ECOTOXICOLOGICAL EFFECTS OF COPPER AND SELENIUM COMBINED POLLUTIONON SOIL ENZYME ACTIVITIES IN PLANTED AND UNPLANTED SOILS

BIN HU, DONGLI LIANG,* JUANJUAN LIU, and JUNYU XIECollege of Natural Resources and Environment, Northwest A&F University, Key Laboratory of Plant Nutrition and the Agri-environment in Northwest

China, Ministry of Agriculture, Shaanxi Province, Yangling, China

(Submitted 18 September 2012; Returned for Revision 26 October 2012; Accepted 19 December 2012)

Abstract—The present study explored the joint effects of Cu and Se pollution mechanisms on soil enzymes to provide references for thephytoremediation of contaminated areas and agricultural environmental protection. Pot experiments and laboratory analyses were carriedout to study the individual and combined influences of Cu and Se on soil enzyme activities. The activities of four soil enzymes (urease,catalase, alkaline phosphatase, and nitrate reductase) were chosen. All soil enzyme activities tested were inhibited by Cu and Se pollution,either individually or combined, in varying degrees, following the order nitrate reductase > urease > catalase > alkaline phosphatase.Growing plants stimulated soil enzyme activity in a similar trend compared with treatments without plants. The joint effects of Cu and Seon catalase activity showed synergism at low concentrations and antagonism at high concentrations, whereas the opposite was observedfor urease activity. However, nitrate reductase activity showed synergism both with and without plant treatments. The half maximaleffective concentration (EC50) of exchangeable fractions had a similar trend with the EC50 of total content and was lower than that of totalcontent. The EC50 values of nitrate reductase and urease activities were significantly lower for both Se and Cu (p < 0.05), which indicatedthat they were more sensitive than the other two enzymes. Environ. Toxicol. Chem. 2013;32:1109–1116. # 2013 SETAC

Keywords—Combined pollution Cu Se Soil enzyme activities EC50

INTRODUCTION

Heavy metals enter soils mainly through atmosphericdeposition and the application of agrochemicals, mining,wastewater, and organic fertilizers [1]. The adsorption, desorp-tion, and interaction between different heavy metals can affecttheir distribution, bioavailability, and toxicity [2]. Therefore, theeffect of combined pollution depends on the constituents of themixture and may vary significantly [3]. According to a survey,the concentration of selenium (Se) in wastewater surroundingcopper (Cu) ores surpasses acceptable levels [4]. Furthermore,suspended solids in the atmosphere over the mining area containlarge amounts of Se, which enters the soil through wet and drydeposition [4]. As a result, Se and Cu content in the soil of themining area is far beyond the background value.

Copper is an enzymatic cofactor in several metabolicprocesses and an essential trace element for crop growth atlow concentrations [5]. However, it is also a common soilcontaminant. In recent years, Cu pollution has become moreserious because of Cu mining and the widespread use of feedadditives, fungicides, organic fertilizer, irrigation, and urbansewage-sludge compost [6]. High accumulations of total Cu inthe topsoil, far in excess of the natural background metalconcentrations, have been observed in planted soils [7]. Somestudies have been conducted on the effects of single Cu pollutionon soil enzyme activities [7,8].

Selenium is an essential metalloid trace element fororganisms, and deficiency of Se leads to Keshan and Kashin-Bek diseases [9]. Seleniummay be released into the environmentthrough natural processes, such as weathering of minerals, or by

anthropogenic activities, such as fossil-fuel combustion andindustrial, agricultural, and metallurgical processes, especiallyfrommining of sulfide ores [10]. Recent studies have shown thatsoils around coal mines and power plants are heavilycontaminated with Se, with content 50 times higher than thebackground standards [11]. Because of the narrow safety margindoses between nutrient and toxic, Se residues in soil can migrateand transform with the changes of soil physical and chemicalproperties, posing a potential danger to drinking water, soil, andeven the food chain [9].

There is strong evidence that heavy metals have toxic effectson soil microbial communities, microbial biomass, and soilenzyme activity [7]. Soil enzymes participate in all biochemicalreactions, material recycling, and energy metabolism in soil, andthe activity levels of enzymes reflect the direction and strength ofthe biochemical processes [12]. Soil enzyme activity is sensitiveto heavy metal contamination; thus, it is widely used as abiological indicator of soil health and in estimating the adverseeffects of various pollutants on soil quality [13,14]. Previousstudies found that Cu and Se could increase soil urease, catalase,and phosphatase activities at low concentrations but inhibit themat higher concentrations [15–17].

