Ecotoxicity assessment of sodium dimethyldithiocarbamate and its micro-sized metal chelates in Caenorhabditis elegans

Article abstract of DOI:10.1016/j.scitotenv.2020.137666

Sodium dimethyldithiocarbamate (SDDC) is a widely used heavy metal chelating agent in harmless treatment of wastewater and hazardous waste, but SDDC and its heavy metal chelates may leak into the environment and bring potential ecological risks. In this study, the model organism Caenorhabditis elegans was used to evaluate the toxic effect of SDDC and its heavy metal Cu, Pb chelates. Multiple endpoints were investigated by subacute exposure to SDDC (0.01–100 mg/L) and micro-sized Cu, Pb chelates of SDDC (1–100 mg/L). Our data indicated that the LC₅₀ value of SDDC was 139.39 mg/L (95% Cl: 111.03, 174.75 mg/L). In addition, SDDC was found that concentration of 1 mg/L is a safe limit value for nematode C. elegans, and concentration above 1 mg/L caused adverse effects on the survival, growth, locomotion behaviors and reactive oxygen species (ROS) production of exposed nematodes. Furthermore, all tested SDDC-Cu and SDDC-Pb chelates had obviously lower toxic effect than untreated Cu, Pb metals. These two chelates also had a lower toxic effect than SDDC agent due to its more stable structure. Moreover, SDDC-Cu had a higher toxic effect than SDDC-Pb at the same concentration. Thus, our results suggest that SDDC as a kind of chelating agent applied in harmless treatment of heavy metals, the safe addition limit should not be exceeded.

Full text of DOI:10.1016/j.scitotenv.2020.137666

Science of the Total Environment 720 (2020) 137666
Contents lists available at ScienceDirect
Science of the Total Environment
journal homepage: www.elsevier.com/locate/scitotenv
Ecotoxicity assessment of sodium dimethyldithiocarbamate and its
micro-sized metal chelates in Caenorhabditis elegans
⁎
Yitian Wang, Han Zhang, Xiangyu Wu, Cheng Xue, Yang Hu, Asim Khan, Fuwen Liu, Lankun Cai
State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, School of Resources and Environmental Engineering, East China University of
Science and Technology, Shanghai 200237, China
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
• SDDC at concentration of 1 mg/L is a safe
limit value for nematode C. elegans.
• SDDC (≥1 mg/L) caused adverse effects
on survival, growth, behaviors and ROS.
• SDDC-Cu and SDDC-Pb chelates had
lower toxic effect than Cu and Pb.
• SDDC metal chelates had a more stable
chemical structure than SDDC.
a r t i c l e i n f o
a b s t r a c t
Article history:
Sodium dimethyldithiocarbamate (SDDC) is a widely used heavy metal chelating agent in harmless treatment of
wastewater and hazardous waste, but SDDC and its heavy metal chelates may leak into the environment and
bring potential ecological risks. In this study, the model organism Caenorhabditis elegans was used to evaluate
the toxic effect of SDDC and its heavy metal Cu, Pb chelates. Multiple endpoints were investigated by subacute
exposure to SDDC (0.01–100 mg/L) and micro-sized Cu, Pb chelates of SDDC (1–100 mg/L). Our data indicated
that the LC50 value of SDDC was 139.39 mg/L (95% Cl: 111.03, 174.75 mg/L). In addition, SDDC was found that
concentration of 1 mg/L is a safe limit value for nematode C. elegans, and concentration above 1 mg/L caused ad-
verse effects on the survival, growth, locomotion behaviors and reactive oxygen species (ROS) production of ex-
posed nematodes. Furthermore, all tested SDDC-Cu and SDDC-Pb chelates had obviously lower toxic effect than
untreated Cu, Pb metals. These two chelates also had a lower toxic effect than SDDC agent due to its more stable
structure. Moreover, SDDC-Cu had a higher toxic effect than SDDC-Pb at the same concentration. Thus, our results
suggest that SDDC as a kind of chelating agent applied in harmless treatment of heavy metals, the safe addition
limit should not be exceeded.
Received 12 December 2019
Received in revised form 28 February 2020
Accepted 29 February 2020
Available online 2 March 2020
Editor: Henner Hollert
Keywords:
Sodium dimethyldithiocarbamate
Heavy metal
Chelating agent
C. elegans
Ecological risk
© 2020 Elsevier B.V. All rights reserved.
