Ecotoxicology DOI 10.1007/s10646-014-1196-8
Risk assessment of petroleum-contaminated soil using soil enzyme activities and genotoxicity to Vicia faba Jun Ma • Jinglong Shen • Qingxing Liu • Fang Fang • Hongsheng Cai • Changhong Guo
Accepted: 16 January 2014 Ó Springer Science+Business Media New York 2014
Abstract Pollution caused by petroleum is one of the most serious problems worldwide. To better understand the toxic effects of petroleum-contaminated soil on the microflora and phytocommunity, we conducted a comprehensive field study on toxic effects of petroleum contaminated soil collected from the city of Daqing, an oil producing region of China. Urease, protease, invertase, and dehydrogenase activity were significantly reduced in microflora exposed to contaminated soils compared to the controls, whereas polyphenol oxidase activity was significantly increased (P \ 0.05). Soil pH, electrical conductivity, and organic matter content were correlated with total petroleum hydrocarbons (TPHs) and a correlation (P \ 0.01) existed between the C/N ratio and TPHs. Protease, invertase and catalase were correlated with TPHs. The Vicia faba micronucleus (MN) test, chromosome aberrant (CA) analyses, and the mitotic index (MI) were used to detect genotoxicity of water extracts of the soil. Petroleum-contaminated samples indicated serious genotoxicity to plants, including decreased index level of MI, increased frequency of MN and CA. The combination of enzyme activities and genotoxicity test via Vicia faba can be used as an important indicator for assessing the impact of TPH on soil ecosystem. Keywords Petroleum-contaminated soil Soil enzyme activities Genotoxicity Vicia assay Total petroleum hydrocarbon
J. Ma J. Shen Q. Liu F. Fang H. Cai C. Guo (&) Key Laboratory of Molecular and Cytogenetics, Heilongjiang Province, College of Life Science and Technology, Harbin Normal University, Harbin 150025, China e-mail: [email protected]
Introduction Soil contamination with petroleum-derived products has becoming an increasingly frequent ecological problem, and this pollution usually exerts adverse effects on organisms indirectly by taking toxic chemicals in the soil. The petroleum-derived products contain large amounts of benzene, phenol and polycyclic aromatic hydrocarbons that can show potential mutagenic and carcinogenic effects on humans and pollute the environment (Gestel et al. 2003; Zhao et al. 2012a, b). Oil seeping into the soil not only changes its physical and chemical properties, but also reduces its productivity, such as qualities, compositions of organic components, water-holding capacity as well as air exchange caused by filling of soil pores (Gogoi et al. 2003; Singh et al. 2005). Soil health plays an important role in terrestrial ecosystem and has profound effect on sustainable development. Petroleum contaminants introduced into the soil not only modify physicochemical properties, which includes the pH, oxygen and nutrient availability of soil, but also alter the populations and activities of soil microflora at different functional levels (Wyszkowska and Kucharski 2000; Maliszewska-Kordybach et al. 2007). Enzymes are responsible for numerous metabolic reactions in microbial cells and, accordingly, their inhibition or activation could be the underlying cause of toxicity (Coleman 1992). Soil enzyme activities have great potential to provide a unique integrative biological assessment of soils and the possibility of assessing the health of the soil biota (Alkorta et al. 2003). Many studies have demonstrated that oil pollutants can influence the enzyme activities of soil, and among which, the oxidoreductase (e.g., dehydrogenase and catalase) and hydrolase (e.g., urease and invertase) activities could be
123
J. Ma et al.
served as a symbol that reflect the degree of soil degradation because of their sensibility to pollution (Stanislaw et al. 2004; Achuba and Peretiemo-Clarke 2008). However, some researchers argued that it had limitations when soil enzymes were considered as indicators for assessed soil pollution, thus, the use of soil enzyme activities as indicators for assessing the degree of soil pollution is not enough, different biological indicators are certainly needed (Trasar-Cepeda et al. 2000; Gil-Sotres et al. 2005). Genotoxicity assays are used specifically to evaluate the genotoxic potential of environmental and industrial effluent samples (Cotelle et al. 1999; Leme et al. 2008; Howcroft et al. 2011, Justino et al. 2012). High plants are recognized as excellent indicators of cytogenetic and mutagenic effects of environmental chemicals and applicable for detection of environmental mutagens (Grant 1999; Wang et al. 2010). Micronucleus test and chromosomal aberration assay are common methods for genetic toxicity assessment. A number of studies show that Vicia faba is appropriate for pursuing the genotoxicity evaluation of the contaminated soil (Be´kaert et al. 1999; Lah et al. 2008; Marcato-Romain et al. 2009), and it has more advantages than the other plants (e.g., Tradescantia paludosa and Allium cepa) (Cotelle et al. 1999). Daqing oil filed, which is one of the largest oil fields in the world, accounts for nearly 25 % of China’s oil production (Tang et al. 2010). However, environmental risk caused by petroleum contamination in this region has not been well investigated. In the present study, a comprehensive ecotoxicological testing of petroleum-contaminated soil from Daqing region in northeastern China was carried out. Soil quality and the main soil enzyme activities were evaluated, and correlations between these factors and TPHs were also generated by performing regression analysis. In addition, Vicia faba micronucleus (MN) test, Chromosome aberrant (CA) analyses and the Mitotic index (MI) were performed to detect the potential genotoxicity of water extracts of the soil.
Materials and methods Soil sample collection and preparation Nine surface soil sub-samples (\20 cm depth), each, were randomly collected from 4 different Petroleum-contaminated sites in the Daqing Oil Field Third Factory Area (a Daqing oil field located in Daqing City, Heilongjiang Province, China) in 2010. These four sites are: S0 (the control, no TPH-polluted industrial soil), S1 (saline-alkali meadow), S2 (a secondary pollution area of oil pipeline leakage), and S3 (an oil polluted central area with historical TPH-polluted industrial soil). To minimize the possibility of
123
cross-contamination, disposable sample equipment was used. Samples were taken from the same depth (about 20 cm depth) of soil. A spade was used to dig a v-shaped hole, and a uniform thick slice of soil from bottom to top of the exposed soil surface was selected and placed in a bucket. Samples were transported to the laboratory and stored in a refrigerated at 4 °C prior to experimental analysis. Physicochemical properties determination A soil sub-sample (approximately 1 kg) was air-dried, passed through a 2 mm sieved and subjected to chemical characterization (Methods of Soil Analysis 1996; Shao et al.2012). Soil pH was determined in a soil: deionised water suspension (1:2.5 w/v), electrical conductivity (EC) in a soil:deionised water suspension (1:5 w/v), and salt content (SC) was measured gravimetrically in a soil:water (1:5 w/v) suspension.Soil organic matter (SOM) was determined by dichromate oxidation and total nitrogen (TN) and available nitrogen (AN) were analyzed using the Kjeldahl method. Total phosphorous (TP) was measured colorimetrically as molybdo-vanadate phosphoric acid. Available phosphorous (AP) and available potassium (AK) were determined using the Egner-Riehm method (Riehm 1958). Total petroleum hydrocarbons were measured following USA EPA 3550 (US EPA Method 3550). All measurements were carried out in triplicate. Soil enzyme activity assays All soil samples were kept refrigerated (4 °C) at their field moisture content. Before analysis, samples were sieved through a 2 mm sieve, and their dry matter content was determined in order to express the enzyme activity on a dry matter basis. Drying soil at 105 °C for 24 h, soil field moisture content was determined on each sample, dry matter content was determined gravimetrically by soil field moisture content. The activity of the following enzymes was determined: urease (Zantua and Bremner 1975), protease (Ladd et al. 1976), invertase (Ohshima et al. 2007), dehydrogenase (Tabatabai 1982), catalase (Achuba and Peretiemo-Clarke 2008) and polyphenol oxidase (Perucci et al. 2000). All measurements were carried out in triplicate. Genotoxicity test Soil aqueous extracts was prepared following the methods described by Marcato-Romain et al. (2009), and modified as follows: 100 g dry soil sample and 500 ml distilled water were mixed and stirred at room temperature for 24 h. The mixture was centrifuged at 10,0009g for 20 min and then the supernatant was stored at 4 °C in the dark.
