads (100 nm) exhibited obvious effects on the biological activity
of biofilms possible through oxidative stress. The inhibited GLU
suggested that carbon cycling, as the most important role of bio-
films in stream ecosystems, might be negatively affected. The in-
hibition of LAP indicated that the nitrogen acquisition of biofilms
may be limited and thus directly affect nitrogen cycling. These re-
sults highlighted the significant risks posed by PS nanoparticles to
biofilms and their ecosystem functions in aquatic environments.
Declaration of competing interest
The authors declare no competing financial interest.
Acknowledgments
We are grateful for the grants for Project supported by the Na-
tional Science Funds for Creative Research Groups of China
(No.51421006), the Key Program of National Natural Science
Foundation of China (No. 91647206), the Fundamental Research
Funds for the Central Universities (No. 2019B14414, No.
2017B20514), the Outstanding Youth Fund of Natural Science
Foundation of Jiangsu, China (BK20160038), the National Natural
Science Foundation of China (51709081, 51979075), and PAPD.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.envpol.2019.113300.
References
Almeida, A.C., Gomes, T., Langford, K., Thomas, K.V., Tollefsen, K.E., 2017. Oxidative
stress in the algae Chlamydomonas reinhardtii exposed to biocides. Aquat.
Toxicol. 189, 50e59.
Battin, T.J., Besemer, K., Bengtsson, M.M., Romani, A.M., Packmann, A.I., 2016. The
ecology and biogeochemistry of stream biofilms. Nat. Rev. Microbiol. 14,
251e263.
Bergami, E., Pugnalini, S., Vannuccini, M.L., Manfra, L., Faleri, C., Savorelli, F.,
Dawson, K.A., Corsi, I., 2017. Long-term toxicity of surface-charged polystyrene
nanoplastics to marine planktonic species Dunaliella tertiolecta and Artemia
franciscana. Aquat. Toxicol. 189, 159e169.
Besemer, K., 2015. Biodiversity, community structure and function of biofilms in
stream ecosystems. Res. Microbiol. 166, 774e781.
Besseling, E., Wang, B., Lurling, M., Koelmans, A.A., 2014. Nanoplastic affects growth
of S. Obliquus and reproduction of D. Magna. Environ. Sci. Technol. 48,
12336e12343.
Bhattacharya, P., Lin, S.J., Turner, J.P., Ke, P.C., 2010. Physical adsorption of charged
plastic nanoparticles affects algal photosynthesis. J. Phys. Chem. C 114,
16556e16561.
Bundschuh, M., Filser, J., Luderwald, S., McKee, M.S., Metreveli, G., Schaumann, G.E.,
Schulz, R., Wagner, S., 2018. Nanoparticles in the environment: where do we
come from, where do we go to? Environ. Sci. Eur. 30, 6.
Calabrese, E.J., Iavicoli, I., Calabrese, V., 2012. Hormesis: why it is important to
biogerontologists. Biogerontology 13, 215e235.
Chae, Y., An, Y.J., 2017. Effects of micro- and nanoplastics on aquatic ecosystems:
current research trends and perspectives. Mar. Pollut. Bull. 124, 624e632.
Chalifour, A., Juneau, P., 2011. Temperature-dependent sensitivity of growth and
photosynthesis of Scenedesmus obliquus, Navicula pelliculosa and two strains
of Microcystis aeruginosa to the herbicide atrazine. Aquat. Toxicol. 103, 9e17.
Chen, X., Zhu, Y., Yang, K., Zhu, L., Lin, D., 2019. Nanoparticle TiO2 size and rutile
content impact bioconcentration and biomagnification from algae to daphnia.
Environ. Pollut. 247, 421e430.
Coburn, K.M., Wang, Q., Rediske, D., Viola, R.E., Hanson, B.L., Xue, Z., Seo, Y., 2016.
Effects of extracellular polymeric substance composition on bacteria disinfec-
tion by monochloramine: application of MALDI-TOF/TOF-MS and multivariate
analysis. Environ. Sci. Technol. 50, 9197e9205.
Fan, W.H., Wang, X.L., Cui, M.M., Zhang, D.F., Zhang, Y., Yu, T., Guo, L., 2012. Dif-
ferential oxidative stress of octahedral and cubic Cu2O micro/nanocrystals to
Daphnia magna. Environ. Sci. Technol. 46, 10255e10262.
Flemming, H.C., Wingender, J., Szewzyk, U., Steinberg, P., Rice, S.A., Kjelleberg, S.,
2016. Biofilms: an emergent form of bacterial life. Nat. Rev. Microbiol. 14,
563e575.
Gigault, J., Pedrono, B., Maxit, B., Ter Halle, A., 2016. Marine plastic litter: the un-
analyzed nano-fraction. Environ. Sci.: Nano 3 (2), 346e350.