Abbreviations: C. elegans, Caenorhabditis elegans; CGC, Caenorhabditis Genetics Center; DA, Dopaminergic; DTC, Dithiocarbamate acid; E. coli OP50, Escherichia coli OP50; MSWI,
Municipal solid waste incineration; NGM, Nematode growth medium; PD, Parkinson’s disease; ROS, Reactive oxygen species; SDDC, Sodium dimethyldithiocarbamate; SEM, Standard
error of the mean; S/S, Solidification/stabilization.
⁎
Corresponding author at: School of Resources and Environmental Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
E-mail address: cailankun@126.com (L. Cai).
https://doi.org/10.1016/j.scitotenv.2020.137666
0048-9697/© 2020 Elsevier B.V. All rights reserved.

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Y. Wang et al. / Science of the Total Environment 720 (2020) 137666
1. Introduction
Cu(II) N Pb(II) N Zn(II) N Mn(II) (Phinney and Bruland, 1997;
Weissmahr and Sedlak, 2000).
Sodium dimethyldithiocarbamate (SDDC), as a kind of dithiocarba-
mate acid (DTC), is widely used as a disinfectant, corrosion inhibitor,
vulcanizing agent, chelating agent and pesticides in water treatment,
rubber industry and agricultural application (Abdollahi and Khaksar,
2014; Lei et al., 2015; Mack et al., 2018; Tilton et al., 2006). Due to its ex-
cellent chelating ability, SDDC has also been commercially used in
harmless treatment of heavy metals such as wastewater, soil and haz-
ardous waste treatment (Liu et al., 2019b; Malerius et al., 2003;
McFarland et al., 2012; Su et al., 2013; Zheng et al., 2019). Especially
in China, as a chelating-stabilizing agent, SDDC is extensively used in so-
lidification/stabilization (S/S) treatment of municipal solid waste incin-
eration (MSWI) fly ash and polluted soils (Chen et al., 2018b; Ma et al.,
2019; Wang et al., 2015).
The SDDC agent in S/S treatment processes of fly ashes and polluted
soils may have risks of leakage into the surrounding environment, such
as in storage, treatment and disposal site, as well as transportation pro-
cess and landfill site (Fig. 1(a)). Herein, amounts of residual SDDC che-
lating agent and its heavy metal complexes are easily released into soils
and groundwater, which will bring potential harm to the environment.
Although most of the SDDC metal complexes are insoluble, they also
have the possibility of dissociation in water. Previous studies indicated
that different metals of SDDC complexes showed different stability con-
stant of inhibiting acid-catalyzed hydrolysis. Among these, stability con-
stant (Log K) of Mn(II), Zn(II), and Cu(II) is b2, 3.7 and 20–24,
respectively (Weissmahr and Sedlak, 2000). The order of capability
inhibiting the dissociation reactions of SDDC complexes are as follows:
SDDC has very high mobility in soil and is hardly adsorbed to
suspended solids or sediment (Abdollahi and Khaksar, 2014). More-
over, SDDC can decompose into toxic secondary products, including
tetramethylthiuram, dimethylamine and thiram, as well as the major
circulatory metabolites, carbon disulfide (CS2), which is a known an-
imal and human neurotoxicant (Abdollahi and Khaksar, 2014;
Matlock et al., 2002). In December 1999, there was a serious accident
associated with the leakage of SDDC in which N1.5 million gallons of
contaminated wastewater with SDDC were discharged into local
state waters, killing a total of 117 tons of fish over a 50 mile stretch
from Anderson to Indianapolis, Indiana, United States (Erven, 2001;
Matlock et al., 2002).
Considering the environmental risk of SDDC agent, some studies
have performed toxicological researches on SDDC for different organ-
isms, such as rabbits, zebrafish, mice and rats (Abdollahi and Khaksar,
2014; Mack et al., 2018; Tilton et al., 2006). Mack et al. (2018) investi-
gated the neurotoxicity of SDDC in mice and found that SDDC resulted
in motor deficits and a transient neurological deficit during the recovery
period after four consecutive days of administration (1 mg/nostril) as
well as caused an increase in reactive oxygen species (ROS) production
within olfactory bulb and striatum. Chou et al. (2008) found that SDDC
selectively damaged dopaminergic (DA) neurons in organisms, and
caused persistent motor deficits as well as mild injury to the
nigrostriatal pathway. Several studies evidenced that SDDC exposure
was related to an increased risk of Parkinson's disease (PD) (Chou
et al., 2008; Dennis and Valentine, 2015; Mack et al., 2018).
Fig. 1. (a) Schematic drawing of environmental risks of SDDC leakage in the process of S/S treatment of MSWI fly ash and polluted soil; (b) The chelating reaction equation of SDDC with
copper and lead.

