Quimiosfera 251 (2020) 126415

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Concentración de metales pesados, evaluación de riesgo ecológico potencial y actividad enzimática en

suelos afectados por un derrame de colas de plomo y zinc en Guangxi, China

Kehui Liu una , si , Chunming Li una , si , Sanqi Tang una , si , Guiduo Shang una , si , Fangming Yu una , C , * * ,
Yi Li una , C , ** **

una Laboratorio clave de ecología de especies raras y en peligro de extinción y protección del medio ambiente (Universidad Normal de Guangxi), Ministerio de Educación, China
si Facultad de Ciencias de la Vida, Universidad Normal de Guangxi, 541004, Guilin, China
C Colegio de Medio Ambiente y Recursos, Universidad Normal de Guangxi, 541004, Guilin, China

Destacar Gráficamente abstracto

El riesgo ambiental ecológico del suelo aumentó

aguas abajo. Pb, Zn y Cd fueron los contaminantes
dominantes.

Las actividades enzimáticas del suelo disminuyeron

aguas abajo, excepto la de la catalasa.

La actividad humana y el origen del suelo fueron las

principales fuentes de contaminación.

información del artículo resumen

Historia del articulo: En 1976, un accidente de colapso de una presa de relaves en el plomo Xingping mi La mina de zinc en la provincia de Guangxi, China, provocó el
Recibido el 11 de diciembre de 2019 Recibido en derrame de aguas residuales mineras y lodos en tierras agrícolas aguas abajo en Side Village. Las concentraciones de metales pesados, la
forma revisada 26 de febrero de 2020 Aceptado el
contaminación del suelo y la actividad de las enzimas del suelo se analizaron para comprender las características de contaminación de las tierras
3 de marzo de 2020 Disponible en línea el 6 de
agrícolas a lo largo de la corriente lateral por este accidente. Se seleccionaron el suelo de colas (TS), el suelo forestal natural sin contaminación por
marzo de 2020
derrames (NFS) y cuatro suelos agrícolas representativos. Los cuatro sitios de suelo agrícola ubicados en la entrada de la corriente al suelo agrícola
(EnS), la región aguas arriba (EE. UU.), La corriente media (MS) y a la salida de la corriente (ExS) de la aldea, respectivamente. Los resultados
Editor de manejo: X. Cao mostraron que los valores de pH del suelo y las concentraciones de metales pesados ​estaban en el orden de la cola del suelo TS> ExS> MS> US>
EnS> NFS La concentración de Pb fue la más alta entre los elementos analizados, seguida de Zn, Cd, Mn y Cu. Las concentraciones de Pb, Zn, Cd
Palabras claves: Mn y Cu 10530.41,
Metales pesados
Patrón de distribución de la contaminación Evaluación 1708.58, 8.32, 885.61 y 104.51 mg kg 1, respectivamente. Las evaluaciones de la contaminación del suelo por índice de contaminación único, índice de
de la contaminación Actividad enzimática del suelo
contaminación de síntesis y análisis de riesgo ecológico potencial individual / integral explicaron que todos los suelos alcanzaron el nivel altamente
contaminado y presentaron grados de riesgo ecológico extremadamente altos. Pb, Zn y Cd fueron los contaminantes dominantes. Las actividades
enzimáticas del suelo de invertasa, proteasa y ureasa

** Dirección de los autores correspondientes: No.15, Yucai Road, Facultad de Medio Ambiente y Recursos,
Universidad Normal de Guangxi, 541004, Guilin, China.
* ** * Autor correspondiente. Laboratorio clave de ecología de especies raras y en peligro de extinción y protección del
medio ambiente (Universidad Normal de Guangxi), Ministerio de Educación, China.

Correos electrónicos: fmyu1215@163.com (F. Yu), liyi412@mailbox.gxnu.edu.cn

(Y. Li).