Risk assessment of petroleum-contaminated soil
as ‘‘not detected’’, were assumed to be equal to the detection limit. Pearson correlation coefficients (r) were calculated between soil physicochemical properties and their enzyme activity. All statistical analysis was performed using SPSS for Windows, version 18.0 (IBM SPSS, Inc., Chicago, 2010).
In the current study, faba beans (Vicia faba.L. cv. Songzi peel, 2n = 12) were used as the test system to evaluate genotoxicity. Faba beans, provided by CCNU (Central China Normal University, Wuhan City, Hubei Province, China), were selected for their sensitivity to environmental genotoxicity (Ma et al. 1995). Fava bean seeds were treated with 10 % NaClO solution for 12 min and grown in distilled water in plates for 24 h. Germinated beans were grown in beakers at 25 ± 0.5 °C in an incubator (EYELA Eyelatron FLI-301NH, Japan). When the length of the primary roots was 2 cm, the beans were exposed to soil aqueous extracts for 24 h followed by a 24 h recovery period and excised. For each experiment, six plants were used as six independent replicates per treatment. Each experiment was carried out in triplicate. The root tips were immersed in Carnoy’s solution (3:1 ethanol: glacial acetic acid) for 24 h, and then stored in 70 % ethanol at 4 °C prior to slide preparation. After being hydrolyzed with 1 M HCl at 60 °C for 10 min, the root rips were rinsed and Feulgen stained. To prepare the slides, the meristematic regions were coated with coverslips and carefully squashed into a drop of 45 % acetic acid. The coverslips were removed using liquid nitrogen and the slides were mounted in synthetic resin for further analysis. All slides were analyzed under light microscopically (Leica, Lelica Microsystems ltd., Germany), first with lowpower magnification and then with high-power (409 or 1009). The MN, CA and MI were scored. The MI and CA frequencies were expressed in percent, while MN frequencies were expressed per 1,000 cells. The scores were carried out according to standard protocols described by Ma et al. (1995) and Guo et al. (2010). Six slides were prepared for each of the six seeds (one slide per root tip) and at least 1,500 cells were counted per root tip. The MN frequency was obtained from at least nine thousand cells per treatment.
Concentrations of TPH were below detection limits in S0 samples and TPH concentration were significant greater (P \ 0.05) in S2 and S3 soil samples compared to S1 soil samples (Table 1). The SOM content was greater (P \ 0.05) in S2 and S3 soil compared to S0 soil, but was not significantly different from SOM in S1 soil. The S1, S2 and S3 soils had significantly greater pH, EC values, C/N ratio and SC values (Table 1) compared with S0 soil (P \ 0.05), while Total P and Available N, P, and K content were significantly decreased compared with S0 soil (P \ 0.05). Urease, protease, invertase, and dehydrogenase activities were reduced in S1, S2, and S3 soils compared to S0 soil (P \ 0.05). Catalase activity was greater in S2 and S3 soil but similar in S1 soil compared to S0 soil (P \ 0.05) and polyphenol oxidase activity was significantly increased in S1, S2, and S3 soils compared to S0 soil (P \ 0.05) (Fig. 1). Chemical parameters (pH, EC, SOM and SC) and TPH concentration were positively correlated (P \ 0.05) and the C/N ratio and TPH concentration was highly positively correlated (P \ 0.01) (Table 2). The protease and invertase activities and TPH concentration were negatively correlated (P \ 0.05) while catalase and TPH concentration were positively correlated (P \ 0.05).
Statistical analysis
Genotoxicity to Vicia faba
Microsoft Office Excel 2007 (Microsoft Corporation, USA) was used to randomize treatment assignments, to enter and store data, to sort data and prepare for statistical analysis. All data were checked for homogeneity of variance and normality (Kolmogorov–Smirnov test) and, differences among treatments were evaluated using a one-way ANOVA. Data not satisfying assumptions for ANOVA were analyzed non-parametrically using Kruskal–Wallis ANOVA by Ranks test. Whenever significant differences were found (P \ 0.05), a post hoc Tukey Honest Significant Difference (HSD) test was used to further elucidate differences among means (P \ 0.05). For statistical purposes, results below the detection limit, although reported
Micronucleus and abnormal mitotic phenomena were observed in Vicia root cells in different stages of the mitosis cycle including interphase, prophase, metaphase, anaphase and telophase using microscopic examination. Micronucleus were one of main aberration types observed in the current study (Fig. 2 a–c). Chromosome break (Fig. 2 f), chromosome loss (Fig. 2 d, g) and chromosome bridge (Fig. 2 h) were main aberrations observed, while chromosome adherence and unoriented chromosomes at anaphase cells (Fig. 2 e, f) were less often observed. The MI % of S1 soils was not significantly different from S0 soils; however, was significantly reduced (P \ 0.05) in S2 and S3 soils. The MN % was significant
Results Changes in soil physicochemical properties and enzyme activities