Y. Wang et al. / Science of the Total Environment 720 (2020) 137666
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The complexes of SDDC and metal ions are suggested to be sta-
ble, however, the complexes of heavy metals have characteristics
of micro-sized polymer and heavy metals, which has the leakage
risk of stabilized heavy metal (Liu et al., 2019c). Chelation is not
equated with detoxification and may not affect bioavailability of
the metal (Harrington et al., 2012). A later study by Tilton et al.
(2006) found that copper(II) treated with SDDC alleviated the se-
vere effect of early growing development compared to copper(II)
and SDDC alone, but still induced the notochord distortions in the
zebrafish. Consequently, systematic toxicity evaluation of SDDC
and its metal chelates on other experimental organisms is
necessary.
Caenorhabditis elegans (C. elegans) is free-living nematodes which
feeds fungi and bacteria in soil. As a small model organism, C. elegans
can perform rapid evaluations of potential chemicals toxins due to
short life cycle, fast-speed reproduction, small size, transparent tis-
sues and extensive homology to mammals at genetic level (Hunt,
2017; Liu et al., 2019a; Weinhouse et al., 2018). C. elegans can alter
reproductive speed, life cycle, behavioral and other properties
when exposed to stresses (Wang and Wang, 2008). Due to the ad-
vantages of C. elegans in toxicological evaluations, we hypothesize
that the SDDC exposure to nematodes can also bring adverse effect
and its metal chelates may alleviate the severe effect of heavy
metal. In this study, the model organism nematode C. elegans was
used to assess the toxicities of SDDC and its metal chelates (Cu and
Pb) after subacute exposure by testing the endpoints of lethality,
growth, locomotion behaviors and ROS production. These results
were helpful to assess the ecotoxicity of SDDC and its metal chelates
in harmless treatment of heavy metals and provide a safety limit
value of SDDC in ecological environment.
with absence of food (E. coli OP50). All exposure experiments
were performed in 12-well sterile tissue culture plates at 20 °C.
2.3. Preparation of exposure solutions
The age synchronous nematodes were exposed to different SDDC
solutions prepared by K-medium (32 mM KCl, 51 mM NaCl): 0 (con-
trol), 0.01, 0.1, 1, 10 and 100 mg/L. The self-made chelates of SDDC
and M2+ metals (Cu, Pb), referred as SDDC-Cu, SDDC-Pb, respec-
tively, were prepared from the reaction of 100 mg/L M2+ metal solu-
tions (Cu, Pb) and 0.75 g/L SDDC for 30 min. During chelating
process, the reaction temperature was maintained at 25 °C with
mild stirring (600 rpm) using a magnetic stirring bar and pH of
7.0. The chelating equation of SDDC with Cu, Pb was illustrated in
Fig. 1 (b). These SDDC metal chelates were filtered and gathered
by the 0.45 μm filter membrane. Subsequently, the gathered metal
chelate samples were washed 3 times by deionized water and then
dried at 80 °C for 12 h. The SDDC-Cu and SDDC-Pb chelates were di-
luted respectively in K-medium to obtain three different concentra-
tions of 1, 10 and 100 mg/L homogeneous suspension solutions by
sonication for 30 min, respectively (Ding et al., 2018; Wu et al.,
2013). Meanwhile, M2+ metal solutions (Cu2+: 4.9 mg/L, Pb2+
:
6.7 mg/L) as heavy metal control groups were also diluted by K-
medium.
2.4. Characterization of SDDC and its metal chelates
In order to study on the physico-chemical characterization of
SDDC and its copper and lead chelates, several analysis methods
were performed. Transmission electron microscopy (TEM) (JEM-
1400, Japan) was used to determine the microstructure and parti-
cles size of SDDC-Cu and SDDC-Pb chelates. Fourier-transform in-
2. Materials and methods
frared spectroscopy (FT-IR) (Fisher Nicolet 6700, 4000–400 cm−1
)
2.1. Chemicals and reagents
was performed to determine the structural identification and func-
tional group of SDDC and its metal chelates. X-ray photoelectron
spectroscopic (XPS) (Thermo Fisher, ESCALAB 250Xi) was used to
determine the full element distribution of two chelates, as well as
Cu(2p) spectra of SDDC-Cu chelate and Pb (4f) spectra of SDDC-
Pb by using monochromatic Al Ka X-ray radiation method.
