Ecotoxicology and Environmental Safety 147 (2018) 266–274

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Ecotoxicology and Environmental Safety

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

Soil properties influence kinetics of soil acid phosphatase in response to T

arsenic toxicity
⁎
Ziquan Wanga, Xiangping Tana, Guannan Lua, Yanju Liub,c, Ravi Naidub,c, Wenxiang Hea,
a
College of Natural Resources and Environment, Northwest A & F University, Key Laboratory of Plant Nutrition and Agro-environment in Northwest China, Ministry of
Agriculture, Yangling, 712100 Shaanxi, China
b
Global Centre for Environmental Research, The Faculty of Science and Information Technology, University of Newcastle, University Drive, Callaghan, NSW 2308,
Australia
c
Cooperative Research Centre for Contamination Assessment and Remediation of the Environment (CRC CARE), Mawson Lakes, SA 5095, Australia

A R T I C L E I N F O A B S T R A C T

Keywords: Soil phosphatase, which plays an important role in phosphorus cycling, is strongly inhibited by Arsenic (As).
Arsenic However, the inhibition mechanism in kinetics is not adequately investigated. In this study, we investigated the
Soil acid phosphatase kinetic characteristics of soil acid phosphatase (ACP) in 14 soils with varied properties, and also explored how
Soil property kinetic properties of soil ACP changed with different spiked As concentrations. The results showed that the
Inhibition constant
Michaelis constant (Km) and maximum reaction velocity (Vmax) values of soil ACP ranged from 1.18 to 3.77 mM
Noncompetitive inhibition
and 0.025–0.133 mM h−1 in uncontaminated soils. The kinetic parameters of soil ACP in different soils changed
differently with As contamination. The Km remained unchanged and Vmax decreased with increase of As con-
centration in most acid and neutral soils, indicating a noncompetitive inhibition mechanism. However, in al-
kaline soils, the Km increased linearly and Vmax decreased with increase of As concentration, indicating a mixed
inhibition mechanism that include competitive and noncompetitive. The competitive inhibition constant (Kic)
and noncompetitive inhibition constant (Kiu) varied among soils and ranged from 0.38 to 3.65 mM and
0.84–7.43 mM respectively. The inhibitory effect of As on soil ACP was mostly affected by soil organic matter
and cation exchange capacity. Those factors influenced the combination of As with enzyme, which resulted in a
difference of As toxicity to soil ACP. Catalytic efficiency (Vmax/Km) of soil ACP was a sensitive kinetic parameter
to assess the ecological risks of soil As contamination.

1. Introduction contaminated soils (Koo et al., 2012; Lyubun et al., 2013).

Arsenic (As) is a metalloid placed in the same family with phos-
phorus (P) in the periodic table of elements. It exists mainly as in-
organic forms with pentavalent and trivalent state dominating in oxi-
dizing and reducing environment, respectively (Bissen and Frimmel,
2003; Duker et al., 2005). The anthropogenic activities, including
mining, application of fertilizer and pesticides, burning of fossil fuels,
have caused the release of As into the environment, which leads to As
pollution worldwide and causes environmental incidents (Mandal and
Suzuki, 2002; Singh et al., 2015). Arsenical compounds not only threat
indirectly human being and animal health, but also have very toxic
effects on microorganisms and soil enzymes through replacing phos-
phorus in molecular and/or interacting with sulfhydryl groups of pro-
teins (Duker et al., 2005). Knowing the mechanisms of As pollution on
soil biochemical processes is useful for identifying its environmental
exposure and providing important information for the remediation of
Soil enzyme participates in important ecosystem processes in soils,
such as the decomposition of organic matter, the formation of soil
humus, and the cycling of nutrients (Burns et al., 2013). Meanwhile,
soil enzyme is known as a sensitive indicator of natural and anthro-
pogenic changes in ecosystems, which is used to assess the impact of
various pollutants including heavy metals contaminated in the soil
(Ciarkowska, 2015; Rao et al., 2014). Heavy metals inhibited enzyme
activity in several ways by masking catalytically active groups, dena-
turing the protein conformation or competing with metal ions (Karaca
et al., 2010). Soil enzyme kinetics can depict the relationship between
enzyme activity and substrate concentration, and provide a rapid ap-
proach for assessing the relative amounts of an enzyme and the cata-
lytic characteristics in soils (Dick, 2011). The Michaelis-Menten equa-
tion was used to describe quantitatively this relationship. In the
equation, the maximal catalytic reaction rate (Vmax) at saturated sub-
strate concentration and enzyme affinity (Km) for its substrate could

⁎
Corresponding author.
E-mail address: wenxiang.he@nwafu.edu.cn (W. He).