Plants affect soil enzyme activities in two different ways.First, plants affect the soil biota by influencing the quantity andquality of organic substrates that reach the soil [18]. The rootexudates and litter inputs of plants may influence soil micro-organisms, and extracellular enzymes are derived mainly fromsoil microorganisms, animals, and plant roots; thus, plants playimportant roles in soil enzyme activities [19]. Second, plantstake up some heavy metals from soil, which may result in thereduction of heavy metal toxicity on soil enzymes [15]. Previousstudies have used the dose–response model to calculate the halfmaximal effective concentration (EC50) between the totalconcentration of heavy metal and adenosine 5'-triphosphateconcentration, soil enzyme activities, or other soil

All Supplemental Data may be found in the online version of this article.� To whom correspondence may be addressed

(dlliang@nwsuaf.edu.cn).Published online 8 February 2013 in Wiley Online Library

(wileyonlinelibrary.com).

Environmental Toxicology and Chemistry, Vol. 32, No. 5, pp. 1109–1116, 2013# 2013 SETAC

Printed in the USADOI: 10.1002/etc.2152

1109

indexes [16,17]. It has been argued that the total concentration ofheavy metal in soil could not predict the ecotoxicological effectof heavy metals [12,20,21].

Many combined metal pollution studies have involved Cuand Cd; Cu, Pb, and Cd; and Pb and Cd metal cations [12–16].Varying degrees of antagonism or detoxification between Se andPb and between Cd, As, and Hg have been observed [22]; butlittle is known about the combined pollution of Cu and Se. Insoil, Cu exists predominantly as Cu2þ and Se mostly asSeO3

2�or SeO42�, which are the main forms absorbed by

plants [23]. Is there any interaction between Cu and Se? Doesthis interaction depend on metal concentration? What are theecological effects of Cu–Se interaction on their mobility,bioavailability, and toxicity?

Therefore, the aim of the present study was to investigate theindividual and joint effects of Cu and Se on four soil enzymeactivities in planted and unplanted soils to explore thebiological mechanisms to Cu and Se (cation–anion) combinedpollution. The EC50 value was calculated from the concen-trations of total and available heavy metals to establish the mostsensitive microbiological index to Cu and Se combinedcontamination.

MATERIALS AND METHODS

Experimental materialsAll reagents used in this study were of analytical grade. The

tested forms of Cu and Se were CuSO4 and Na2SeO3,respectively. The seeds of pakchoi (Brassica chinensis L.,Qinbai 2) were provided by Northwest A&F University SeedsCompany. Noncontaminated soil was collected at a depth of 0 to20 cm from the Northwest A&F University (34816'N, 108804'E)farm in Shaanxi Province, China. The basic physicochemicalproperties of the soil are shown in Table 1. These properties weredetermined according to the methods described by Bao [24].Soil pH was determined in water extracts using a soil-to-solutionratio of 1:2.5. Organic matter content was measured with hotK2Cr2O4 oxidation and FeSO4 titration. Cation exchangecapacity (CEC) was determined using the NH4OAC method,and the total content of soil heavy metal elements wasdetermined using atomic absorption spectroscopy after digestionwith aqua regia.

Experimental designThe soil was air-dried at room temperature, homogenized,

and allowed to pass through a 5-mm sieve. Chemical fertilizers,

including 100 mg kg�1 N (urea), 75 mg kg�1 P2O5 (calciumsuperphosphate), and 75 mg kg�1 K2O (potassium chlorine),were mixed thoroughly with 1.0 kg air-dried soil (bulk soil) inplastic pots (10 cm in height and 15 cm in diameter). A two-factor complete test was designed with four Cu2þ concentrations(0, 200, 400, and 800 mg kg�1 soil, added as CuSO4) and fourSe4þ concentrations (0, 2.5, 10, and 20 mg kg�1 soil, added asNa2SeO3), for a total of 16 treatments (Table 2). Solutions of Cuand Se were individually spiked in soil to set the contaminationlevels. The spiked soil was then aged for 14 d at 258C and 50%humidity. All treatments were arranged in a complete randomdesign with eight replicates, four planted with pakchoi and fourwithout. Ten pakchoi seeds were sowed in each pot (15 cm), andseedlings were thinned to five in each pot after 10 d. The soilmoisture content was kept at 70% water holding capacity (18%)by quantitative watering once every 2 d. Plants were harvestedafter 30 d.

