Science of the Total Environment 794 (2021) 148629

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Science of the Total Environment

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

Heavy metals in soil from gangue stacking areas increases children health
risk and causes developmental neurotoxicity in zebrafish larvae
Fenglong Yang, Yang Yun, Guangke Li ⁎, Nan Sang
College of Environment and Resource, Research Center of Environment and Health, Shanxi University, Taiyuan, Shanxi 030006, PR 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

• Heavy metals vary according to the

stacking characteristics of coal gangue.
• Long-term stacking of gangue enhances
the non-carcinogenic risks to local chil-
dren.
• Village soil leachate alters the locomotor
behavior of the zebrafish larvae.
• Village soil leachate inhibits the expres-
sion of neurodevelopment-associated
genes

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

Article history: Coal is the main energy source in China, with 4.5 billion metric tons of coal gangue accumulating near the mining
Received 7 April 2021 areas in the process of coal mining. The objectives of the present study were to identify the health risks to children
Received in revised form 16 June 2021 from soil pollution caused by coal gangue accumulation and to clarify the possible developmental neurotoxicity
Accepted 19 June 2021
caused by this accumulation using zebrafish (Danio rerio) as a model. The results reveal that As and seven other
Available online 22 June 2021
heavy metals in soil samples from the gangue dumping area to the downstream villages exhibited distance-
Editor: Jay Gan dependent concentration variations and posed substantial potential non-carcinogenic risks to local children. Ad-
ditionally, soil leachate could affect the key processes of early neurodevelopment in zebrafish at critical windows,
mainly including the alterations of cytoskeleton regulation (α1-tubulin), axon growth (gap43), neuronal
Keywords: myelination (mbp) and synapse formation (sypa, sypb, and psd95), eventually leading to hypoactivity in the
Coal gangue stacking zebrafish larvae. These findings suggest the possible health risks of soil pollution in the coal gangue stacking
Heavy metal-containing soil areas to children, particularly affecting their early neurodevelopment.
Non-carcinogenic risk © 2021 Elsevier B.V. All rights reserved.
Zebrafish embryos
Developmental neurotoxicity

1. Introduction
The solid waste produced and discharged in the process of coal min-
ing and dressing constitutes 10–15% of raw coal production and is called
coal gangue (Zhou et al., 2014). Coal gangue is usually stacked near
⁎ Corresponding author at: College of Environment and Resource, Shanxi University,
Taiyuan, Shanxi 030006, PR China.
E-mail address: liguangke@sxu.edu.cn (G. Li).
mines in the form of cones and valley dumps adjacent to residential
areas. The Chinese government has already promulgated several poli-
cies and regulations regarding the management and utilization of coal
gangue, but the comprehensive level of utilization of coal gangue is
only 15% (Yun et al., 2017). A large amount of coal gangue has accumu-
lated on the earth's surface via air-soil exchange from contaminated air,
dry/wet deposition, and surface runoff (Bandowe et al., 2018; Jiang
et al., 2014), releasing several toxic and harmful substances (polycyclic
aromatic hydrocarbons, trace elements, heavy metals, and other organic
pollutants) into the surrounding environment (Fan et al., 2013).

https://doi.org/10.1016/j.scitotenv.2021.148629
0048-9697/© 2021 Elsevier B.V. All rights reserved.
F. Yang, Y. Yun, G. Li et al.
Average concentrations of Mn, Fe, Ti, As, Zn, Nb, Pb, Fe, Rb, Zr, and Sr in
the soils from mine drainage and the surrounding agricultural fields of the
Barapukuria Coal Basin, Bangladesh, have exceeded the world averages
(Bhuiyan et al., 2010). Moreover, Cd, Cu, Pb, Zn, Ni, Mn, Fe, and Cr have
been detected in groundwater samples from the Dingji Coal Mine in the
Huainan field in China (Zhang et al., 2016a). Owing to bio-
accumulation, the content of Pb (0.38 mg/kg) in rice in the vicinity of
coal gangue piles in the Guqiao Coal Mine, China, has exceeded the max-
imum permissible limit of 0.2 mg/kg (Wang et al., 2013). Another study
observed that the concentrations of Sr, Se, and Ni are elevated in female
southern toads collected from the coal combustion waste-contaminated
area and that high levels of Cu, Pb, Sr, and Se are transferred to their
eggs (Metts et al., 2013). These strong evidences demonstrate that resi-
dents living in regions with coal gangue contamination will have indirect
or direct exposure to environmental heavy metals in the mining area
through eating contaminated crops and drinking contaminated water
and through hand-to-mouth oral ingestion during outdoor recreational
activities (Gu and Gao, 2018). Recent studies have demonstrated that
heavy metal contaminants in soils derived from mining areas pose a
high non-carcinogenic risk to children living in the vicinity (Li et al.,
2014).