https://doi.org/10.1016/j.chemosphere.2020.126415
0045-6535 / © © 2020 Publicado por Elsevier Ltd.
2 K. Liu et al. / Chemosphere 251 (2020) 126415
exhibió el patrón de distribución opuesto al de las concentraciones de metales pesados, mientras que los resultados inversamente se
observaron para la actividad de la catalasa.
1. Introducción
Como un problema ambiental global, la contaminación del suelo se está intensificando
continuamente. fi ed por fenómenos antropogénicos como la minería ( Li, 2006 ; Shi et al., 2019 ) Se
producen grandes cantidades de desechos mineros y luego se depositan en la superficie terrestre,
cubriendo vastas áreas y, en consecuencia, transformando tierras fértiles y cultivadas en tierras
baldías ( Li, 2006 ) Se debe prestar más atención a las grandes cantidades de aguas residuales
contaminadas de la mina, incluidas las aguas residuales de lavado de minas, fl otación de aguas
residuales y aguas residuales de fundición, debido a sus altas concentraciones de contaminantes y fl propiedades
piscina de colas y Side Village es de aproximadamente 6 km. Las elevaciones de la piscina de
de uidez ( Pavlowsky et al., 2017 ) Además de la descarga de aguas residuales que contienen
metales, la deposición atmosférica de partículas metalenriquecidas produce una contaminación
severa del suelo alrededor de las fábricas de fundición ( Li et al., 2015 ; Pavlowsky et al., 2017 ; Xiao et
al., 2019 ) De hecho, la deposición atmosférica y las aguas residuales de la mina son las dos rutas
principales a través de las cuales se contaminan los suelos alrededor de las minas ( Helali et al., 2016 ;
Pavlowsky et al., 2017 ; Shi et al., 2019 ) Sin embargo, los accidentes inesperados de contaminación
ambiental ( Grimalt et al., 1999 ; Lacal et al., 2003 ; Liu et al., 2005 ; Qu et al., 2008 ;
Martín Peinado et al., 2015 ) puede provocar problemas más graves que el riego de aguas residuales
que contienen metales, la aplicación de fertilizantes o la deposición atmosférica. Además, el
sedimento bajo un accidente puede no ser consistente con la regla de deposición anterior, es decir,
que las concentraciones de metal disminuyen con la distancia de la fuente de contaminación ( Li et
al., 2015 ; Pavlowsky et al., 2017 ; Ciarkowska, 2018 )
La contaminación ambiental resultante de las actividades mineras causa problemas ecológicos y
sociales. La creciente evidencia muestra que la contaminación por metales pesados ​en las zonas
afectadas por la minería ha causado daños a la salud de los residentes locales ( Roba et al., 2016 ; Nawab were to (1) quantify the concentrations of the heavy metals Pn, Zn, Mn, copper (Cu) and Cd in the
et al., 2016 ;
Wang et al., 2017 ; Liu et al., 2018 ; Nuapia et al., 2018 ) así como la salud del propio suelo ( Wahsha et
al., 2017 ; Wu et al., 2018 ) Por ejemplo, nuestro estudio anterior sobre suelo agrícola alrededor de
una mina de manganeso (Mn) en la provincia de Guangxi mostró que el suelo estaba severamente
contaminado por los elementos Mn, plomo (Pb), zinc (Zn) y cadmio (Cd) ( Liu et al., 2018 ) La
evaluación de la contaminación del suelo mostró además que todos los suelos estudiados se
clasificaron como muy contaminados, y los cultivos cultivados en esos suelos también estaban muy
contaminados, lo que representa un peligro ecológico potencial para los agricultores locales ( Liu et
al., 2018 )
Las enzimas del suelo son un grupo de enzimas que generalmente se encuentran en el suelo y
que continuamente juegan un papel importante en el mantenimiento de la ecología del suelo, las
propiedades físicas y químicas, la fertilidad y la salud ( Carreira et al., 2008 ; Zornoza et al., 2009 ) Son
críticos para los procesos bioquímicos que involucran el ciclo de nutrientes y la degradación de
contaminantes orgánicos y son sensibles a los cambios y tensiones ambientales ( Wahsha et al.,
2014 ) Por lo tanto, el monitoreo de la estabilidad funcional de la enzima del suelo proporciona una
idea de cómo los procesos microbianos resisten y se recuperan de la perturbación ( Xian et al., 2015 ; Hagmann
et al., 2015 ;
Wahsha et al., 2017 ; Duan et al., 2018 ) Antes de planificar un programador de remediación del suelo
costoso y lento, se deben realizar muestreos y análisis de laboratorio para controlar las
concentraciones de elementos tóxicos, las propiedades microbianas del suelo, como la actividad
enzimática del suelo, y su relación ( Xian et al., 2015 ; Khosravi et al., 2018 )
La provincia de Guangxi es una famosa provincia no ferrosa en el sur
© © 2020 Publicado por Elsevier Ltd.
China; y los inventarios de Pb y Zn son 175,29 y 676,75 diez mil toneladas, respectivamente ( Chen,
2000 ) La mina Xingping Pn-Zn se encuentra en el sur de la región de Guilin de la provincia de
Guangxi, classi fi ed como una de las minas Pn-Zn de tamaño mediano en la provincia de Guangxi.
Las actividades mineras comenzaron en la década de 1950 y se detuvieron en 2012. Las aguas
residuales de procesamiento de minerales fl baja a una piscina de relaves sin tratamiento. Las aguas
residuales almacenadas en la piscina de residuos presentan un riesgo de fl debido a la corriente de la
montaña y fi Finalmente llega a la corriente lateral. La corriente lateral es una pequeña corriente fl debido
a Side Village. Los residentes suelen llevar a cabo el riego con agua del arroyo. La distancia entre la
relaves y el pueblo son de aproximadamente 770 my 250 m, respectivamente. En 1976, un evento de
fuertes lluvias duró tres días, y las fuertes lluvias causaron el colapso de la presa de relaves. Las
aguas residuales residenciales retenidas por la presa se precipitaron a lo largo del valle y finalmente
llegaron a Side Stream. Las aguas residuales, que contienen
fi ne partículas de colas (incluyendo metales pesados), dispersas a lo largo del arroyo, causando que
una gran área de tierras agrícolas fl ood Side Village se encuentra dentro de la llanura Fenlin (bosque
pico), formada por depresiones kársticas; por lo tanto, la fl las aguas residuales permanecieron allí
durante mucho tiempo (aproximadamente tres días) antes de fl La carne retrocedió, lo que resultó en
una contaminación de la tierra agrícola a gran escala. Después del accidente, los residentes
adoptaron principalmente tecnología de torneado; es decir, enterraron el lodo profundo y excavaron
el suelo profundo para reemplazarlo en el subsuelo. Durante las últimas décadas, los residentes
continuaron cultivando en el suelo contaminado hasta que la conciencia sobre la seguridad ambiental
aumentó en los últimos años. Sin embargo, la cantidad de contaminación causada por el accidente
que ocurrió hace más de 40 años (especialmente en tierras agrícolas), los patrones de
contaminación a lo largo del arroyo fl ow, and soil microbial characteristics such as enzyme activity will
differ in different polluted soils and different horizons. Hence, the main purposes of the present study
polluted agricultural soils along the stream and in the tailing pool; (2) determine the main metal
pollution types; (3) assess the potential ecological risk of heavymetals; (4) evaluate soil enzyme
activity in different sampled polluted sites and soil horizons; (5) determine the relationships between
the soil pH, heavy metal concentrations and soil enzyme activity; and (6) analyse other possible
pollution sources for this kind of pollution in this area.
2. Materials and methods
2.1. Site description and soil sampling
The study area (N24 59 0 9 00- N25 0 0 21 00, E110 33 0 25 00-
E110 36 0 35 00) ( Fig. 1 ) lies in the south of the Guilin region of Guangxi Province, China, which is well
known for its spectacular karst landforms developed mainly in Devonian and Carboniferous limestone
( Deng et al., 1988 ). The area presents a sub-tropical monsoon climate with an annual average
temperature of 19.9 C, average rainfall of 1636 mm and average humidity of 78% ( Liu et al., 2018 ).
The geomorphology consists of hilly terrain. The distance between the tailing pool and the entrance of
the stream in the Side agricultural area is approximately 6 km. The difference between the
 K. Liu et al. / Chemosphere 251 (2020) 126415 3

Fig. 1. Sampling site location and elevation altitude.

elevations of the tailing pool and the Side agricultural area is approximately 500 m. Four agricultural soil sites were chosen along the fl ow direction of the Side Stream, which were all fl ooded
by the tailing dam collapse accident. The fi rst site was located at the stream entrance in the
Soil samples were collected onMay 19th, 2018. Six sampled sites were selected in this study. agricultural lands (EnS); the second lay upstreamof the agricultural
One site was located at tailing soil (TS).
4 K. Liu et al. / Chemosphere 251 (2020) 126415

lands (US); the third was located in the middle stream in the agricultural lands (MS); and the
fourthwas located at the exit of the stream from Side Village (ExS). The stream fl ows out of the village
at this location and crosses a souterrain. A natural forest soil (NFS) site that was not subjected to
tailing wastewater fl ooding was also selected as the control site, which was located approximately 3
km from the tailing pool, and the difference in elevations between the tailing pool and NFS was
approximately 230 m. The details of the sampled sites are shown in Table 1 and Fig. 1 . The soil
samples were collected based on the S-curve sampling method, plum blossom point method or
random sampling method according to the conditions at each site. A total of 54 points were selected;
at each point, soil was collected at depths of 0 e 5 cm and 5 e 20 cm, and a total of 108 soil samples
were obtained. Each soil sample was obtained by pooling 9 sub-samples, and this mixture was then
divided into three parallel samples. All soil samples were sealed in clean polyethylene plastic bags
and brought to the laboratory for measurements.
distilledwater were added. After shaking, the fl asks were incubated at 37 C for 24 h. Finally, 1 ml of
the supernatant was withdrawn, and the quantity of reducing sugars was estimated via the
colorimetric 3,5-dinitrosalicylic acid method at 508 nm. Enzyme activity was expressed as the weight
in mg of glucose released per gram of soil per 24 h of incubation.
Protease activity was measured via the colorimetric ninhydrin method. A 5 g sample of sieved
air-dried soil was placed in a 50 ml conical fl ask, and 1 ml of toluene and 20 ml of 1% casein were
added. After shaking, the fl asks were incubated at 37 C for 24 h. At the end of incubation, 2 ml of 0.1
M 1/2H 2 SO 4 and 12 ml of 20% sodium sulfate were added for protein deposition. After certi fi cation at
6000 r min 1, 2 ml of the supernatant was transferred to a 50 ml conical fl ask, and 1 ml ninhydrin and a
few millilitres of distilled water (to wash the inner wall of the fl ask) were then added. The extraction
mixture was incubated in boiling water for 10 min, then cooled to room temperature, diluted to 50 ml
and measured with a spectrophotometer at 500 nm. Protease activity was expressed as the weight in
mg of amine N produced by the decomposition of casein per gram of soil per 24 h of incubation.

