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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,
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
Global Centre for Environmental Research, The Faculty of Science and Information Technology, University of Newcastle, University Drive, Callaghan, NSW 2308,
Cooperative Research Centre for Contamination Assessment and Remediation of the Environment (CRC CARE), Mawson Lakes, SA 5095, Australia


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).

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

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

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. Ecotoxicology and Environmental Safety 147 (2018) 266–274

provide information of enzyme intrinsic properties. Exploring the ki- using the pipette method (Kettler et al., 2001). Soil organic matter
netic characteristics of soil enzymes can provide a mechanistic under- (SOM) was determined by potassium dichromate digesting and ferrous
standing of how various biological processes occur and are regulated in sulfate titration method (Walkley and Black, 1934). Total P was di-
heavy metal contaminated soils (Dick, 2011). For instance, after gested with melting sodium hydroxide and determined using mo-
studying the kinetics of soil enzyme in loquat orchards polluted by Cu lybdenum blue colorimetric method (Bao, 2000). Cation exchange ca-
pollution, Fu et al. (2009) found that urease Vmax and Vmax/Km were pacity (CEC) was determined with ammonium acetate pH 7.0 (Sumner
negatively correlated while invertase Km was positively correlated with and Miller, 1996). Amorphous Fe oxide was extracted by ammonium
Cu concentration, indicating smaller amount of soil urease and lower oxalate and determined by flame atomic absorption spectrometry (Bao,
affinity of invertase for substrate in Cu polluted soil than in un- 2000). The soil samples were digested using nitrohydrochloric acid and
contaminated soil. background total arsenic was determined by atomic fluorescence
Soil phosphorus cycling is one of the most important biological spectroscopy method (AFS) (GB/T, 22105.2, 2008).
processes in soil environment. Soil phosphatase catalyzes the hydrolysis
of ester–phosphate bonds, breaking down the organic phosphorus into 2.2. Experimental design
inorganic form which is easily taken up by plants or microorganisms
(Nannipieri et al., 2011). Therefore, phosphatase plays an important Heavy metal reactions with enzyme are rapid processes. The
role in phosphorus cycling. Soil phosphatases are classified in acid changes in proliferation and enzyme synthesis by microbes can mask
phosphatase, neutral phosphatase and alkaline phosphatase according the toxicity if measured over a long period of exposure to heavy metal.
to the optimum pH (Nannipieri et al., 2011). Phosphatases are sensitive Thus a shorter exposure time (e.g., 30 min) was suitable for de-
to heavy metals (Nannipieri et al., 2011). Many studies have suggested termining the toxicity of heavy metal to soil enzyme (Matyja et al.,
that As has inhibitory effect on soil phosphatase activity (Bhattacharyya 2016).
et al., 2008; Das et al., 2013; Speir et al., 1999; Wang et al., 2017). Three gram of air-dried soils were spiked with 3 ml As solutions
Phosphatase activity then has been used as a bio-indicator to assess As (Na3AsO4·12H2O,AR, Sinopharm Chemical Reagent Co., Ltd, Shanghai
toxicity (Nannipieri et al., 2011; Speir et al., 1999). However, these China) to obtain final soil As concentrations of 0, 10, 25, 50, 100, 200,
studies mainly focused on enzyme activities, the processes of As com- 400 and 600 mg kg−1 soil (Weight(soil): Volume(As)=1:1) in a 50 ml
bination with enzyme and inhibition mechanism have not been re- flask then the flask were shaken to homogenize the soils and solutions.
searched thoroughly. Enzyme kinetics reflects the process of a reaction. After 30 min of equilibration, the soil ACP activity was measured.
Identification of changes for kinetic properties of soil phosphatase Triplicate samples were prepared for each treatment.
under As pollution could help in understanding how As effect on soil
phosphatase and clarifying its environmental exposure risks (Dick, 2.3. Determination of soil ACP activity
2011). Furthermore, the toxicity based on enzyme activity may vary
with the substrate type and concentration used to measure the enzyme Soil ACP activity was determined by the following procedure (Guan,
activity (Tan et al., 2017a, 2017b). In addition, soil properties can in- 1986; Zavišić et al., 2016): 20 ml disodium phenyl phosphate (1, 2.5, 5,
fluence As toxicity to soil phosphatase (Speir et al., 1999). The complex 10 mM) prepared in pH 5.0 acetate buffer were added to the soil mix-
soil properties may affect the inhibition kinetics and reaction process tures that treated with As in the flasks, then the flasks were capped and
resulting in different toxicity of As to soil phosphatase. To our knowl- incubated at 37 °C for 4 h. After incubation, the released phenol was
edge, the kinetic characteristics of soil phospahtase-catalyzed reaction determined colorimetrically at 510 nm, using 4-aminoantipyrine and
in response to As pollution and the influence of soil properties on As potassium ferricyanide as coloring reagents. A substrate control
toxicity have not been characterized clearly. Thus, it is of importance to (without soil) and an optional abiotic control (without substrate) were
clarify the mechanism and reaction process of As inhibition on soil simultaneously incubated. The concentration of phenol was calculated
phosphatase to get an better understanding of soil quality and phos- using a standard curve (Guan, 1986).
phorus cycling in As contaminated soils.
Therefore, this study has focused on soil acid phosphatase (ACP, EC 2.4. Data analysis because of its wide spread presence in acid and neutral soils
and in a certain amounts of alkaline soils from decomposition of plant 2.4.1. Soil enzyme kinetics without As treatment
debris (Nannipieri et al., 2011). We investigated the kinetic properties The two kinetic parameters, the Michaelis constant (Km) and max-
of soil ACP under exogenous As stress using soil samples representing imum reaction velocity (Vmax) were determined by nonlinear regression
14 different agriculture soil types in China. The objectives of this study of Michaelis-Menton equation:
were to 1) investigate the reaction process and mechanism of As in-
V = Vmax S /(Km + S ) (1)
hibition on soil acid phosphatase among various soils, and 2) determine
the feasibility of using kinetic parameters to assess As toxicity and 3) Where V is soil ACP activity, S is substrate concentration.
reveal how soil properties affect As toxicity to soil ACP through enzyme When Km and Vmax were determined, the catalytic efficiency was
kinetic study. calculated by the ratio of Vmax/Km.