The nervous system is known to be very sensitive to many sub-
stances during the early stages of development, and heavy metals
have proven to be one of the key culprits (Rice and Barone Jr, 2000;
von Stackelberg et al., 2015). Previous epidemiological studies have
shown the relationship between heavy metals in coal mine environ-
ments and abnormal neural development (Huang et al., 2011). A previ-
ous study has demonstrated that excess concentration of Cd in the blood
(0.54 μg/L) of pregnant women living near a coal combustion factory
could lead to a high prevalence of neural tube defects (Jin et al., 2014).
A prospective cohort study of 455 Mexican children between 1 and
3 years of age has found that the interactions between Mn and Pb at
high blood levels increased neurodevelopmental deficiencies in early
childhood, particularly during critical windows of development (Claus
Henn et al., 2012). In view of the increasingly severe pollution caused
by coal gangue accumulation in China, a comprehensive assessment of
the potential health risks and neurodevelopmental toxicity in gangue
accumulation areas is urgently needed.
In this study, we determined the possible health risks of heavy
metals in soil samples from gangue stacking areas to children, and fur-
ther clarified the neurodevelopmental disruption of village soil samples
at the critical windows using a zebrafish (Danio rerio) model. The work
aimed to identify the status of soil ecological pollution and estimate the
possible health risks of the coal gangue stacking areas.
2. Materials and methods
2.1. Field sampling and sample processing
Three areas were selected for the present study: Qinyuan County
(QY, 36°34′12.24″N, 12°8′55.67″E), Gujiao City (GJ, 37°59′18.38″N,
112°4′37.80″E) and Yangquan City (YQ, 37°52′14.33″N, 113°27′47.90″E),
these are the key coal production bases in the Shanxi Province. We
collected samples from three coal gangue hills and from the representative
downstream soils in these three areas. These three coal gangue sampling
sites represented a long-abandoned, recently abandoned, and functioning
coal gangue hill, respectively. Details regarding sample collection are
provided in Text S1.
The sampling was initiated from the gangue hills adjacent to the vil-
lage, and the downstream soil samples were collected according to the di-
rection of flow of the local river, as shown in Fig. S1. We collected a total of
19 coal gangue samples or soil samples from the three study areas. At
each sampling site, the surface soil was removed and 0–20 cm of the top-
soil was collected using a small plastic shovel. Each soil sample was com-
posed of three subsamples, which were thoroughly mixed to obtain a
representative soil sample. After natural drying at room temperature,
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Science of the Total Environment 794 (2021) 148629
the gravel was removed from all the soil samples; the samples were
then passed through a 100-mesh sieve (<150 μm) (Gu and Gao, 2018).
Following pre-processing, all the samples were packed in polyethylene
bags and stored in a cool-dry place for subsequent analysis.
The village soil leachate composed of 100 mL deionized water and
100 g village soil sample (1 g/mL) was shaken at 145 rpm on a thermo-
static oscillator at 28 °C (3 d). The mixture was then centrifuged at
3000 rpm for 10 min after standing overnight. The supernatant was fil-
tered using a sterilized filter to avoid any contamination due to expo-
sure to microorganisms, and the filtrate was stored at 4 °C until
subsequent experiments.
