Science of the Total Environment 660 (2019) 1144–1154

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Review

Impact of engineered nanoparticles on microbial transformations of

carbon, nitrogen, and phosphorus in wastewater treatment
processes – A review
Shubiao Wu a,b,⁎, Haiming Wu c, Mark Button d, Dennis Konnerup a,b, Hans Brix b
a
Aarhus Institute of Advanced Studies, Aarhus University, Aarhus 8000C, Denmark
b
Department of Bioscience, Aarhus University, Aarhus 8000C, Denmark
c
College of Resources and Environment, Northwest A&F University, Yangling, Shaanxi 712100, PR China
d
Department of Earth and Environmental Sciences, University of British Columbia - Okanagan, Kelowna, British Columbia, Canada

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Increasing concern regarding negative

effect of ENPs on bioprocesses formed
this review.
• Exposure of low level ENP shows no ef-
fect on heterotrophics for organic re-
moval.
• Impacts of ENPs on microbial nitrogen
transformations have been well re-
ported.
• No negative effect on P removal is re-
ported after short-term nor long-term
exposure.
• Inhibition effects are overestimated in
cultivated toxicity test compared to
real wastewater.

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

Article history: Concern regarding the potential negative impacts of released engineered nanoparticles (ENPs) on pollutant re-
Received 12 November 2018 moval performance of wastewater treatment systems has received booming attention in recent years. However,
Received in revised form 7 January 2019 the conclusions drawn from different studies often lead to fragmented overall knowledge, some of which are
Accepted 10 January 2019
even contradictory. This scenario shows the necessity for a comprehensive review of the interactions of ENPs
Available online 11 January 2019
in wastewater treatment systems, particularly on the impacts of ENPs on microbial processes of carbon (C), nitro-
Editor: Damia Barcelo gen (N), and phosphorus (P) removal in water treatment systems. This review introduced the impact of 6 often
reported ENPs in 5 types of treatment systems. We found that exposure to most of the investigated ENPs at low
Keywords: concentrations doesn't adversely influence the growth of the heterotrophic microbes, which are responsible for
Wastewater treatment organic matter removal. The impacts of ENPs on various microbial nitrogen transformation processes have been
Engineered nanoparticles investigated. Dosing of ENPs often causes acute microbial nitrogen removal inhibition at various concentrations,
Inhibitory effect but does not influence long-term operation due to microbial adaption. No significant negative effects on biolog-
Exposure ical phosphorus removal in different wastewater treatment processes have been reported after both short-term
Microbial processes
and long-term exposure (except copper nanoparticles). Environmentally relevant concentrations of ENPs have
been reported to enhance the photosynthetic capacity of wetland plants, whereas chronic inhibition to photosyn-
thesis was found in exposure to high concentrations of ENPs. Inhibition effects are often overestimated in pure
cultivated toxicity test assays compared to testing with artificially prepared wastewater containing various ingre-
dients or with real wastewater. Potential ligands in real wastewater can bind with ENPs and lower their

⁎ Corresponding author at: Aarhus Institute of Advanced Studies, Aarhus University, Aarhus 8000C, Denmark.
E-mail address: wushubiao@gmail.com (S. Wu).

https://doi.org/10.1016/j.scitotenv.2019.01.106
0048-9697/© 2019 Elsevier B.V. All rights reserved.
 S. Wu et al. / Science of the Total Environment 660 (2019) 1144–1154 1145

dissolution. Some challenges exist regarding detection and quantification techniques of ENPs at environmental
concentrations, modeling of engineered nanomaterial release on a worldwide scale, and inhibitory mechanisms
to microbial transformations.
© 2019 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1145
2. Influence of ENPs on microbial C transformation in water treatment systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1146
3. Influence of ENPs on microbial N transformation in water treatment systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1146
4. Influence of ENPs on P removal in water treatment systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1148
5. Influence of ENPs exposure on wetland plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1149
6. Challenges to understand the impact of ENPs in wastewater treatment systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1150
7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1152
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1152
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1152

1. Introduction decades. However, understanding of the interactions among C, N, and

Nanotechnology is one of the fastest developing technologies today.
Engineered nanoparticles (ENPs) are typically defined as particles with
at least one dimension between 1 and 100 nanometres (nm) in size. The
physicochemical and electronic properties, e.g., considerably higher
specific surface area, surface reactivity, and increased quantum effect,
associated with materials in this size range substantially differentiate
these materials from their bulk counterparts (Kumar et al., 2017).
Owing to these unique properties, the production and use of ENPs
have grown rapidly over the last two decades (Kwak and An, 2016)
and ENPs have been widely incorporated in numerous consumer and
industrial products, e.g. dyes, health care products, cosmetics and food
packing materials (Roco, 2011; Vance et al., 2015).
Daily water use and disposal may be not free of ENPs, particularly
certain metal-oxide nanoparticles. For example, textiles, plastics, and
cosmetics are commonly used household products that often contain
some ENPs, such as silver nanoparticles (Ag NPs), zinc oxide nanoparti-
cles (ZnO NPs) and titanium dioxide nanoparticles (TiO2 NPs). The ENPs
are released during washing and end up in the wastewater that flows
into wastewater treatment systems (Lorenz et al., 2012; Windler et al.,
2012). Although the data in terms of nanoparticle concentrations in
real sewage are lacking, there is strong evidence exhibiting that ENPs
have already been present in municipal wastewater. For example,
Westerhoff et al. (2011) found that the concentration of TiO2 NPs in
wastewater effluents of 10 municipal facilities in the USA varied from
b2 to 20 μg/L. The concentrations of Ag NPs in the influents of nine
wastewater treatment plants in German vary in the range of 0.06–1.5
μg/L (Li et al., 2013).
Although the estimated concentration of most ENPs in wastewater
treatment plants is only at the μg/L level, some treatment plants may ex-
perience much higher concentrations in their influent when receiving
periodic ENP discharges from industries (Shafer et al., 1998;
Kunhikrishnan et al., 2015). The special properties that make these
nanoparticles unique and useful may also make them hazardous to mi-
croorganisms and the environment under certain conditions. Thus, the
use and potential environmental risks of ENPs are subjects of increasing
concern, social debate and several government reports. Accordingly,
concern regarding the impact and possible risks of these ENPs has led
to multiple investigations on their dynamic transport and fate in the
aquatic environment (Yang et al., 2014b).
Extensive investigations on microbial transformations of carbon (C),
nitrogen (N) and phosphorus (P) in various wastewater treatment sys-
tems have been performed and have undoubtedly enabled the mecha-
nisms of such transformations to be more evident in the last few
P might still be insufficient, owing to the simultaneous complex compe-
tition among electron donors and acceptors in their transformation
pathways. Besides, the incorporation of ENPs would make their rela-
tionship more complex owing to different susceptibilities of the various
functional microbial communities to ENP exposure. The publication re-
cord under the scheme of this topic in the Web of Science shows a
booming tendency in the last five years, indicating increasing concern
from scientists, and shows the increasing transparency of the knowl-
edge “black box”, etc. However, the conclusions drawn from different
studies often lead to fragmented overall knowledge, some of which
are even contradictory. The recently published reviews in the similar
topic have mainly described the advances on the analytical develop-
ments of ENPs and discussed the fate of ENPs in the drinking water
treatment and wastewater treatment processes (Wang and Chen,
2016; Park et al., 2017; Joo and Aggarwal, 2018). The information on
the specific interactions of often reported ENPs among microbial trans-
formations of C, N, and P in different centralized wastewater treatment
systems (e.g. activated sludge systems and sequencing batch reactor)
and decentralized eco-treatment systems (e.g. constructed wetlands)
is still insufficient. This scenario profoundly shows the necessity of this
review.
Moreover, some issues exist that confound the understanding of the
impact of ENPs on the performance of biological wastewater processes.
First, to demonstrate the potential risk of ENPs and their negative effect
on microbial transformations in biological wastewater treatment sys-
tems, experiments are often performed by dosing high concentrations
of ENPs without real environmental relevance. The toxicity of ENPs to
microbial communities might be overestimated because the occurrence
probability of high concentrations of ENP in discharge and the bioavail-
ability of the ENPs in wastewater treatment systems remain highly
questionable. It has been well evidenced that the processes of aggrega-
tion, sedimentation, sulfidation, and adsorption significantly influence
the fate of ENPs in water treatment systems and only a small fraction
of ENPs may be present in the liquid discharge. To some extent, ENPs
may even be providing trace elements, functioning as stimulants of bio-
mass growth. However, changing the preconceived notion that the
presence of ENPs always has an adverse effect continues to be difficult.
Therefore, it appears to be time to rethink the research development on
the relationship between ENP exposure and the microbial transforma-
tions of C, N, and P in the wastewater treatment process in addition to
addressing the current challenges to such research.
The fate and transport of ENPs in constructed wetlands (CWs) for
wastewater treatment have been poorly studied, yet CWs have the po-
tential for remediating ENP-contaminated water (Hu et al., 2018). We
1146 S. Wu et al. / Science of the Total Environment 660 (2019) 1144–1154