Sodium dimethyldithiocarbamate (SDDC) (C3H6NNaS2·2H2O) (CAS
No. 128-04-1, purity ≥ 98%) was purchased from Shanghai Yuanye Bio-
technology Co., Ltd. (Shanghai, China). Analytical purity grade lead chlo-
ride (PbCl2) and copper sulfate (CuSO4·5H2O) were purchased from
Shanghai Titan Technology Co., Ltd. (Shanghai, China). The rest of
chemicals and reagents used in this study were also analytical purity
grade, which were purchased from Shanghai Titan Technology Co.,
Ltd. (Shanghai, China) or Shanghai Ling Feng Chemical Reagent Co.,
Ltd. (Shanghai, China).
2.5. Evaluation of physiological indicators
2.5.1. Lethality assay
In this study, the lethal toxicity test was classified into two proto-
cols, the acute LC50 determination (young adult stage nematodes,
24-h) and subacute lethality test (L-1 larva stage nematodes, 72-h).
To determine the LC50 value of SDDC, 30 worms were exposed in
1000, 500, 100, 50, 10 and 1 mg/L SDDC solutions and in subacute le-
thality test, worms were exposed as described above. Both lethality
tests were evaluated by the percentage of survival worms during
the exposure stage (De Almeida Fagundez et al., 2015; Lenz et al.,
2017; Wu et al., 2013), and a tiny metal wire was used to touch inac-
tive nematodes to judge whether dead or not (Chen et al., 2018a). 30
worms were examined and each experiment was performed for at
least 3 times.
2.2. Nematode strains and exposure conditions
Nematode strain used was the wild-type Bristol N2, which was
attained from the Caenorhabditis Genetics Center (CGC). The nema-
todes were cultivated on the nematode growth medium [NGM:
3 g/L NaCl, 2.5 g/L peptone, 17 g/L agar, 25 mM potassium phos-
phate, 1 mM CaCl2, 1 mM MgSO4, and 1 mL cholesterol in ethanol
(5 mg/ml)] plates seeded with Escherichia coli OP50 at 20 °C as pre-
viously described (Brenner, 1974). The gravid nematodes were
washed by M9 Buffer (22.06 mM KH2PO4, 42.25 mM Na2HPO4,
85.56 mM NaCl and 1 mM MgSO4) and added with solution of alka-
line bleach (1.6 M NaOH + 3.3% NaClO) to attain nematode eggs
(Stiernagle, 2006). The synchronous cultures of C. elegans eggs
were washed for 3 times by M9 Buffer and then maintained in a
new NGM plate for 12 h to obtain L-1 larva stage nematodes and
for 3 days to obtain young adult stage nematodes as described pre-
viously (Lenz et al., 2017; Yang et al., 2015). The subacute exposure
condition was applied in this study (L-1 larva to adult day-1, 72 h)
with presence of food (E. coli OP50). The acute exposure condition
was applied in the acute lethality test (young adult stage, 24 h)
2.5.2. Growth assay
Growth was evaluated by body length, and the exposed nematodes
were killed by heat and observed by microscope (Nikon Eclipse 80i)
equipped with a graduated eyepiece as previously described (Wang
et al., 2018; Zhou et al., 2016). Subsequently, gathered data were ana-
lyzed by the ImageJ@ software. Triplicate independent experiments
were performed and 30 nematodes were examined in per exposure
experiments.

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Y. Wang et al. / Science of the Total Environment 720 (2020) 137666
2.5.3. Locomotion behavior assay
2.6. Reactive oxygen species (ROS) test
The expression of locomotion behaviors can represent the neurobe-
havioral deficit and reflection of the neurotoxicity condition. The head
thrashes and body bends indicators were acted as endpoints of locomo-
tion behaviors. The definition of head thrashes and body bends was de-
scribed by the previous studies (Wu et al., 2013; Zhao et al., 2020).
Before the test, the exposed nematodes were washed 3 times by M9
Buffer and transferred into a clean NGM plate without food. The head
thrashes were counted for 1 min and body bends were scored in an in-
terval of 20 s. Triplicate independent experiments were performed and
30 nematodes were examined in per exposure experiments.
The activated oxidative damage can be quantified by the ROS produc-
tion. The exposed nematodes after washed 3 times by M9 Buffer were
transferred to 100 μL M9 Buffer containing 1 μM CM-H2DCFDA and culti-
vated for 2 h at 20 °C as described (Cao et al., 2019; Wu et al., 2013). The
laser scanning confocal microscope (Nikon Eclipse 80i, PMT/A1R, Japan)
at 488 nm of excitation wavelength and 525 nm of emission filter was
used to measure the fluorescence intensities by semi-quantified measure-
ment. Triplicate independent experiments were performed and 30 nem-
atodes were examined in per exposure experiments.