http://dx.doi.org/10.1016/j.ecoenv.2017.08.050
Received 15 May 2017; Received in revised form 18 August 2017; Accepted 21 August 2017
Available online 14 September 2017
0147-6513/ © 2017 Elsevier Inc. All rights reserved.
Z. Wang et al.
provide information of enzyme intrinsic properties. Exploring the ki-
netic characteristics of soil enzymes can provide a mechanistic under-
standing of how various biological processes occur and are regulated in
heavy metal contaminated soils (Dick, 2011). For instance, after
studying the kinetics of soil enzyme in loquat orchards polluted by Cu
pollution, Fu et al. (2009) found that urease Vmax and Vmax/Km were
negatively correlated while invertase Km was positively correlated with
Cu concentration, indicating smaller amount of soil urease and lower
affinity of invertase for substrate in Cu polluted soil than in un-
contaminated soil.
Soil phosphorus cycling is one of the most important biological
processes in soil environment. Soil phosphatase catalyzes the hydrolysis
of ester–phosphate bonds, breaking down the organic phosphorus into
inorganic form which is easily taken up by plants or microorganisms
(Nannipieri et al., 2011). Therefore, phosphatase plays an important
role in phosphorus cycling. Soil phosphatases are classified in acid
phosphatase, neutral phosphatase and alkaline phosphatase according
to the optimum pH (Nannipieri et al., 2011). Phosphatases are sensitive
to heavy metals (Nannipieri et al., 2011). Many studies have suggested
that As has inhibitory effect on soil phosphatase activity (Bhattacharyya
et al., 2008; Das et al., 2013; Speir et al., 1999; Wang et al., 2017).
Phosphatase activity then has been used as a bio-indicator to assess As
toxicity (Nannipieri et al., 2011; Speir et al., 1999). However, these
studies mainly focused on enzyme activities, the processes of As com-
bination with enzyme and inhibition mechanism have not been re-
searched thoroughly. Enzyme kinetics reflects the process of a reaction.
Identification of changes for kinetic properties of soil phosphatase
under As pollution could help in understanding how As effect on soil
phosphatase and clarifying its environmental exposure risks (Dick,
2011). Furthermore, the toxicity based on enzyme activity may vary
with the substrate type and concentration used to measure the enzyme
activity (Tan et al., 2017a, 2017b). In addition, soil properties can in-
fluence As toxicity to soil phosphatase (Speir et al., 1999). The complex
soil properties may affect the inhibition kinetics and reaction process
resulting in different toxicity of As to soil phosphatase. To our knowl-
edge, the kinetic characteristics of soil phospahtase-catalyzed reaction
in response to As pollution and the influence of soil properties on As
toxicity have not been characterized clearly. Thus, it is of importance to
clarify the mechanism and reaction process of As inhibition on soil
phosphatase to get an better understanding of soil quality and phos-
phorus cycling in As contaminated soils.
Therefore, this study has focused on soil acid phosphatase (ACP, EC
3.1.3.2) because of its wide spread presence in acid and neutral soils
and in a certain amounts of alkaline soils from decomposition of plant
debris (Nannipieri et al., 2011). We investigated the kinetic properties
of soil ACP under exogenous As stress using soil samples representing
14 different agriculture soil types in China. The objectives of this study
were to 1) investigate the reaction process and mechanism of As in-
hibition on soil acid phosphatase among various soils, and 2) determine
the feasibility of using kinetic parameters to assess As toxicity and 3)
reveal how soil properties affect As toxicity to soil ACP through enzyme
kinetic study.
2. Materials and methods
2.1. Soil sampling and physicochemical analysis
Fourteen uncontaminated farmland soils with varied properties
were sampled from different provinces in China (Fig. 1). The soils were
selected to be representative of the major soil types and the distribution
of soil pH of agricultural soils in China. Soil samples were taken from 0
～20 cm depth and air-dried in the laboratory. Soils were ground and
passed through a 1 mm nylon sieve before use. Soil physical chemical
properties were determined using methods illustrated in Bao (2000)
and presented in Table 1. Briefly, soil pH was determined by a pH
electrode with a water: soil ratio of 2.5:1. Soil texture was measured
267
Ecotoxicology and Environmental Safety 147 (2018) 266–274
using the pipette method (Kettler et al., 2001). Soil organic matter
(SOM) was determined by potassium dichromate digesting and ferrous
sulfate titration method (Walkley and Black, 1934). Total P was di-
gested with melting sodium hydroxide and determined using mo-
lybdenum blue colorimetric method (Bao, 2000). Cation exchange ca-
pacity (CEC) was determined with ammonium acetate pH 7.0 (Sumner
and Miller, 1996). Amorphous Fe oxide was extracted by ammonium
oxalate and determined by flame atomic absorption spectrometry (Bao,
2000). The soil samples were digested using nitrohydrochloric acid and
background total arsenic was determined by atomic fluorescence
spectroscopy method (AFS) (GB/T, 22105.2, 2008).
2.2. Experimental design
Heavy metal reactions with enzyme are rapid processes. The
changes in proliferation and enzyme synthesis by microbes can mask
the toxicity if measured over a long period of exposure to heavy metal.
Thus a shorter exposure time (e.g., 30 min) was suitable for de-
termining the toxicity of heavy metal to soil enzyme (Matyja et al.,
2016).
Three gram of air-dried soils were spiked with 3 ml As solutions
(Na3AsO4·12H2O，AR, Sinopharm Chemical Reagent Co., Ltd, Shanghai
China) to obtain final soil As concentrations of 0, 10, 25, 50, 100, 200,
400 and 600 mg kg−1 soil (Weight(soil): Volume(As)=1:1) in a 50 ml
flask then the flask were shaken to homogenize the soils and solutions.
After 30 min of equilibration, the soil ACP activity was measured.
Triplicate samples were prepared for each treatment.
2.3. Determination of soil ACP activity
Soil ACP activity was determined by the following procedure (Guan,
1986; Zavišić et al., 2016): 20 ml disodium phenyl phosphate (1, 2.5, 5,
10 mM) prepared in pH 5.0 acetate buffer were added to the soil mix-
tures that treated with As in the flasks, then the flasks were capped and
incubated at 37 °C for 4 h. After incubation, the released phenol was
determined colorimetrically at 510 nm, using 4-aminoantipyrine and
potassium ferricyanide as coloring reagents. A substrate control
(without soil) and an optional abiotic control (without substrate) were
simultaneously incubated. The concentration of phenol was calculated
using a standard curve (Guan, 1986).
2.4. Data analysis
2.4.1. Soil enzyme kinetics without As treatment
The two kinetic parameters, the Michaelis constant (Km) and max-
imum reaction velocity (Vmax) were determined by nonlinear regression
of Michaelis-Menton equation:
V = Vmax S /(Km + S ) (1)
Where V is soil ACP activity, S is substrate concentration.
When Km and Vmax were determined, the catalytic efficiency was
calculated by the ratio of Vmax/Km.
2.4.2. Soil enzyme kinetics with As treatment
Kinetic parameters will change at the presence of heavy metal in-
hibitors. Inhibitors of single substrate soil enzyme-catalyzed reactions
can often be grouped as either simple competitive (unchanged Vmax but
increased Km), simple noncompetitive (decreased Vmax but unchanged
Km) inhibitors (Dick, 2011), and a linear mixed (decreased Vmax and
increased Km) inhibitors (Cornish-Bowden, 2012). The competitive in-
hibition constant (Kic) can be estimated by a linear plot of
app
Km/appVmax against As concentration (Eq. (2)), thus Kic = intercept/
slope (Cornish-Bowden, 2012).
app
K m/app Vmax = Km/ Vmax + Km/ Vmax *I / Kic (2)
app app
where Km and Vmax are the apparent Michaelis constant and the
Z. Wang et al. Ecotoxicology and Environmental Safety 147 (2018) 266–274