2. Materials and methods 2.4.2. Soil enzyme kinetics with As treatment

Kinetic parameters will change at the presence of heavy metal in-
2.1. Soil sampling and physicochemical analysis hibitors. Inhibitors of single substrate soil enzyme-catalyzed reactions
can often be grouped as either simple competitive (unchanged Vmax but
Fourteen uncontaminated farmland soils with varied properties increased Km), simple noncompetitive (decreased Vmax but unchanged
were sampled from different provinces in China (Fig. 1). The soils were Km) inhibitors (Dick, 2011), and a linear mixed (decreased Vmax and
selected to be representative of the major soil types and the distribution increased Km) inhibitors (Cornish-Bowden, 2012). The competitive in-
of soil pH of agricultural soils in China. Soil samples were taken from 0 hibition constant (Kic) can be estimated by a linear plot of
~20 cm depth and air-dried in the laboratory. Soils were ground and app
Km/appVmax against As concentration (Eq. (2)), thus Kic = intercept/
passed through a 1 mm nylon sieve before use. Soil physical chemical slope (Cornish-Bowden, 2012).
properties were determined using methods illustrated in Bao (2000) app
K m/app Vmax = Km/ Vmax + Km/ Vmax *I / Kic (2)
and presented in Table 1. Briefly, soil pH was determined by a pH
app app
electrode with a water: soil ratio of 2.5:1. Soil texture was measured 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 fitted, an ecological dose (ED) could be calculated. For instance, ED50,
equilibrium constant of enzyme-inhibitor (EI) complex. In non- the concentration of an inhibitor that causing 50% inhibition, equals to
competitive and mixed inhibition, the noncompetitive inhibition con- 1/b for model 1 and (1–a/b)/(b–a) for model 2. Since ED50 value is too
stant (Kiu) was calculated by rearrangement of following equation: large for environmental pollution assessment, a lower dose of ED10 (the
app concentration of an inhibitor causing 10% inhibition) which would be
V max = Vmax /(1 + I / Kiu ) (3)
more meaningful is also calculated (Speir et al., 1999).
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 2.4.4. Statistical analysis
and Kiu) are important factors determining the toxicity of an inhibitor to The goodness of fit of Michaelis-Menton model and dose-response
enzyme. The Kic and Kiu could represent the affinity of an inhibitor to model was examined by F test. Linear regression also was performed to
enzyme or enzyme-substrate (ES) complex (Marangoni, 2003). depict the relationships between kinetic parameters and As concentra-
tion where it was appropriate. Pearson correlation analysis was used to
2.4.3. Calculation of ecological dose reveal the relationship between soil properties and kinetic parameters
Two models are used to fit the dose-response relationship between of As inhibition on soil ACP. Stepwise regression analysis was also
kinetic parameters and As concentrations (Speir et al., 1999): carried out to investigate the relationships between soil properties and
ED50 values. All analyses were done using SPSS 18.0 software (SPSS
y = c /(1 + bx ) model 1 (4)
Inc., Chicago, USA).
y = c (1 + ax )/(1 + bx ) model 2 (5)
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

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.