Fig. 2. Physicochemical properties of two prepared SDDC-Cu and SDDC-Pb chelates. (a) TEM image of SDDC-Cu, (b) TEM image of SDDC-Pb, (c) wide XPS spectra of SDDC-Cu, (d) wide XPS
spectra of SDDC-Pb, (e) Cu(2p) XPS spectra of SDDC-Cu and (f) Pb(4f) XPS spectra of SDDC-Pb.

Y. Wang et al. / Science of the Total Environment 720 (2020) 137666
5
2.7. Statistical analyses
cuprous (Cu(I)) species, and the major lead chemical form in SDDC-Pb
chelate was Pb(II) specie, which are in good accordance with observa-
tion in other studies (Dobrowolski et al., 2019; Fairthorne et al., 1997).
In this study, the LC50 value of SDDC was calculated by using
OriginPro@ software. The one-way ANOVA followed by Dunnett's post
hoc tests was used to determine the significance of differences between
the groups. All of data were expressed as the mean standard error of
the mean (SEM) and p values of b0.05 and b0.01 were considered statis-
tically significant.
3.2. Effects of SDDC and its copper and lead chelates on lethality
The dose–response curve analysis of acute lethal toxicity of SDDC in-
dicated that the acute-24 h LC50 for SDDC was 139.39 mg/L (95% Cl:
111.03, 174.75 mg/L) in C. elegans nematodes (Fig. 3 (a)). Fig. 3
(b) showed that concentrations of SDDC b 1 mg/L did not obviously af-
fect the survival of C. elegans. In contrast, high concentrations of SDDC
(10 and 100 mg/L) significantly (p b .01) reduced the survival of
C. elegans compared to the control group. Furthermore, in Fig. 3 (c),
the result shows that SDDC-Cu chelate at concentration of 100 mg/L
had significant (p b .05) effect on the survival of nematodes and all
tested SDDC-Pb had no significant effect (p N .05), as well as both che-
lates had lower lethal effect than SDDC alone at the same concentration
of 100 mg/L.
3. Results
3.1. Characterization of SDDC and its copper and lead chelates
The results of TEM show that the SDDC-Cu and SDDC-Pb chelates
were micro-sized particles and microstructure of these chelates were
totally different. The polygonal (mainly hexagonal) structure was ob-
served in SDDC-Cu and stick-like structure was observed in SDDC-Pb
(Fig. 2 (a)–(b)). In addition, the particle size of SDDC-Cu and SDDC-Pb
were in the range of 389 95 nm and 637 204 nm after sonication,
respectively, and aggregate size of SDDC-Cu and SDDC-Pb were 1.4
3.3. Effects of SDDC and its copper and lead chelates on growth
0.1 μm and 1.6
0.3 μm in K-medium, respectively. The FT-IR spec-
trums of SDDC and its Cu, Pb metal chelates were shown in Fig. S1 and
exhibited that these two metal chelates had the same characteristic
bands (e.g. C\\N, C_S). Fig. 2 (c) and (d) shows the elemental compo-
sition of SDDC-Cu and SDDC-Pb, and we can find these two chelates had
the same inorganic elements (C1s, S2p and N1s). Moreover, The results
of Cu(2p) and Pb(4f) XPS spectrum show that the major copper chem-
ical form in SDDC-Cu chelate was the mixture of cupric (Cu(II)) and
Fig. 4 (a) and (b) shows the effects of SDDC and its Cu, Pb metal che-
lates on body length of C. elegans. Results show that, in Fig. 4 (a), high
concentrations of SDDC (1, 10 and 100 mg/L) caused more severe
body length defects and concentrations b 1 mg/L had no effect on the
growth of C. elegans. On the other hand, Fig. 4 (a) and Table S1 showed
that body length of 0.1 mg/L SDDC was slightly higher than the control
group (slightly longer 25 μm). Furthermore, in Fig. 4(b) and Table S2, it
Fig. 3. Acute and subacute lethal toxicity of SDDC and its copper and lead chelates to C. elegans nematodes: (a) SDDC acute 24-h LC50 = 139.39 mg/L (95% Cl: 111.03, 174.75 mg/L);
(b) Effects of SDDC on survival of nematodes (subacute 72-h, L-1 larva to adult day-1); (c) Effects of SDDC-Cu and SDDC-Pb on survival of nematodes (subacute 72-h, L-1 larva to
adult day-1; Cu2+: 4.9 mg/L, Pb2+: 6.7 mg/L). All data represent mean SEM. “*” (p b .05) and “**” (p b .01).