do not doubt that these ENPs will inevitably find their way to enter
aquatic environments from many sources and then ultimately have
some uncertain impacts to the function of the ecosystem and human
health. However, in eco-treatment systems, such as CWs, whether the
same effects of ENPs on functional bacteria and wetland plants as well
as influences on microbial C, N and P cycling in activated sludge waste-
water treatment system exist remains a question. Compared to the acti-
vated sludge treatment processes, the presence of the matrix in CWs,
e.g., sand/gravel and associated biofilm, may improve the buffering ca-
pacity of the system to the adverse effects of ENPs (Hu et al., 2018).
However, accumulation of ENPs on the surface of the sand or gravel dur-
ing long-term operation may pose a serious threat to the bacteria. Thus,
knowledge regarding the influence of ENPs in eco-treatment systems,
e.g., CWs, is still insufficient and should be summarized.
In this review, the influence of 6 often reported ENPs (Ag NPs, ZnO
NPs, TiO2 NPs, CeO2 NPs, CuO NPs, and Al2O3 NPs) on microbial C, N,
and P transformations in 5 types of treatment systems (sequencing
batch reactor, constructed wetlands, upflow anaerobic sludge blanket
reactor, membrane bioreactor, and activated sludge system) will be
discussed based on the present knowledge. The impact of exposure to
ENPs on wetland plant biomass production and the fate of ENPs associ-
ated with the roots, uptake, and translocation will be summarized. Fi-
nally, issues challenging the understanding of the impact of ENPs in
wastewater treatment systems, including the analysis of ENPs in com-
plex wastewaters, modeling extensions for ENP research, as well as
mechanisms of ENP inhibition in treatment systems will be discussed.
2. Influence of ENPs on microbial C transformation in water
treatment systems
case in membrane bioreactor. The knowledge on the impact of ENPs ex-
posure to other treatment systems is still insufficient. Moreover, expo-
sure to most of the investigated ENPs at low concentrations barely
affects the removal of organic matter by heterotrophic microorganisms.
Only exposure at high ENP concentrations (e.g. 30–60 mg/L) slightly re-
duced their performance. Besides, the investigations reported from the
current available literature are only performed in lab-scale wastewater
treatment systems. It is still a question that whether the findings from
these lab-scale systems can be repeated in pilot scale or full scale treat-
ment systems. Yet exposure to these high ENP concentrations in waste-
water may not occur often or in the long term. Therefore, more
investigations are needed.
Regarding the adverse effects of ENPs on organic matter removal, He
et al. (2017) found that exposure to 5, 10, and 20 mg/L zinc oxide nano-
particles (ZnO NPs) actually stimulated COD (Chemical Oxygen De-
mand) removal in an investigation of a sequencing batch reactor for
an operational period of 180 days. Higher tolerance of heterotrophic or-
ganisms in wastewater treatment process to ENPs has been reported
frequently, which may be due to their higher microbial population
and/or their protective barrier formed with organic matter in the sub-
strate (Fabrega et al., 2009). A strong interaction between ENPs and or-
ganic material in wastewater exists. For example, due to the
complexation of Ag NPs with humic or other organic acids in sludge,
majority of Ag NPs present in the wastewater treatment system are
transferred to the sludge (Shafer et al., 1998). Thus, considering the po-
tential occurrence of ENPs at an environmentally relevant concentra-
tion, the impact of these ENPs on the removal of organic matter may
be overlooked and whether should we put much concern on future
monitoring may need consideration.

The impact of ENPs on the removal of organic matter is an important
monitoring focus in wastewater treatment systems, owing to the regu-
lation of the organic matter content in treated effluent. However, as
shown in Table 1, the tested treatment systems currently were only lim-
ited to sequencing batch reactors, constructed wetlands as well as one
3. Influence of ENPs on microbial N transformation in water
treatment systems
It is well known that N removal from wastewater is often the
results of complex microbial transformations, including traditional

Table 1
Effect of engineered nanoparticles (ENPs) on the removal of organic matter in different wastewater treatment systems.