123
J. Ma et al. Table 1 Characteristics of soil at the different sites used in the study EC (lS cm-1)
SOM (g kg-1)
TN (g kg-1)
C/N
687 ± 13.60a
3.02 ± 0.13a
1.84 ± 0.05d
0.96 ± 0.06a
0.71 ± 0.05d
916.5 ± 13.47
b
2.9 ± 0.11
a
c
b
0.37 ± 0.06a
1043.25 ± 25.22
c
3.21 ± 0.12
b
0.65 ± 0.05
c
3.16 ± 0.12
0.44 ± 0.06b
0.59 ± 0.04a
4.73 ± 0.20d
0.51 ± 0.10c
Station
pH
S0
7.85 ± 0.07a
S1
8.25 ± 0.11
b
S2
8.65 ± 0.12
c
S3
9.06 ± 0.05d
1352.32 ± 8.64d
5.28 ± 0.18c
0.89 ± 0.06
b
TP (g kg-1)
1.24 ± 0.17
Station
AN (mg kg-1)
AP (mg kg-1)
AK (mg kg-1)
SC (%)
TPH (g kg-1)
S0
146.00 ± 9.42d
84.00 ± 6.76c
235.97 ± 14.25d
0.58 ± 0.07a
ND
S1 S2
128.00 ± 11.59c 56.00 ± 10.03b
31.10 ± 4.28b 15.50 ± 3.37a
137.24 ± 12.44b 162.80 ± 9.97c
0.92 ± 0.09b 1.25 ± 0.17c
0.02 ± 0.02a 11.85 ± 0.59b
S3
37.00 ± 6.16a
12.70 ± 2.49a
112.40 ± 11.93a
3.15 ± 0.19d
23.54 ± 0.41c
The used data mean and standard deviation presentation Means within each column marked with the same letter are not significantly different (Tukey HSD test, P [ 0.05) EC electrical conductivity, SOM soil organic matter, TN total nitrogen, C/N soil organic carbon and total nitrogen ratio, TP total phosphorous, AN available nitrogen, AP available phosphorous, AK available potassiumm, SC salt content, TPH pseudo-total petroleum hydrocarbons content, ND not detected
Fig. 1 Soil enzyme activities of the samples from four different sites in Daqing. a Urease activity; b protease activity; c invertase activity; d dehydrogenase activity; e catalase and f polyphenol oxidase
123
Risk assessment of petroleum-contaminated soil Table 2 Pearson’s correlation coefficients between soil physicochemical properties and their enzyme activities pH
EC
SOM
TN
C/N
TP
EC
0.988**
OM
0.817
0.870
1.000
TN
-0.886
-0.857
-0.492
1.000
0.882
-0.749
1.000
0.019
0.815
-0.229
C/N
0.968*
AN
AP
AK
1.000
0.953*
TP
-0.463
-0.442
AN
-0.965*
-0.920*
-0.749
0.825
-0.972*
0.353
1.000
AP
-0.892
-0.860
-0.497
1.000**
-0.759
0.805
0.837
AK
-0.835
-0.867
-0.606
0.915*
-0.682
0.776
0.680
0.930*
-0.169
-0.828
-0.654
-0.737
0.996**
-0.150
-0.949*
-0.701
-0.644
SC
0.905*
TPH
0.946*
0.948* 0.939*
0.981**
-0.650
0.915*
-0.691
1.000 1.000 0.907*
1.000
Ure
-0.895
-0.894
-0.580
0.981**
-0.753
0.797
0.790
0.978*
0.975*
Prot
-0.975*
-0.932*
-0.738
0.860
-0.966*
0.412
0.998**
0.871
0.722
Inver
-0.994**
-0.979*
-0.845
0.836
-0.990**
0.367
0.979*
0.844
0.771
DHA
-0.977*
-0.942*
-0.676
0.949*
-0.914*
0.591
0.961*
0.955*
0.837
CAT PPO
0.966* 0.978*
0.931* 0.941*
0.805 0.679
-0.787 -0.943*
0.989** 0.919*
-0.799 -0.950*
-0.666 -0.827
SC
TPH
Ure
Prot
Invert
SC
1.000
TPH
0.945*
-0.286 -0.579 DHA
-0.725
-0.705
1.000
Prot
-0.827
-0.939*
0.828
Inver
-0.918*
-0.974*
0.839
0.982*
DHA CAT
-0.795 0.869
-0.875 0.974*
0.925 -0.765
0.977* -0.990*
0.960* -0.986*
1.000 -0.942
0.880
-0.918
-0.980*
-0.962*
0.947
0.796
CAT
PPO
1.000
Ure
PPO
-0.995** -0.965*
1.000 1.000 1.000 -1.000**
1.000
Marked correlations are significant at: *P \ 0.05; **P \ 0.01 EC electrical conductivity, OM soil organic matter, TN total nitrogen, C/N soil organic carbon and total nitrogen ratio, TP total phosphorous, AN available nitrogen, AP available phosphorous, AK available potassium, SC salt content, TPH pseudo-total petroleum hydrocarbons content, Ure urease, Prot protease, Inver invertase, DHA dehydrogenase, CAT catalase, PPO polyphenol oxidase
less (P \ 0.05) in S0 soils compared to soils collected at the other 3 sites, while the CA % decreased significantly (P \ 0.05) in S0 soils (Table 3).
Discussion In the present study, by taking petroleum-contaminated soil from Daqing region in Northeast China, the physicochemical properties, enzyme activities and genoticixity to Vicia faba were investigated. Enzyme activities are commonly used as indicators of soil health because they play an important role in nutrient cycling (Gianfreda et al. 2005), and can be sensitive indicators of pollution with hydrocarbons, heavy metals or pesticides (Bayer et al. 1982; Dick 1997; Top et al. 1999, Margesin et al. 2000a, Baran et al. 2004, Yao et al. 2012). Protease is the enzyme that catalyses protein hydrolysis to
peptides and aminoacids (Alef and Nannipieri 1995), and urease catalyses the hydrolysis of urea to carbon dioxide and ammonium, and it is widely distributed in microorganisms, plants and animals (Nannipieri et al. 2002), these enzymes are both involved in the N-cycle. Invertase is known to be a very stable and persistent enzyme and its association with soil components is well documented (Kiss et al. 1978; Sannino and Gianfreda 2001), it is involved in process of C-cycle in the soil. Invertase can facilitate the hydrolysis process of the decomposition of organic matter, which suggests their important roles in increasing the soluble nutrients in soil. The present study showed suppressive trends of these enzyme activities, and the inhibition is probably due to changes in the molecular structure of the enzyme caused by the inhibitors as illustrated by former studies (Verrhiest et al. 2002; Kolesnikov et al. 2007; Wyszkowska and Wyszkowski 2010). For petroleum hydrocarbons, it is assumed that they may denature the