Fig. 1. The location of soil sampling sites.

maximum reaction velocity in the presence of As respectively. Kic is
equilibrium constant of enzyme-inhibitor (EI) complex. In non-
competitive and mixed inhibition, the noncompetitive inhibition con-
stant (Kiu) was calculated by rearrangement of following equation:
app
V max = Vmax /(1 + I / Kiu )
Kiu is equilibrium constant of enzyme-substrate-inhibitor (ESI)
complex. When Kic = Kiu, the inhibition is pure noncompetitive
(Cornish-Bowden, 2012). The equilibrium constants of EI and ESI (Kic
and Kiu) are important factors determining the toxicity of an inhibitor to
enzyme. The Kic and Kiu could represent the affinity of an inhibitor to
enzyme or enzyme-substrate (ES) complex (Marangoni, 2003).
2.4.3. Calculation of ecological dose
Two models are used to fit the dose-response relationship between
kinetic parameters and As concentrations (Speir et al., 1999):
y = c /(1 + bx ) model 1
y = c (1 + ax )/(1 + bx ) model 2
where y is catalytic efficiency; x is As concentration; a, b, c are re-
gression parameters and always positive with b > a. When models are
fitted, an ecological dose (ED) could be calculated. For instance, ED50,
the concentration of an inhibitor that causing 50% inhibition, equals to
1/b for model 1 and (1–a/b)/(b–a) for model 2. Since ED50 value is too
large for environmental pollution assessment, a lower dose of ED10 (the
concentration of an inhibitor causing 10% inhibition) which would be
(3)
more meaningful is also calculated (Speir et al., 1999).
2.4.4. Statistical analysis
The goodness of fit of Michaelis-Menton model and dose-response
model was examined by F test. Linear regression also was performed to
depict the relationships between kinetic parameters and As concentra-
tion where it was appropriate. Pearson correlation analysis was used to
reveal the relationship between soil properties and kinetic parameters
of As inhibition on soil ACP. Stepwise regression analysis was also
carried out to investigate the relationships between soil properties and
ED50 values. All analyses were done using SPSS 18.0 software (SPSS
(4)
Inc., Chicago, USA).
(5)

Table 1
Soil physical chemical properties.

Soil sample Location Soil type Clay % pH SOM ( g kg−1) TP (g kg−1) CEC (cmol kg−1) Amorous Fe (g kg−1) Total As (mg kg−1)

S1 Hunan Argi-Udic Ferrosols 42.91 4.90 15.52 0.47 10.85 1.71 4.35
S2 Liaoning Hapli-Udic Argosols 17.32 5.74 25.84 0.73 12.19 2.14 4.61
S3 Chongqing Dystric Purpli-Udic Cambosols 24.96 5.74 17.48 0.55 21.34 2.14 2.17
S4 Yunnan Hapli-Udic Ferralosols 27.52 5.92 34.26 0.81 11.10 1.97 1.92
S5 Jiangxi Argi-Udic Ferrosols 36.51 6.01 11.69 0.52 8.70 1.76 1.18
S6 Anhui Ferri -Udic Argosols 16.84 6.25 20.04 0.35 19.08 2.34 1.25
S7 Heilongjiang Hapli-Udic Isohumosols 19.33 6.27 35.69 0.48 28.59 1.96 1.06
S8 Jilin Hapli-Udic Isohumosols 30.18 6.82 32.85 0.35 31.11 1.84 1.34
S9 Jiangsu Typic Gleyi-Stagnic Anthrosols 45.94 6.93 47.69 0.69 26.20 2.43 12.4
S10 Shaanxi Earth -cumuli -Orthic Anthrosols 26.01 7.90 16.49 0.98 22.37 1.20 0.54
S11 Xinjiang Calci-Orthic Aridosols 9.57 8.12 19.43 0.78 25.25 1.19 0.39
S12 Tianjin Marinic Aqui-Orthic Halosols 7.59 8.29 22.02 0.92 24.67 1.60 0.23
S13 Shandong Ochri-Aquic Cambosols 17.11 8.65 11.84 0.97 13.09 0.92 0.52
S14 Inner Mongolia Argic Calci-Ustic Isohumosols 10.51 8.80 16.30 0.38 11.61 0.72 0.17

SOM, soil organic matter; TP, total phosphorus; CEC, cation exchange capacity.