Treatment ENPs Low level High level Duration (d) COD removal performance References
system
Conc. Effect Conc. Effect Short Long

SBR TiO2 NPs b10 × 10–60 √ – 290√ b10 mg/L: COD removal kept stable at 92%; (Li et al., 2017)
10–60 mg/L: decreased from 92% to 88%.
Ag NPs 0.1 × – – 15× – No statistical difference as compared to control. (Hou et al., 2012)
0.5 × – – 15× – No statistical difference as compared to control. (Hou et al., 2012)
b2 × 2–30 √ – 290√ b2 mg/L: COD removal kept stable at 92%; (Xu et al., 2017a, 2017b)
2–30 mg/L: decreased from 92% to 88%.
0.1–0.5 × – – 15× – No statistical difference as compared to control. (Chen et al., 2014)
0.5–1.5 × – – 27× – No adverse effect on COD removal efficiency. (Wang et al., 2012)
ZnO NPs b5 × 5–60 √ – 180√ b5 mg/L: COD removal kept stable at 92%; (Wang et al., 2016b)
5–60 mg/L: decreased from 92% to 87%.
1–5 × – – 15× – No statistical difference as compared to control. (Chen et al., 2014)
CeO2 NPs b5 × 10–60 √ – 290√ b5 mg/L: COD removal kept stable at 92%; (Wang et al., 2016a)
5–60 mg/L: decreased from 92% to 87%.
Ag NPs 1 × – – 20× – Effluent COD concentrations before and after nanosilver (Liang et al., 2010)
shock load were 10.2 ± 4.4 mg/L and 10.2 ± 3.1 mg/L.
MBR Ag NPs 0.1 × – – – 60× No significant difference in effluent COD before and (Zhang et al., 2014)
after nanosilver exposure with an average permeate
COD concentration of 24 ± 10 mg/L.
CW CeO2 NPs 1 × 50 × 5× 60√ Only under chronic exposure, COD removal efficiency (Hu et al., 2018)
was only slightly reduced.
Ag NPs 0.02 × – – – 128× The average wastewater effluent COD concentrations (Huang et al., 2018b)
before and after AgNPs exposure were 6.32 ± 0.65 mg/L
and 6.75 ± 0.81 mg/L. No significant negative influence.
0.1 × – – – 120× Before adding Ag NPs, the average removal efficiencies (Huang et al., 2018a)
of COD were 80%–87%. After adding Ag NPs, the
removal efficiencies were stabilized at about 93%.
No significant adversary effects.

Note: 1. Abbreviations: ENPs, engineered nanoparticles; SBR, sequencing batch reactor; CW, constructed wetland; Conc., concentration; MBR, membrane bioreactor.
2. The unit for Conc. is mg/L.
3. × means no negative effect and √ means negative effect. – means lack of observations.
 S. Wu et al. / Science of the Total Environment 660 (2019) 1144–1154 1147

nitrification, denitrification and also emerging shortcut N transforma-
tions (e.g., partial nitrification, anammox) (Zhang et al., 2018a). There-
fore, microbial population diversity and active community structures
play essential roles in achieving high-rate biological N removal. The
lower resistance to ENPs by N transformation microorganisms as com-
pared to the heterotrophs responsible for organic matter removal can
be seen from the current literature review in Table 2. Ammonia-
oxidizing bacteria (AOB) had been reported to be more vulnerable to
ENP exposure, as compared to nitrite-oxidizing bacteria (NOB) (Jeong
et al., 2014; Yang et al., 2014a). Microbial community structure analysis
indicated that AOB and the NOB, Nitrospira, experienced population de-
crease easily after shock loading. However, most denitrification
processes in activated sludge under an anoxic environment have a
much stronger resistance to ENPs than those involving microorganisms
under aerobic conditions (Jeong et al., 2012). Considering that most of
the denitrifying bacteria are heterotrophs, this result is consistent with
findings from Section 2 showing that ENPs may not adversely impact
the growth of heterotrophs (Jeong et al., 2012).
As compared to the recorded investigations performed on the
impact of ENPs on organic matter removal in wastewater treatment sys-
tems, the influence of ENPs exposure to nitrogen removal performance
attracted more research focus (Table 2). The used type of experimental
treatment systems has been extended to upflow anaerobic sludge
blanket reactor (UASB). Moreover, the recent research interest is also

Table 2
Effect of engineered nanoparticles on the removal of nitrogen in different wastewater treatment systems.

Treatment ENPs Low level High level Duration (d) Nitrogen removal performance References
system
Conc. Effect Conc. Effect Short Long

SBR TiO2 NPs b10 × 10–60 × – 290× 0–60 mg/L: NH+ 4 -N removal maintained at 98%. (Li et al., 2017)
Ag NPs b5 × 30 × – 290× b30 mg/L: NH+ 4 -N removal maintained at 98%. (Xu et al., 2017a, 2017b)
0.1–0.5 × – – 15× – No statistical difference as compared to control. (Hou et al., 2012)
0.1–0.5 × – – 15× – No statistical difference as compared to control. (Chen et al., 2014)
ZnO NPs b10 × 10–60 × – 180× b10 mg/L: NH+ 4 -N removal 99%; (Wang et al., 2016b)
10–60 mg/L: NH+ 4 -N removal 98%.
+
5 √ – – 15√ – NH4 -N removal was only slightly repressed. (Chen et al., 2014)
1 × 10, 50 √ – – 1 mg/L of ZnO NPs showed no measurable effect on (Zheng et al., 2011b)
both nitrogen and phosphorus removal.
Compared with the absence of ZnO NPs, the
presence of 10 and 50 mg/L of ZnO NPs decreased
total nitrogen removal efficiencies from 81.5% to
75.6% and 70.8%, respectively.
CeO2 NPs b30 × 60 × – 290× b30 mg/L: NH+ 4 -N removal maintained at 99%. (Wang et al., 2016a)
60 mg/L: NH+ 4 -N removal at 98%.
1 √ – – – 30√ NH+ 4 -N removal efficiency decreased from almost (Qiu et al., 2016)
100% to around 70% after 6 days of continuous
exposure to 1.0 mg/L of CeO2 NPs, followed by a
gradual recovery until a stable value of around
90% after 20 days.
UASB Ag NPs 1 × 10, 50 × – 120× The reactor inoculated with mature anammox granules. (Zhang et al., 2018a)
Nitrogen removal efficiency was not influenced by
adding Ag NPs and stably maintained at 87.9%.
ZnO NPs 1–5 × 10 √ – 140√ The reactor inoculated with mature anammox granules. (Zhang et al., 2018b)
The introduction of 1–5 mg/L ZnONPs did not affect
reactor performance, but 90% of the nitrogen removal
capacity was deprived by a shock of
10 mg/L ZnONPs within 3 days.
AS CuO NPs 1 √ – – – 180√ The average effluent concentration of NH+ 4 -N during (Wang et al., 2017)
the last 30 days after CuO NPs exposure was 6.8 mg/L,
remarkably higher than that in the control (0.3 mg/L).
TiO2 NPs 1 × 50 ×√ 1× 70√ 1 and 50 mg/L TiO2 NPs had no acute effects on TN removal. (Zheng et al., 2011a)
However, 50 mg/L TiO2 NPs was observed to significantly
decrease TN removal efficiency from 80.3% to 24.4%
after long-term exposure.
Ag NPs 1 √ – – 20√ – Batch respirometric assays, nitrification inhibition 13.5% (Liang et al., 2010)
Al2O3 NPs 1 × 50 ×√ 1× 70√ Short-term exposure to 1 and 50 mg/L induced (Chen et al., 2012b)
marginal influences on wastewater nitrification,
denitrification. The prolonged exposure to 50 mg/L
was observed to decrease the TN removal
efficiency from 80.4% to 62.5%.
MBR Ag NPs 0.1 × – – – 60× The average concentrations of total inorganic nitrogen (Zhang et al., 2014)
before and after nanosilver loading were 12.5 ± 2.5 mg
N/L and 13.2 ± 1.5 mg N/L. No significant change.
CW CeO2 NPs 1 √ 50 √ 5√ 60√ Under both acute and chronic exposure, the removal (Hu et al., 2018)
efficiencies of NH+ 4 -N and TN were significantly reduced.
Ag NPs 0.02 √ – – – 128√ Average removal efficiencies decreased by 16% and (Huang et al., 2018b)
11% for NH4 + -N and TN respectively compared
with before adding AgNPs.
0.1 √ – – – 120√ The removal efficiency of NH+ 4 -N also had a significant (Huang et al., 2018a)
decrease after adding AgNPs into wastewater,
with reducing by 35.6% and 27.1% for CW1 (unplanted)
and CW2 (planted) respectively compared to
before exposing AgNPs.