Sampling and analysisSoil sampling and preparation. Soil samples were collected

from all treatments, with or without pakchoi. After the removalof plant and detrital materials, the soil samples were air-dried for1 to 2 d and ground. Part of each sample was sieved through 2-mm mesh for analysis of soil enzyme activity, and the other partwas ground to pass through a 0.15-mm mesh nylon sieve forchemical analyses of soil Cu and Se concentrations [24].

Total and available Se and Cu concentrations. The total Cuconcentration in the soil samples was analyzed by digesting thesoils with HCl (10 ml), HNO3 (15 ml), HF (5 ml), and HClO4

(10 ml) at 3008C. The total Se concentration was analyzed bydigesting the soils with HNO3:HClO4 (8 ml:2 ml, respectively)at 1708C. The concentration of Cu or Se was determined byatomic absorption spectroscopy or atomic fluorescence spec-troscopy at the corresponding wavelengths recommended by thespectrophotometer manufacturer [24].

The available Cu was determined by adding 1 g of soil and10 ml of 0.1 mol L�1 NH4HAc into a centrifuge tube. The tubewas shaken for 2 h at 258C and then centrifuged for 5 min. Theavailable Se was determined by adding 1 g of soil and 10 ml of0.7 mol L�1 KH2PO4 in a centrifuge tube. The tube was shakenfor 1 h at 258C and then centrifuged for 5 min. The supernatantfluid of Cu or Se was determined by the same method as total Cuand Se concentration.

Table 1. Basic physicochemical properties of unpolluted soil

Property Value

pH 7.95CEC 23.34 Cmol kg�1

Clay 39.5%CaCO3 54.99 g kg�1

Organic matter 16.33 g kg�1

Amorphous Fe 845.3 mg kg�1

Complex Al 25.4 mg kg�1

Complex Fe 24.0 mg kg�1

Cu 25.56 mg kg�1

Se 0.221 mg kg�1

Zn 56 mg kg�1

Cd 0.02 mg kg�1

Cr 47 mg kg�1

Hg 0.02 mg kg�1

As 4.4 mg kg�1

Pb 19.4 mg kg�1

Table 2. Heavy metal content in single and combined pollution treatments

Treatment

Concentration (mg kg�1)

Cu Se

CK 0 0Cu200 200 0Cu400 400 0Cu800 800 0Se2.5 0 2.5Cu200, Se2.5 200 2.5Cu400, Se2.5 400 2.5Cu800, Se2.5 800 2.5Se10 0 10Cu200, Se10 200 10Cu400, Se10 400 10Cu800, Se10 800 10Se20 0 20Cu200, Se20 200 20Cu400, Se20 400 20Cu800, Se20 800 20

1110 Environ. Toxicol. Chem. 32, 2013 B. Hu et al.

Soil enzyme activity. Soil catalase, urease, alkaline phospha-tase, and nitrate reductase activities were measured by themethods of Guan [25]. Soil catalase activity was measured usingthe potassium permanganate (KMnO4) titration method. Soilsamples (5 g) were added to 40 ml deionized water and 5 ml0.3% hydrogen peroxide solution. The mixture was incubatedfor 20 min at 378C. After incubation, the reaction was stoppedby adding 5 ml of 1.5 mol L�1 sulfuric acid. The mixture wasfiltered and titrated using 0.1 mol L�1 KMnO4. The amount of0.1 mol L�1 KMnO4 consumed per gram of dried soil was usedto measure catalase activity [25].

Urease activity was measured by mixing 5 g air-dried soilsamples, 1 ml toluene, 10 ml urea solution (10%), and 20 mlcitrate buffer (pH 6.7) in a reaction flask. The mixture wasincubated for 24 h at 378C. The indophenol colorimetric methodwas used to measure the NH4

þ released by enzymatic hydrolysisof urea. Indophenol was determined colorimetrically at 578 nm.Controls were prepared without substrate to determine theammonium produced in the absence of urea. This method wasbased on the determination of NH4

þ released and expressed asmilligrams of NH3–N per kilogram hourly [25].

Alkaline phosphatase activity was measured by mixing 5 gsoil, 1 ml toluene, 5 ml 0.675% p-nitrophenyl disodiumphosphate, and 5 ml borate buffer (pH 10) in hermeticallysealed flasks and incubating for 12 h at 378C. The mixture wasfiltered and determined by colorimetry at a wavelength of570 nm. Soil alkaline phosphatase activity was expressed asmicrograms of p-nitrophenol produced per gram of soil [25].