2.2. Content analysis of the elements
First, 0.1 g of the sample was weighed into a pre-cleaned Teflon micro-
wave tube. The samples were then digested overnight with a typical con-
centrated acid mixture (HNO3-HCl-HF-HClO4) using a microwave
digestion instrument. After cooling, the digestion solution was diluted
with 1% HNO3 and filtered through a membrane filter (0.45 μm). Follow-
ing a modified procedure by (Hadzi et al., 2019), the filtrates were mon-
itored by inductively coupled plasma-mass spectrometry (NexION350X;
PerkinElmer, USA); the contents of eight elements (Cd, Cr, Cu, Ni, Pb, V,
As, and Zn) in the coal gangue samples and soil samples were determined.
2.3. Quality assurance and quality control
The China National Standard Reference Soil (GBW07401, GSS-1) was
used for quality assurance and quality control purposes, and the test re-
sults were consistent with the standard values. Reagent blanks were
used throughout the analysis and applied to correct the analytical results.
The accepted recoveries of the eight elements ranged from 89% to 109%.
The detection limits of Cd, Cr, Cu, Ni, Pb, V, As, and Zn were 0.0025,
0.0175, 0.005, 0.0175, 0.015, 0.0025, 0.0025, and 0.09 mg/kg, respectively.
2.4. Assessment of the non-carcinogenic risks
The heavy metals in soils pose serious health risks to humans pri-
marily via three routes of exposure, namely ingestion, inhalation, and
dermal exposure (Gu et al., 2016a). Based on an assessment method de-
veloped by the United States Environmental Protection Agency
(USEPA), we assessed the non-carcinogenic health risks to the local res-
idents in the coal gangue dumping districts after chemical exposure and
quantified the risk using the hazard quotient (HQ) (USEPA, 1989; Zhang
et al., 2016b). The total potential non-carcinogenic health risks of all
metals via the three routes are represented by the total exposure hazard
index (HIt). If HQ or HIt ≤ 1, the non-carcinogenic health risks can be ig-
nored; if HQ or HIt > 1, it indicates the presence of potential non-
carcinogenic risks (Cao et al., 2015; USEPA, 1989). The probability of
non-carcinogenic risks tends to increase as the HQ or HIt value increases
(Gu et al., 2016b). The formulae for HQ and HIt are presented in Text S3.
2.5. Zebrafish husbandry
Mature wild-type zebrafish (AB strain, 6 months) obtained from the
China Zebrafish Resource Center (CZRC) were kept in a 14/10 h light/
dark cycle in a continuous flow-through system at 28 ± 0.5 °C. Normally
developed embryos were immediately selected after fertilization (≤ 2 h
post fertilization (hpf)) and randomly divided into different treatment
groups. The E3 culture medium (containing 0.17 mM KCl, 5 mM NaCl,
0.33 mM MgSO4, and 0.33 mM CaCl2; pH = 7.4) was used for the dilu-
tion of the soil leachate and the control group.
2.6. Real-time quantitative reverse transcription-polymerase chain reaction
For gene transcription analysis, the zebrafish embryos were exposed
to the village soil leaching solutions at various concentrations (V leachate:
F. Yang, Y. Yun, G. Li et al.
V E3 culture medium = 1:27, 1:9, 1:3, or 1:1). The treatment and control
groups were collected at 24, 48, and 72 hpf, respectively. Each treatment
concentration contained six replicates, and each replicate had 30 nor-
mally developed embryos; 50% of the exposure solution was renewed
with fresh solution every day. Details regarding the extraction, purifica-
tion, and quantification of total RNA from the zebrafish embryos/larvae
and the first-strand cDNA synthesis are provided in Text S4. β-actin was
chosen as the internal reference gene in the present study (Tang et al.,
2007; Xu et al., 2019). The primer sequences for each selected gene are
provided in Table S3.
2.7. Locomotor activity assay
We quantified the locomotor activities of the zebrafish larvae at
120 hpf using a Video-Track system (Danio Vision; Noldus Information
Technology, Netherlands), as described previously (He et al., 2011). Be-
fore monitoring, the larvae were maintained in 96-well plates at 28 ±
0.5 °C for 10 min. The swimming behavior was monitored based on
the larval responses to dark-to-light transition stimulation (10 min
dark, 10 min light, 10 min dark). The data (distance moved, velocity,
and the cumulative duration of movement) were collected every 60 s.