123
J. Ma et al.
Fig. 2 Microphotograph of the fava bean root tip aberrant cells induced by the petroleum-contaminated soil at different stages of mitosis. a Micronucleated cell in interphase; b MN of small size in interphase; c MN in prophase; d anaphase with chromosome loss and
unoriented chromosome; e disturbed anaphase with unoriented chromosomes; f chromosome break; g anaphase with chromosome loss; h chromosome bridge and late-separating chromosomes in telophase. Scale in figure 10 lm
Table 3 Genotoxic effects on Vicia faba tip cells of the soil at different sites
activity, which was followed by a subsequent increase in enzymatic activity, possibly due to adaptation of the indigenous microorganisms. In some cases, a stimulatory activity was observed (Margesin et al. 2000b). Polyphenol oxidase plays an important role in the process that convert the aromatic organic compounds into humus in soil and it is an important measure of the soil microflora’s capacity to degrade potentially recalcitrant organic ones (Gianfreda et al. 2005). Therefore, the presence of petroleum hydrocarbons can result in an activated in polyphenol oxidase activity. The influence of PAHs on the enzymatic activity depends on a significant level on soil properties like total organic carbon content and pH (Baran et al. 2004). Organic C is the main constituent of the soil organic matter, and as such it may represent a source of enzyme production but also a substrate for enzyme degradation (Gianfreda et al. 2005). Gianfreda et al. (2005) showed that the measured organic C contents can be mostly attributed to the presence of organic wastes, rather than to organic biomass, in the soils polluted by petroleum and PAHs. These results could indicate that the high organic C content does not necessarily reflect corresponding increases of enzyme activities. In a general way, soil enzyme activities and soil pH are significantly correlated, and positive, negative or no correlations have been reported (Kang and Freeman 1999; Acosta-Martinez and Tabatabai 2000; Canet et al. 2000, Gianfreda et al. 2005). In our situation, dehydrogenase, polyphenol oxidase, catalase
Station
Mitotic index (MI %)
Micronucleus rate (MN %)
Chromosome aberration rate (CA %)
S0
14.61 ± 2.10c
6.11 ± 1.16a
6.34 ± 0.96a
S1
12.22 ± 1.67c
7.44 ± 1.02b
8.15 ± 0.87b
S2
b
9.40 ± 0.92
c
10.39 ± 1.01c
11.74 ± 1.39
d
13.84 ± 0.96d
S3
7.24 ± 1.40
a
3.60 ± 1.39
The used data mean and standard deviation presentation The small letters in the same vertical line show the statistical significance between different at level of P \ 0.05 (Tukey HSD test)
entire protein structure (Shen and Sali 2006). Dehydrogenase, which is also involved in process of C-cycle in the soil, is almost exclusively intracellular to the majority of organisms and used as an indicator of the oxidative potential of a soil. Contamination with PAHs and alkanes inhibited the dehydrogenase activity were reported in several soils (Menharaj et al. 2000; Ihra et al. 2003; Gianfreda et al. 2005, Wyszkowska and Wyszkowski 2010). A similar tendency was observed in our study. The influence of PAHs on soil enzyme depends mainly on the amount and type of pollutants introduced into the environment (Boopathy 2000). Claassens et al. (2006) showed that the addition of the petroleum hydrocarbons initially resulted in a decrease in the potential enzyme
123
Risk assessment of petroleum-contaminated soil
activity and soil pH are significantly correlated (P \ 0.05), whereas protease (P \ 0.05), invertase (P \ 0.01) activity and soil pH are negatively correlated. These results suggest that enzymes commonly present as an extracellular enzymes that combine inorganic or organic soil colloids, and some enzyme activities are pH dependent (Gianfreda et al. 2005). Soil enzymes activities are usually correlated with TN contents or C/N (Deng and Tabatabai 1997; Acosta-Martinez and Tabatabai 2000; Dodor and Tabatabai 2002, Taylor et al. 2002). Our survey also showed the positively correlated relationship between urase, dehydrogenase activity and TN contents (P \ 0.05), and negatively correlated relationship between polyphenol oxidase activity and TN contents (P \ 0.05). Previous studies consistently showed that among the hydrocarbons, the PAHs, present in crude oil, are some of the most dangerous environmental contaminants due to their toxic, carcinogenic, and mutagenic effect (Aina et al. 2006). The cytotoxicity levels of a bioindicator can be determined by the increase or decrease in the MI (Linnainmaa et al. 1978; Romansik et al. 2007). Our findings are in agreement with the earlier studies (Leme et al. 2008), the interaction of TPH with the proteins essential for cell cycle progression may be the cause of inhibition of MI. Findings of the present study reflecting the MI of Vicia can be considered as a reliable method to determine the presence of cytotoxic agents in the environment, and thus can be considered as a sensitive test to estimate petroleum pollution levels. The present data showed a significant MN number in the roots exposed to the petroleum-contaminated soils and MN presented in these root cells showed different sizes. The MN may result from acentric fragments (aneugenic agent) or whole chromosomes (clastogenic agent) that were not incorporated to the main nucleus during the cell cycle (Fenech 2000). Petroleum, which composed of a complex mixture of hydrocarbons, can have both clastogenic and aneugenic actions. Our results confirmed these two actions, and coincide with the previous findings (Kirsch-Volders et al. 2002; Aina et al. 2006; Leme et al. 2008). The types of mitotic chromosomal abnormalities induced by the petroleum hydrocarbons in Vicia root cells can be grouped into two classes. The first class comprises those which indicate that the mutagens are either spindle inhibitors or clastogens, represented by chromosome lagging and unoriented chromosomes at anaphase cells and micronuclei. The second comprises chromosomal adherence, chromosomal bridges and late-separating chromosomes. Chromosomal adherence, which usually leads to chromosomal bridges and, thereby, chromosomal breaks (Fiskesjo¨ and Levan 1993; Marcano et al. 2004; Leme et al. 2008), is a common sign of genotoxicity and may cause irreversible effects on the cell, triggering the cell death
process. In our study this distortion phenomenon was observed, the presence of this abnormality can explain of chromosomal bridges and breaks in these roots.
Conclusion The data from this study provides useful information on the impact of TPHs on the soil ecosystem in Daqing region in Northeastern China. The soil enzymatic activities changed significantly in petroleum-contaminated soil compared to control soil. Moreover, positive and negative correlations between soil properties, TPHs and soil enzymatic activities were observed. Petroleum-contaminated samples indicated serious cytotoxicity and genotoxicity to plants, including decreased index level of MI, increased frequency of MN and CA. The present study reveals that combination of enzymatic activities and genotoxicity test via Vicia faba can be used as an important indicator for assessing the risk of TPH on soil ecosystem. Acknowledgments We thank Dr. Richard S Halbrook and Dr. JiDong Gu for their invaluable assistance in reviewing the manuscript. This study was supported by the National Natural Science Foundation of China (Grant No. 31170479), Programs for Science and Technology Development of Heilongjiang Province, China (Grant No. GC12B304), and Aid program for Science and Technology Innovative Research Team in Higher Educational Institutions of Heilongjiang Province (2010TD10) and Harbin Normal University (KJTD2011-2). Conflict of interest of interest.