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.

Z. Wang et al. 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.

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


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

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-

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

(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

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

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

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.

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

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

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-

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

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

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


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

Z. Wang et al. 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.

belonged to noncompetitive or linear mixed inhibition depend on soil current knowledge and future directions. Soil Biol. Biochem. 58, 216–234.
Ciarkowska, K., 2015. Enzyme activities in soils contaminated with heavy metals in
types. The catalytic efficiency is a sensitive index to assess As toxicity. varying degrees. In: Sherameti, I., Varma, A. (Eds.), Heavy Metal Contamination of
Regression analysis showed soil SOM and CEC were the major con- Soils: Monitoring and Remediation. Springer International Publishing, Cham, pp.
trolling factors affecting As toxicity to soil ACP (ED50 determined by 145–158.
Cornish-Bowden, A., 2012. Fundamentals of Enzyme Kinetics. John Wiley & Sons,
Kic), through affecting the combination of As with enzyme. This study Weinheim.
suggested that the kinetic properties of enzyme give more subtle in- Das, S., Jean, J.-S., Kar, S., Chakraborty, S., 2013. Effect of arsenic contamination on
formation about enzyme-catalytic process changes in respond to heavy bacterial and fungal biomass and enzyme activities in tropical arsenic-contaminated
soils. Biol. Fertil. Soils 49, 757–765.
metal pollutions and were necessary to derive toxicity of heavy metals Dick, W.A., 2011. Kinetics of soil enzyme reactions. In: Dick, R.P. (Ed.), Methods of Soil
by the inhibition on soil enzyme kinetics. 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.
This work was supported by the National Natural Science GB/T22105. 2, 2008. Soil Quality - Analysis of Total Mercury, Arsenic and Lead Contents
Foundation of China [Nos. 41571245, 41603116]; the Basic Scientific in Soils - Atomic Fluorescence Spectrometry - Part 2: Analysis of Total Arsenic
Contents in Soils. State Environmental Protection Administration of China.
Research Foundation of Northwest A & F University [No. ZD2013012]. Guan, S., 1986. Soil Enzyme and its Research Method. China Agricultural Press, Beijing
(in Chinese).
References 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
Bao, S., 2000. Soil Agro-chemistrical Analysis. China Agriculture Press, Beijing (in enzyme activities. In: Sherameti, I., Varma, A. (Eds.), Soil Heavy Metals. Springer-
Chinese). Verlag, Heidelberg, pp. 237–262.
Bhattacharyya, P., Tripathy, S., Kim, K., Kim, S.-H., 2008. Arsenic fractions and enzyme Kettler, T.A., Doran, J.W., Gilbert, T.L., 2001. Simplified method for soil particle-size
activities in arsenic-contaminated soils by groundwater irrigation in West Bengal. determination to accompany soil-quality analyses. Soil Sci. Soc. Am. J. 65, 849–852.
Ecotoxicol. Environ. Saf. 71, 149–156. Koo, N., Lee, S.-H., Kim, J.-G., 2012. Arsenic mobility in the amended mine tailings and
Bissen, M., Frimmel, F.H., 2003. Arsenic – a review. Part I: occurrence, toxicity, specia- its impact on soil enzyme activity. Environ. Geochem. Health 34, 337–348.
tion, mobility. Acta Hydrochim. Hydrobiol. 31, 9–18. Lyubun, Y.V., Pleshakova, E.V., Mkandawire, M., Turkovskaya, O.V., 2013. Diverse ef-
Burns, R.G., DeForest, J.L., Marxsen, J., Sinsabaugh, R.L., Stromberger, M.E., Wallenstein, fects of arsenic on selected enzyme activities in soil–plant–microbe interactions. J.
M.D., Weintraub, M.N., Zoppini, A., 2013. Soil enzymes in a changing environment: Hazard. Mater. 262, 685–690.