Each treatment concentration was tested in six replicates (eight larvae
per replicate, 48 larvae per treatment concentration). The data were an-
alyzed using the custom Open Office. Org v.2.4 software.
2.8. Statistical analyses
All data were analyzed using SPSS v.23.0, and the results are pre-
sented as the mean ± standard error. One-way analysis of variance
(ANOVA), followed by Fisher's least significant difference (LSD) test
was used to evaluate the mean differences between the treatment
groups and the control group. Statistically significant differences were
established when p < 0.05.
3. Results
3.1. As and other heavy mentals from gangue stacking areas pose non-
carcinogenic risks to children
We first determined total contents of the eight elements in gangue
and soil samples around the coal gangue dumps with different accumu-
lation characteristics, and the contents are presented in Fig. S2. To con-
firm whether As and other seven heavy metals in the village soil
samples posed health risks to local children, we evaluated the non-
Fig. 1. Non-carcinogenic risk analysis for children residing in the coal gangue stacking areas (A) Qinyuan, (B) Gujiao and (C) Yangquan.
a Hazard quotient (HQ) represents the potential non-carcinogenic risk of a single element via the ingestion, inhalation, and dermal exposure pathways.
b Total exposure hazard index (HIt) represents the total potential non-carcinogenic effects of all the eight elements from the three exposure pathways.
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Science of the Total Environment 794 (2021) 148629
carcinogenic health risks by calculating the HQ. The HIt values in gangue
from QY, GJ, and YQ were 3.01, 4.07, and 5.83, respectively, and those in
village soils from QY, GJ, and YQ were 4.81, 5.95, and 5.50, respectively
(Fig. 1). The HIt values for children were approximately 3–6
(3.01–5.95) times higher than the maximum acceptable level (1.0), sug-
gesting that As and other heavy metals from village soils around gangue
stacking areas in QY, GJ, and YQ pose potential non-carcinogenic risks to
local children. The HIt values for adults in QY, GJ, and YQ were lower
than the maximum acceptable level (1.0), indicating that As and other
heavy metals in coal gangue samples and village soil samples had no
significant non-carcinogenic risk to adults. Among the eight elements,
the HQ value of Cd for children ranged between 1.45 and 2.91, which
exceeded the typical risk index of 1.0. The HIt values for the adults are
presented in Table S4.
3.2. Soil leachate affects locomotor activities of zebrafish larvae
Children's bodies are in a stage of dynamic development; they are
more vulnerable to harm from exposure to environmental factors than
adults. Previous studies have demonstrated associations between expo-
sure to environmental pollutants and several health outcomes in chil-
dren, including increased risk of cardiovascular and respiratory
diseases, skin diseases, allergies, and neurodevelopmental disorders
(Ait Bamai et al., 2020; Haryanto, 2020; Oliveira et al., 2019; Zeng
et al., 2020). The developing nervous system is extremely sensitive to
xenobiotics, and neurodevelopmental abnormalities are considered im-
portant indicators of health risk in children (Grandjean and Landrigan,
2006; Tsuji et al., 2015). Thus, we used zebrafish larvae as a model to de-
tect locomotor activities using a photoperiod stimulation test. Locomo-
tor traces and average swimming speed of the larvae are shown in Fig. 2.
The swimming activity of the larvae was much slower during the light
period than during the dark period (Fig. 2A-C). In addition, the soil
leachate from QY at a concentration of 1:27 resulted in the hyperactivity
of larvae whereas higher doses caused sedation. The swimming activity
of larvae exposed to high concentrations of soil leachate from QY, GJ,
and YQ increased much more slowly than that of larvae in the control
group during the transition from light to dark period.
The average swimming speed of larvae exposed to 1:1 soil leachate
from QY, GJ, and YQ was significantly slower than that of larvae in the
control group during the first and the second dark periods (Fig. 2D–F).
Specifically, the average swimming speed of the larvae exposed to 1:1
soil leachate from QY, GJ, and YQ significantly decreased to 0.63-,
0.45-, and 0.35-fold of that of the control larvae, respectively, during
the first dark period and significantly decreased to 0.59-, 0.53-, and
F. Yang, Y. Yun, G. Li et al.
Fig. 2. Locomotor behavior of the zebrafish larvae after exposure to the village soil leachate for 120 h. (A-C) Locomotor traces and (D\
represented as the mean ± standard error of the mean (SE) and expressed as the fold change relative to the control. *p < 0.05, **p < 0.01, and ***p < 0.001 versus the control group.