268
Z. Wang et al. Ecotoxicology and Environmental Safety 147 (2018) 266–274

Table 2 29.8% and 43.8% of the variation in Vmax, Km and Vmax/Km respec-
Kinetic parameters of soil ACP in unspiked soils. tively.
Soil sample Vmax mM h−1 Km mM Vmax/Km ×10−2 h−1

S1 0.108 ± 0.006 2.26 ± 0.28 4.78 ± 0.65 3.2. Kinetic properties of soil ACP in the presence of As
S2 0.108 ± 0.004 1.34 ± 0.13 8.00 ± 0.77
S3 0.073 ± 0.003 1.18 ± 0.11 6.21 ± 0.63
When As was added into the soils, the values of Vmax decreased with
S4 0.126 ± 0.007 2.17 ± 0.25 5.80 ± 0.75
S5 0.056 ± 0.003 1.19 ± 0.16 4.68 ± 0.67
the increase of As concentration, indicating that the rate of ES complex
S6 0.133 ± 0.006 2.00 ± 0.18 6.69 ± 0.68 breakdown to enzyme and product was slowed down by As inhibition
S7 0.070 ± 0.003 2.05 ± 0.20 3.41 ± 0.38 (Fig. 3a, b). The values of Vmax decreased sharply as As concentration
S8 0.063 ± 0.004 2.36 ± 0.33 2.66 ± 0.42 increased from 0 to 100 mg kg−1, then the decrease slowed down with
S9 0.070 ± 0.007 3.19 ± 0.56 2.19 ± 0.44
the continuing increase of As concentration (Fig. 3a, b). Except for S4
S10 0.037 ± 0.002 3.77 ± 0.37 0.98 ± 0.11
S11 0.039 ± 0.001 3.29 ± 0.22 1.18 ± 0.08 soil (Hapli-Udic Ferralosols), the decreases of Vmax in acid soil were
S12 0.042 ± 0.001 2.43 ± 0.15 1.72 ± 0.12 over 30% at As concentration of 600 mg kg−1. The largest decrease was
S13 0.025 ± 0.001 3.18 ± 0.26 0.80 ± 0.07 observed in S1 soil (Argi-Udic Ferrosols) with Vmax from 0.11 mM h−1
S14 0.053 ± 0.002 3.08 ± 0.16 1.74 ± 0.11 decreased to 0.047 mM h−1, followed by S5 soil (Argi-Udic Ferrosols)
Average 0.072 2.39 3.63
CV/% 47.91 34.42 65.51
with a reduction of 46.8%. However, the Vmax of ACP in five alkaline
soils (S10–S14) decreased by no more than 25% which were less than
Values are mean ± standard error. those for acid soils. The Vmax of S11 soil (Calci-Orthic Aridosols) was
the least affected with a reduction of 15.3%.
3. Results The Km of soil ACP responded to As contamination in two strategies
(Fig. 3c, d). Except for S4 and S5 soils (Hapli-Udic Ferralosols and Argi-
3.1. Kinetic properties of soil ACP in unspiked soils Udic Ferrosols), the Km of ACP for acid and neutral soils (S1–S9) re-
mained unchanged as the As concentration increased. However, for
The kinetic parameters of soil ACP varied among different types of alkaline soils (S10–S14) and two acid soils (S4 and S5), the Km of ACP
soils (Table 2). The values of Vmax, Km, Vmax/Km ranged from 0.025 to increases linearly with increasing As concentrations. The S5 soil was the
0.133 mM h−1, 1.18–3.77 mM and 0.80–8.00 ×10−2 h−1, with an most affected for Km increased from 1.19 to 3.12 mM as As con-
average of 0.072 mM h−1, 2.39 mM and 3.63 ×10−2 h−1 respectively. centration increased from 0 to 600 mg kg−1. This indicated that the
For Km, the values in soils S9–S14 were higher than those in other soils, affinity of ACP for the substrate remain unchanged or decreased mainly
indicating a lower affinity of enzyme for substrate (Table 2). The Linear depend on soil pH under As contamination.
regression analysis showed that pH and amorphous Fe were the con- The ratio of Vmax/Km represents catalytic efficiency of an enzyme
trolling factors determining the variation of soil ACP kinetic char- catalytic reaction. Fig. 3e,f shows that the catalytic efficiency of soil
acteristics (Fig. 2) (other soil properties did not show significant in- ACP varies among soils and is inhibited by As. The Vmax/Km for S5 soil
fluence and results are not shown). Soil pH explained about 54.8%, (Argi-Udic Ferrosols) decreased from 4.68×10−2 h−1 to
−2 −1
50.8% and 66.1% of the variation in Vmax, Km and Vmax/Km for the 14 0.96×10 h , with a largest reduction of 79.5%, followed by S1
soils respectively. While soil amorphous Fe explained about 41.2%, (Argi-Udic Ferrosols) and S14 (Argic Calci-Ustic Isohumosols) soils with
the decrease of 65.7% and 53.4% respectively. The decrease of Vmax/Km

Fig. 2. Relationships between ACP kinetic parameters and soil pH

or amorphous Fe.