2.2. Sample analysis

To estimate urease activity, 5 g of sieved air-dried soil was placed in a 50 ml conical fl ask, and
1.0 ml of toluene was added, followed by incubation for 15 min. Then, 10 ml (w/v) of urea solution and
2.2.1. Soil pH and heavy metal concentration
20 ml of 0.1 M citrate buffer (pH 6.7) were also added, followed by thorough mixing. The fl ask was
After screening out large gravels, weeds, plant roots and various other materials, the soil
stoppered and incubated at 37 C for 24 h. A blank was maintained by adding 10 ml of distilled water
samples were air dried and sieved through a 2 mm sieve. The soil pH (sieved through a 2 mm sieve)
to each sample instead of the urea solution. After incubation, the suspension was diluted to 50 ml with
was measured by glass electrode method using a pH metre (Leici pHS-
distilled water, mixed thoroughly and immediately fi ltered through a

25, China) at a soil:water ratio of 1:2.5 after shaking for 0.5 h and equilibration for 0.5 h. After sieving
with a 0.15 mm sieve, the airdried soil was used to analyse the total concentration of metals.
Approximately 0.25 g of air-dried soil was weighed and placed in a cleaned 50 ml digestion tube, and
0.45 m m sieve. Three millilitres of the fi ltrate was used to estimate ammonia via the colorimetric
then 8 ml of a digestant mixture (HCl:HNO 3, v:v ¼ 1:1) was added for overnight soaking. Digestion
indophenol blue method at 578 nm. The amount of ammonia N that formed was calculated using a
with the mixture was performed for 8 e 10 h at temperatures of 140 e 160 C depending on the reaction
standard curve prepared with an ammonia solution. Soil urease activity was expressed as the weight
process. When a white precipitate was generated at the bottom of the tube, the digestion
in g of ammonia per g of soil per 24 h.
procedurewas stopped, and the digested liquid was cooled to room temperature. The residuum was
dissolved in 1% HNO 3, transferred to a volumetric fl ask, diluted to 50 ml and mixed. The
concentrations of Mn, Pb, Zn, Cu and Cd were measured using an atomic absorption spectrometer
To estimate invertase activity, 1 g of soil was placed in a 50 ml Erlenmeyer fl ask, and 5 ml of
(PinAAcle 900H, PerkinElmer, USA). The dried and sieved plant materials (0.25 g) were digested with
1/15 M (v/v) phosphate buffer (pH
5ml of HNO 3 and 1 ml of HClO 4 for 4 h. The detailed digestion and measurement procedures were
5.5), 5 drops of toluene, 15 ml of 8% sucrose solution and 3 ml of distilledwater were added. After
similar to those for the soil. All data were corrected relative to blank sample values from mixed
shaking, the fl asks were incubated at 37 C for 24 h. Finally, 1 ml of the supernatant was withdrawn,
digestants prepared in the same way ( Liu et al., 2018 ; Yu et al., 2019 ).
and the quantity of reducing sugars was estimated via the colorimetric 3,5-dinitrosalicylic acid method
at 508 nm. Enzyme activity was expressed as the weight in mg of glucose released per gram of soil
per 24 h of incubation.

Catalase activity in the soils was determined using the KMnO 4

titrimetric method modi fi ed according to Guo et al. (2018) . Five grams of soil was placed in a 100 ml
conical fl ask, and 40 ml of distilled water and 5 ml of 0.3% H 2 O 2 solution were added. The mixturewas
2.2.2. Soil enzyme measurement
shaken thoroughly for 20min. Then, 5ml of 3MH 2 SO 4
After sieving with a 2 mm sieve, the air-dried soil was stored at 4 C until soil enzyme activity was
measured. Soil enzymes were measured according to the methods of Sivakumar et al. (2012) . To
was added, and the mixture was fi ltered. Finally, 25 ml of fi ltrate was titratedwith 0.05MKMnO 4. Enzyme
estimate invertase activity, 1 g of soil was placed in a 50 ml Erlenmeyer fl ask, and 5 ml of 1/15 M (v/v)
activity was calculated by the volume of KMnO 4 consumed.
phosphate buffer (pH

5.5), 5 drops of toluene, 15 ml of an 8% sucrose solution and 3 ml of

Table 1
Geographical coordinates, elevations and soil characteristics of each site.

Soil Sampling site description Geographical coordinates Elevations Distance from the tailing pool

NFS natural forest soil without tailing wastewater fl ooding N24 59 0 19 00 , E110 35 0 16 00 540 m 3 km
EnS the entrance of the stream to the agricultural area N24 58 0 38 00 , E110 33 0 37 00 250 m 6 km
US the upstream of the agricultural area N24 58 0 41 00 , E110 33 0 32 00 248 m 6.5 km
MS the middle-stream of the agricultural area N24 58 0 56 00 , E110 33 0 28 00 245 m 7 km
ExS the exits of stream from the Side Village N24 59 0 9 00 , E110 33 0 25 00 240 m 8 km
TS tailing pool N25 0 0 21 00 , E110 36 0 35 00 770 m

The distance was calculated along the stream path.

 K. Liu et al. / Chemosphere 251 (2020) 126415 5

2.3. Soil pollution assessment 3. Results

2.3.1. Assessment of the degree of heavy metal contamination 3.1. Soil pH and heavy metal concentration
The single contamination index ( P i) ( Li et al., 2007 ; Lei et al., 2015 ) and the Nemerow multi-factor
index ( P com) ( Ogunkunle and Fatoba, 2013 ) are usually used to assess the contamination levels of soil The soil pH values and heavymetal concentrations were highest in TS, followed by ExS, MS, US
or food. P i and P com were calculated according to Eqs. EnS and NFS ( Table 2 ). The average soil pH value in the 0 e 5 cm horizonwas 6.36 (ranging from 4.36
to
(1) and (2 ), respectively. 7.31), which was higher than that in the 5 e 20 cm soil horizon of
6.16 (ranging from 5.31 to 6.78). The concentration of Pb ranked highest among these analysed
P i ¼ C i= S i (1) elements, with an average of
10530.41 mg kg 1, which were approximately 540.02 times their background values. The corresponding
P i: single contamination index; C i: measured concentration of examined metal i in the soil; and S i: background
values for Zn, Cd, Mn and Cu were 1708.58, 8.32, 885.61 and 104.51 mg kg 1, which were
concentration of metal i. The evaluation results are divided into 5 grades: P i approximately 22.60, 31.16, 5.03 and 3.76 times their background values, respectively. All of
0.7, thesemetal concentrations were over the background values for soil in Guangxi Province (China ’ s
safe; 0.7< P i 1.0, warning; 1< P i 2, light pollution; 2< P i 3, Environmental Protection Agency, 1996; Environmental Research Institution of Guangxi, 1992) and
moderate pollution; and P i > 3, heavy pollution. the national standards for soil quality (Grade 2, GB15618-1995, MEP, 1995). In general, the heavy

. metal concentrations in the 0 e 5 cmhorizonwere higher than those in the 5 e 20 cm horizon, with a few

P com ¼ h P 2 þ P 2 2 i 1=2 (2) exceptions. Bivariate correlation analysis showed that there were signi fi cant positive correlations
max
between soil pH and Pb ( P < 0.01), Zn ( P < 0.01), Cd ( P < 0.01), and Mn ( P < 0.05) and between these
metals ( Table S1 ).
P com: composite contamination index; P: average value of the single-factor index; and P max: maximum
value of the single-factor index. The evaluation results are divided into 5 grades: P com 0.7, safe; 0.7< P com
1.0, warning; 1< P com 2, light pollution; 2< P com 3, moderate pollution; and P com> 3, heavy pollution.