0.36-fold of that of the control larvae, respectively, during the second
dark period. During the 10 min light period, the average swimming
speed of the larvae exposed to 1:9, 1:3, and 1:1 soil leachate from QY
significantly decreased to 0.34-, 0.40-, and 0.24-fold of that of the con-
trol larvae, respectively, and that of the larvae exposed to soil leachate
from GJ significantly decreased to 0.43-, 0.30-, and 0.15-fold of that of
the control larvae, respectively. Additionally, during this period, the av-
erage swimming speed of the larvae exposed to 1:3 and 1:1 soil leachate
from YQ significantly decreased to 0.40- and 0.21-fold that of the control
larvae. The above results showed neurobehavioral alterations in
zebrafish larvae upon exposure to soil leachate. Hence, the heavy metals
in the soil from the coal gangue stacking areas may have a risk of devel-
opmental neurotoxicity.
3.3. Soil leachate affects the expression of neurodevelopment-related genes
To clarify the possibility of developmental neurotoxicity caused by
heavy metals in village soil samples, we detected the expression levels
of neurodevelopment-related genes in zebrafish embryos from 24 to
72 hpf. α1-tubulin, growth associated protein 43 (gap43), and myelin
basic protein (mbp) were selected to clarify the effect on the growth
of axons after village soil leachate exposure. Our results confirmed
that α1-tubulin levels decreased in a concentration-dependent and ex-
posure duration-dependent manner after all sample treatments
(Fig. 3A). The levels of α1-tubulin in 24 and 48 hpf larvae exposed to
1:1 concentration from QY significantly decreased to 0.75- and 0.66-
fold of those in the control larvae, respectively, and those in 24, 48,
and 72 hpf larvae exposed to 1:1 concentration from GJ significantly de-
creased to 0.63-, 0.76-, and 0.62-fold of those in the control larvae, re-
spectively. Following exposure to 1:3 and 1:1 soil leachate from YQ,
the expression level of α1-tubulin in 48 hpf larvae significantly de-
creased to 0.73- and 0.74-fold of that in the control larvae, respectively,
and its expression level in 72 hpf larvae significantly decreased to 0.70-
and 0.71-fold of that in the control larvae, respectively. After exposure
to soil leachates from QY, GJ, and YQ at 24 hpf, the mRNA level of
gap43 was slightly increased in a concentration-dependent manner
but not significantly (Fig. 3B). At 48 and 72 hpf, the levels of gap43 de-
creased in a concentration-dependent manner after all sample treat-
ments. Especially after exposure to soil leachate from GJ, the levels of
gap43 in 1:3 and 1:1 leachate samples significantly decreased to 0.57-
4
Science of the Total Environment 794 (2021) 148629
\F) average swimming speed of the larvae. Values are
and 0.64-fold of those in the control larvae at 48 hpf and those in the
1:1 group significantly decreased to 0.64-fold of those in the control lar-
vae at 72 hpf. The expression of mbp remained unchanged at the early
neurodevelopmental stages (24 and 48 hpf), but it significantly de-
creased at 72 hpf (Fig. 3C). At 1:9 and 1:1 concentrations, the expression
levels in the larvae exposed to QY soil leachate were reduced to 0.69-
and 0.63-fold of those in the control larvae, respectively, and the levels
in the larvae exposed to YQ soil leachate reduced to 0.75- and 0.65-fold
of those in the control larvae, respectively. At 1:1, the levels in the larvae
exposed to GJ soil leachate were significantly reduced to 0.55-fold of
those in the control larvae.