269
Z. Wang et al.
for other tested soils were no more than 50%. The Vmax/Km for S9 soil
(Typic Gleyi-Stagnic Anthrosols) was the least affected with a decrease
less than 25%.
The Kic values ranged from 0.38 to 3.65 mM, indicating large var-
iation between soils (Table 3). The Kic for S9 soil (Typic Gleyi-Stagnic
Anthrosols) was the largest, followed by S7 soil (Hapli-Udic Iso-
humosols). The smallest value was for S5 soil (Argi-Udic Ferrosols)).
Both the former two soils had high content of SOM, but the latter had
the lowest, indicating that SOM may affect the affinity of As for ACP.
The Kiu values were from 0.84 to 7.43 mM (Table 3). The Kiu for al-
kaline soils (S10–S14) were larger than those for acid soils (S1–S9)
except for S4 soil (Hapli-Udic Ferralosols), suggesting that the combi-
nation of As with ES complex is more difficult in alkaline soils than in
acid soils. Furthermore, the Kiu was also greater than Kic for alkaline
soils, indicating that ESI complex was less stable than EI complex. For
acid soils, there was no identical relationship between Kiu and Kic, in-
dicating that the inhibition in acid soil is more complicated. In most soil
samples, the Kic values were smaller than the Km values in the absence
of As, indicating a higher affinity of As than substrate with soil ACP.
3.3. Ecological doses
Catalytic efficiency is an effective parameters assessing As toxicity
since it is very sensitive to As pollution. Two kinetic models described
previously were used to regress the relationship of catalytic efficiency
with As concentration. Both the two models showed good fitness
(Table 4). The model 2 had higher values of determination coefficient
(R2), suggesting a higher degree of fitting. The ED10 and ED50 values
ranged from 14.0 to 206.5 mg kg−1 and 126.1–1858.7 mg kg−1 for
270
Ecotoxicology and Environmental Safety 147 (2018) 266–274
Fig. 3. Kinetic properties of soil ACP in the presence of As.
Regressions are performed to depict the relationships between
kinetic parameters with As concentration. Regression lines are
drawn in each graph for each soil. For Vmax (a, b) and Vmax/Km (e,
f), model 2 is used; for Km (c, d), linear fitting is used.
model 1, and 5.7–43.1 mg kg−1 and 51.1–388.3 mg kg−1 for model 2
respectively. The ED values for model 2 were apparently lower than
those for model 1. The ED values of S5 (Argi-Udic Ferrosols) for the two
models were very low suggesting high toxicity of As. The S7-9 soils
(Hapli-Udic Isohumosols and Typic Gleyi-Stagnic Anthrosols) had low
ED values for model 2 but high values for model 1. Actually, the cat-
alytic efficiency for these soils was less affected by As than other soils
(Fig. 3e, f). This controversial result for the two models is caused by the
difference in calculation of ED values which was discussed in Speir et al.
(1999).
3.4. Factors influencing As toxicity to soil ACP
The results of correlation analysis suggested that there was a strong
positive correlation between ED50 and Kic, and both the two parameters
were positive correlated with SOM and CEC (Table 5). The Kiu was
significantly and positively correlated with pH and TP (Table 5).
However, the correlation analysis showed that none of these parameters
significantly correlated with amorphous Fe content and soil texture
(Table 5).
Stepwise regression analysis showed that Kic explained 94.7% of
variation in ED50, indicating that Kic was the determinative factor of As
toxicity to soil ACP (Fig. 4a). Among soil properties, SOM and CEC were
the major factor affecting As toxicity to soil ACP kinetics, explaining
28.5% and 72.4% of the total variation in ED50 respectively (Fig. 4c, d).
Furthermore, these two properties explained 37.5% and 69.3% of var-
iation in Kic respectively (Fig. 4e, f), indicating that SOM and CEC were
the major factors affecting the combination of As with soil ACP. The
variation in Kiu was mainly explained by soil pH and TP, suggesting the
Z. Wang et al. Ecotoxicology and Environmental Safety 147 (2018) 266–274

combination of As with enzyme-substrate complex was significantly

0.88 ± 0.10
4.19 ± 1.33
S14 affected by soil pH and TP.

4. Discussion
1.64 ± 0.32
5.30 ± 1.18

4.1. Mechanism of As inhibition on soil ACP

S13

Heavy metals inhibit enzyme activity in several ways: (1) by

2.17 ± 0.16
3.85 ± 0.52

masking catalytically active groups; (2) denaturing the protein con-

formation, or; (3) competing with metal ions that are needed to form
enzyme-substrate complexes (Karaca et al., 2010). The inhibition can
S12

be classified as competitive, noncompetitive, uncompetitive and mixed

inhibition in kinetics (Cornish-Bowden, 2012; Dick, 2011). Competitive
2.27 ± 0.41
5.01 ± 0.81

inhibitors are those that compete with the substrate for the active site of
the enzyme resulting in increase in Km but no change of Vmax. Non-
S11

competitive inhibitor reacts to the non-active site and generally causes

a change in the shape of the enzyme, resulting in a change of the ef-
2.33 ± 0.41
4.31 ± 0.83

fective concentration of the enzyme so that the Vmax value is decreased

(Cornish-Bowden, 2012; Dick, 2011). A mixed-model inhibitor is one
that exhibits both competitive and noncompetitive characteristics
S10

(Cornish-Bowden, 2012).
Our study showed that As inhibition on soil ACP varied among
3.65 ± 0.82
4.49 ± 1.11

different soil types. ACP in alkaline soils were less sensitive to As for the
lower inhibition of catalytic efficiency than in acid soils (Fig. 3e, f). The
S9

kinetic properties responded differently to As stress. In all the tested

soils, the Vmax of ACP decreased to different extents (Fig. 3a, b), sug-
2.26 ± 0.54
2.21 ± 0.50

gesting the rate of transformation of ES complex into enzyme and

product was inhibited by As. The enzyme affinity for substrate (Km) was
not affected in most acid soils, but decreased (increased Km) in alkaline
S8

soils (Fig. 3c, d). In majority of acid and neutral soils (S1–S9, except for
S4 and S5 soils), the Vmax decreased but Km did not change with As
3.10 ± 0.61
2.90 ± 0.39

addition, suggesting the inhibition was noncompetitive. While in al-

kaline soils (S10–S14) and the other 2 acid soils (S4 and S5), it showed
S7

mixed inhibition (including competitive and noncompetitive inhibition)

because the Vmax decreased and Km increased with the increase of As
1.55 ± 0.18
1.39 ± 0.22

concentrations (Cornish-Bowden, 2012). Since the inhibition of Vmax

was greater than the inhibition of Km by As in most soils, the non-
competitive inhibition type was the dominated one.
S6

Comparing the inhibition of As on pure ACP which is competitive or

0.38 ± 0.05
1.58 ± 0.49

mixed of competitive and noncompetitive inhibition (Vincent et al.,

1991), the kinetics of As inhibition on soil ACP varied. This may be due
to the immobilization effect of soil enzymes onto soil particles. The
S5

immobilization alters enzyme conformation structure which may result

in different response to heavy metal stresses (Huang and Shindo, 2000).
1.67 ± 0.10
7.43 ± 2.46

For instance, the kinetics and thermodynamics of mineral-immobilized

alkaline phosphatase (ALP) in response to As was different from the
S4

response of pure ALP and immobilization of enzyme on clay minerals

alleviated the inhibition of ALP activity by As (Wang et al., 2017).
2.40 ± 0.22
1.79 ± 0.45

In a previous work, we found that the type of As inhibition on soil

ALP is competitive or mixed inhibition (competitive dominated) (Wang
et al., 2017), which is different from the inhibition on soil ACP (non-
S3

competitive dominated) in this study. This suggests that the kinetics of

Inhibition constants of As inhibition on soil ACP.

different types of soil phosphatase respond differently to As pollution.