The authors declare that they have no conflict
References Achuba FI, Peretiemo-Clarke BO (2008) Effect of spent engine oil on soil catalase and dehydrogenase activities. Int Agrophys 22:1–4 Acosta-Martinez V, Tabatabai MA (2000) Enzyme activities in a limed agricultural soil. Biol Fertl Soils 31:85–91 Aina R, Palin L, Citterio S (2006) Molecular evidence for benzo[a]pyrene and naphthalene genotoxicity in Trifolium repens L. Chemosphere 65:666–673 Alef K, Nannipieri P (1995) Methods in applied soil microbiology and biochemistry. Academic Press, London Alkorta I, Aizpurua A, Riga P, Albizu I, Ame´zaga I, Garbisu C (2003) Soil enzyme activities as biological indicators of soil health. Rev Environ Health 18:65–73 Baran S, Bielin´ska JE, Oleszczuk P (2004) Enzymatic activity in an airfield soil polluted with polycyclic aromatic hydrocarbons. Geoderma 118:221–232 Bayer H, Mitterer M, Schinner F (1982) Der einfluss von insektiziden auf mikrobiogene prozesse in Ah-materialien eines landwirtschaftlich genutzten bodens. Pedobiologia 23:311–319 Be´kaert C, Rast C, Ferrier V, Bispo A, Jourdain MJ, Vasseur P (1999) Use of in vitro (Ames and Mutatox tests) and in vivo (Amphibian Micronucleus test) assays to assess the genotoxicity of leachates from a contaminated soil. Org Geochem 30:953–962 Boopathy R (2000) Factors limiting bioremediation technologies. Bioresource Technol 74:64–67
123
J. Ma et al. Canet R, Albiach R, Pomares F (2000) Indexes of biological activity as tools for diagnosing soil fertility in organic farming. In: Garcia C, Herna´ndez T (eds) Research and perspectives of soil enzymology in Spain. Cebas-CSIC, Murcia, pp 27–39 Claassens S, Van Rensburg L, Riedel KJ, Bezuidenhout JJ, Jansen Van Rensburg PJ (2006) Evaluation of the efficiency of various commercial products for the bioremediation of hydrocarbon contaminated soil. Environmentalist 26:51–62 Coleman JE (1992) Zinc proteins: enzymes, storage proteins, transcription factors, and replication proteins. Annu Rev Biochem 61:897–946 Cotelle S, Masfaraud JF, Fe´rard JF (1999) Assessment of the genotoxicity of contaminated soil with the Allium/Vicia-micronucleus and the Tradescantia-micronucleus assays. Mutat Res Fund Mol M 426:167–171 Deng SP, Tabatabai MA (1997) Effect of tillage and residue management on enzyme activities in soils: III phosphatases and arylsulfatase. Biol Fertl Soils 24:141–146 Dick RP (1997) Soil enzyme activities as integrative indicators of soil health. In: Panhurst CE, Double BM, Gupta VV (eds) Biological indicators of soil health. Commonwealth Agricultural Bureaux International, Oxford, pp 121–157 Dodor DE, Tabatabai MA (2002) Effects of cropping system and microbial biomass on arylamidase activity in soils. Biol Fertl Soils 35:253–261 Fenech M (2000) The in vitro micronucleus technique. Muta Res Fund Mol M 455:81–95 Fiskesjo¨ G, Levan A (1993) Evaluation of the first ten MEIC chemicals in the Allium test. ATLA 21:139–149 Gestel KV, Mergaert J, Swings J, Coosemans J, Ryckeboer J (2003) Bioremediation of diesel oil-contaminated soil by composting with biowaste. Environ Pollut 125:361–368 Gianfreda L, Rao MA, Piotrowska A, Palumbo G, Colombo C (2005) Soil enzyme activities as affected by anthropogenic alterations: intensive agricultural practices and organic pollution. Sci Total Environ 341:265–279 Gil-Sotres F, Trasar-Cepeda C, Leiros MC, Seoane S (2005) Different approaches to evaluating soil quality using biochemical properties. Soil Biol Biochem 37:877–887 Gogoi BK, Dutta NN, Goswami P, Krishna Mohan TR (2003) A case study of bioremediation of petroleum-hydrocarbon contaminated soil at a crude oil spill site. Adv Environ Res 7:767–782 Grant WF (1999) Higher plants assays for the detection of chromosomal aberrations and gene mutations: a brief historical background on their use for screening environmental chemicals. Mutat Res Fund Mol M 426:107–112 Guo D, Ma J, Li R, Guo G (2010) Genotoxicity effect of nitrobenzene on soybean (Glycine max) root tip cells. J Hazard Mater 178:1030–1034 Howcroft CF, Gravato C, Amorim MJB, Novais SC, Soares AMVM, Guilhermino L (2011) Biochemical characterization of cholinesterases in Enchytraeus albidus and assessment of in vivo and in vitro effects of different soil properties, copper and phenmedipham. Ecotoxicology 20(1):119–130 Ihra N, Slet J, Petersell V (2003) Effect of heavy metals and PAH on soil assessed via dehydrogenase assay. Environ Int 28:779–782 Justino CI, Pereira R, Freitas AC, Rocha-Santos TA, Panteleitchouk TS, Duarte AC (2012) Olive oil mill wastewaters before and after treatment: a critical review from the ecotoxicological point of view. Ecotoxicology 21(2):615–629 Kang H, Freeman C (1999) Phosphatase and arylsulphatase activities in wetland soils: annual variation and controlling factors. Soil Biol Biochem 31:449–454 Kiss S, Dragan-Bularda M, Radulescu D (1978) Soil polysaccharidases: activity and agricultural importance. In: Burns RG (ed) Soil enzymes. Academic Press, London, pp 117–147
123
Kolesnikov SI, Tatosyan ML, Aznaur’yan DK (2007) Change in enzymatic activity of common chernozem polluted with crude oil and its products in model experiments. Russ Agric Sci 33:318–320 Krisch-Volders M, Vanhauwaert A, De Boeck M, Decordier I (2002) Importance of detecting numerical versus structural chromosome aberrations. Mutat Res Fund Mol M 504:137–148 Ladd JN, Brisbane PG, Butler JHA, Amato M (1976) Studies on soil fumigation. 3. Effects on enzyme-activities, bacterial numbers and extractable ninhydrin reactive compounds. Soil Biol Biochem 8:255–260 Lah B, Vidic T, Glasencnik E, Cepeljnik T, Gorjanc G, MarinsekLogar R (2008) Genotoxicity evaluation of water soil leachates by Ames test, comet assay, and preliminary Tradescantia micronucleus assay. Environ Monit Assess 139:107–118 Leme DM, de Angelis DF, Marin-Morales MA (2008) Action mechanisms of petroleum hydrocarbons present in waters impacted by an oil spill on the genetic material of Allium cepa root cells. Aquat Toxicol 88:214–219 Linnainmaa K, Meretoja T, Sorsa M, Vainto H (1978) Cytogenetic effects of styrene and styrene oxide. Mutat Res Gen Tox En 58:277–286 Ma T-H, Xu Z, Chengen Xu, McConnell H, Valtierra Rabago E, Adriana Arreola G, Zhang H (1995) The improved Allium/Vicia root tip micronucleus assay for clastogenicity of environmental pollutants. Mutat Res Gen Tox En 334:185–195 Maliszewska-Kordybach B, Klimkowicz-Pawlas A, Smreczak B, Janusauskaite D (2007) Ecotoxic effect of phenanthrene on nitrifying bacteria in soils of different propertie. J Environ Qual 36:1635–1645 Marcano L, Carruyo I, Del Campo A, Montiel X (2004) Cytoxicity and mode of action of maleic hydrazide in root tips of Allium cepa L. Environ Res 94:221–226 Marcato-Romain CE, Guiresse M, Cecchi M, Cotelle S, Pinelli E (2009) New direct contact approach to