Nitrate reductase activity was measured by mixing 1 g soilsamples, 1 ml 0.8 mmol L�1 2,4-dinitrophenol solution, 1 ml0.05% KNO3 solution, 1 ml 1% glucose solution, and 7 mlanaerobic deionized water. After incubation for 24 h at 308C, themixture was filtered and analyzed colorimetrically at awavelength of 520 nm. Activity was expressed as milligramsof NO2�–N per kilogram daily [25].

Control tests without soils or substrates were carried out toevaluate the spontaneous or abiotic transformation of substratesin all enzyme activities analyzed. The results were expressed asan average of three replicates in analysis.

Data analysisJoint effect. Scientific information on qualitative statements

of the joint effect of heavy metals on soil enzyme activity is veryscarce. According to Bliss’s definition [26], we confirmed thejoint effects of Cu and Se on soil enzyme activities by a newparameter,DT, which was calculated by determining the enzymeactivity inhibition rate (T) (Eqn. 1); then, DT can be obtainedfrom Equation 2

T ¼ ð1� Ei

ECKÞ � 100% ð1Þ

DT ¼ TCuþSe � TCu � TSe ð2Þ

where T is the inhibition ratio of every treatment, Ei is theenzyme activity in single or combined pollution, and ECK isactivity in unpolluted soil.

If DT ¼ 0, the joint effect of Cu and Se is additive, whereasDT > 0 and DT < 0 represent synergism and antagonism,respectively.

The EC50 value. The EC50 value is useful in describing thetoxicity of heavy metals (Eqn. 3). In addition, EC50 could beused to establish the microbiological index most sensitive toheavy metal contamination [27]. The mathematical equation for

the dose–responsemodel is widely used for the parameters testedto analyze heavy metal pollution [28]

y ¼ A

1þe½pðlogX��logEC50Þ� ð3Þ

where y is the response variable, X is the exogenous heavymetal concentration, A is the uninhibited value of y, and p is theslope factor [28]. This logistic curve model represents therelationship between the enzyme activity measured and theconcentration of toxicant.

Statistical analysis. All results were reported as the mean offour replicates, and all data were subjected to two-way analysis ofvariance using SPSS 13.0 statistical software (SPSS). The leastsignificant difference test was used to detect significant differ-ences among the means of the different treatments (p < 0.05).

RESULTS

Effects of Cu and Se combined pollution on soil enzyme activityCatalase activity. In single pollution (Fig. 1A), increasing soil

Se progressively reduced catalase activity by 6.4, 5.1, and 11.6%from 2.5 to 20 mg kg�1 Se. The addition of Cu (200–800 mg kg�1) further reduced catalase activity by 12.4, 19.3,and 25.4% under Cu 200, 400, and 800 mg kg�1, respectively.

In combined Cu and Se pollution, when the Se concentrationsin soil were the same, the inhibition rate of catalase activity

a

a

a

a

b

b b a

ab

ab

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Cu0 Cu200 Cu400 Cu800

Cal

atas

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(ml/

0.1

mol

L-1

KM

nO4)

Treatment (mg kg-1)

Planted Se0 Se2.5 Se10 Se20

A

a a a

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c c a

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nO4)

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Unplanted

Se0 Se2.5 Se10 Se20

B

Fig. 1. Effects of Cu and Se combined pollution on soil catalase activity inplanted (A) and unplanted (B) soil. Data are given as means � standarddeviation, and different letters represent 0.05 significance level.

Ecotoxicological effects of Cu and Se combined pollution on soil enzyme activity Environ. Toxicol. Chem. 32, 2013 1111

increased with increasing exogenous Cu concentration. Themaximum inhibition rate reached 30.1% in the Cu800 þ Se10treatment. For Cu 200 and 400 treatments, the catalase activityshowed an upward trend after the drop trend, with the highestpeak at the Se20 treatment. For Cu 800 treatment, the catalaseactivity increased with Se concentration. Catalase activity in soilwithout plants was slightly lower than in soil with plants andfollowed the same trend as planted soil (Fig. 1B). The interactionof Cu and Se on soil catalase was synergism when Cu was200 mg kg�1 and antagonism at Cu �400 mg kg�1 for plantedsoil (except Cu400 þ Se10). In contrast, it was synergism at Cu�400 mg kg�1 and antagonism when Cu was 800 mg kg�1 forunplanted soil (Table 3).