1.45 ± 0.23
2.09 ± 0.52

Soil ACP and ALP are the main types of phospahtase in acid and alka-
line soils respectively. Due to the difference in inhibition kinetics, the
S2

microbes in acid and alkaline soils may have different strategies to

Values are mean ± standard error.

adapt to As stress for phosphorus nutrition. In acid soils, the microbes

0.56 ± 0.05
0.84 ± 0.13

may synthesize and secrete more ACPs to soils to meet the need of
phosphorus nutrition. However, in alkaline soils, the microbes can se-
crete ALPs that have high affinity for substrate as well as synthesize
S1

more amount of phosphatase to meet the needs. So, analysis of the acid
and alkaline soils collected from long-term As contaminated fields
Soil sample

would provide further information.

Table 3

Kiu
Kic

271
Z. Wang et al. Ecotoxicology and Environmental Safety 147 (2018) 266–274

Table 4
Ecological doses for As inhibition on catalytic efficiency (Vmax/Km) of soil ACP.

Soil sample Model 1 Model 2

ED10 ED50 R2 F p ED10 ED50 R2 F p

S1 32.3 291.0 0.906 57.63 < 0.001 19.0 171.0 0.915 26.82 < 0.001
S2 74.0 666.0 0.913 62.57 < 0.001 19.7 177.0 0.964 67.31 < 0.001
S3 142.8 1285.6 0.946 104.21 < 0.001 28.1 252.5 0.978 111.57 < 0.001
S4 58.7 528.7 0.953 121.58 < 0.001 36.7 330.7 0.957 55.89 < 0.001
S5 14.0 126.1 0.931 80.95 < 0.001 6.6 59.8 0.958 56.57 < 0.001
S6 89.0 801.3 0.954 123.02 < 0.001 43.1 388.3 0.965 68.41 < 0.001
S7 178.7 1608.5 0.802 24.30 < 0.001 12.4 111.3 0.880 18.35 < 0.001
S8 113.4 1020.4 0.756 18.55 < 0.001 5.7 51.1 0.941 39.97 < 0.001
S9 206.5 1858.7 0.794 23.15 < 0.001 20.8 187.6 0.887 19.65 < 0.001
S10 133.8 1204.6 0.904 34.29 < 0.001 39.4 354.4 0.932 56.28 < 0.001
S11 129.2 1162.8 0.882 44.91 < 0.001 37.2 335.0 0.910 25.45 < 0.001
S12 91.7 825.4 0.966 170.92 < 0.001 43.0 387.0 0.977 107.70 < 0.001
S13 82.6 743.8 0.894 50.50 < 0.001 18.0 162.1 0.964 66.28 < 0.001
S14 47.6 428.7 0.977 258.24 < 0.001 26.9 242.5 0.987 193.50 < 0.001

ED10, ED50 represent As concentrations at where the catalytic efficiency decreased by 10% and 50% of its initial value respectively.

4.2. Kinetic parameter as index to assess As pollution
Soil enzyme activity is commonly used as index to assess heavy
metal pollution. However, there is a drawback for using this index. Soil
enzyme activity is determined on the basis of saturated substrate con-
centration which is far more higher than the substrate concentration in
natural soil (Burns et al., 2013). In competitive inhibition, increase of
substrate concentration can offset inhibition resulting in under-
estimation of heavy metal toxicity. The kinetic parameter of catalytic
efficiency (Vmax/Km) is an integrated index and independent of sub-
strate concentration and proved to be very sensitive to As (Fig. 3e, f).
The effect of the heavy metal on soil enzyme activity can be quantified
by determining the ecological dose parameter (Karaca et al., 2010). For
instances, ED10 and ED50, the concentration of heavy metal at which the
enzyme activity, or some other biological activity, is reduced by 10%
and 50% of its uninhibited value, are suitable indicators of heavy metal
pollutions (Karaca et al., 2010). The ED10 values calculated from the
inhibition of catalytic efficiency by As ranged from 14.0 to
206.5 mg kg−1 (model 1) (Table 4), which differentiate among dif-
ferent soil types with various soil properties. This also certified catalytic
efficiency was a sensitive and ideal index to assess As pollution.
As discussed above, the mechanisms of As inhibition on soil ACP
and ALP are different. The differences of inhibition in kinetics may
cause varied toxicity to soil ACP and ALP. For instance, we used the
same method to determine As toxicity on ALP in the same soils and got
the results that the ED50 for model 1 ranged from 66.9～335.6 mg kg−1
(data not published yet), which is lower than the values of this study
(126.1～1858.7 mg kg−1 for model 1). This suggests that As inhibition
on soil ACP is weaker than on soil ALP.
(Table 5, Fig. 4c, d). Several studies also confirmed that SOM and CEC
had certainly affected on heavy metal toxicity on soil enzymes (Moreno
et al., 2001; Speir et al., 1995; Tan et al., 2014; Xian et al., 2015). SOM
plays an important role in metal binding, control of metal solubility,
and mobility (Karaca et al., 2010; Martínez-Toledo et al., 2017). Soil
with high SOM content had huge buffer capacity to protect soil enzyme
against different interferences and provided a better nutritional con-
dition for microorganisms to resist ambient environmental changes
(Karaca et al., 2010; Tan et al., 2017b). Thus higher content of SOM
would alleviated heavy metal toxicity. In this study, S9 soil (Typic
Gleyi-Stagnic Anthrosols) had the highest SOM with a highest ED value
whereas the opposite for S5 (Argi-Udic Ferrosols) with the lowest ED
value (Tables 1, 4). CEC indicates soil cation exchange ability and also
plays an important role in heavy metal availability in soil. The sig-
nificant positive correlation between ED50 and CEC indicated a low As
toxicity in soils with high CEC value (Table 5). The strong positive
correlation between ED50 and Kic suggested that As toxicity on soil ACP
was determined by the ability of As combination with enzyme. Large Kic
value suggests a weak combination of enzyme-inhibitor (EI) complex,
resulting in a lower toxicity. Soil SOM and CEC were both significant
positively correlated to Kic (Table 5; Fig. 4e, f), revealing that these soil
properties may affect As toxicity to soil ACP through functioning on the
combination of As with enzyme. Many researches also revealed that pH
is a control factor affecting heavy metal toxicity on soil enzyme
(Martínez-Toledo et al., 2017; Tan et al., 2014). In this study, the cor-
relation between pH and ED50 was not significant. However, there was
significant positive correlation between pH and Kiu, suggesting the
dissociation of enzyme-substrate-inhibitor (ESI) complex was affected
by soil pH, which may influence the toxicity of As to soil ALP.