Note: 1. Abbreviations: ENPs, engineered nanoparticles; AS, activated sludge; SBR, sequencing batch reactor; CW, constructed wetland; Conc., concentration; UASB, upflow anaerobic
sludge blanket reactor; MBR, membrane bioreactor; TN, total nitrogen.
2. The unit for Conc. is mg/L.
3. × means no negative effect and √ means negative effect. – means lack of observations.
1148 S. Wu et al. / Science of the Total Environment 660 (2019) 1144–1154
expanded from the conventional nitrogen removal process, e.g. nitrifi-
cation and denitrification, to emerging shortcut N transformations, e.g.
Anammox (Zhang et al., 2018a, 2018b). However, the investigations
under this research topic are also only performed in lab-scale wastewa-
ter treatment systems. More investigations should verify these findings
in pilot scale or full scale treatment systems.
Chen et al. (2012a, 2012b) did not find significantly different N re-
moval performances under exposure to 1 and 50 mg/L aluminum
oxide nanoparticles (Al2O3 NPs) as compared to those in the absence
of NPs, indicating the negligible acute negative effects of Al2O3 NPs at
these tested concentrations on biological N removal from wastewater
(Chen et al., 2012a, 2012b). However, the long-term exposure to
50 mg/L Al2O3 NPs did not show any inhibition effect to ammonium re-
moval from wastewater, but negatively affected the denitrification of ni-
trate. This result is contrary to most of the others and might need to be
explored further.
Acute inhibition is often observed at the beginning of ENP dosage to
the experimental system, but the system usually recovers successfully
after a long-term operation. Thus, recent investigations tend to focus
on the long-term effects of ENPs under real-world conditions. Lower in-
hibition effect in artificially prepared wastewater with various ingredi-
ents or in real sewage than that observed in pure cultivated toxicity
test assays has been reported often. That is because of the potential li-
gands in real sewage that can bind with ENPs and decrease their disso-
lution and bactericidal effects of ENPs (Anderson et al., 2014). These
ligands comprise various organic compounds and inorganic ions, e.g.
Cl− and S2−.
Anaerobic ammonium oxidation (anammox) is a relatively new N
removal process. The potential impact of engineered Ag nanoparticles
on this process in wastewater treatment was investigated by Zhang
et al. (2018a). However, no adverse effect was seen in this study either
on N removal performance or on the relative abundance of Candidatus
Kuenenia inhabiting granules under exposure of Ag NPs at concentra-
tions of 1, 10, 50 mg/L. The strong resistance by anammox granules to
Ag NPs in this study was linked to the defensive function of extracellular
polymeric substances and higher abundances of Ag+ efflux protein SilA
secreted from anammox consortia. This research team also investigated
the impact of ZnO NPs to anammox reactor at various loadings. They
found that the addition of ZnO NPs with concentration of 1–5 mg/L
did not create any negative influence to the reactor operation, but
shock loading of 10 mg/L ZnO NPs only lasted for 3 days (Zhang et al.,
2018b).
The impact of ENPs on partial nitrification (PN) and completely au-
totrophic N removal over nitrite (CANON) processes as part of autotro-
phic N removal has also been investigated (Zhang et al., 2017a; Zhang
et al., 2017b). The results interestingly showed that CuO NPs at low con-
centration (b5 mg/L) can enhance ammonia oxidation by improving
both the bioactivity and relative abundance of AOB. A suppression effect
was only found at the threshold of 30 mg/L in short-term exposure and
10 mg/L in long-term exposure, owing to the adsorption and accumula-
tion of NPs in the sludge. Similarly, in the CANON process, exposure to
1 mg/L ZnO NPs was profitable, whereas only a high concentration ex-
posure, such as 10 mg/L, suppressed the bioactivities of both AOB and
anaerobic ammonia-oxidizing bacteria (AAOB). This growth promotion
effect by ZnO NPs has previously been reported for denitrifying bacteria
at moderate concentrations of b20 mg/L (Wang et al., 2015). However,
knowledge regarding promotional and inhibitory effects is still insuffi-
cient and should be investigated further.
4. Influence of ENPs on P removal in water treatment systems
The consequences of exposure to various ENPs on P removal in dif-
ferent wastewater treatment systems are summarized in Table 3. The
influence of some popular ENPs [e.g., Ag NPs, TiO2 NPs, ZnO NPs, cerium
(IV) oxide (CeO2 NPs)] on biological P removal in sequencing batch re-
actors has well been studied. In addition, a few studies have reported on
the treatment performance and bacterial communities' response to
ENPs in activated sludge systems and CWs as ecosystems. However,
the results therein are still quite divergent, which might be strongly de-
pendent on the treatment system type, nature and characteristics of
ENPs as well as exposure concentrations. The mechanism of influence
of ENP exposure on P removal in different treatment systems is
discussed as follows, based on different types of ENPs.
For biological P removal in sequencing batch reactors, the impacts of
TiO2 NPs, Ag NPs, ZnO NPs, and CeO2 NPs have been investigated. No
acute effect on P removal from wastewater by these ENPs under low
concentration dosage was found, either with short-term or long-term
exposure (Zheng et al., 2011a, 2011b; Wang et al., 2016a, 2016b; Xu
et al., 2017a, 2017b). However, the soluble orthophosphate removal ef-
ficiency decreased from 75.9 ± 1.5% to 58.0 ± 4.8% with the increase of
Ag NP concentration from 5 to 30 mg/L (Xu et al., 2017a, 2017b). Simi-
larly, the soluble orthophosphate removal efficiency decreased from
69.8 ± 1.4% to 65.5 ± 0.6% with the increase of ZnO NP concentration
from 5 to 60 mg/L, indicating the inhibitive effect of ZnO NP on phos-
phate release and phosphate uptake of polyphosphate-accumulating
organisms (PAOs) in a sequencing batch reactor (Wang et al., 2016a,
2016b).
In activated sludge wastewater treatment systems, the potential
negative impact of copper oxide nanoparticles (CuO NPs) on treatment
performance, bacterial response, and the molecular ecological network
was investigated by Wang et al. (2017). Their results show that P re-
moval efficiency decreased under exposure of CuO NPs at concentration
of 1 mg/L in a long-term operation. This was supposed to be caused by
the inhibited enzyme activities of exopolyphosphatase, and
polyphosphate (Wang et al., 2017). Moreover, the bacterial diversity
was significantly decreased and the overall bacterial community struc-
ture was also altered. Molecular ecological network analysis showed
that CuO NPs markedly changed network interactions among different
phylogenetic populations (Wang et al., 2017).
Constructed wetlands, as decentralized and cost-effective eco-
treatment systems, are widely used to treat various wastewaters,
some of which potentially contain ENPs. Contaminants can be removed
in CWs through simultaneously occurring physical, chemical and bio-
logical processes among cooperation between media, plants and micro-
organism. As an inorganic element, ENPs have high possibility to be
taken up by the wetland plants. Besides, ENPs can also be adsorbed
onto the plant root surface and/or the media in the wetland. Thus, the
above-discussed investigations regarding the impact of ENPs on perfor-
mance of biological wastewater treatment systems (e.g., sequencing
batch reactors) might be difficult to compare to those regarding CWs.
Hu et al. (2018) examined the acute and chronic impacts of CeO2 NPs
(size = 70–100 nm) on the treatment performance of nine sequencing
batch CW microcosms. They compared the exposures to 1 mg/L CeO2
NPs as an example of environmentally relevant concentration and
50 mg/L CeO2 NPs as a high concentration and found that the removal
of total phosphorus (TP) was neither influenced by acute (5 days) nor
chronic (60 days) exposure. This is because that TP was mainly removed
by matrix adsorption in CWs, not like the biological process of PAOs in
conventional activated sludge systems.
However, the adverse effect on P removal in vertical flow CWs under
exposure to Ag NPs has been reported in many studies (Huang et al.,
2017; Cao et al., 2018; Huang et al., 2018a). For example, Huang et al.
(2018b) investigated the performance of a pilot-scale vertical-flow
CW during 128 days exposure to 20 μg/L Ag NPs (size = 10–40 nm).
Compared to the results for the experimental period without Ag NP ad-
dition, the removal rate of TP declined by 11% after Ag NP exposure. It is
well known that a higher content of Mg2+, Fe2+, Al3+, and especially
Ca2+ in the soil materials of wetlands may contribute to a better positive
P removal performance (Zhu et al., 1997). Moreover, the ionic strength
of Ca2+ was also reported to be positively correlated to the removal of
Ag NP (Bae et al., 2013). Thus, the impact of exposure to Ag NPs on P re-
moval in these vertical wetlands was thought to be due to the physical
 S. Wu et al. / Science of the Total Environment 660 (2019) 1144–1154 1149