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

Mandal, B.K., Suzuki, K.T., 2002. Arsenic round the world: a review. Talanta 58, Sumner, M.E., Miller, W.P., 1996. Cation exchange capacity and exchange coefficients. In:
201–235. Sparks, D.L., Page, A.L., Helmke, P.A., Loeppert, R.H. (Eds.), Methods of Soil Analysis
Marangoni, A.G., 2003. Enzyme Kinetics: A Modern Approach. John Wiley & Sons, Part 3—Chemical Methods. Soil Science Society of America, American Society of
Hoboken, New Jersey. Agronomy, Madison, WI, pp. 1201–1229.
Martínez-Toledo, Á., Montes-Rocha, A., González-Mille, D.J., Espinosa-Reyes, G., Torres- Tan, X., Kong, L., Yan, H., Wang, Z., He, W., Wei, G., 2014. Influence of soil factors on the
Dosal, A., Mejia-Saavedra, J.J., Ilizaliturri-Hernández, C.A., 2017. Evaluation of en- soil enzyme inhibition by Cd. Acta Agric. Scand. Sect. B-Soil Plant Sci. 64, 666–674.
zyme activities in long-term polluted soils with mine tailing deposits of San Luis Tan, X., Liu, Y., Yan, K., Wang, Z., Lu, G., He, Y., He, W., 2017a. Differences in the
Potosí, México. J. Soils Sediment. 17, 364–375. response of soil dehydrogenase activity to Cd contamination are determined by the
Matyja, K., Małachowska-Jutsz, A., Mazur, A.K., Grabas, K., 2016. Assessment of toxicity different substrates used for its determination. Chemosphere 169, 324–332.
using dehydrogenases activity and mathematical modeling. Ecotoxicology 25, Tan, X., Wang, Z., Lu, G., He, W., Wei, G., Huang, F., Xu, X., Shen, W., 2017b. Kinetics of
924–939. soil dehydrogenase in response to exogenous Cd toxicity. J. Hazard. Mater. 329,
Moreno, J.L., García, C., Landi, L., Falchini, L., Pietramellara, G., Nannipieri, P., 2001. 299–309.
The ecological dose value (ED50) for assessing Cd toxicity on ATP content and de- Vincent, J.B., Crowder, M.W., Averill, B.A., 1991. Spectroscopic and kinetics studies of a
hydrogenase and urease activities of soil. Soil Biol. Biochem. 33, 483–489. high-salt-stabilized form of the purple acid phosphatase from bovine spleen.
Nannipieri, P., Giagnoni, L., Landi, L., Renella, G., 2011. Role of phosphatase enzymes in Biochemistry 30, 3025–3034.
soil. In: Bünemann, E., Oberson, A., Frossard, E. (Eds.), Phosphorus in Action. Walkley, A., Black, I.A., 1934. An examination of the Edgtjareff method for determining
Springer, Heidelberg, pp. 215–243. soil organic matter and a proposed modification of the chromic acid titration method.
Rao, M.A., Scelza, R., Acevedo, F., Diez, M.C., Gianfreda, L., 2014. Enzymes as useful Soil Sci. 37, 29–37.
tools for environmental purposes. Chemosphere 107, 145–162. Wang, Z.Q., Li, Y.B., Tan, X.P., He, W.X., Xie, W., Megharaj, M., Wei, G.H., 2017. Effect of
Singh, R., Singh, S., Parihar, P., Singh, V.P., Prasad, S.M., 2015. Arsenic contamination, arsenate contamination on free, immobilized and soil alkaline phosphatases: activity,
consequences and remediation techniques: a review. Ecotoxicol. Environ. Saf. 112, kinetics and thermodynamics. Eur. J. Soil Sci. 68, 126–135.
247–270. Xian, Y., Wang, M., Chen, W., 2015. Quantitative assessment on soil enzyme activities of
Speir, T.W., Kettles, H.A., Parshotam, A., Searle, P.L., Vlaar, L.N.C., 1995. A simple kinetic heavy metal contaminated soils with various soil properties. Chemosphere 139,
approach to derive the ecological dose value, ED50, for the assessment of Cr(VI) 604–608.
toxicity to soil biological properties. Soil Biol. Biochem. 27, 801–810. Zavišić, A., Nassal, P., Yang, N., Heuck, C., Spohn, M., Marhan, S., Pena, R., Kandeler, E.,
Speir, T.W., Kettles, H.A., Parshotam, A., Searle, P.L., Vlaar, L.N.C., 1999. Simple kinetic Polle, A., 2016. Phosphorus availabilities in beech (Fagus sylvatica L.) forests impose
approach to determine the toxicity of AS[V] to soil biological properties. Soil Biol. habitat filtering on ectomycorrhizal communities and impact tree nutrition. Soil Biol.
Biochem. 31, 705–713. Biochem. 98, 127–137.