3.2. Soil pollution assessment

2.3.2. Soil potential ecological risk assessment

Soil potential ecological risk assessments or ecological risk indices serve as direct indicators of
soil safety. In this paper, the monomial potential ecological risk factor ( E i) and potential ecological risk
index ( RI) are used. RI, proposed by Hakanson (1980) , integrates the concentrations of heavy metals
with respect to their ecological effect, environmental effect and toxicity and has been used to assess
heavymetal pollution and ecological hazards in soils. The detailed calculation processes are based on
Eqs. (1), (3) and (4)
( Hakanson, 1980 ; Rehman et al., 2018 ; Nuapia et al., 2018 ).
Ei¼ T Pi
3.2.1. Assessment of the degree of heavy metal contamination
The results of the evaluation of P i for each single heavy metal (Mn, Cd, Cu, Pb and Zn) and the
multi-factor index ( P com) of each soil and horizon based on the background values for soil in Guangxi
Province (China ’ s Environmental Protection Agency, 1996; Environmental Research Institution of
Guangxi, 1992) are listed in
Table 3 . The P i values of the sampled sites exhibited the same change trend as the soil pH and heavy
metal concentrations. Regarding the speci fi c elements, the values of P Pb were highest at the sampled
sites, followed by the P i values for Cd z Zn >Mn z Cu. The P i values for Pb, Zn and Cd were all greater
than 3, which is the standard for the heavy pollution level. The P com values were all far greater than 3,
indicating a high degree of contamination of these soils.
(3)

RI ¼ X E i (4)

E i: monomial potential ecological risk factor; the evaluation results are divided into 5 grades: Er < 40,
low potential ecological risk; 40 Er < 80, moderate potential ecological risk; 80 Er < 160, considerable
potential ecological risk; 160 Er < 320, high potential ecological risk; and Er 320, very high potential
ecological risk. T i:
metal toxicity factor of metal i; the standardized values for Cd, Pb, Cu, Zn, Mn are 30, 5, 5, 1, and 1
mg kg 1, respectively. P i: single contamination index. RI: potential ecological risk index; the evaluation
results are divided into 5 grades: RI < 150, low ecological risk; 150 RI < 300, moderate ecological risk;
300 RI < 600, considerable ecological risk; 600 RI < 1200, very high ecological risk; and RI
3.2.2. Soil potential ecological risk assessment
The E r values of Zn, Mn and Cu were all lower than 40 except for TS, indicating that the potential
ecological risks of these elements were negligible, while the values for Pb and Cd were much higher
than 320 except for that of NFS in 5 e 20 cm horizon ( Table 4 ), indicating high ecological risk of these
two elements in these soils. With exception of NSF, all the RI values of the sampled soils were
beyond 1200, indicating high potential ecological risk in these soils. There was a signi fi cant ( P < 0.01)
positive relationship between the heavy metal concentrations and P com, especially for the element Pb,
for which the value of r was as high as 1.000 ( Table S1 ). Similar relationships were found between
heavy metal concentrations and

1200, extremely high ecological risk.

2.4. Data and statistical analysis
Data were processed with Microsoft Excel 2003 and SPSS 13.0 software. Figures were drawn
with Origin pro 9.1 (32 Bit). All the values were expressed as themean ± standard deviation (SD) of
the three replicates. Data were statistically analysed using one-way ANOVA with P < 0.05 considered
signi fi cant according to Duncan ’ s test. Pearson correlation analyses were also used to analyse the
relationship between the two parameters.
RI, especially for Pb, where the value of r was 0.941 ( Table S1 ). These results indicate that Pb plays
a decisive role in determining the degree of contamination and potential ecological risk of the
agricultural soils in Side Village. Regarding the two examined soil horizons, the patterns of Pi, Pcom,
Er and RI were consistent with those of the heavy metal concentrations.
3.3. Soil enzyme activity
The invertase, protease, urease and catalase activities of the six studied sites are shown in Fig.
2 . Soil enzyme activity varied with
6 K. Liu et al. / Chemosphere 251 (2020) 126415

Table 2
Soil pH and heavy metal concentrations (mg kg 1) in the tailing pool and agricultural soils in Side Village of Guangxi Province, China (n ¼ 36).

Soil depth Soil pH Pb Zn Cd Mn Cu

0 e 5 cm NFS 4.63 ± 0.02d 5279.90 ± 300.66e 855.86 ± 90.92d 4.59 ± 0.27c 251.81 ± 28.90c 66.36 ± 3.15cd
EnS 5.19 ± 0.07c 4017.59 ± 801.72f 878.38 ± 152.55d 4.01 ± 0.68c 109.38 ± 15.95c 51.21 ± 9.47d
US 6.86 ± 0.43b 6961.49 ± 334.76d 1188.21 ± 60.31cd 4.61 ± 1.64c 156.36 ± 90.87c 70.34 ± 2.61cd
MS 7.05 ± 0.03a 11438.23 ± 418.97c 1246.57 ± 64.60c 5.87 ± 0.90bc 112.72 ± 9.83c 101.95 ± 5.58b
ExS 7.06 ± 0.11a 17892.56 ± 279.41b 2627.69 ± 49.65b 7.27 ± 0.11b 625.26 ± 190.03b 270.96 ± 24.44a
TS 7.31 ± 0.02a 30264.24 ± 474.3a 3708.5 ± 394.03a 25.46 ± 1.96a 3597.99 ± 207.98a 80.00 ± 9.62c
5 e 20 cm NFS 5.31 ± 0.02d 989.83 ± 29.2e 146.58 ± 12.59d 1.65 ± 0.45c 682.2 ± 234.45b 54.97 ± 0.60d
EnS 5.62 ± 0.06cd 4708.35 ± 496.58d 1076.6 ± 85.60c 4.48 ± 1.72bc 99.67 ± 7.03c 57.24 ± 4.77d
US 6.41 ± 0.75 ab 5624.08 ± 910.33d 1231.94 ± 36.18c 4.79 ± 1.64bc 192.31 ± 16.32c 72.28 ± 10.69c
MS 6.81 ± 0.04a 11407.72 ± 450.97c 1241.46 ± 207.94c 5.80 ± 0.50bc 129.37 ± 40.02c 101.12 ± 3.63b
ExS 5.95 ± 0.03bc 24780.55 ± 1271.01b 2604.76 ± 124.67b 7.73 ± 0.38b 524.3 ± 114.79b 234.21 ± 9.18a
TS 6.78 ± 0.20a 30023.35 ± 164.45a 3696.32 ± 169.66a 23.57 ± 5.76a 3713.86 ± 155.01a 93.51 ± 5.12b
Background value of 5.3* 19.5** 75.6* 0.267* 176** 27.8*
soil in Guangxi
Province*
GB15618-1995 (MEP, < 6.5 250 200 0.3 e 50
1995)

(1) "*": data cited fromChina ’ s Environmental Protection Agency (1990); "**": data cited from the Environmental Research Institution of Guangxi (1992). (2) NFS, EnS, US, MS, ExS and TS represent the soil samples from natural forest soil without
tailing wastewater fl ooding (CK), the entrance of the stream to the agricultural area, upstream of the agricultural area, the middle-stream in the agricultural area, the exit of the stream from Side Village and the tailing pool, respectively. (3) Values
were means ± S.D. (n ¼ 3), one-way ANOVA (Analysis of Variance) was performed for each parameter, and different lowercase letters within the same column in the same horizon indicated a signi fi cant difference at P < 0.05 by Duncan ’ s
multi-range test.