Table 3
Effect of engineered nanoparticles on the removal of phosphorus in different wastewater treatment systems.

Treatment ENPs Low level High level Duration (d) Phosphorus removal performance References
system
Conc. Effect Conc. Effect Short Long

SBR TiO NPs – – 5 √ – 290 Reduced from 88% to 68% at 5 mg/L and then gradually increased to (Li et al., 2017)
96% at 60 mg/L.
1 × 50 × 1× 70× N99% phosphorus removal and no influence. (Zheng et al., 2011a)
Ag NPs b5 × – – – Almost all SOP was removed no matter what the tested Ag NPs (Chen et al., 2012a,
concentrations were. 2012b)
b5 × 5–30 √ – 290 b5 mg/L: SOP removal slightly reduced. (Xu et al., 2017a)
5–30 mg/L: SOP removal reduced from 76%–58%
ZnO NPs b5 × 5–60 √ – 180√ b5 mg/L: SOP removal stable at 70% (Wang et al., 2016b)
5–60 mg/L: decreased from 70% to 66%
1 × 10, 50 √ – – The effluent phosphorus concentrations increased from (Zheng et al., 2011b)
nondetectable to 10.3 and 16.5 mg/L, respectively, which were higher
than the influent phosphorus (9.8 mg/L)
CeO2 NPs 0.1 × 20 √ 0.3√ – At concentration of 0.1 mg/L, CeO2 NPs posed no impacts on total (Xu et al., 2017b)
phosphorus (TP) removal. While at 20 mg/L, TP removal efficiency
reduced from 85.16% to 59.62%.
AS CuO NPs 1 √ – – – 180√ The average TP removal efficiency during the last 30 days after CuO NP (Wang et al., 2017)
exposure was 63%, remarkably lower than that in the control (91%)
Al2O3 NPs 1 × 50 × 1× 70× No significant influence for both short- and long-term exposure. (Chen et al., 2012b)
CW CeO2 NPs 1 × 50 × 5× 60× Under both acute and chronic exposure, the removal efficiencies of TP (Hu et al., 2018)
were not significantly affected.
Ag NPs 0.02 √ – – – 128√ After Ag NPs exposure, the average removal efficiency of TP decreased (Huang et al., 2018b)
by 11% compared with no Ag NPs adding.
0.1 √ – – – 120√ Removal efficiency of TP decreased dramatically from 71.3% to 45.7% (Huang et al., 2018a)
in CW1 (unplanted) and about 77.9% to 51.7% in CW2 (planted) when
exposing 0.1 mg/L AgNPs for about 90 days.