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Y. Wang et al. / Science of the Total Environment 720 (2020) 137666
Fig. 4. Effects of (a) different concentrations of SDDC and (b) SDDC-Cu, Pb metal chelates on growth of C. elegans after subacute exposure (72-h, L-1 larva to adult day-1; Cu2+: 4.9 mg/L, Pb2
+: 6.7 mg/L). All data represent mean SEM. “*” (p b .05) and “**” (p b .01).
was clearly shown that heavy metals (Cu, Pb) after chelating treatment
with SDDC had significantly (p b .01) lower effect on growth of C. elegans
than untreated heavy metals Cu, Pb (Cu: 4.9 mg/L, Pb: 6.7 mg/L), respec-
tively. Interestingly, the result of the specific growth parameters
(Table S2) showed that C. elegans exposed to different concentrations
(1–100 mg/L) of SDDC-Cu and SDDC-Pb still showed obviously de-
creased (10 mg/L was the most visible) in body length compared to
the control group. Additionally, SDDC-Pb had a lower effect than
SDDC-Cu at the same concentration.
3.4. Effects of SDDC and its copper and lead chelates on locomotion
behaviors
Fig. 5(a) shows that SDDC at concentration of 1–100 mg/L signifi-
cantly (p b .01) reduced both body bends and head thrashes of C. elegans
compared to the control group. Contrarily, concentrations b 1 mg/L had
no effect on the locomotion behaviors of nematodes. Furthermore, Fig. 5
(a) and Table S1 also showed that a concentration of 0.1 mg/L SDDC
showed a little good performance on head thrashes indicator than the
Fig. 5. Effects of (a) different concentrations of SDDC and (b) SDDC-Cu, Pb chelates on locomotion behaviors (head thrashes and body bends) of C. elegans after subacute exposure (72-h, L-
1 larva to adult day-1; Cu2+: 4.9 mg/L, Pb2+: 6.7 mg/L). All data represent mean SEM. “*” (p b .05) and “**” (p b .01).

Y. Wang et al. / Science of the Total Environment 720 (2020) 137666
7
Fig. 6. Toxicity evaluation of (a) different concentrations of SDDC and (b) SDDC-Cu, Pb metal chelates (100 mg/L) on ROS production of nematodes after subacute exposure (72-h, L-1 larva
to adult day-1; Cu2+: 4.9 mg/L, Pb2+: 6.7 mg/L). All data represent mean SEM. “*” (p b .05) and “**” (p b .01).
control group (without SDDC). Meanwhile, in Fig. 5(b) and Table S2, re-
sults show that heavy metals (Cu, Pb) after chelating treatment with
SDDC significantly (p b .01) increased both body bends and head
thrashes of nematodes compared to untreated heavy metals exposure.
Meanwhile, compared to the control group, SDDC-Cu at 100 mg/L still
significantly affected the locomotion behaviors of C. elegans. However,
all the tested SDDC-Pb concentrations did not obviously affect the loco-
motion behaviors, as well as both chelates had lower effect than the
SDDC (100 mg/L) alone.
SDDC was found that it has toxic effects at concentrations beyond a
safe limit value. Previous works have evidenced that SDDC can lead to
neurological and motion deficits in mice, and speculated that SDDC
has associated with an increased risk of PD (Chou et al., 2008; Mack
et al., 2018). The ecotoxicity effects of different concentrations of
SDDC on nematodes were assessed. Initially, the result of acute lethal
toxicity showed that the 24 h-LC50 value for SDDC was 139.39 mg/L
(95% Cl: 111.03, 174.75 mg/L), which is the first report of acute lethal
toxicity of SDDC chelating agent in this model organism as far as we
know. The LC50 is a reliable ecotoxicological endpoint in characterizing
the toxicity of chemicals (Chen et al., 2018a; Roh et al., 2007). Compared
with aquatic organisms, LC50 value for Oncorhynchus mykiss (rainbow
trout) is 32.2 mg/L for 24 h, and LC50 value for Daphnia magna is
1.5 mg/L for 48 h (Abdollahi and Khaksar, 2014), which was lower
than several times or even several orders of magnitude in nematodes.
This suggests that C. elegans may not be as sensitive to SDDC compared
to those aquatic species, which was consistent with recent studies for
C. elegans on other environmental toxicants (Lenz et al., 2017).
Subsequently, based on the acute LC50 value, several lower concen-
trations (0.01–100 mg/L) were selected for subacute exposure of
SDDC and found that SDDC at the concentration of 1 mg/L is a safe
limit value for C. elegans nematodes. The concentrations below 1 mg/L
of SDDC did not obviously affect the survival rate, body length, locomo-
tion behaviors and ROS production of nematodes, besides, concentra-
tion of 0.1 mg/L SDDC showed a better performance on body length
and movement than the control group. The possible reasons are that
the body length and movement indicators of 0.1 mg/L SDDC concentra-
tion are still within the normal growth indicators range of C. elegans.