evaluate soil genotoxicity using the Vicia faba micronucleus test. Chemosphere 77:345–350 Margesin R, Walder G, Schinner F (2000a) The impact of hydrocarbon remediation (diesel oil and polycyclic aromatic hydrocarbons) on enzyme activities and microbial properties of soil. Acta Biotechnol 20:313–333 Margesin R, Zimmerbauer A, Schinner F (2000b) Monitoring of bioremediation by soil biological activities. Chemosphere 40:339–346 Megharaj M, Singleton I, McClure NC, Naidu R (2000) Influence of petroleum hydrocarbon contamination on microalgae and microbial activities in a long-term contaminated soil. Arch Environ Con Tox 38:439–445 Methods of Soil Analysis. (1996) In: Sparks DL (ed) Part 3 chemical methods. SSA book series, vol 5. Soil Science Society of America and American Society of Agronomy, Madison Nannipieri PB, Kandler E, Ruggiero P (2002) Enzyme activity and microbial and biochemical processes in soil. In: Burns RG, Dick RP (eds) Enzymes in the environment: activity, ecology and applications. Marcel Dekker, New York Ohshima T, Tamura T, Sato M (2007) Influence of pulsed electric field on various enzyme activities. J Electrostat 65:156–161 Perucci P, Casucci C, Dumontet S (2000) An improved method to evaluate the o-diphenol oxidase activity of soil. Soil Biol Biochem 32:1927–1933 Riehm H (1958) Die ammonium lactates sigsaure-methode zur bestimmung der leichtloeslichen phosphosaure in karbonathaltigen boden. Agrochimica 3:49–65 Romansik EM, Reilly CM, Kass PH, Moore PF, London CA (2007) Mitotic index is predictive for survival for canine cutaneous mast cell tumors. Vet Pathol 44:335–341 Sannino F, Gianfreda L (2001) Pesticide influence on soil enzymatic activities. Chemosphere 45:417–425
Risk assessment of petroleum-contaminated soil Shao Y, Zhang W, Liu Z, Sun Y, Chen D, Wu J, Fu S (2012) Responses of soil microbial and nematode communities to aluminum toxicity in vegetated oil-shale-waste lands. Ecotoxicology 21(8):2132–2142 Shen M, Sali A (2006) Statistical potential for assessment and prediction of protein structures. Protein Sci 15(11):2507–2524 Singh A, Ward OP, Kuhad RC (2005) Feasibility studies for microbial remediation hydrocarbon-contaminated soil. Monit Assess Soil Bioremediation 5:131–153 Stanislaw B, Bielin´ska JE, Oleszczuk P (2004) Enzymatic activity in an airfield soil polluted with polycyclic aromatic hydrocarbons. Geoderma 118:221–232 Tabatabai MA (1982) Soil enzymes. In: Page AL, Millar EM, Keeney DR (eds) Methods of soil analysis. ASA and SSSA, Madison, pp 501–538 Tang X, Baosheng Z, Mikael H, Lianyong F (2010) Forecast of oil reserves and production in Daqing oilfield of China. Energy 35:3097–3102 Taylor JP, Wilson B, Mills MS, Burns RG (2002) Comparison of microbial numbers and enzymatic activities in surface soils and subsoils using various techniques. Soil Biol Biochem 34:387–401 Top EM, Maila MP, Clerinx M, Goris J, De Vos P, Verstraete W (1999) Methane oxidation as method to evaluate the removal of 2,4-D from soil by plasmid-mediated bioaugumentation. FEMS Microbiol Ecol 28:203–213 Trasar-Cepeda C, Leiro´s MC, Seoane S, Gil-Sotres F (2000) Limitations of soil enzymes as indicators of soil pollution. Soil Biol Biochem 32:1867–1875 US EPA Method 3550 Ultrasonic Extraction. http://www.epa.gov/sw846/pdfs/3550c.pdf. Accessed 27 Jan 2014
Verrhiest GJ, Cle´ment B, Volat B, Montuelle B, Perrodin Y (2002) Interactions between a polycyclic aromatic hydrocarbon mixture and the microbial communities in a natural freshwater sediment. Chemosphere 46:187–196 Wang C, Tian Y, Wang X, Geng J, Jiang J, Yu H, Wang C (2010) Lead-contaminated soil induced oxidative stress, defense response and its indicative biomarkers in roots of Vicia faba seedlings. Ecotoxicology 19(6):1130–1139 Wyszkowska J, Kucharski J (2000) Biochemical properties of soil contaminated by petrol. Pol J Environ Stud 9:479–485 Wyszkowska J, Wyszkowski M (2010) Activity of soil dehydrogenases, urease, and acid and alkaline phosphatases in soil polluted with petroleum. J Toxicol Env Health 73:1202–1210 Yao Y, Lu Z, Min H, Gao H, Zhu F (2012) The effect of tetrahydrofuran on the enzymatic activity and microbial community in activated sludge from a sequencing batch reactor. Ecotoxicology 21(1):56–65 Zantua MI, Bremner JM (1975) Preservation of soil samples for assay of urease activity. Soil Biol Biochem 7:297–299 Zhao Z, Zhuang YX, Gu JD (2012a) Abundance, composition and vertical distribution of polycyclic aromatic hydrocarbons in sediments of the Mai Po Inner Deep Bay of Hong Kong. Ecotoxicology 21:1734–1742 Zhao ZY, Chu YL, Gu JD (2012b) Distribution and sources of polycyclic aromatic hydrocarbons in sediments of the Mai Po Inner Deep Bay Ramsar Site in Hong Kong. Ecotoxicology 21:1743–1752
123
Risk assessment of petroleum-contaminated soil using soil enzyme activities and genotoxicity to Vicia faba Jun Ma • Jinglong Shen • Qingxing Liu • Fang Fang • Hongsheng Cai • Changhong Guo
Accepted: 16 January 2014 Ó Springer Science+Business Media New York 2014
Abstract Pollution caused by petroleum is one of the most serious problems worldwide. To better understand the toxic effects of petroleum-contaminated soil on the microflora and phytocommunity, we conducted a comprehensive field study on toxic effects of petroleum contaminated soil collected from the city of Daqing, an oil producing region of China. Urease, protease, invertase, and dehydrogenase activity were significantly reduced in microflora exposed to contaminated soils compared to the controls, whereas polyphenol oxidase activity was significantly increased (P \ 0.05). Soil pH, electrical conductivity, and organic matter content were correlated with total petroleum hydrocarbons (TPHs) and a correlation (P \ 0.01) existed between the C/N ratio and TPHs. Protease, invertase and catalase were correlated with TPHs. The Vicia faba micronucleus (MN) test, chromosome aberrant (CA) analyses, and the mitotic index (MI) were used to detect genotoxicity of water extracts of the soil. Petroleum-contaminated samples indicated serious genotoxicity to plants, including decreased index level of MI, increased frequency of MN and CA. The combination of enzyme activities and genotoxicity test via Vicia faba can be used as an important indicator for assessing the impact of TPH on soil ecosystem. Keywords Petroleum-contaminated soil Soil enzyme activities Genotoxicity Vicia assay Total petroleum hydrocarbon
J. Ma J. Shen Q. Liu F. Fang H. Cai C. Guo (&) Key Laboratory of Molecular and Cytogenetics, Heilongjiang Province, College of Life Science and Technology, Harbin Normal University, Harbin 150025, China e-mail: [email protected]