Gil-Allue, C., Schirmer, K., Tlili, A., Gessner, M.O., Behra, R., 2015. Silver nanoparticle
effects on stream periphyton during short-term exposures. Environ. Sci. Tech-
nol. 49, 1165e1172.
Gonz
alez-Pleiter, M., Tamayo-Belda, M., Pulido-Reyes, G., Amariei, G., Legane
s, F.,
Rosal, R., Ferna
ndez-Pin~ as, F., 2019. Secondary nanoplastics released from a
biodegradable microplastic severely impact freshwater environments. Environ.
Sci.: Nano 6 (5), 1382e1392.
Han, X., Wang, Z., Wang, X., Zheng, X., Ma, J., Wu, Z., 2016. Microbial responses to
membrane cleaning using sodium hypochlorite in membrane bioreactors: cell
integrity, key enzymes and intracellular reactive oxygen species. Water Res. 88,
293e300.
Han, X., Wang, Z., Chen, M., Zhang, X., Tang, C.Y., Wu, Z., 2017. Acute responses of
microorganisms from membrane bioreactors in the presence of NaOCl: pro-
tective mechanisms of extracellular polymeric substances. Environ. Sci. Technol.
51, 3233e3241.
Huffer, T., Praetorius, A., Wagner, S., von der Kammer, F., Hofmann, T., 2017.
Microplastic exposure assessment in aquatic environments: learning from
similarities and differences to engineered nanoparticles. Environ. Sci. Technol.
51, 2499e2507.
Jaikumar, G., Baas, J., Brun, N.R., Vijver, M.G., Bosker, T., 2018. Acute sensitivity of
three Cladoceran species to different types of microplastics in combination with
thermal stress. Environ. Pollut. 239, 733e740.
Koelmans, A.A., Nor, N.H.M., Hermsen, E., Kooi, M., Mintenig, S.M., De France, J.,
2019. Microplastics in freshwaters and drinking water: critical review and
assessment of data quality. Water Res. 155, 410e422.
Lambert, S., Wagner, M., 2016. Characterisation of nanoplastics during the degra-
dation of polystyrene. Chemosphere 145, 265e268.
Li, J., Liu, H., Chen, J.P., 2018. Microplastics in freshwater systems: a review on
occurrence, environmental effects, and methods for microplastics detection.
Water Res. 137, 362e374.
Liu, S.Q., Wang, C., Hou, J., Wang, P.F., Miao, L.Z., Li, T.F., 2018. Effects of silver sulfide
nanoparticles on the microbial community structure and biological activity of
freshwater biofilms. Environ. Sci.-Nano 5, 2899e2908.
Miao, L., Wang, C., Hou, J., Wang, P., Ao, Y., Li, Y., Lv, B., Yang, Y., You, G., Xu, Y., 2016.
Effect of alginate on the aggregation kinetics of copper oxide nanoparticles
(CuO NPs): bridging interaction and hetero-aggregation induced by Ca(2.).
Environ. Sci. Pollut. Res. Int. 23, 11611e11619.
Miao, L.Z., Wang, P.F., Wang, C., Hou, J., Yao, Y., Liu, J., Lv, B.W., Yang, Y.Y., You, G.X.,
Xu, Y., Liu, Z.L., Liu, S.Q., 2018. Effect of TiO2 and CeO2 nanoparticles on the
metabolic activity of surficial sediment microbial communities based on oxygen
microelectrodes and high-throughput sequencing. Water Res. 129, 287e296.
Nel, A., Xia, T., Madler, L., Li, N., 2006. Toxic potential of materials at the nanolevel.
Science 311, 622e627.
Nolte, T.M., Hartmann, N.B., Kleijn, J.M., Garnaes, J., van de Meent, D., Hendriks, A.J.,
Baun, A., 2017. The toxicity of plastic nanoparticles to green algae as influenced
by surface modification, medium hardness and cellular adsorption. Aquat.
Toxicol. 183, 11e20.
Pikuda, O., Xu, E.G., Berk, D., Tufenkji, N., 2019. Toxicity assessments of micro- and
nanoplastics can Be confounded by preservatives in commercial formulations.
Environ. Sci. Technol. Lett. 6, 21e25.
Prata, J.C., da Costa, J.P., Lopes, I., Duarte, A.C., Rocha-Santos, T., 2019. Effects of
microplastics on microalgae populations: a critical review. Sci. Total Environ.
665, 400e405.
Samarajeewa, A.D., Velicogna, J.R., Princz, J.I., Subasinghe, R.M., Scroggins, R.P.,
Beaudette, L.A., 2017. Effect of silver nano-particles on soil microbial growth,
activity and community diversity in a sandy loam soil. Environ. Pollut. 220,
504e513.