4.3. Influences of soil properties on As toxicity 5. Conclusions

The results of this study revealed that As toxicity to soil ACP eval- This study showed the kinetic properties of soil ACP in soils with
uated by ED values was strongly influenced by soil properties. varied properties and the changes of kinetics under exogenous As pol-
Correlation analysis and regression analysis suggested that SOM, and lution. The results suggested that soil ACP kinetic characteristics varied
CEC were the major soil factors affecting As toxicity to soil ACP among different soil types. The mechanism of As inhibition on soil ACP

Table 5
Pearson's correlation coefficients between soil properties and inhibition kinetic parameters.

pH SOM TP CEC Amorphous Fe Clay ED50 Kic

* **
ED50 0.135 0.628 0.134 0.824 0.314 0.053 1.000
Kic 0.171 0.710** 0.233 0.836** 0.328 0.017 0.973** 1.000
Kiu 0.536* 0.260 0.636* 0.001 −0.369 −0.237 0.159 0.291

SOM, soil organic matter; TP, total phosphorus; CEC, cation exchange capacity. R0.05 = 0.532, R0.01 = 0.661, n = 14. *, ** Significance at p < 0.05 and p < 0.01 level respectively.

272
Z. Wang et al.
belonged to noncompetitive or linear mixed inhibition depend on soil
types. The catalytic efficiency is a sensitive index to assess As toxicity.
Regression analysis showed soil SOM and CEC were the major con-
trolling factors affecting As toxicity to soil ACP (ED50 determined by
Kic), through affecting the combination of As with enzyme. This study
suggested that the kinetic properties of enzyme give more subtle in-
formation about enzyme-catalytic process changes in respond to heavy
metal pollutions and were necessary to derive toxicity of heavy metals
by the inhibition on soil enzyme kinetics.
Acknowledgement
This work was supported by the National Natural Science
Foundation of China [Nos. 41571245, 41603116]; the Basic Scientific
Research Foundation of Northwest A & F University [No. ZD2013012].
References
Bao, S., 2000. Soil Agro-chemistrical Analysis. China Agriculture Press, Beijing (in
Chinese).
Bhattacharyya, P., Tripathy, S., Kim, K., Kim, S.-H., 2008. Arsenic fractions and enzyme
activities in arsenic-contaminated soils by groundwater irrigation in West Bengal.
Ecotoxicol. Environ. Saf. 71, 149–156.
Bissen, M., Frimmel, F.H., 2003. Arsenic – a review. Part I: occurrence, toxicity, specia-
tion, mobility. Acta Hydrochim. Hydrobiol. 31, 9–18.
Burns, R.G., DeForest, J.L., Marxsen, J., Sinsabaugh, R.L., Stromberger, M.E., Wallenstein,
M.D., Weintraub, M.N., Zoppini, A., 2013. Soil enzymes in a changing environment:
273
Ecotoxicology and Environmental Safety 147 (2018) 266–274
Fig. 4. Relationships between ED50, Kic, Kiu and soil properties.
Data were log-transformed prior to analysis.
current knowledge and future directions. Soil Biol. Biochem. 58, 216–234.
Ciarkowska, K., 2015. Enzyme activities in soils contaminated with heavy metals in
varying degrees. In: Sherameti, I., Varma, A. (Eds.), Heavy Metal Contamination of
Soils: Monitoring and Remediation. Springer International Publishing, Cham, pp.
145–158.
Cornish-Bowden, A., 2012. Fundamentals of Enzyme Kinetics. John Wiley & Sons,
Weinheim.
Das, S., Jean, J.-S., Kar, S., Chakraborty, S., 2013. Effect of arsenic contamination on
bacterial and fungal biomass and enzyme activities in tropical arsenic-contaminated
soils. Biol. Fertil. Soils 49, 757–765.
Dick, W.A., 2011. Kinetics of soil enzyme reactions. In: Dick, R.P. (Ed.), Methods of Soil
Enzymology. Soil Science Society of America, Madison, WI, pp. 57–69.
Duker, A.A., Carranza, E.J.M., Hale, M., 2005. Arsenic geochemistry and health. Environ.
Int. 31, 631–641.
Fu, L., Yang, W., Wei, Y., 2009. Effects of copper pollution on the activity of soil invertase
and urease in loquat orchards. Chin. J. Geochem. 28, 76–80.
GB/T22105. 2, 2008. Soil Quality - Analysis of Total Mercury, Arsenic and Lead Contents
in Soils - Atomic Fluorescence Spectrometry - Part 2: Analysis of Total Arsenic
Contents in Soils. State Environmental Protection Administration of China.
Guan, S., 1986. Soil Enzyme and its Research Method. China Agricultural Press, Beijing
(in Chinese).
Huang, Q., Shindo, H., 2000. Effects of copper on the activity and kinetics of free and
immobilized acid phosphatase. Soil Biol. Biochem. 32, 1885–1892.
Karaca, A., Cetin, S.C., Turgay, O.C., Kizilkaya, R., 2010. Effects of heavy metals on soil
enzyme activities. In: Sherameti, I., Varma, A. (Eds.), Soil Heavy Metals. Springer-
Verlag, Heidelberg, pp. 237–262.