Moreover, SDDC as a kind of organic salt containing inorganic elements
such as nitrogen and sulfur, when SDDC was in a very low concentration
range, the toxic effect of anionic group of SDDC could be negligible and
inorganic elements (e.g., nitrogen, sodium and sulfur) can be noticed as
trace supplements which are beneficial to C. elegans growth.
3.5. Effects of SDDC and its copper and lead chelates on ROS production
In Fig. 6(a), result shows that concentration b 1 mg/L of SDDC had no
effect on ROS production, but SDDC at concentrations of 10 mg/L and
100 mg/L significantly (p b .01) induced the ROS production in C. elegans
compared to control group. The quantified ROS production data
(Table S3) clearly show that the mean fluorescence intensities increased
with the increase of SDDC concentrations range from 0.1–100 mg/L. More-
over, in Fig. 6(b) and Table S4, it was clearly shown that all tested heavy
metals (Cu, Pb) displayed obviously increase of fluorescence intensities.
However, after SDDC chelating treatment, the significant (p b .01) reduc-
tion in the level of ROS production was observed in nematodes, compared
to untreated heavy metal Cu, Pb exposure. The results also showed that
SDDC-Cu, Pb chelates (100 mg/L) did not significantly (p N .05) induce
the ROS production compared to control group. However, in Table S4,
the mean fluorescence intensity of SDDC-Cu was larger than the control
group and SDDC-Pb showed no difference to the control group.
4. Discussion
In S/S treatment of MSWI fly ash and polluted soil, SDDC as a tradi-
tional chelating agent, had environmental risks of SDDC leakage to the
soil and groundwater of surrounding environments. In this study,

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Y. Wang et al. / Science of the Total Environment 720 (2020) 137666
In contrast, higher concentration of SDDC (10, 100 mg/L) caused sig-
(Tilton et al., 2006). The locomotion behaviors and ROS production ef-
fect of SDDC-Cu, Pb was lower than SDDC. The possible interpretation
of these results is that metals Cu and Pb can reduce the production of
toxicity secondary products of SDDC (e.g., carbon disulfide). The reac-
tion process between heavy metals (Cu and Pb) and SDDC was shown
in following Eqs. (3) and (4) (Weissmahr and Sedlak, 2000):
nificant defects on the survival rate, body length, locomotion behaviors
and ROS production of nematodes compared to the control group, and
SDDC at level of 1 mg/L also had adverse effect on the body length and lo-
comotion behaviors. These results suggest that nematodes exposed at
higher concentrations (≥1 mg/L) of SDDC cannot maintain the normal so-
matic functions, such as survival, growth level, basic locomotion ability
and oxidative stress, especially in locomotion ability, which are consistent
with the observation of SDDC toxicological performance in mice (Mack
et al., 2018). One possibility is that SDDC has a higher toxicity than others
DTCs and can easily release toxic secondary products. SDDC has a lower
molecular weight and none of benzene rings (Segovia et al., 2002) could
be decomposed in water under different pH conditions (Humeres et al.,
1998). Among these, the mechanism of decomposition under neutral
and acidic conditions can be described as Eq. (1), and under alkaline con-
dition can be described as Eq. (2) (Lei et al., 2015):
Â
Ã
Â
Ãþ
−
ðCH3Þ NCS2 þ M2þ→M ðCH3Þ NCS2
ð3Þ
ð4Þ
2
2
Â
Ã
Â
Ã
Â
Ã
M ðCH3Þ NCS2 þ þ ðCH3Þ NCS2 −→M ðCH3Þ NCS2
0
2
2
2
2
Due to the chelating reaction with metals, the metals can slow the
hydrolysis reactions of SDDC in water, which would reduce the produc-
tion of toxic organic hydrolysates (Weissmahr and Sedlak, 2000).
Namely, the chelate of SDDC with heavy metals has a more stable chem-
ical structure than SDDC and reduces the production of toxic secondary
products (e.g., carbon disulfide). Considering the difference in chemical
characteristic of central atom in SDDC-Cu and SDDC-Pb chelate, and the
result indicated that lead(II) showed a lower toxic effect than copper(II)
in units of mass/volume (mg/L), which was consistent with other study
(Anderson et al., 2001). Taken together, the chelate SDDC-Pb had lower
toxic effect than SDDC-Cu.