Note: 1. Abbreviations: ENPs, engineered nanoparticles; AS, activated sludge; SBR, sequencing batch reactor; CW, constructed wetland; Conc., concentration; SOP, soluble orthophosphate;
TP, total phosphorus.
2. The unit for Conc. is mg/L.
3. × means no negative effect and √ means negative effect. – means lack of observations.

competition between TP and Ag NP removals in the CWs (Huang et al.,
2018b). Similarly, Huang et al. (2018a) compared the impact of Ag NPs
(100 μg/L, size = 10–40 nm) on P removal in two vertical-flow CW mi-
crocosms: one unplanted and one planted (Huang et al., 2018a). In a
90 days experimental operation, they found a significant decrease of
TP removal from 71.2% to 45.7% in the unplanted wetland and from
about 77.8% to 51.7% in the planted wetland when shock exposed to
100 μg/L Ag NPs. However, the long-term TP removal between the
two experimental wetlands after Ag NP loading did not differ (Huang
et al., 2018a), due to the minor role of plants on Ag retention
(Auvinen et al., 2016).
5. Influence of ENPs exposure on wetland plants
Constructed wetlands are commonly used as eco-systems for
decentralized wastewater treatment in rural areas due to their robust
operation and low-maintenance requirement (Wu et al., 2014; Wu
et al., 2018). In addition to their well-established use for domestic sew-
age treatment, a large variety of industrial effluents, which may contain
various ENPs, are treated in CWs (Wu et al., 2015). The possible trans-
formation processes of different ENPs and the environmental factors af-
fecting these processes in CWs have previously been reviewed and
discussed by Auvinen et al. (2017). In brief, the aggregation and subse-
quent sedimentation of ENPs with suspended organic compounds can
significantly decrease their mobility in CWs by restricting their distribu-
tion mainly in the first section of wetlands (as shown in Fig. 1). Adsorp-
tion to substrate and sulfidation (defined as the reaction of ENPs with
sulfide) in CWs have also been shown to be important (Auvinen et al.,
2017). However, the interactions between ENPs with wetland plants
might be a little bit more complex, since plants can uptake some ENPs
to enhance ENPs retention in wetlands, while ENPs can also accumu-
lated in the root surface and then cause toxic effect to the plants. After
the review by Auvinen et al. (2017), there emerged numerous new pub-
lications discussing the interactions of ENPs with plants. Thus, the influ-
ence of exposure to ENPs on wetlands plants in this review can be seen
as supplementary to Auvinen's review.
Most previous studies mainly focused on the potential toxicity of
ENPs to food-related plants, e.g., rice, barley, radish, rape, ryegrass, let-
tuce, corn, spinach, and cucumber (Yang et al., 2007; Battke et al.,
2008; Ma et al., 2010). Information on the interactions between ENPs
and wetland plants is still insufficient. The impact of ENPs on the growth
of food-related plants is well investigated and found to be quite variable,
i.e., enhancement, inhibition, or no effect (Ma et al., 2010). However, no
significant negative influence to biomass production in wetland plants
under exposure to ENPs has been reported (Jacob et al., 2013). Even
though most ENPs are not considered essential plant nutrients, some re-
searchers indeed observed a positive stimulating effect to the growth of
wetland plants. Hu et al. (2018) examined the acute and chronic re-
sponses of Phragmites australis in sequencing-batch CWs to CeO2 NPs.
They observed a higher photosynthesis rate of P. australis and a margin-
ally increased biomass production under exposure to CeO2 NPs at con-
centration of 1 mg/L. However, exposure to 50 mg/L CeO2 NPs
significantly reduced the photosynthesis rate and decreased the bio-
mass production by 29% in dry weight. Besides, the tissue content of C,
N, and P of P. australis in this experiment under exposure to high con-
centration of CeO2 NPs was also decreased by 20–30%.
Cyrusova et al. (2017) examined the response of the wetland plant
Carex vulpina to exposure of copper and iron nanoparticles. Under expo-
sure to copper nanoparticles at concentration of 100 mg/L, the growth,
root characteristics, and leaf photosynthetic pigment contents of C.
vulpina were significantly negatively affected. Yet, the same concentra-
tion of iron nanoparticles did not lead to reductions in any of the mea-
sured items (Cyrusova et al., 2017).
The associated sorption of ENPs to root surfaces has been observed
in many studies (Ma et al., 2010; Kumar and Yadav, 2009; Lin and
Xing, 2008), as evidenced by the higher concentration of ENPs in the
rinse solutions after exposure to the ENPs. It is interesting that the pres-
ence of Fe2+ and iron oxide nanoparticles (γ-Fe2O3; 20–100 nm) in
wastewater can significantly stimulate the formation of iron plaque on
the root surface of P. australis (Pardo et al., 2016). Owing to the reactive
characteristics of γ-Fe2O3 nanoparticles in the intensively formed iron
plaque, the adsorption removal of arsenic (As) in wastewater by roots
1150 S. Wu et al. / Science of the Total Environment 660 (2019) 1144–1154
Fig. 1. Schematic illustration of the flow of ENPs in horizontal subsurface constructed wetlands and associated sorption of ENPs to root surfaces and partial uptake.
(Modified from (Kadlec and Wallace, 2009) and (Auvinen et al., 2017)).
of P. australis was strongly improved (Fig. 2). It has been reported that
exposure to iron nanoparticles (Fe3O4) can decrease root size and mod-
ify root architecture, reduce root water content of Lactuca sativa
(Trujillo-Reyes et al., 2014). However, no adverse effects to P. australis
were observed in the study by Pardo et al. (2016), which may be due
to the higher tolerance capacity of P. australis to many metal
contaminants.
Uptake of ENPs by plants is not a new knowledge and has been re-
ported for decades. However, the form of these ENPs entering the tis-
sues is still hard to differentiate, as they can be either the ion released
from the ENPs or the ENPs themselves. ENPs dosed experimentally to
plants were found to be mainly retained in the root part of the plants.
For example, Hu et al. (2018) detected 91.6–95.2% of applied cerium
(Ce) (size = 70–100 nm) in the roots of P. australis in a series of se-
quencing batch CWs, whereas only 3.6–5.8% in the stems and 1.2–2.5%
in the leaves. If we assume that the size of a plant's cell wall pore in
Fig. 2. Concentrations of As adsorbed onto stimulated iron plaque (extracted with
dithionitecitrate-bicarbonate) in the root surface of P. australis (n = 4) and visual
appearance of the roots under different treatments. CT-N: nutritive solution; 1/2FeII + III:
Fe2+ and nFe2O3 combination at half dose; FeII + III: Fe2+ and nFe2O3 combination.
(Modified from Pardo et al., 2016).
the range of approximately 5 nm in diameter, the ENPs used in this ex-
periment with size bigger than 5 nm may not be able to pass directly
through the cell wall. However, the transmission electron microscopy
(TEM) images from Hu et al. (2018) showed the presence of CeO2 NPs
not only in the root epidermis, but also in the interior of root cells.
This observation may indicate the uptake of CeO2 NPs to plant roots
via symplastic pathways, such as endocytosis pathways.
Furthermore, the uptake of ENPs may also be influenced by the com-
position of the wastewater. The investigation by Jacob et al. (2013)
showed that titanium (Ti) uptake in Elodea canadensis significantly de-
creased with increasing P in the water. This trend was also observed
in Triglochin maritima, as an example of another wetland plant
(Sunwar, 2011). However, other crop species, for example, chickpea
and lentil, showed a positive correlation of higher Ti uptake with addi-
tional more application of P (Gunes et al., 2009). This phenomenon
has been speculated to be the effect of binding, or competition for ad-
sorption sites between ENPs and P; however, the mechanism of ENP
and P interaction should be further explored.
Most wetland plants can translocate ionic metal elements from their
roots to their shoots. Accumulation of ENP-originated metal elements in
shoots has been well reported (Jacob et al., 2013). However, more inves-
tigations are needed to verify the presence of ENPs in plant tissues, as
only a few studies have shown that plants, e.g., Arabidopsis thaliana
(Kurepa et al., 2010) and Nicotiana tabacum (Ghosh et al., 2010), can
translocate TiO2 NPs into their shoots. Huang et al. (2018b) found that
55.1% of Ag NPs (20 μg/L, 10–40 nm) absorbed by Iris pseudacorus
were transported to the aboveground portion of the plant. The transport
of Ag NPs resulted in the accumulation of Ag in leaves of I. pseudacorus,
which was only slightly lower than that in roots. However, in another of
their experiments, using a simulated 100 μg/L Ag NP (10–40 nm) influ-
ent, a significantly higher concentration of Ag was found in the roots
than in the stems and leaves. This is in line with the results from
Auvinen et al. (2016), showing that P. australis-associated Ag NPs
(90.7 ± 7.9 nm) was mainly retained by the roots, and only a maximum