Schug, H., Isaacson, C.W., Sigg, L., Ammann, A.A., Schirmer, K., 2014. Effect of TiO2
nanoparticles and UV radiation on extracellular enzyme activity of intact het-
erotrophic biofilms. Environ. Sci. Technol. 48, 11620e11628.
Silva, A.B., Bastos, A.S., Justino, C.I.L., da Costa, J.A.P., Duarte, A.C., Rocha-
Santos, T.A.P., 2018. Microplastics in the environment: challenges in analytical
chemistry A review. Anal. Chim. Acta 1017, 1e19.
Sjollema, S.B., Redondo-Hasselerharm, P., Leslie, H.A., Kraak, M.H.S., Vethaak, A.D.,
2016. Do plastic particles affect microalgal photosynthesis and growth? Aquat.
Toxicol. 170, 259e261.
Song, Y.K., Hong, S.H., Jang, M., Han, G.M., Jung, S.W., Shim, W.J., 2017. Combined
effects of UV exposure duration and mechanical abrasion on microplastic
fragmentation by polymer type. Environ. Sci. Technol. 51 (8), 4368e4376.
Su, L., Xue, Y., Li, L., Yang, D., Kolandhasamy, P., Li, D., Shi, H., 2016. Microplastics in
Taihu Lake, China. Environ. Pollut. 216, 711e719.
Sun, X.M., Chen, B.J., Li, Q.F., Liu, N., Xia, B., Zhu, L., Qu, K.M., 2018. Toxicities of
polystyrene nano- and microplastics toward marine bacterium Halomonas
alkaliphila. Sci. Total Environ. 642, 1378e1385.
Tallec, K., Huvet, A., Di Poi, C., Gonzalez-Fernandez, C., Lambert, C., Petton, B., Le
Goic, N., Berchel, M., Soudant, P., Paul-Pont, I., 2018. Nanoplastics impaired
oyster free living stages, gametes and embryos. Environ. Pollut. 242,
1226e1235.
Tang, J., Zhu, N.Y., Zhu, Y., Liu, J.Z., Wu, C.X., Kerr, P., Wu, Y.H., Lam, P.K.S., 2017.
Responses of periphyton to Fe2O3 nanoparticles: a physiological and ecological
 L. Miao et al. / Environmental Pollution 255 (2019) 113300
basis for defending nanotoxicity. Environ. Sci. Technol. 51, 10797e10805.
Tang, J., Wu, Y., Esquivel-Elizondo, S., Sorensen, S.J., Rittmann, B.E., 2018. How mi-
crobial aggregates protect against nanoparticle toxicity. Trends Biotechnol. 36,
1171e1182.
Thompson, R.C., Olsen, Y., Mitchell, R.P., Davis, A., Rowland, S.J., John, A.W.,
Russell, A.E., 2004. Lost at sea: where is all the plastic? Science 304 (5672), 838-
838.
Triebskorn, R., Braunbeck, T., Grummt, T., Hanslik, L., Huppertsberg, S., Jekel, M.,
Knepper, T.P., Krais, S., Muller, Y.K., Pittroff, M., Ruhl, A.S., Schmieg, H., Schur, C.,
Strobel, C., Wagner, M., Zumbulte, N., Kohler, H.R., 2019. Relevance of nano- and
microplastics for freshwater ecosystems: a critical review. Trac. Trends Anal.
Chem. 110, 375e392.
Xu, H., Xu, M., Li, Y., Liu, X., Guo, L., Jiang, H., 2018. Characterization, origin and
aggregation behavior of colloids in eutrophic shallow lake. Water Res. 142,
9
176e186.
Zhang, C., Chen, X., Wang, J., Tan, L., 2017. Toxic effects of microplastic on marine
microalgae Skeletonema costatum: interactions between microplastic and
algae. Environ. Pollut. 220, 1282e1288.
Zhang, Q., Qu, Q., Lu, T., Ke, M.J., Zhu, Y.C., Zhang, M., Zhang, Z.Y., Du, B.B., Pan, X.L.,
Sun, L.W., Qian, H.F., 2018. The combined toxicity effect of nanoplastics and
glyphosate on Microcystis aeruginosa growth. Environ. Pollut. 243, 1106e1112.
Zhang, S.L., Wang, J.Q., Liu, X., Qu, F.J., Wang, X.S., Wang, X.R., Li, Y., Sun, Y.K., 2019.
Microplastics in the environment: a review of analytical methods, distribution,
and biological effects. Trac. Trends Anal. Chem. 111, 62e72.
Zhu, N., Wang, S., Tang, C., Duan, P., Yao, L., Tang, J., Wong, P.K., An, T.,
Dionysiou, D.D., Wu, Y., 2019. Protection mechanisms of periphytic biofilm to
photocatalytic nanoparticle exposure. Environ. Sci. Technol. 53, 1585e1594.