Table 3
Assessment of the concentrations of heavy metals in the tailing pool and agricultural soils in Side Village of Guangxi Province, China (n ¼ 36).

Soil depth Soil Single contamination index ( Pi) Nemerow multi-factor index ( P com)

Pb Zn Cd Mn Cu

0 e 5 cm NFS 270.76 H 11.32 H 17.19 H 1.43 L 2.39 M 196.20 H

EnS 206.03 H 11.62 H 15.02 H 0.62 C 1.84 L 149.43 H
US 357.00 H 15.72 H 17.27 H 0.89 C 2.53 M 258.49 H
MS 586.58 H 16.49 H 21.99 H 0.64 C 3.67 H 424.21 H
ExS 1552.01 H 34.76 H 27.23 H 3.55 H 9.75 H 1121.31 H
TS 917.57 H 49.05 H 95.36 H 20.44 H 2.88 M 666.72 H
5 e 20 cm NFS 50.76 H 1.94 L 6.18 H 3.88 H 1.98 L 37.04 H
EnS 241.45 H 14.24 H 16.78 H 0.57 C 2.06 M 175.11 H
US 288.41 H 16.30 H 17.94 H 1.09 L 2.60 M 209.10 H
MS 585.01 H 16.42 H 21.72 H 0.74 C 3.64 H 423.08 H
ExS 153.86 H 34.45 H 28.95 H 2.98 M 8.42 H 113.50 H
TS 1270.80 H 48.89 H 88.28 H 21.10 H 3.36 H 921.14 H

(1) P i: single contamination index, the evaluation results are divided into 5 grades: P i 0.7, safe; 0.7< P i 1.0, warning; 1< P i 2, light pollution; 2< P i 3, moderate pollution;
P i > 3, heavy pollution. (2) P com: Nemerow multi-factor index, the evaluation results are divided into 5 grades: P com 0.7, safe; 0.7< P com 1.0, warning; 1< P com 2, light pollution; 2< P com 3, moderate pollution; P com> 3, heavy pollution. (3) The superscripts C,
L, M and H represent clean, light pollution, medium pollution and heavy pollution, respectively.

Table 4
Potential ecological risk assessment of the tailing pool and agricultural soils in Side Village in Guangxi Province, China (n ¼ 36).

Soil depth Soil Individual potential ecological risk factor ( Ei) Ecological risk ( RI)

E Pb E Zn E Cd E Mn E Cu

0 e 5 cm NFS 1353.82 VH 11.32 L 515.73 VH 1.43 L 11.94 L 1894.24 VH

EnS 1030.15 VH 11.62 L 450.56 VH 0.62 L 9.21 L 1502.16 VH
US 1785.00 VH 15.72 L 517.98 VH 0.89 L 12.65 L 2332.23 VH
MS 2932.88 VH 16.49 L 659.55 VH 0.64 L 18.34 L 3627.90 VH
ExS 7760.06 VH 34.76 L 816.85 VH 3.55 L 48.73 M 8663.96 VH
TS 4587.84 VH 49.05 M 2860.67 VH 20.44 L 14.39 L 7532.40 VH
5 e 20 cm NFS 253.80 H 1.94 L 185.39 H 3.88 L 9.89 L 454.90 VH
EnS 1207.27 VH 14.24 L 503.37 VH 0.57 L 10.29 L 1735.74 VH
US 1442.07 VH 16.30 L 538.20 VH 1.09 L 13.00 L 2010.66 VH
MS 2925.06 VH 16.42 L 651.69 VH 0.74 L 18.19 L 3612.09 VH
ExS 769.32 VH 34.45 L 868.54 VH 2.98 L 42.12 M 1717.42 VH
TS 6353.99 VH 48.89 M 2648.31 VH 21.10 L 16.82 L 9089.11 VH

(1) E i: monomial potential ecological risk factor, standards: Er < 40, low potential ecological risk (L); 40 Er < 80, moderate potential ecological risk (M); 80 Er < 160, considerable potential ecological risk (C); 160 Er < 320, high potential ecological risk
(H); Er 320, very high potential ecological risk (VH). (2) T i: metal toxicity factor, the standardized values for Cd, Pb, Cu, Zn, andMn are 30, 5, 5, 1, and 1, respectively. (3) Pi: single contamination index; (5) RI: potential ecological risk index; the
evaluation results are divided into 5 grades: RI < 150, lowecological risk (L); 150 RI < 300, moderate ecological risk (M); 300 RI < 600, considerable ecological risk (C); 600 RI < 1200, very high ecological risk (VH); RI

1200, extremely high ecological risk (EH).

 K. Liu et al. / Chemosphere 251 (2020) 126415 7

Fig. 2. Soil enzyme activity in the tailing pool and agricultural soils in Side Village of Guangxi Province, China (n ¼ 36). (1) NFS, EnS, US, MS, ExS and TS represent the soil samples from natural forest soil without tailing wastewater fl ooding
(CK), the entrance of the stream to the agricultural area, upstream of the agricultural area, the middle-stream in the agricultural area, the exit of the stream from the Side Village and the tailing pool, respectively. (2) (A), (B), (C) and (D) represent
the soil enzymes invertase, protease, urease and catalase, respectively. (3) Values are means ± SD (n ¼ 3); one-way ANOVA (analysis of variance) was performed for each parameter, and different lowercase letters for the 0 e 5 cm horizon and
capital letters for the 5 e 20 cm horizon between soils from different locations indicate a signi fi cant difference at P < 0.05 by Duncan ’ s multi-range test.

the soil source and soil horizon. In general, the activities of invertase, protease and urease exhibited controlling the chemical forms of these materials ( Avila et al.,
the opposite trends as the heavy metal concentrations: TS < ExS <MS < US < EnS < NFS, with the 2016). In general, the grade of the soil pHwas neutral-weakly acidic in this study. The highest soil pH
exception of invertase activity in the 0 e 5 cm horizon. Catalase activity ranked highest, followed by the occurred in TS and the lowest in NFS. The pH values of the agricultural soils fl ooded by the tailing
activities of protease, invertase and urease. The values for invertase were 0.68 e 1.41 and wastewater were all signi fi cantly ( P < 0.05) higher than those of NFS and were also higher than
background soil pH values (4.10 e 5.16) ( Liu et al., 2000 ), indicating that the mining activities increased
the soil pH. This area developed from karst landforms, and two of the principal inherent properties of
0.32 e 2.36 mg glucose g 1 24 h 1 in the 0 e 5 cm and 5 e 20 cm soil horizons, respectively. The this type of soil are that it is alkali and enriched in calcium ( Yuan, 2001 ). Furthermore, sulfur (S) is
corresponding values were 1.04 e 1.12 and 0.90 e 1.55 mg NH 2 e N g 1 24 h 1, 0.02 e 0.43 and 0.02 e 0.41 very limited in the Xingping Pb e Zn mine ( Ning, 1992 ), which may explain the signi fi cant difference
mg NH 3 e N g 1 24 h 1, and 4.70 e 18.00 and 2.36 e 18.16 ml g 1 h 1 for protease, urease and catalase, between NFS and the other soils. These results were different from those of our previous study
respectively. There was no obvious difference in the distribution pattern between the surface and conducted in soil from the Pingle agricultural area in Guilin, Guangxi Province, where mining activities
subsurface soil horizons. had no substantial effect on the soil pH ( Liu et al., 2018 ). These results were also different from those
of research performed at the Huangjiang Pb e Znmine of Guangxi Province by Qu et al. (2008) , where
a tailing dam collapse accident at the mine signi fi cantly decreased the soil pH value. The Huangjiang
Pb e Zn mine is rich in the element S, mainly in the forms of FeS and ZnS; reduced sulfur is oxidized to