Urease activity. Under Cu pollution alone, the Cu treatmentsof 200, 400, and 800 mg kg�1 significantly reduced ureaseactivity by 31.5, 38.1, and 59.6%, respectively, compared withthe control (p < 0.05). Under Se pollution alone, the 2.5, 10, and20 mg kg�1 treatments decreased urease activity by 4.4, 5.4, and3.6%, respectively (Fig. 2A).

In combined pollution when the soil Se concentrations werethe same, increasing exogenous Cu concentrations inhibitedurease activity, with a maximum inhibition rate of 60.0%(Cu800 þ Se2.5). At Cu �400 mg kg�1, urease activity initiallyincreased, then declined, with the highest inhibition seen at theSe2.5 treatment; when Cu concentration was 800 mg kg�1, thehighest urease activity was at the Se20 treatment. The groupswithout plants showed slightly lower rates than those with plantsand followed the same trend as those with plants (Fig. 2B). Theeffect on urease activity in unplanted soil represented antago-nism but represented synergism at high concentrations in plantedsoil (Table 3).

Alkaline phosphatase activity. In single pollution (Fig. 3A),increasing soil Se progressively reduced catalase activity by 3.8,11.2, and 12.1% from 2.5 to 20 mg kg�1 Se. The addition of Cu200, 400, and 800 mg kg�1further reduced catalase by 15.4,36.6, and 20.6%, respectively.

Under the combined pollution treatments when the Se soilconcentrations were the same, increasing exogenous Cuconcentration inhibited alkaline phosphatase activity, with themaximum inhibition rate at 41.3% (Cu800 þ Se2.5). On the otherhand, when the Cu concentrations were the same, alkalinephosphatase activity declined after an initial increase, with thelowest at the Se10 treatment. The groups without plants showedslightly lower rates than the groups with plants and followed thesame trend as that in treatments with plants (Fig. 3B). Thetreatments at Cu400 and Se10 showed antagonism on soil alkalinephosphatase activity in soils with and without plants; the other

treatments produced synergism (Table 3). The treatments atCu400 and Se10 showed antagonism on soil alkaline phosphataseactivity in both planted and unplanted soils; the other treatmentsproduced synergism.

Nitrate reductase activity. As seen in Figure 4A, both singleand combined pollution strongly inhibited soil nitrate reductaseactivity. Under Cu pollution alone, the Cu treatments of 200,400, and 800 mg kg�1 in soil significantly reduced nitrate

Table 3. DT values of four soil enzymes under combined pollutiona

DT(%)Treatment

Catalase Urease Alkaline phosphatase Nitrate reductase

Planted Unplanted Planted Unplanted Planted Unplanted Planted Unplanted

Cu200 Se2.5 4.45 � 0.22 6.89 � 0.34 �9.04 � �0.45 �12.49 � �0.62 10.12 � 0.50 12.82 � 0.64 �15.06 � �0.75 3.85 � 0.19Se10 3.70 � �0.04 1.45 � 0.16 �0.47 � �0.20 �2.20 � �0.12 �9.1 � �0.51 �20.54 � �0.60 �22.7 � �0.76 �9.54 � �1.11Se20 �6.51 � �0.52 6.83 � �0.39 1.26 � 0.16 �7.39 � �0.25 13.35 � 1.15 10.17 � 1.44 �34.08 � �0.92 �17.67 � �0.03

Cu400 Se2.5 �0.76 � 0.18 3.31 � 0.07 �4.01 � �0.02 �2.46 � �0.11 �10.27 � �0.45 �12 � �1.02 �15.32 � �1.13 �22.29 � �0.47Se10 3.01 � 0.15 �2.26 � �0.11 10.27 � 0.51 8.69 � 0.43 �21.53 � �1.07 �20.14 � �1 �30.23 � �1.51 �35.73 � �1.78Se20 �4.89 � �0.43 5.25 � �0.94 7.81 � 0.31 �2.18 � �0.40 �15.37 � �0.78 �14.24 � �0.43 �33.91 � �1.59 �26.45 � �0.24

Cu800 Se2.5 �10.47 � �0.32 �7.95 � 0.34 3.25 � 0.06 �5.1 � �0.36 23.03 � 0.66 28.89 � 1.51 �18.58 � �1.71 �0.77 � �0.88Se10 �8.72 � �0.24 �18.97 � 0.26 6.32 � 0.39 �8.02 � �0.11 �15.75 � �0.76 �8.74 � �0.71 �31.95 � �1.69 �4.87 � �1.32Se20 �16.35 � �0.81 �8.82 � �0.44 0.79 � 0.03 �14.98 � �0.74 13.64 � 0.68 16.27 � 0.81 �40.03 � �2.03 �10.26 � 0.51

aData are given as mean � standard error; when DT¼ 0: additive, DT> 0: synergism, DT< 0: antagonism.