Next, we tested whether synaptogenesis-associated genes, including
synaptophysin a (sypa), synaptophysin b (sypb), and postsynaptic den-
sity protein 95 (psd95) were restricted (Fig. 4). At 24 hpf, the expression
level of sypa was slightly elevated upon exposure to the YQ sample, sig-
nificantly increased to 1.38-fold of control level at 1:1 by the QY sample,
and significantly decreased to 0.69-fold of control level at 1:3 by the GJ
sample. At 24 hpf, the expression level of sypb significantly attenuated
to 0.62- and 0.59-fold of control level at 1:3 and 1:1, respectively, by
the GJ sample. The levels of psd95 significantly increased to 1.34-fold
of control level at 1:1 by QY, 1.29-fold of control level at 1:3 by YQ,
and significantly decreased to 0.53-fold of control level at 1:1 by GJ. At
48 and 72 hpf, the expression levels of sypa, sypb, and psd95 decreased
following exposure to the QY, GJ, and YQ samples. Specifically, the levels
of psd95 in 48 hpf larvae significantly decreased to 0.58-, 0.59-, 0.68-and
0.65-fold, respectively, of the control level and those in 72 hpf larvae sig-
nificantly decreased to 0.60-, 0.56-, 0.57-, and 0.58-fold, respectively, of
the control level by the GJ sample.
3.4. Heavy metals in the soil leachate activate metallothioneins (MTs)
MTs are metal-binding proteins that are induced by heavy metal
toxicity, and alteration of MT levels could be a useful bioindicator for
heavy metal exposure in organisms (Kim and Kang, 2017). To confirm
the possible developmental toxicity of metals from soil, we exposed
zebrafish embryos to the soil leachates from different gangue stacking
areas and detected the mRNA expression of mt at early developmental
stages (24 hpf). The results showed that mt levels were elevated in a
concentration-dependent manner after all sample treatments. Specifi-
cally, the content markedly increased to 1.37- and 1.50-fold of the
F. Yang, Y. Yun, G. Li et al. Science of the Total Environment 794 (2021) 148629

Fig. 3. The expression levels of various axon growth-associated genes at different developmental windows in the zebrafish larvae. Values are represented as the mean ± SE and expressed
as the fold change relative to the control. *p < 0.05 and **p < 0.01 versus the control group.

control level at 1:1 concentration of the QY and YQ samples and in-
creased to 1.26-, 1.33-, and 1.44-fold of the control level at 1:9, 1:3,
and 1:1 concentrations of the GJ samples, respectively (Fig. 5). These
data suggest that heavy metals in the soil samples combined with MTs
and activated mt expression, ultimately causing neurodevelopmental
toxicity.
4. Discussion
Coal gangue has become a chief solid waste in China. Heavy metals
are the main hazardous components of coal gangue, which can be
discharged into the surrounding environment, causing ecological dam-
age and exhibiting potential health risks to the local residents (Fan et al.,

Fig. 4. The expression levels of various synaptogenesis-associated genes at different developmental windows in the zebrafish larvae. Values are represented as the mean ± SE and
expressed as the fold change relative to the control. *p < 0.05, **p < 0.01, and ***p < 0.001 versus the control group.

5
F. Yang, Y. Yun, G. Li et al.
Fig. 5. The expression levels of metallothionein (mt) in the zebrafish embryos after
exposure to the village soil leachate. Values are represented as the mean ± SE and
expressed as the fold change relative to the control. *p < 0.05 and **p < 0.01 versus the
control group.
2013). After years of weathering, the lattice structure of minerals (such
as the silicate minerals) in the coal gangue is destroyed, producing a
large amount of metal oxides that can easily release heavy metals
(Yang et al., 2018). In addition, metals in coal gangue are easily precip-
itated under weakly acidic conditions (pH = 5) (Hua et al., 2018).
Therefore, the distribution characteristics of heavy metals in down-
stream soils varied among the three study areas, which can be attrib-
uted to the different accumulation times and pH values of the gangue
piles (da Silva et al., 2018) (Fig. S3). Numerous epidemiological and ex-
perimental studies have indicated that, in typical rural communities of
coal mining areas, the level of heavy metal exposure is usually higher
in children than in adults owing to their higher outdoor activities,
body surface areas, respiratory rates, and gastrointestinal absorption
of toxic elements (Jiang et al., 2017; Li et al., 2014; Rout et al., 2013).