3.4. Soil enzyme activities correlation matrix sulfate in air, inducing a decrease in the soil pH, and the polluted agricultural soil pH was only 2.50 e 3.65
( Qu et al., 2008 ). Although the above three mines all lie within Guangxi Province and were formed

As shown in Fig. 3 , invertase, protease and urease were found from Devonian and Carboniferous limestone, their pH levels are very different. These results showed

negatively correlated with heavy metal concentrations, especially by that of Zn. The r values were 0.576, that factors such as the kind of mine and the content of S affect the soil pH together. In this study, the

0.455 and 0.877 for invertase, protease and urease, respectively. In general, soil pH had negative soil pH values at the surface were generally higher than those at the subsurface, supporting the idea

effects on soil enzyme activities, especially for urease ( P < 0.01) and protease ( P < 0.05) in this study. that the soil pH is more in fl uenced by the tailing dam collapsed accident or anthrophonic sediments

Catalase activity exhibited the opposite pattern as compared to above three soil enzyme activities, than by inherent properties of soils originating from soil parent materials or vegetation cover.

i.e., it had strong correlations ( P < 0.01) with Zn ( r ¼ 0.791) and Mn ( r ¼ 0.764).

4. Discussion

4.1. Soil pH and heavy metal concentrations

Soil pH is considered a master variable in soil because it controls many chemical processes that
take place in soil. It speci fi cally affects the availability of plant nutrients and toxic substances by
In general, the detected soil heavy metal concentrations were in
8 K. Liu et al. / Chemosphere 251 (2020) 126415

Fig. 3. Heat map of correlation between soil pH, heavy metal concentration, P com and soil enzyme activity in soil samples base on Pearson correlation coef fi cients (n ¼ 36). (1) The colors of yellow, white and blue present strong positive
correlation, weak correlation and strong negative correlation, respectively. (2) “*” and “**” means signi fi cant correlations at
P < 0.05 and at P < 0.01, respectively. (3) The data in the parentheses was the r values. (For interpretation of the references to color in this fi gure legend, the reader is referred to the Web version of this article.)

order of TS > ExS > MS > US > EnS > NFS; i.e., the heavy metal concentrations increased and the soil parental composition, especially the former.
downstream. Our results are similar to those of a study from Kien et al. (2010) , who reported that the
principal source of the detected pollution was sediment in fl ow owing to the collapse of a dike. This
4.2. Soil pollution assessment
kind of metal pollution distribution pattern is different from that associated with atmospheric
deposition; i.e., the heavy metal concentrations will decrease with increasing distance from the mine
A higher concentration of one speci fi c element does not always indicate a greater degree of
or tailing pool. For example, Li et al. (2015) found decreases in potentially toxic element levels with
pollution or a higher ecological health risk. Therefore, contamination assessment and potential
increasing distance from a Pb e Zn smelter up to 10 km.
ecological risk assessment approaches have been used in previous studies ( Hakanson, 1980 ; Rehman
et al., 2018 ; Memoli et al., 2019 ) as well as in this study. Evaluation of the P i of heavy metals (Mn, Cd,
Cu, Pb and Zn) in soils was carried out for the fi ve heavy metals examined in this study based on the
background values for soil in Guangxi Province (China ’ s Environmental Protection Agency, 1996;
Pavlowsky et al. (2017) also reported that metal concentrations decline downstream from mine
Environmental Research Institution of Guangxi, 1992). The values of P Pb
sources. The strong positive correlations between these metals and the negative correlation between
the metals and the distance from the main roundabout in town indicated their anthropogenic origin ( Ciarkowska,
2018 ). Among all the investigated heavy metals, Pb exhibited the highest absolute concentration,
followed by Zn, similar to the results of previous studies conducted in other Pb e Zn mine soils ( Pavlowsky
were highest at the sampled sites, followed by the P i values for Cd z Zn, Mn z Cu ( Table 3 ). The values
et al., 2017 ; Ciarkowska, 2018 ; Khosravi et al., 2018 ;
of P i for Pb, Zn and Cd were all higher than 3, while the values of P i for Mn and Cu were close to or
greater than 3, indicating that the agriculture soils have been seriously contaminated with heavy
metals, especially Pb, Zn and