Kettler, T.A., Doran, J.W., Gilbert, T.L., 2001. Simplified method for soil particle-size
determination to accompany soil-quality analyses. Soil Sci. Soc. Am. J. 65, 849–852.
Koo, N., Lee, S.-H., Kim, J.-G., 2012. Arsenic mobility in the amended mine tailings and
its impact on soil enzyme activity. Environ. Geochem. Health 34, 337–348.
Lyubun, Y.V., Pleshakova, E.V., Mkandawire, M., Turkovskaya, O.V., 2013. Diverse ef-
fects of arsenic on selected enzyme activities in soil–plant–microbe interactions. J.
Hazard. Mater. 262, 685–690.
Z. Wang et al.
Mandal, B.K., Suzuki, K.T., 2002. Arsenic round the world: a review. Talanta 58,
201–235.
Marangoni, A.G., 2003. Enzyme Kinetics: A Modern Approach. John Wiley & Sons,
Hoboken, New Jersey.
Martínez-Toledo, Á., Montes-Rocha, A., González-Mille, D.J., Espinosa-Reyes, G., Torres-
Dosal, A., Mejia-Saavedra, J.J., Ilizaliturri-Hernández, C.A., 2017. Evaluation of en-
zyme activities in long-term polluted soils with mine tailing deposits of San Luis
Potosí, México. J. Soils Sediment. 17, 364–375.
Matyja, K., Małachowska-Jutsz, A., Mazur, A.K., Grabas, K., 2016. Assessment of toxicity
using dehydrogenases activity and mathematical modeling. Ecotoxicology 25,
924–939.
Moreno, J.L., García, C., Landi, L., Falchini, L., Pietramellara, G., Nannipieri, P., 2001.
The ecological dose value (ED50) for assessing Cd toxicity on ATP content and de-
hydrogenase and urease activities of soil. Soil Biol. Biochem. 33, 483–489.
Nannipieri, P., Giagnoni, L., Landi, L., Renella, G., 2011. Role of phosphatase enzymes in
soil. In: Bünemann, E., Oberson, A., Frossard, E. (Eds.), Phosphorus in Action.
Springer, Heidelberg, pp. 215–243.
Rao, M.A., Scelza, R., Acevedo, F., Diez, M.C., Gianfreda, L., 2014. Enzymes as useful
tools for environmental purposes. Chemosphere 107, 145–162.
Singh, R., Singh, S., Parihar, P., Singh, V.P., Prasad, S.M., 2015. Arsenic contamination,
consequences and remediation techniques: a review. Ecotoxicol. Environ. Saf. 112,
247–270.
Speir, T.W., Kettles, H.A., Parshotam, A., Searle, P.L., Vlaar, L.N.C., 1995. A simple kinetic
approach to derive the ecological dose value, ED50, for the assessment of Cr(VI)
toxicity to soil biological properties. Soil Biol. Biochem. 27, 801–810.
Speir, T.W., Kettles, H.A., Parshotam, A., Searle, P.L., Vlaar, L.N.C., 1999. Simple kinetic
approach to determine the toxicity of AS[V] to soil biological properties. Soil Biol.
Biochem. 31, 705–713.
274
Ecotoxicology and Environmental Safety 147 (2018) 266–274
Sumner, M.E., Miller, W.P., 1996. Cation exchange capacity and exchange coefficients. In:
Sparks, D.L., Page, A.L., Helmke, P.A., Loeppert, R.H. (Eds.), Methods of Soil Analysis
Part 3—Chemical Methods. Soil Science Society of America, American Society of
Agronomy, Madison, WI, pp. 1201–1229.
Tan, X., Kong, L., Yan, H., Wang, Z., He, W., Wei, G., 2014. Influence of soil factors on the
soil enzyme inhibition by Cd. Acta Agric. Scand. Sect. B-Soil Plant Sci. 64, 666–674.
Tan, X., Liu, Y., Yan, K., Wang, Z., Lu, G., He, Y., He, W., 2017a. Differences in the
response of soil dehydrogenase activity to Cd contamination are determined by the
different substrates used for its determination. Chemosphere 169, 324–332.
Tan, X., Wang, Z., Lu, G., He, W., Wei, G., Huang, F., Xu, X., Shen, W., 2017b. Kinetics of
soil dehydrogenase in response to exogenous Cd toxicity. J. Hazard. Mater. 329,
299–309.
Vincent, J.B., Crowder, M.W., Averill, B.A., 1991. Spectroscopic and kinetics studies of a
high-salt-stabilized form of the purple acid phosphatase from bovine spleen.
Biochemistry 30, 3025–3034.
Walkley, A., Black, I.A., 1934. An examination of the Edgtjareff method for determining
soil organic matter and a proposed modification of the chromic acid titration method.
Soil Sci. 37, 29–37.
Wang, Z.Q., Li, Y.B., Tan, X.P., He, W.X., Xie, W., Megharaj, M., Wei, G.H., 2017. Effect of
arsenate contamination on free, immobilized and soil alkaline phosphatases: activity,
kinetics and thermodynamics. Eur. J. Soil Sci. 68, 126–135.
Xian, Y., Wang, M., Chen, W., 2015. Quantitative assessment on soil enzyme activities of
heavy metal contaminated soils with various soil properties. Chemosphere 139,
604–608.
Zavišić, A., Nassal, P., Yang, N., Heuck, C., Spohn, M., Marhan, S., Pena, R., Kandeler, E.,
Polle, A., 2016. Phosphorus availabilities in beech (Fagus sylvatica L.) forests impose
habitat filtering on ectomycorrhizal communities and impact tree nutrition. Soil Biol.
Biochem. 98, 127–137.