Introduction Soil contamination with petroleum-derived products has becoming an increasingly frequent ecological problem, and this pollution usually exerts adverse effects on organisms indirectly by taking toxic chemicals in the soil. The petroleum-derived products contain large amounts of benzene, phenol and polycyclic aromatic hydrocarbons that can show potential mutagenic and carcinogenic effects on humans and pollute the environment (Gestel et al. 2003; Zhao et al. 2012a, b). Oil seeping into the soil not only changes its physical and chemical properties, but also reduces its productivity, such as qualities, compositions of organic components, water-holding capacity as well as air exchange caused by filling of soil pores (Gogoi et al. 2003; Singh et al. 2005). Soil health plays an important role in terrestrial ecosystem and has profound effect on sustainable development. Petroleum contaminants introduced into the soil not only modify physicochemical properties, which includes the pH, oxygen and nutrient availability of soil, but also alter the populations and activities of soil microflora at different functional levels (Wyszkowska and Kucharski 2000; Maliszewska-Kordybach et al. 2007). Enzymes are responsible for numerous metabolic reactions in microbial cells and, accordingly, their inhibition or activation could be the underlying cause of toxicity (Coleman 1992). Soil enzyme activities have great potential to provide a unique integrative biological assessment of soils and the possibility of assessing the health of the soil biota (Alkorta et al. 2003). Many studies have demonstrated that oil pollutants can influence the enzyme activities of soil, and among which, the oxidoreductase (e.g., dehydrogenase and catalase) and hydrolase (e.g., urease and invertase) activities could be
123
J. Ma et al.
served as a symbol that reflect the degree of soil degradation because of their sensibility to pollution (Stanislaw et al. 2004; Achuba and Peretiemo-Clarke 2008). However, some researchers argued that it had limitations when soil enzymes were considered as indicators for assessed soil pollution, thus, the use of soil enzyme activities as indicators for assessing the degree of soil pollution is not enough, different biological indicators are certainly needed (Trasar-Cepeda et al. 2000; Gil-Sotres et al. 2005). Genotoxicity assays are used specifically to evaluate the genotoxic potential of environmental and industrial effluent samples (Cotelle et al. 1999; Leme et al. 2008; Howcroft et al. 2011, Justino et al. 2012). High plants are recognized as excellent indicators of cytogenetic and mutagenic effects of environmental chemicals and applicable for detection of environmental mutagens (Grant 1999; Wang et al. 2010). Micronucleus test and chromosomal aberration assay are common methods for genetic toxicity assessment. A number of studies show that Vicia faba is appropriate for pursuing the genotoxicity evaluation of the contaminated soil (Be´kaert et al. 1999; Lah et al. 2008; Marcato-Romain et al. 2009), and it has more advantages than the other plants (e.g., Tradescantia paludosa and Allium cepa) (Cotelle et al. 1999). Daqing oil filed, which is one of the largest oil fields in the world, accounts for nearly 25 % of China’s oil production (Tang et al. 2010). However, environmental risk caused by petroleum contamination in this region has not been well investigated. In the present study, a comprehensive ecotoxicological testing of petroleum-contaminated soil from Daqing region in northeastern China was carried out. Soil quality and the main soil enzyme activities were evaluated, and correlations between these factors and TPHs were also generated by performing regression analysis. In addition, Vicia faba micronucleus (MN) test, Chromosome aberrant (CA) analyses and the Mitotic index (MI) were performed to detect the potential genotoxicity of water extracts of the soil.
Materials and methods Soil sample collection and preparation Nine surface soil sub-samples (\20 cm depth), each, were randomly collected from 4 different Petroleum-contaminated sites in the Daqing Oil Field Third Factory Area (a Daqing oil field located in Daqing City, Heilongjiang Province, China) in 2010. These four sites are: S0 (the control, no TPH-polluted industrial soil), S1 (saline-alkali meadow), S2 (a secondary pollution area of oil pipeline leakage), and S3 (an oil polluted central area with historical TPH-polluted industrial soil). To minimize the possibility of
123
cross-contamination, disposable sample equipment was used. Samples were taken from the same depth (about 20 cm depth) of soil. A spade was used to dig a v-shaped hole, and a uniform thick slice of soil from bottom to top of the exposed soil surface was selected and placed in a bucket. Samples were transported to the laboratory and stored in a refrigerated at 4 °C prior to experimental analysis. Physicochemical properties determination A soil sub-sample (approximately 1 kg) was air-dried, passed through a 2 mm sieved and subjected to chemical characterization (Methods of Soil Analysis 1996; Shao et al.2012). Soil pH was determined in a soil: deionised water suspension (1:2.5 w/v), electrical conductivity (EC) in a soil:deionised water suspension (1:5 w/v), and salt content (SC) was measured gravimetrically in a soil:water (1:5 w/v) suspension.Soil organic matter (SOM) was determined by dichromate oxidation and total nitrogen (TN) and available nitrogen (AN) were analyzed using the Kjeldahl method. Total phosphorous (TP) was measured colorimetrically as molybdo-vanadate phosphoric acid. Available phosphorous (AP) and available potassium (AK) were determined using the Egner-Riehm method (Riehm 1958). Total petroleum hydrocarbons were measured following USA EPA 3550 (US EPA Method 3550). All measurements were carried out in triplicate. Soil enzyme activity assays All soil samples were kept refrigerated (4 °C) at their field moisture content. Before analysis, samples were sieved through a 2 mm sieve, and their dry matter content was determined in order to express the enzyme activity on a dry matter basis. Drying soil at 105 °C for 24 h, soil field moisture content was determined on each sample, dry matter content was determined gravimetrically by soil field moisture content. The activity of the following enzymes was determined: urease (Zantua and Bremner 1975), protease (Ladd et al. 1976), invertase (Ohshima et al. 2007), dehydrogenase (Tabatabai 1982), catalase (Achuba and Peretiemo-Clarke 2008) and polyphenol oxidase (Perucci et al. 2000). All measurements were carried out in triplicate. Genotoxicity test Soil aqueous extracts was prepared following the methods described by Marcato-Romain et al. (2009), and modified as follows: 100 g dry soil sample and 500 ml distilled water were mixed and stirred at room temperature for 24 h. The mixture was centrifuged at 10,0009g for 20 min and then the supernatant was stored at 4 °C in the dark.