Duan et al., 2018 ; Shara fi et al., 2018 ). In general, the concentrations of heavy metals were similar
Cd. These results are consistent with previous studies conducted in other Pb e Zn mines ( Pavlowsky et
between the 0 e 5 cm and 5 e 20 cm horizons, with the exception of Pb, Zn Cd, and Mn in NFS. The
al., 2017 ; Ciarkowska, 2018 ;
obviously higher concentrations of Pb, Zn and Cd at the surface in NFS compared to those at the
Khosravi et al., 2018 ; Duan et al., 2018 ; Shara fi et al., 2018 ). P com
subsurface might be due to atmospheric deposition from the Pb e Zn mine. The distance fromNFS to
allows the assessment of soil quality at a much more comprehensive level than the evaluation of a
TS is only 3 km. An opposite result was observed for Mn, possibly because this element is a vital
single element ( Brady et al., 2015 ). The assessment results for P com further proved that the sampled
nutrient for plant growth ( Salman et al., 2017 ) and is absorbed by roots ( Zuazo et al., 2011 ), resulting
soils were severely polluted, as the values were far greater than 5, which is the threshold for the
in a decrease in its concentration.
heavy pollution level. In this study, the values of P i and P com increased downstream. Regarding the
soil horizons, the values of P com were generally higher in the 0 e 5 cm horizon, indicating that the
surface soil was more polluted than the subsurface soil, which agreed with the sediment
characteristics ( Ciarkowska, 2018 ).
In addition, it is worth noting that the analysed metal concentrations in the 5 e 20 cm horizon in
NFS were much higher than the background values for soil in Guangxi Province and were even
higher than the standards for national soil quality in some cases (data given in Table 2 ). However, this
site was not fl ooded by the previously mentioned accident or atmospheric deposition. One reasonable
E r represents the ecological risk of each element. The E r values of Zn, Mn and Cu were lower
explanation for these high concentrations is the soil origin. The soil exhibits a high inherent heavy
than 30 with some exceptions, while the values of Pn and Cd were much higher than 320 ( Table 4 ),
metal concentration due to the soil parent materials, especially for Pb, Cd and Mn ( Table 2 ).
indicating that the elements Pb and Cd played a vital role in soil pollution. The heavymetal
Therefore, the main reasons for the high heavy metal concentrations in the Side agricultural soils
concentrations in the soil will ultimately be hazardous to human health, as the most likely metal
were human activities
transportation pathway is from the soil to roots, roots to shoots and plants to humans via consumption
( Bermudez et al., 2012 ). Special
 K. Liu et al. / Chemosphere 251 (2020) 126415
attention should be paid to Pb because of its decisive role in the assessment of soil contamination
and potential ecological risk in this study ( Table S1 ). Furthermore, Pb is not an essential element but,
rather, is a toxic element for life and can be incorporated into organisms ( Kirkham, 2006 ).
Epidemiologic studies have shown that even at low concentrations, chronic exposure to Pb can result
in adverse effects in the blood and central nervous system, especially in young children ( Wallace et
al., 1974 ). In 2013, it was estimated that Pb pollution was responsible for 0.6% of the world ’ s disease
burden ( Needleman, 2004 ) and 853,000 deaths ( WHO, 2016 ). The average Pb levels in blood are
higher in Chinese children than in children in other countries ( Han et al., 2018 ). In addition, Cd should
be monitored with great vigilance due to the high values of Cd according to the single contamination
index and soil potential ecological risk assessments. Cadmium is also not an essential element but,
rather, is a toxic element to life, similar to Pb ( Bermudez et al., 2012 ). Excess Cd in the body is
harmful to the kidneys, liver, lungs, brain, prostate, testes, muscles, heart, and immune and central
nerve systems ( Ghosh and Indra, 2018 ). Furthermore, the residence time of Cd in the body is as long
as 20 e 30 years.
RI represents the sensitivity of the biological community to toxic substances and illustrates the
potential ecological risk caused by overall contamination. To quantify the overall potential ecological
risk of heavy metals in the agriculture soils, the values of RI were calculated ( Table 4 ). The RI values
of the sampled soils were all above 600 except for the 5 e 20 cm soil horizon in NFS, which was not
affected by the collapse accident or atmospheric deposition. The results indicated that the agriculture
soils in Side Village presented a very high potential ecological risk. Similar results were found at the
Chenzhou Lead-Znmine in Hunan Province, China ( Liu et al., 2005 ) and the Aznalcollar Mine in
Seville, Spain ( Grimalt et al., 1999 ; Lacal et al., 2003 ), as these two sites have both been subject to
tailing dam collapse accidents. Regarding the soil horizons, the values of E r and RI were similar for all
heavy metal elements, with the exception of Pn and Cd in NFS. The high ecological health risk
observed in the 0 e 5 cm horizon in NFS may be caused by high atmospheric deposition because this
location is only approximately 3 km from the tailing pool.
4.3. Soil enzyme activity and correlation matrix
Under heavymetal pollution, microorganisms are the fi rst group in the biota that experiences
direct and indirect impacts of available forms of heavy metals in soil solution or adsorbed on soil
colloids ( Brookes, 1995 ; Wu et al., 2018 ). Soil enzymes are mainly produced by microbes, which play
important roles in maintaining soil physical and chemical properties, fertility, and health ( Zornoza et
al., 2009 ). They are critical to biochemical processes involving nutrient cycling and degradation of
organic pollutants and are sensitive to environmental changes and stresses ( Burns et al., 2013 ;
Wahsha et al., 2014 ; Rao et al., 2014 ). Furthermore, the measurement of soil enzyme activities is
rapid, simple, and inexpensive in comparison with other methods ( Yang et al., 2016 ). Thus, the
investigation of soil enzyme activities under heavy metal pollution is critical for understanding changes
in soil ecological functioning ( Wang et al., 2019 ) and pollution status ( Xian et al., 2015 ; Hagmann et
al., 2015 ; Wahsha et al., 2017 ; Duan et al., 2018 ). Soil enzyme activity varied with the soil source and
soil horizon in this study ( Fig. 2 ). In general, the activities of invertase, protease and urease exhibited
the same trend as the heavy metal concentrations, i.e., TS < ExS < MS < US < EnS < NFS, with the
exception of invertase activity in the 0 e 5 cm horizon. Bivariate correlation analysis showed that the
activities of these three enzymes were negatively affected by heavy metals, especially Zn, with r values
9
of 0.572, 0.457 and 0.876 for invertase, protease and urease, respectively ( Fig. 3 and Table S1 ).
These results were similar to those of previous studies. For example, the results form Carreira et al.
(2008) showed soil enzyme activities (dehydrogenase, bglucosidase, urease, arylsulfatase, acid and
alkaline phosphatase) were strongly inhibited in eroded areas where pollutant residues appeared in
the surface. Meng et al. (2018) reported that urease, saccharase, protease, catalase and polyphenol
oxidase (POA) activities were signi fi cantly negatively correlated with heavy metals in soils, especially
Zn and Cu. Fluorescein diacetate hydrolase activities were found to be signi fi cantly affected by Ni, the
primary ore processing collector, sodium butyl xanthate (SBX) and a Ni/SBX mixture ( Li et al., 2018 ).
Polyphenoloxidase was found to be the soil enzymewhose activitywas themost sensitive andwas signi fi
cantly correlated with almost all detected metals (Pb, Zn Cd and As) ( P < 0.05) ( Yang et al., 2016 ). A
study from Ciarkowska (2018)
showed that the environmental impacts of urbanization and tourismwere mainly observed in the
surface horizons of Zakopane soils on the basis of decreases in activity of single enzymes
(dehydrogenase, invertase and urease activity) as well as the enzyme activity index (calculated as the
geometric mean, a general index for integrating information from variables that possess different units
and ranges of variation). A direct reason for the decrease in enzyme activity observed mainly in
central soils was probably the accumulation of Pb, Cd, Zn and Cu, which are positively correlated with
each other and negatively correlated with enzymes ( Ciarkowska, 2018 ). One possibility is that the
presence of metals results in lower rather than higher enzyme activities ( Kuperman and Carreiro,
1997 ) by causing protein denaturation, forming a complex with the substrate, interacting with the
residues involved in enzyme function, or reacting with the enzyme substrate complex ( Hemida et al.,
1997 ; Ciarkowska and Gambu s, 2005 ). Other papers have reported that lower enzyme activities are
more closely related to a lack of nutrients than to long-term heavy metal accumulation ( Ciarkowska et
al., 2016 ; Ciarkowska, 2017 ). In our study, catalase activity exhibited the opposite pattern to the
