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3.6 Analysis of the toxicity mechanisms of the TiO
2
-NPs
To determine the deep-seated toxic mechanisms of the four TiO
2
-NPs, we first
used the molecular fluorescent dyes SYTO® 9 and propidium iodide combined with
CLSM to assess the damage to the bacterial cytoplasmic membrane
in situ
. As shown
in Fig. 5A, the four TiO
2
-NPs (50 mg/L) all caused significant (p < 0.05) damage to
the microbe cells in the sludge; the toxicities of pristine TiO
2
-A and TiO
2
-R were most
prominent, with lethality rates of 48.4% and 41.9%, respectively (Fig. S2). The
toxicity was mainly due to the leakage or fragmentation of the bacterial plasma
membranes, which led to the extensive seepage of intracellular substances such as
LDH. LDH leakage was associated with the stronger photocatalytic activity of the
TiO
2
-NPs, which led to necrosis resulting from substantial acellular ROS production
under simulated sunlight (Fig. 1B) (Li et al., 2019a; Qian et al., 2017). TiO
2
-A and
TiO
2
-R caused a significant elevation of LDH release (p < 0.05) with respect to the
groups exposed to the aged NPs and the untreated sludge groups (Fig. 6A). The
phenomena described above may have resulted from lipid peroxidation and direct
contact damage to the cell membrane caused by the TiO
2
-NPs, which is presumably
closely related to the photoactivity and water environment stability of the NPs, i.e.,
the particle size range of the nanoscale effects, and the impact of aging or inclusion.
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The ultrastructure of the sludge cells was determined by TEM (Fig. 5Ba), which
revealed the clear biological phases, and the smooth morphology and integrity (green
arrows) of the control cells. In Fig. 5Bb to Fig. 5Be, the black areas (purple arrows)
correspond to electrodense material (i.e., the TiO
2
-NPs) inferred from the previous
reference (Li et al., 2019a), confirmed by the high and wide peaks of Ti in EDS (Fig.
5Bb1 to Fig. 5Be1), by contrast, the content of Ti element in the unexposed
acclimated activated sludge was less than the detection limit of plasma-mass
spectrometer (ICP-MS), just like the control smooth cells without black mark in TEM
(Fig. 5Ba),
not interfered by TiO
2
-NPs. The bacterial cells in the exposed groups had
rough morphologies, with necrosis and lysis particles (red arrows), which suggests
that the microbial community in the sludge suffered from great oxidative stress,
regardless of the crystal form or aging status of the NPs. Notably, Fig. 6B shows the
crystalline-dependent extracellular and intracellular titanium concentration in the
activate sludge, which was consistent with our previous short-term research on the
interaction between TiO
2
-NPs and activated sludge, with discrepant distribution of
rutile and anatase NPs
in vivo
and
in vitro
in the sludge layers (Li et al., 2019a). In the
present study, compared to the aged TiO
2
-NPs, there was more intracellular and
extracellular titanium (p < 0.05) from the pristine NPs, implying that TiO
2
-A and
TiO
2
-R were preferentially bio-adsorbed and deposited on the surface of the sludge.
This increased the possibility of killing superficial cells with minimal loss of
photocatalytic activity (Fig. 5A and Fig. S2) (Li et al., 2019b). These results could
explain why the pristine TiO
2
-NPs, particularly TiO
2
-A, were more likely to
agglomerate in the sludge suspension (Fig. 1Bc) and exert stronger oxidation pressure
on the permeability of the cytoplasmic membranes (Fig. 1Bb and Fig. 6A), and were
more prevalent on the cell surfaces, enabling them to penetrate the sludge (Fig. 6B).
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In contrast, it should be noted that aging did not seem to reduce or eliminate the
differences in toxicity stresses between the anatase and rutile NPs, such as EPS
secretion (Fig. 3) and elemental titanium distribution in the sludge layer (Fig. 6B).
However, in terms of toxicity indicators including LDH release (Fig. 6A) and cell
mortality (Fig. S2), there were still significant differences between the aging anatase
and rutile NPs when they were used at a concentration of 50 mg/L. Furthermore,
contrary to the toxicity results of two pristine NPs, aTiO
2
-R was more toxic than
aTiO
2
-A. This might have been due to the stronger photoactivity of aTiO
2
-R than that
of aTiO
2
-A (Fig. 1Bb) (the red shift in light absorption was due to the decrease in the
band gaps) (Fig. 1Ba), and its relative stability and lower hydrodynamic particle
diameter (Fig. 1Bc), resulting in greater intracellular titanium accumulation (Fig. 6B)
after aging. Summarily, the complicated and crystalline-dependent behaviors of the
sludge microbes in defending themselves from the stresses arising from 72-h exposure
to NPs included a decrease in decontamination capability, the response to EPS
resistance, the change in the functional structure of the community, the removal of P
metabolites, and damage at the cellular level. Natural aging may alleviate the
oxidative stress caused by NPs to some extent, but it might also reverse the toxicity of
the different crystalline NPs. However, in terms of the toxicity mechanism of
TiO
2
-NPs, in addition to the differences caused by the initial characteristics of
different crystal types, such as energy band gap and crystal surface index (Wang et al.,
2015), the aging transformation of the sewage environment of nanoparticles and the
stability of the nanoscale
comprehensively controlled their ecological toxicity effects
(Li et al., 2019a). Summarily, the results of the present study provide a better
understanding of the risks to the water environment, enabling an evaluation of policy
control with regard to nanoparticles. Furthermore, it is important to consider the
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effects of aging on the NPs.
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