a

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Ure

ase

(m

g N

H4+ –

N k

g–1 h

–1)

Treatment (mg kg-1)

Planted

Se0 Se2.5 Se10 Se20

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ase

(m

g N

H4+ –

N k

g–1 h

–1)

Treatment (mg kg-1)

Unplanted

Se0 Se2.5 Se10 Se20

B

Fig. 2. Effects of Cu and Se combined pollution on soil urease activity inplanted (A) and unplanted (B) soil. Data are given as means � standarddeviation, and different letters represent 0.05 significance level.

1112 Environ. Toxicol. Chem. 32, 2013 B. Hu et al.

reductase activity by 26.5, 69.3, and 86.1%, respectively,compared with the control (p < 0.05). Under Se pollution alone,the 2.5, 10, and 20 mg kg�1 treatments decreased nitratereductase activity by 10.2, 20.7, and 34.9%, respectively(p < 0.05).

Under the combined pollution treatments, when the soil Seconcentrations were the same, increasing exogenous Cuconcentration inhibited nitrate reductase activity, with themaximum inhibition rate at 89.4% (Cu800 þ Se20). When theCu concentrations were the same, nitrate reductase activitydecreased after the initial upward trend, with the highest at theSe2.5 and Se10 treatments. The groups without plants showedslightly lower rates than the groups with plants, following thesame trend as that in treatments with plants (Fig. 4B). The effectof combined Cu and Se on soil nitrate reductase was antagonismin treatments both with and without plants (Table 3).

EC50 value of four enzymes in soil with and without plantsThe concentrations of total and available forms of the heavy

metals were used to calculate the EC50 value (Table 4). TheEC50 values for the activities of the following three enzymeswere in the order of nitrate reductase < urease < catalase.Alkaline phosphatase activity could not fit Equation 3 at a

significant level (p < 0.05). The same trend was found for theEC50 value on total and available Se and Cu contents, but theEC50 values were much smaller for both available Cu and Seconcentrations compared with the total heavy metal content. TheEC50 value of catalase and nitrate reductase increasedwith the Seconcentration in both soils with and without plants. Themaximum value of catalase activity was achieved at the Se20treatment, whereas that of nitrate reductasewas at Se10. The EC50value of catalase increased with Se concentration in both soilswith andwithout plants. The EC50 values inmost treatments withplants were higher than in those without (by 4–11%).

DISCUSSION

Soil enzyme activities under single and combined pollutionHeavy metal in soils can inhibit enzyme activity through

many pathways: (1) by reducing the production of enzymesthrough its toxic effect on soil microflora, (2) by combining withthe active groups of the enzyme, (3) through complexation of thesubstrate, and (4) by reacting with the enzyme–substratecomplex [28]. However, different metals affect soil enzymesin different ways. Soil catalase, alkaline phosphatase, and nitratereductase activities diminished with increasing Cu or Seconcentrations. These findings are in agreement with earlierreports that soil enzymatic activities diminished with the risingof available concentrations of Cu and Se [18,23]. Moreover, soilurease activity was also inhibited with rising Cu concentration,but no significant inhibition was observed for Se-amendedsoil [8,15]. The significant inhibition of Cu could be explained asabove, while the insignificant effects of Se may be due to the factthat Se could replace sulfur in the unstable active centers of theseenzymes. Thus, varied speed or degrees of the disintegration ofthe enzyme substrate complex may lead to different effects onenzymatic reactions.

Combined heavymetal pollution is more complex than singleheavy metal pollution. Combined Cu and Se pollution producedvarying degrees of influence on the four soil enzymes ranked inthe following order: nitrate reductase > urease > catalase >alkaline phosphatase. According to recent research, urease,catalase, and alkaline phosphatase activities are significantlyinhibited by Cu [7,29] and nitrate reductase and urease activitiesare sensitive to Se pollution [15]. Accordingly, nitrate reductaseand urease activities are more sensitive to Cu and Se combinedpollution than the other two enzymes. Catalase is an intracellularenzyme that is involved in microbial oxidoreductasemetabolism [30]. Our finding is in contrast with the results ofBelyaeva et al. [31], who found that catalase activity was notmarkedly inhibited by Cu at concentrations ranging from 0 to100 mg kg�1, which were much lower than ours (0–800 mg/kg). Copper is an essential trace element and could enhance plantgrowth at lower concentrations, but it is toxic and inhibits plantgrowth at high concentrations [5]. Hinojosa et al. [32] alsoobserved that phosphatase was less sensitive at tracing heavymetal effects than the others. The observations on catalase andphosphatase could be explained as these soil enzymes actingdifferently under different heavy metal concentrations and indifferent soils.