Our results confirm that both the coal gangue and the village soil from
the three gangue areas with different stacking characteristics have po-
tential non-carcinogenic risks to local children. Specifically, the highest
non-carcinogenic risk presented by the village soil sample from GJ was
attributed to the high content of heavy metals in the village soil. The rel-
atively high risk level of the YQ village soil sample might be attributed to
the high content of heavy metals in village soil owing to the short dis-
tance between the village and the gangue dump. In addition, as gangue
waste piles (in QY and GJ) have been used for a long time before being
abandoned, heavy metals have entered the downstream soil via
weathering and leaching for many years, and the non-carcinogenic
risks of village soil are higher than those of coal gangue. YQ represents
a functioning coal gangue hill where the stacking time is relatively
short, heavy metals have not penetrated the downstream soil, and the
non-carcinogenic risks of village soil are lower than those of coal
gangue.
Cd in the village soils from QY, GJ, and YQ pose a high non-
carcinogenic risk to children (2.18–2.91); this may be because accumu-
lation of Cd in the soil was the highest near the gangue hills. Similar to
our results, Tian et al. (Tian et al., 2012) indicated that emissions of Cd
in China were derived from coal burning and were highly concentrated
in provinces in the northern and eastern regions, such as Shanxi, Hebei,
and Shandong. Pan et al. (Pan et al., 2016) also reported that Cd accumu-
lation in soil samples collected from Xiangfen County, Shanxi, was at-
tributed to anthropogenic sources. This may be the main reason for
the excessive Cd content in the soil environments of the three study
areas in Shanxi Province.
Given that heavy metals in village soil samples around the coal
gangue stacking area have a high health risk for children, developmen-
tal neurotoxicity is considered to be an important indicator of health
risk in children (Tsuji et al., 2015). Therefore, we investigated the
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Science of the Total Environment 794 (2021) 148629
neurodevelopmental toxicity of the village soil samples. The locomotor
behavior of zebrafish is generally considered an important indicator of
neural development, which can indicate the toxic effects of environ-
mental pollutants on nerve development (Drapeau et al., 2002). At
120 hpf, zebrafish larvae are almost fully developed and can move freely
and respond to changes in the surrounding environment (Brustein et al.,
2003; Saint-Amant and Drapeau, 1998). In the current study, swimming
behavior was monitored as the larval response to dark-to-light transi-
tion stimulation periods. A notable decrease in swimming speed was
observed, which is indicative of the developmental neurotoxicity of
soil leachate from the coal gangue stacking area. Our results are consis-
tent with those of previous studies in which zebrafish larvae were ex-
posed to heavy metals and accompanied by neurobehavioral changes
(Chen et al., 2012; Zhu et al., 2016).
Previous studies have shown that alterations in locomotor behavior
can be the result of changes in gene expression in the central nervous
system (Irons et al., 2013). Following the above results, we used
zebrafish as the model and selected several neurodevelopment-
related genes to determine the potential neurodevelopmental toxicity
induced by heavy metals in soil samples surrounding the gangue stack-
ing areas. The gene α1-tubulin encodes an intermediate filament pro-
tein that forms the microtubule cytoskeleton during the development
or regeneration of dendrites and axons (Baas, 1997). α1-tubulin is exclu-
sively expressed in the nervous system of early developing zebrafish
larvae (Brosamle and Halpern, 2002). Soil leachate significantly down-
regulated the transcription of α1-tubulin, implicating interference with
cytoskeletal regulation and axonal growth and affecting brain structure
and function (Guo et al., 2018).
gap-43, a nerve tissue-specific glycoprotein, guides the growth of
axons for regeneration after damage (Ren et al., 2014). A previous
study showed that the expression of gap-43 is a good biomarker of neu-
ronal damage in the developing CNS in zebrafish (Shi et al., 2018). The
upregulation of gap43 expression after soil leachate exposure at
24 hpf might be an adaptive increase to maintain overall brain growth
and offset the direct effects of the toxicant. However, at later
neurodevelopmental stages of zebrafish embryos (48 and 72 hpf), the
downregulated gap43 level indicates that its function might disrupt
the process of neurite formation, regeneration, and plasticity in re-
sponse to the treatment.