above three analysed enzymes; i.e., catalase activity increased as the heavy metal concentration
increased ( Fig. 2 ), especially for Zn and Mn. The r values between catalase activity and Pb, Zn, Cd,
Mn and Cu were 0.318, 0.791, 0.094, 0.754, 0.294, respectively ( Table S1 ). The opposite trend in soil
enzyme activity has also been demonstrated by other studies. Yang et al. (2016) showed that
amylase (for C cycling), acid phosphatase (for P cycling) and catalase (for redox reaction) activities
presented signi fi cantly positive correlations ( P < 0.05) with Pb, Cd, Zn and As contents. The highest
enzymatic activities of alkaline phosphatase, cellobiohydrolase, and L- leucine-aminopeptidase were
observed at the site with the greatest soil metal loads (for As, Pb, chromium (Cr), Cu, Zn, and
vanadium (V)), and a particularly strong relationship was detected between enzyme activity and the
metals V and Cr in another study ( Hagmann et al., 2015 ). Ciarkowska et al. (2014) even reported that
invertase activity was much higher in polluted soil samples than in unpolluted forest soil. The reason
for these fi ndings might be that the long-term presence of the heavy metals in the soil led to diversi fi cation
of the microbial population resistant to the toxic action of the heavy metals ( Kizylkaya et al., 2004 ) or
that high organic matter concentrations and a neutral soil pH transform toxic metals into biologically
inactive forms ( Finkenbein et al., 2013 ).
Hagmann et al. (2015) suggested that high enzymatic activities found at sites with particularly high
metal concentrations may not act as catalytically important cofactors or activators, or that the
metal-containing soil itself (or some component of it) may serve as an activator of enzymatic activities,
or that the effect of salinitymay be relevant. The effect of metals on enzyme function may partly
10 K. Liu et al. / Chemosphere 251 (2020) 126415
depend on the length of time that the soil-metal system has been allowed to “ adjust and adapt ”, and
these interactions at the study site highly polluted by heavy metals has been occurring for over 40
years ( Hagmann et al., 2015 ). In summary, enzymes related to the C (invertase) or N (protease and
urease) cycle decreased as the heavy metal concentrations increased, while catalase (related to
antioxidant reactions) increased in this study. A study from Satapute et al. (2019) also showed that
two bacterial strains, SJPS_KUD54 and KUD-MBBT4, exhibited tolerance to As and Cd at 10 ppm.
These two heavy metals caused signi fi cant increases in stress-associated enzymes related to
antioxidant reactions such as superoxide dismutase, catalase and glutathione S-transferase in
SJPS_KUD54 compared to KUD-MBBT4, and these enzymes are positively involved in prevention or
repair mechanisms that reduce the risks caused by heavy metal stress ( Satapute et al., 2019 ). Based
on fi eld investigation and microcosm simulation analysis, Yang et al. (2016)
indicated that the activity of oxidoreductases rather than a speci fi c enzyme should be used as the “ core
enzyme ” activity. Therefore, considering that plants use their antioxidant system to eliminate reactive
oxygen species (ROS, e.g., O 2$ and H 2 O 2 ) by scavenging under heavy metal stress by increasing the
activities of superoxidase (SOD), catalase (CAT) and peroxidase (POD), we speculate that this
process may also exist in the soil body. Themechanisms by which plant antioxidant enzymes alleviate
environmental stress are as follows: SOD acts as the fi rst line of defence against ROS produced by
lipid peroxidation, and SOD then catalyses the transformation of superoxide (O 2$-) radicals to H 2 O 2 and
oxygen, whereas CAT plays an important role in scavenging H 2 O 2 in peroxisomes ( Choudhary et al.,
2007 ; Sidhu et al., 2017 ). Therefore, the combination of SOD, CAT, and POD plays an important role
in the resistance of a plant to environmental stress ( Patel et al., 2005 ; Liu et al., 2016 ; Yadav et al.,
2018 ). Thus, the determination of whether the antioxidant system also exists in the soil microbial body
and is induced under heavy metal stress is our next study question.
4.4. Using soil enzyme activity as a bio-indicator for soil pollution assessment
As mentioned above, soil enzymes are sensitive to environmental changes and stresses;
furthermore, their measurement is much easier than other biome analyses. Naturally, researchers use
soil enzymes as an important index to assess soil pollution or predict soil pollution and soil health ( Yang
et al., 2016 ; Wahsha et al., 2017 ; Ciarkowska, 2018 ) or even for the evaluation of soil
bio-rehabilitation ( Ciarkowska et al., 2014 ). Forty years ago, two scientists from the former Soviet
Union, Grigoryan and Galstyan (1979) reported that invertase could be used as an index in the
assessment of heavy metal soil pollution assessment. They later indicated that phosphatase activity
could be used in a forest grown in meadow soil with heavy metal pollution ( Grigoryan and Galstyan,
1986 ). Urease and invertase activity were also used for predicting heavy metal pollution by Alkorra et
al. (2003) . However, the total number of enzymes in soil is greater than 60, and the utility of only one
or a few kinds of enzymes for the assessment of heavy metal pollution is limited. Hence, some
researchers have used methods such as principal component analysis (PCA) ( Ciarkowska, 2018 ),
cluster analysis, redundancy analysis (RDA) or Pearson correlation analysis ( Yang et al., 2016 ) for
the study of soil enzyme activity and to assess the relationship between the soil enzyme activity and
soil pollution patterns. In addition, some researchers have also proposed the use of the enzyme
activity index (EAI) 1 instead of single enzyme activity to assess soil heavy metal
p
1 EAI ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
3
DHA,Ure,IA , dehydrogenase (DHA), urease (Ure) and invertase (IA).
pollution ( Ciarkowska, 2018 ). For example, Li et al. (2014) used the total analysed soil enzyme
activity (summing the values for all soil enzymes) to assess soil pollution. However, this EAI is only
suitable in a situation inwhich all the soil enzyme activities are positively or negatively associated with
heavy metal stress. If some of the enzyme activities are positively associated with heavy metal stress
while others show the opposite pattern, as in the present study, this kind of EAI would not be suitable
for the assessment of soil heavy metal pollution. Hence, a suitable EAI or modules should be studied
in the future to assess soil pollution more ef fi ciently.
5. Conclusion
The heavymetal concentrations were high in these studied sites, and the elements Pb, Zn and
Cd were the dominant pollutants with the values of 10530.41, 1708.58 and 8.32 mg kg 1, respectively.
The assessment methods for these studied soils by P i, P com, E i and ER
indicated the agricultural lands in Side Village reached the heavily polluted level and presents an
extremely high ecological risk grade. Soil enzyme activity decreased downstream except for that of
catalase. In general, there were signi fi cant relationships between heavy metal concentrations and soil
enzyme activities, especially for the element of Pb. Among the enzymes, the activities of urea were
more sensitive to heavy metals. The pollution distribution pattern shows downstream increases; and
mining activities and the soil origin are the twomain reasons for this situation, especially the former.
Declaration of competing interest
I would like to declare on behalf of my co-authors that the work described was original research
that has not been published previously, and not under consideration for publication elsewhere, in
whole or in part. All the authors listed have approved the manuscript that is enclosed. We declare that
we have known competing
fi nancial interest or personal relationships that could have appeared to in fl uence the work reported in
this paper.
CRediT authorship contribution statement
Kehui Liu: Conceptualization, Methodology, Formal analysis, Writing - original draft. Chunming
Li: Software, Writing - original draft. Sanqi Tang: Data curation, Investigation. Guiduo Shang:
Visualization, Software. Fangming Yu: Conceptualization, Methodology, Funding acquisition. Yi Li: Project
administration, Writing
- review & editing.
Acknowledgements
This project was supported by National Key Research and Development Program of China
(2017YFD0801500), National Science Foundation of China (41661077; 41967019), Guangxi Science
and Technology Development Project of Major Projects of Guangxi (Guike AA17204047-3), and
Guangxi Natural Science Foundation (2018JJA150018). Thank Shaoling Peng and Yanjiang Cai for
their encouragement during the original manuscript formation. We thank American Journal Experts
(AJE) for editing the manuscript and Mr Xiaolong Lin of Beijing Ruimeide Environmental Remediation
(BRER) Co., LTD for sampling the soil.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.chemosphere.2020.126415 .
 K. Liu et al. / Chemosphere 251 (2020) 126415 11

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