3% of Ag were translocated to the leaves.
6. Challenges to understand the impact of ENPs in wastewater
treatment systems
With the increasing concern on the release of ENPs into aquatic en-
vironment, numerous investigations have been performed in the recent
years to understand its potential impact on microbial transformations of
C, N and P in wastewater treatment process as well as plants in the
 S. Wu et al. / Science of the Total Environment 660 (2019) 1144–1154
wetland treatment system. However, knowledge in this field is still in-
sufficient, due to the challenges that the scientists face to analysis of
ENPs in complex wastewater samples and the experimental design to
test the toxicity of ENPs in real wastewater treatment systems. There-
fore, this section of the review is trying to give a general overview of
the current research challenges in the impact of ENPs in wastewater
treatment systems.
Challenges that analytical chemists face concerning the detection of
ENPs in various wastewaters not only include the advanced detection,
identification and quantification of ENPs in wastewater treatment sys-
tems, but also the inherent characteristics and preparation of the sam-
ples (Table 4). Due to the variable composition of different
wastewaters, many inorganic salts (e.g. SO42−, S2−, Cl−, and PO43−)
and organic humic substances (e.g. poly-alcohols, amines, carboxylic
acids) in these wastewaters may act as ligands to complex ENPs, creat-
ing further difficulties on the speciation of ENPs in wastewater and
sludge/soil samples (Choi et al., 2009). Particularly for the samples
taken from the real world containing ENPs with environmental relevant
concentrations, lack of adequate techniques for efficient detection and
quantification of these trace ENPs in colloidal samples often hampers
the understanding their fate and ecotoxicological effects (Von der
Kammer et al., 2012). But it may be possible to tailor some new strate-
gies for analysis of ENPs in wastewater treatment systems to account
for different physical and chemical characteristics of ENPs. The tailored
approach should require the use of multiple techniques to provide a
comprehensive understanding that takes into account all the important
nanoparticle characteristics outlined above. As such, there exists a
greater need to select most important characteristics as the focus of in-
dividual studies on a case by case basis. As a baseline, strategies could in-
clude analysis of particle morphology (size, shape) in combination with
quantification of the particle number, mass and dissolved concentra-
tion. Combining Imaging techniques such as transmission electron mi-
croscopy (TEM) with mass spectroscopic approaches such as single
particle inductively coupled plasma mass spectrometry (SP-ICP-MS)
may represent a suitable baseline approach.
Besides to the detection and quantification of various ENPs in waste-
waters, there is also a lack of research to instruct us on how to collect,
preserve and store samples from wastewater systems. It has been
shown that the ENPs in the water/soil samples are susceptible to the
perturbations driven by factors, e.g. pH, ionic strength, sunlight, biofilm
growth (Christian et al., 2008). Even in some cases, chemical properties
can only be measured in the samples that have been stored due to the
temporally dynamic nature of colloidal systems. However, this require-
ment often creates problems for toxicological investigations, where the
samples are generally not allowed to be stored (Von der Kammer et al.,
2012).
There is also a lack of efficient analytical techniques to differentiate
naturally occurring nanomaterials from ENPs, thus little is known
about the real environmental concentrations of ENPs (Von der
Kammer et al., 2012). Modeling of predicted environmental concentra-
tions (PEC) of ENPs have been performed by many researchers and also
proved to be necessary and valuable substitute for measurement studies
(Gottschalk et al., 2010). According to the literature, to date, only a few
Table 4
Challenges to analysis of ENPs in wastewater treatment systems.
Characteristic Preparation Identification
• Low-level content • Sample • Rapid and sensitive
• Inherent sample collection determination
heterogeneity • Preservation • Quality-controlled validation
• Inherent property • Storage • Multiple lines of evidence
discontinuity • X-ray-based techniques
• Formation of complex • Imaging-based techniques
ligands • Mass-spectrometry-based
• Variable wastewater techniques
composition • Biological sensors
1151
modeling studies have presented quantitative estimations of the aquatic
environmental concentrations of ENPs (Table 5) (Hendren et al., 2013;
Gottschalk et al., 2015). Even though different processes involving
ENP production, application, and release affect the modeled values, an
overall estimate on the order of magnitude of the environmental con-
centrations (at the μg/L level) still can be reached. Therefore, the conclu-
sions drawn on the negative impacts of ENPs in wastewater treatment
process under exposure to extreme high concentrations tested in the
laboratory should be carefully considered.
To gain better knowledge regarding the influence of ENPs on micro-
bial transformations in the wastewater treatment process, understand-
ing the ENP form that shows higher toxicity to microorganisms is
essential, though largely unknown. The toxicity mechanisms associated
with ENPs can be driven either by the dissolved metal ions from disso-
lution of ENPs or the ENPs themselves. Thus, discussion on the inhibi-
tion of microorganisms by ENPs generally involves the basic
characteristics of ENPs, including the size and surface area and the ion
releasing capacity of the ENPs. However, some of these basic character-
istic information is not presented in the published articles.
Release of metal ions is suggested as one of the important ENP tox-
icity mechanism, e.g., Ag+ released from Ag NP (Kennedy et al., 2010).
Similarly, copper NPs are reported to be less toxic than ionic copper
(Griffitt et al., 2008; Ganesh et al., 2010). However, there is still no con-
sistent statement on whether the inhibitory effect of ENPs is due to the
release of ions or the particles themselves. For example, it is well ac-
cepted that ionic silver inhibits the growth of Pseudomonas fluorescens
more than Ag NP does (Matzke et al., 2014). However, some respiro-
metric assays also found that at 1 mg/L of total Ag, nitrification inhibi-
tion by Ag NPs (average size = 1–29 nm) and Ag+ ions was 41.4%
and 13.5%, respectively, indicating a higher toxicity of nanosilver to ni-
trifying bacteria in activated sludge than silver ions (Liang et al.,
2010). Choi et al. (2008) also observed a higher inhibitory effect to res-
piration rate (86%) by nanosilver than by ionic silver (42%). To date, it is
still very difficult to draw general conclusions on these observed differ-
ences in toxic effects, due to the variations of the experimental condi-
tions, e.g. dosed concentrations, duration of the experiment as well as
monitored parameters.
Toxicity of ENPs to functional microorganisms in wastewater treat-
ment processes might also be related to the stability of the used ENPs.
Modification of ENP surfaces by coating materials can increase their sta-
bility and result in discrepancies in toxicity kinetics and their mecha-
nisms (Joo and Aggarwal, 2018). If the coating targets the control of
metal ion release, the negative effect of coated ENPs on microorganisms
might be minimized. However, recent studies also show that the coating
of ENPs with materials such as nickel ions, cobalt, leaf extracts, or chito-
san can enhance the antibacterial activity of un-coated ENPs with re-
spect to pathogenic bacteria (Sundrarajan et al., 2015; Esmaeili and
Farrahi, 2016; Joo and Aggarwal, 2018). Thus, research gaps in exploring
the impact of ENP coating on microbial transformations in wastewater
treatment processes remain.
The potential for biofilm-bound microbial communities to develop
resistance to ENP toxicity represents a further challenge to accurate un-
derstanding of the toxicity of ENPs in wastewater treatment systems.
Enhanced resistance to Ag NP toxicity has been observed in CW micro-
bial communities with a history of prior exposure (Button et al., 2016)
and in engineered facultative bacteria found in wastewater treatment
systems (E. coli) (Joshi et al., 2012). The exact mechanisms behind the
development of resistance to ENP toxicity are not entirely clear but
may involve increased production of extracellular polymeric substances
(EPS) (Joshi et al., 2012), horizontal gene transfer, and gene expression
(Lenz et al., 2008; Gunawan et al., 2013), or simply an environment con-
ducive to the building and maintenance of a viable biofilm by the intrin-
sic microbial community (Button et al., 2016). In the long term, the
development of resistance and the chronic accumulation of ENPs may
become competing factors in the ongoing performance of wastewater
treatment systems.
1152 S. Wu et al. / Science of the Total Environment 660 (2019) 1144–1154