The accumulation of mbp is an essential part of the myelination of
axons in the CNS of developing zebrafish (Brosamle and Halpern,
2002). In general, both synaptogenesis and myelination occur during
later stages of nervous system development (Fan et al., 2010). In our
study, mbp expression showed a concentration-dependent decrease in
later embryonic development, suggesting that exposure causes defects
in myelination, thereby destroying the physiological functions of
neurons.
During the process of neurotransmitter release and synaptogenesis,
PSD-95, SYPa, and SYPb are chosen as markers of synapse formation
(Sarnat and Born, 1999; Zheng et al., 2012). For QY and YQ samples,
the increased psd95 and syp expression during synaptic formation dur-
ing early neurodevelopmental stages in zebrafish (24 hpf) might be due
to the dramatic increase in presynaptic synaptic vesicles induced by vil-
lage soil leachate, which stimulates the presynaptic membranes, re-
leases a large number of excitatory glutamate transmitters, and
induces excitotoxicity. With prolonged exposure time, more toxic sub-
stances penetrated the chorion and significantly inhibited the gene ex-
pression of psd95 and syp, which might have an effect on
synaptogenesis, neurotransmitter release, and neuronal differentiation
(Sánchez-Martín et al., 2013; Shi et al., 2018; Yuki et al., 2014). More-
over, at 24 hpf following exposure to low doses of GJ soil leachate,
the expression levels of psd95 and syp were significantly reduced, veri-
fying that synapse formation was disrupted during the early
neurodevelopmental stages. These data indicate that the neurotoxicity
of GJ village soil leachate might be greater than that of the soil leachates
from QY and YQ. The thiol groups (-SH) of MT cysteine residues have a
F. Yang, Y. Yun, G. Li et al.
high affinity for bonding to particular heavy metals; MT can combine
with metals intracellularly, thereby reducing its toxicity and contribut-
ing to the maintenance of cellular metal ion homeostasis, detoxification
of heavy metals, and scavenging of free radicals (Amiard et al., 2006;
Juarez-Rebollar et al., 2017). To clarify the above neurodevelopmental
toxicity attributed to heavy metals from soil leachate, we detected the
activation of MTs. The results indicate that heavy metals could penetrate
the chorion in the early developmental stages of zebrafish, and mt che-
lated metal ions in eggs to decrease the acute effects of metal exposure
during this period.
Interestingly, we observed negative correlations between the heavy
metals and neurodevelopment-related genes and behavioral function of
zebrafish larvae, with the exception of Pb (Table S5). Among them, Cr,
Cu, As, and Zn exhibited a higher correlation, and showed major inhibi-
tory effects on the neurodevelopment. This observation further con-
firmed that the heavy metals found in the village soils in the vicinity of
the coal gangue stacking areas from QY, GJ, and YQ contributed to the de-
velopmental neurotoxicity in zebrafish larvae by interfering with critical
developmental processes during the brain growth spurt, such as cytoskel-
eton regulation, neuronal myelination and synapse formation, and which
finally led to the decreased locomotor activities of the zebrafish larvae.
5. Conclusions
This study revealed that As and seven other heavy metals exhibited
distance-dependent concentration variations in the soil samples from
the gangue stacking areas to the downstream villages and posed poten-
tial non-carcinogenic risks to the local children. Additionally, the soil
leachate interfered with the critical developmental processes related
to early neurodevelopment in zebrafish at the critical windows, such
as cytoskeleton regulation (α1-tubulin), axon growth (gap43), neuronal
myelination (mbp) and synapse formation (sypa, sypb, and psd95),
eventually leading to hypoactivity in the zebrafish larvae. These findings
indicate the potential health risks of soil pollution in the coal gangue
stacking areas, especially to the early neurodevelopment in children.
CRediT authorship contribution statement
G. L. conceived and designed the experiments. N. S. provided a lot of
work for the revision of the manuscript. Y. Y. collected the coal gangue
and soil samples. F. Y. drafted the manuscript and conducted the exper-
iments. All authors read and approved the final manuscript.
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.
Acknowledgements
This study was supported by the National Natural Science Founda-
tion of China (22076108), Shanxi Provincial Key Research and Develop-
ment Project (201903D321078), Research Project for Shanxi Young
Sanjin Scholarship of China.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2021.148629.
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