ð1Þ
5. Conclusions
The model organism C. elegans nematode was used in this study to
assess the ecotoxicity of SDDC and its metal chelates. Our data demon-
strate that the LC50 value of SDDC in nematodes was 139.39 mg/L (95%
Cl: 111.03, 174.75 mg/L), and concentration below 1 mg/L of SDDC did
not cause toxic effects to nematodes. However, SDDC concentration
above 1 mg/L caused obvious defect on the survival, growth, locomotion
behaviors and increasing ROS production of exposed nematodes. Be-
sides, the mean fluorescence intensities increased with increase of
SDDC concentrations range from 0.1–100 mg/L. Heavy metals Cu, Pb
after SDDC chelating treatment can greatly reduce the toxic effect of
heavy metals and these chelates still perturb early growth development
process in nematodes. On the other side, both chelates had a lower toxic
effect than SDDC because they have a more stable chemical structure
and production of toxic secondary pollutants is reduced, in addition
SDDC-Pb had lower toxic effect than SDDC-Cu.
ð2Þ
According to the above equations, the toxic products (e.g., CS2, (CH3)
2NH) of SDDC could be found after exposure to C. elegans, because the de-
composition of SDDC in water and biological metabolism can double im-
pact on the production of carbon disulfide (Abdollahi and Khaksar, 2014;
Humeres et al., 1998), resulting in neurobehavioral and nervous system
deficit in nematodes. Moreover, the results showed that higher concentra-
tion of SDDC (N1 mg/L) caused more toxic effect on movement than that
on growth, suggesting that the main toxic secondary product of SDDC
after nematodes exposure is CS2 because CS2, as a well-established
neurotoxicant, was found to be less toxic on organism growth develop-
ment (Tilton et al., 2006). PD involves the progressive loss of dopaminergic
(DA) neurons as a result of DA metabolism and the formation of ROS (Cao
et al., 2005; Mor et al., 2017). The conducted experiment proved that SDDC
can induce the movement disorders and increase of ROS resulted from ox-
idative damage, suggesting that DA metabolism altered may result in DA
neurodegeneration in nematodes after exposure of SDDC.
Due to wide existence of Cu(II) and Pb(II) in leaching solution of so-
lidified/stabilized fly ash (Du et al., 2018; Zacco et al., 2014; Zhang et al.,
2016), the toxicity of SDDC-Cu, SDDC-Pb chelates and heavy metals (Cu,
Pb) as control groups were also assessed. SDDC-Cu and SDDC-Pb che-
lates had the same organic functional groups and inorganic elements
except for the central atom (Cu, Pb). These chelates were all belonged
to the micro-sized particles after sonication treatment, which these
micro-sized particles can be easily ingested by nematodes and mainly
distributed in lumen and pharynx (Acosta et al., 2018). These metal che-
lates (1–100 mg/L), heavy metals (Cu, Pb) after SDDC chelating treat-
ment, had significantly lower effect on body length, locomotion
behaviors and ROS production in nematodes compared to the untreated
heavy metals (Cu, Pb) exposure. In addition, both chelates SDDC-Cu and
SDDC-Pb had lower effect on survival, locomotion behaviors and ROS
production of nematodes than SDDC alone at the same concentration
(100 mg/L), and SDDC-Cu had more noticeable effect on survival,
body length and locomotion behaviors of nematodes than SDDC-Pb at
the same concentration. Additionally, these two chelates at high con-
centrations (10, 100 mg/L) can perturb the growth development pro-
cess in nematodes, which was consistent with study on zebrafish
CRediT authorship contribution statement
Yitian Wang:Conceptualization, Methodology, Software, Investiga-
tion, Writing - original draft.Han Zhang:Validation, Formal analysis, Vi-
sualization, Writing - review & editing.Xiangyu Wu:Validation, Formal
analysis, Visualization.Cheng Xue:Resources, Writing - review &
editing, Data curation.Yang Hu:Resources, Writing - review & editing,
Data curation.Asim Khan:Writing - review & editing.Fuwen Liu:Re-
sources, Writing - review & editing.Lankun Cai:Supervision, Project ad-
ministration, Funding acquisition, Writing - review & editing.
Acknowledgements
This study was financially supported by the enterprise grant [B200-
81639] from Donghua Environmental Protection Technology Co., Ltd.
(Changzhou, China). In addition, the authors thank Research Center of
Analysis and Test of East China University of Science and Technology
for the help on the TEM, XPS and FT-IR characterization analysis.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.

Y. Wang et al. / Science of the Total Environment 720 (2020) 137666
9
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2020.137666.
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