Table 5
Predicted concentration of ENPs in the aquatic environment.

ENPs Unit Domain References

TiO2 ZnO Si Ag

1 2–18 μg/L Europe River (Blaser et al., 2008)

2 3.47 0.432 0.045 μg/L Europe STP effluent (Gottschalk et al., 2009)
3 0.7 0.03 μg/L Switzerland Water (Mueller and Nowack, 2008)
4 4.28 0.441 0.039 μg/L Switzerland STP effluent (Gottschalk et al., 2009)
5 4.3 0.039 μg/L Switzerland STP effluent (Gottschalk et al., 2010)
6 13 0.5 μg/L Denmark STP effluent (Gottschalk et al., 2015)
7 1 μg/L UK STP effluent (Johnson et al., 2011)
8 4.91 1.91 1.20 0.32 μg/L UK STP influent (Tiede et al., 2012)
9 1.9–510 ng/L US STP effluent (Hendren et al., 2013)
13 1.75 0.3 0.021 μg/L US STP effluent (Gottschalk et al., 2009)

STP, sewage treatment plant.

7. Conclusions
• The exposure to most of the investigated ENPs at low concentrations
seems not to be negatively affecting the growth of heterotrophic mi-
croorganisms, which are responsible for organic matter removal in
wastewater treatment systems. Only exposure at high concentrations
inhibited their performance, yet exposure to these high concentra-
tions of ENPs in wastewater may neither occur regularly nor in the
long term.
• The impact of ENPs on various microbial N transformation processes,
including traditional nitrification and denitrification and emerging
shortcut N transformations (e.g., partial nitrification, anammox),
have been investigated. Acute inhibition is often observed at the be-
ginning of exposure to ENPs, but does not last in a long-term opera-
tion. No significant negative effects on biological P removal in the
wastewater treatment process were found after short-term or long-
term exposure.
• Environmentally relevant concentrations of ENPs may enhance the
photosynthesis of wetland plants, whereas chronic exposure to a
high concentration of ENPs can inhibit photosynthesis. Higher con-
centrations of ENPs in the rinse solutions after exposure to ENPs
have been observed in many studies, indicating the associated sorp-
tion to root surfaces. The presence of Fe2+ and iron oxide nanoparti-
cles in wastewater can significantly stimulate the formation of iron
plaque on the root surface of P. australis.
• Inhibition effects are often overestimated in pure cultivated toxicity
test assays compared to that in artificially prepared wastewater with
various ingredients or in real wastewater. The potential ligands in
real wastewater can bind with ENPs and lower the dissolution of
ENPs. The discussion on inhibition of microorganisms by ENPs gener-
ally involves the basic characteristics of ENPs, including the size, sur-
face area and ion releasing capacity of the ENPs. However, there is
still no consistent statement on whether the inhibitory effect of
ENPs is due to the release of ions or the NPs themselves.
• Advances in the understanding of the environmental fate, and ecotox-
icological effects of ENPs have been challenged by a lack of adequate
techniques for the detection and quantification of ENMs at environ-
mentally relevant concentrations in complex media such as soils, sed-
iments, and biological tissues.
Acknowledgments
We would like to give thanks to the AIAS-COFUND Fellowship
Programme, which is funded by the European Union Seventh
Framework Programme for Research, Technological Development and
Demonstration under grant agreement no. 609033. We are also
thankful to reviewers for their valuable suggestions for improving this
article.
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