Joint effects under Cu and Se combined pollutionBliss [26] first put forward the definition of joint effects

between numerous pollutants, which were described as additive,antagonistic, or synergistic. Zhou introduced an equationdescribing the joint effect of urease activity under Cd and Hgpollution, which has been quoted by other researchers [13,14].

a

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Alka

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phos

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ase

(m

g PN

P kg

-1 h

-1)

Treatment (mg kg-1)

Planted

Se0 Se2.5 Se10 Se20

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a

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ase

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Treatment (mg kg-1)

Unplanted

Se0 Se2.5 Se10 Se20

B

Fig. 3. Effects of Cu and Se combined pollution on soil alkaline phosphataseactivity in planted (A) and unplanted (B) soil. Data are given as means �standard deviation, and different letters represent 0.05 significance level.

Ecotoxicological effects of Cu and Se combined pollution on soil enzyme activity Environ. Toxicol. Chem. 32, 2013 1113

However, the previous studies mostly concentrated on thetoxicity of combined pollution, ignoring the relative change ratiocompared with the control treatment, and scientific informationon the joint effect of heavy metals on soil enzyme activity is veryscarce. The present study confirmed a new modified equation(Eqn. 2), based on the equation used by previous researchers, inwhich the rate of enzyme activities in all treatments (includingcontrol) was considered. The DT values in Table 3 are mostlynegative, which means that the major joint effect under Cu and

Se combined pollution is antagonistic. This is similar to the resultfound between Cu, Ag, Hg, Zn, Ni, Pb, Cd, and Cr [22].

However, some combinations of metals exhibit a synergisticeffect, while others do not. Khan et al. [33] investigated soilenzyme activities (catalase, alkaline phosphatase, and dehydro-genase) under Cd and Pb combined pollution, in which asynergistic effect was observed. Liu et al. [34] found thatantagonistic effects may be ascribed to reductions in ion activityin the medium when Cd and As are both present, therebyreducing bioavailability. The interaction between two metalson soil colloids, such as adsorption–adsorption, oxidation–reduction, and coordination–chelation, could alter the speciation ofmetals and affect bioavailability and toxicity [28]. In the presentstudy, Cu was added as Cu2þ, while Se was mostly SeO3

2�.Varying degrees of antagonism or detoxification between Se andPb or Cd, As, and Hg have been described [23]; therefore, theantagonistic effect of combined Cu and Se on soil enzymeactivities could also be explained in this way.

Sensitive indicators under Cu and Se combined pollutionIt has been concluded that the EC50 value may be a sensitive

tool for assessing ecotoxicological effects to soil biochemicalparameters [16]. Previous studies have used the model tocalculate the EC50 between the total concentration of heavymetal and soil enzyme activities or other soil indexes [14,16,27].It has been argued that the total concentration of heavy metal insoil could not predict the environmental effect of heavy metal,which is related to other factors (pH, CEC, texture) that influencebioavailability [6]. The extractable fraction of heavy metals isconsidered to represent a more bioavailable fraction of metals insoil, so the concentration of this fraction has been used forecological risk assessment in recent studies [35]. In the presentstudy, the same trend was found for the EC50 values for total andavailable Se and Cu contents, which showed that nitratereductase and urease were the most sensitive among the foursoil enzymes.

Considering that catalase is sensitive to Cu pollution,previous research has suggested it as a monitoring indicator ofCu pollution. However, it was not sensitive to Cu and Secombined pollution in the present study (Fig. 1). Alkalinephosphatase showed resistance under heavy metal stress andcould not fit the equation provided to quantify enzymesensitivity [32]. The EC50 value of urease was higher thanthat of nitrate reductase, but urease activity, which has beenshown by many authors, was sensitive to both single Cu and Sepollution [7,15,29]. Thus, soil urease and nitrate reductase aremore sensitive to Cu and Se combined pollution.

Table 4. Median effective concentration (EC50) values of four enzymes in planted and unplanted soil

Treatment

Catalase Urease Alkaline phosphatase Nitrate reductase

Total Available Total Available Total Available Total Available

PlantedSe0 1,610 � 59 342 � 21 592 � 25 74 � 9 NS NS 123 � 2 6 � 1Se2.5 26,257 � 7,670 5,428 � 411 557 � 38 71 � 3 NS NS 137 � 1 9 � 2Se10 9,850 � 2,674 2,793 � 181 484 � 12 52 � 2 NS NS 135 � 2 8 � 3Se20 4,725 � 522 1,371 � 9 542 � 7 71 � 3 NS NS 178 � 7 16 � 2

UnplantedSe0 1,355 � 96 306 � 19 505 � 16 64 � 5 NS NS 205 � 25 14 � 3Se2.5 7,721 � 1,617 3,452 � 338 525 � 59 70 � 2 NS NS 207 � 2 18 � 1Se10 256,721 � 478 599,011 � 687 404 � 18 40 � 3 NS NS 266 � 26 22 � 2Se20 8,657 � 827 2,689 � 68 527 � 11 65 � 2 NS NS 243 � 40 24 � 1

NS ¼ not significant.

a

b

c d

b

a

b b

c

ab

a a

d

ab

bc c

0

20

40

60

Cu0 Cu200 Cu400 Cu800

Nitr

ate

redu

ctas

e

(mg

NO

2-N

kg-

1 d-1

)

Treatment (mg kg-1)

Planted

Se0 Se2.5 Se10 Se20

A

a

a

b a

a

b

a

a

ab

b

a

b

b

b

b c

0

20

40

60

Cu0 Cu200 Cu400 Cu800

Nitr

ate

redu

ctas

e

(mg

NO

2-N

kg-1

d-1

)

Treatment (mg kg-1)

Unplanted

Se0 Se2.5 Se10 Se20

B

Fig. 4. Effects of Cu and Se combined pollution on soil nitrate reductaseactivity in planted (A) and unplanted (B) soil. Data are given asmeans � standard deviation, and different letters represent 0.05 significancelevel.

1114 Environ. Toxicol. Chem. 32, 2013 B. Hu et al.

Effect of soil enzyme activities with and without plantsCompared with unplanted soil, soils with plants have been

reported to have higher rates of microbial activity due to thepresence of additional surfaces for microbial colonization and tothe organic compounds released by the plant roots [36]. Variousstudies have frequently confirmed the release of specific sugarsand amino acids into the rhizosphere by plants, which couldstimulate the colonization of beneficial rhizobacteria [12,31,37].In the present study, the enzyme activities, as well as EC50values, in soil with plants were higher than those without in mosttreatments. This is consistent with the results of Gao et al. [14],who found that soil enzyme activities in treatments with plantswere significantly higher than those in treatments without plantsunder the combined stress of Cd and Pb. Li et al. [38] showedthat soil with grass had increased activities of urease, acidphosphatase, alkaline phosphatase, and phenol oxidase. Wanget al. [36] found a similar phenomenon in the rhizosphere of aCu-accumulator plant.

This could be explained as follows: On the one hand, rootscould secrete certain substances that could form a microrhizo-sphere environment and provide carbon and nitrogen tomicroorganisms [18]; on the other hand, the organic acidssecreted from roots under heavy metal stress would affect thephysical and chemical properties of soil (pH and ion activity) andsubsequently alter bioavailability [2,28]. It is suggested thatstudies on soil enzyme activity under heavy metal stress shouldnot be focused only on the incubation conditions; instead, theeffects of crops on soil enzyme activity should also be considered.

CONCLUSIONS

The activities of the four soil enzymes studied were allinhibited by single Cu or Se and Cu and Se combined pollutionin different degrees, ordered as nitrate reductase > urease >catalase > alkaline phosphatase. The joint effects of Cu and Seon catalase activity showed synergism at low concentrations andantagonism at high concentrations, whereas it was the oppositefor urease activity. However, only synergism was exhibited fornitrate reductase activity regardless of the presence or absence ofcrops. The EC50 values of nitrate reductase activity and ureaseactivity were significantly lower (p < 0.05). Therefore, thesetwo enzyme activities are more sensitive to Cu and Se combinedpollution in soil.

To be a monitoring indicator, more studies need to be done ondifferent soil types, plant species, and pollution types. Inaddition, further research is needed to improve the currentunderstanding of the mechanisms underlying the differentconcentrations of Se and Cu in soils.

SUPPLEMENTAL DATA

Table S1. (250 KB DOC).

Acknowledgement—The present study was supported by the NationalNatural Science Foundation of China (41171379) and the InnovativeResearch Team Program of Northwest A&F University.

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