Risk assessment of petroleum-contaminated soil
as ‘‘not detected’’, were assumed to be equal to the detection limit. Pearson correlation coefficients (r) were calculated between soil physicochemical properties and their enzyme activity. All statistical analysis was performed using SPSS for Windows, version 18.0 (IBM SPSS, Inc., Chicago, 2010).
In the current study, faba beans (Vicia faba.L. cv. Songzi peel, 2n = 12) were used as the test system to evaluate genotoxicity. Faba beans, provided by CCNU (Central China Normal University, Wuhan City, Hubei Province, China), were selected for their sensitivity to environmental genotoxicity (Ma et al. 1995). Fava bean seeds were treated with 10 % NaClO solution for 12 min and grown in distilled water in plates for 24 h. Germinated beans were grown in beakers at 25 ± 0.5 °C in an incubator (EYELA Eyelatron FLI-301NH, Japan). When the length of the primary roots was 2 cm, the beans were exposed to soil aqueous extracts for 24 h followed by a 24 h recovery period and excised. For each experiment, six plants were used as six independent replicates per treatment. Each experiment was carried out in triplicate. The root tips were immersed in Carnoy’s solution (3:1 ethanol: glacial acetic acid) for 24 h, and then stored in 70 % ethanol at 4 °C prior to slide preparation. After being hydrolyzed with 1 M HCl at 60 °C for 10 min, the root rips were rinsed and Feulgen stained. To prepare the slides, the meristematic regions were coated with coverslips and carefully squashed into a drop of 45 % acetic acid. The coverslips were removed using liquid nitrogen and the slides were mounted in synthetic resin for further analysis. All slides were analyzed under light microscopically (Leica, Lelica Microsystems ltd., Germany), first with lowpower magnification and then with high-power (409 or 1009). The MN, CA and MI were scored. The MI and CA frequencies were expressed in percent, while MN frequencies were expressed per 1,000 cells. The scores were carried out according to standard protocols described by Ma et al. (1995) and Guo et al. (2010). Six slides were prepared for each of the six seeds (one slide per root tip) and at least 1,500 cells were counted per root tip. The MN frequency was obtained from at least nine thousand cells per treatment.
Concentrations of TPH were below detection limits in S0 samples and TPH concentration were significant greater (P \ 0.05) in S2 and S3 soil samples compared to S1 soil samples (Table 1). The SOM content was greater (P \ 0.05) in S2 and S3 soil compared to S0 soil, but was not significantly different from SOM in S1 soil. The S1, S2 and S3 soils had significantly greater pH, EC values, C/N ratio and SC values (Table 1) compared with S0 soil (P \ 0.05), while Total P and Available N, P, and K content were significantly decreased compared with S0 soil (P \ 0.05). Urease, protease, invertase, and dehydrogenase activities were reduced in S1, S2, and S3 soils compared to S0 soil (P \ 0.05). Catalase activity was greater in S2 and S3 soil but similar in S1 soil compared to S0 soil (P \ 0.05) and polyphenol oxidase activity was significantly increased in S1, S2, and S3 soils compared to S0 soil (P \ 0.05) (Fig. 1). Chemical parameters (pH, EC, SOM and SC) and TPH concentration were positively correlated (P \ 0.05) and the C/N ratio and TPH concentration was highly positively correlated (P \ 0.01) (Table 2). The protease and invertase activities and TPH concentration were negatively correlated (P \ 0.05) while catalase and TPH concentration were positively correlated (P \ 0.05).
Statistical analysis
Genotoxicity to Vicia faba
Microsoft Office Excel 2007 (Microsoft Corporation, USA) was used to randomize treatment assignments, to enter and store data, to sort data and prepare for statistical analysis. All data were checked for homogeneity of variance and normality (Kolmogorov–Smirnov test) and, differences among treatments were evaluated using a one-way ANOVA. Data not satisfying assumptions for ANOVA were analyzed non-parametrically using Kruskal–Wallis ANOVA by Ranks test. Whenever significant differences were found (P \ 0.05), a post hoc Tukey Honest Significant Difference (HSD) test was used to further elucidate differences among means (P \ 0.05). For statistical purposes, results below the detection limit, although reported
Micronucleus and abnormal mitotic phenomena were observed in Vicia root cells in different stages of the mitosis cycle including interphase, prophase, metaphase, anaphase and telophase using microscopic examination. Micronucleus were one of main aberration types observed in the current study (Fig. 2 a–c). Chromosome break (Fig. 2 f), chromosome loss (Fig. 2 d, g) and chromosome bridge (Fig. 2 h) were main aberrations observed, while chromosome adherence and unoriented chromosomes at anaphase cells (Fig. 2 e, f) were less often observed. The MI % of S1 soils was not significantly different from S0 soils; however, was significantly reduced (P \ 0.05) in S2 and S3 soils. The MN % was significant
Results Changes in soil physicochemical properties and enzyme activities
123
J. Ma et al. Table 1 Characteristics of soil at the different sites used in the study EC (lS cm-1)
SOM (g kg-1)
TN (g kg-1)
C/N
687 ± 13.60a
3.02 ± 0.13a
1.84 ± 0.05d
0.96 ± 0.06a
0.71 ± 0.05d
916.5 ± 13.47
b
2.9 ± 0.11
a
c
b
0.37 ± 0.06a
1043.25 ± 25.22
c
3.21 ± 0.12
b
0.65 ± 0.05
c
3.16 ± 0.12
0.44 ± 0.06b
0.59 ± 0.04a
4.73 ± 0.20d
0.51 ± 0.10c
Station
pH
S0
7.85 ± 0.07a
S1
8.25 ± 0.11
b
S2
8.65 ± 0.12
c
S3
9.06 ± 0.05d
1352.32 ± 8.64d
5.28 ± 0.18c
0.89 ± 0.06
b
TP (g kg-1)
1.24 ± 0.17
Station
AN (mg kg-1)
AP (mg kg-1)
AK (mg kg-1)
SC (%)
TPH (g kg-1)
S0
146.00 ± 9.42d
84.00 ± 6.76c
235.97 ± 14.25d
0.58 ± 0.07a
ND
S1 S2
128.00 ± 11.59c 56.00 ± 10.03b
31.10 ± 4.28b 15.50 ± 3.37a
137.24 ± 12.44b 162.80 ± 9.97c
0.92 ± 0.09b 1.25 ± 0.17c
0.02 ± 0.02a 11.85 ± 0.59b
S3
37.00 ± 6.16a
12.70 ± 2.49a
112.40 ± 11.93a
3.15 ± 0.19d
23.54 ± 0.41c
The used data mean and standard deviation presentation Means within each column marked with the same letter are not significantly different (Tukey HSD test, P [ 0.05) EC electrical conductivity, SOM soil organic matter, TN total nitrogen, C/N soil organic carbon and total nitrogen ratio, TP total phosphorous, AN available nitrogen, AP available phosphorous, AK available potassiumm, SC salt content, TPH pseudo-total petroleum hydrocarbons content, ND not detected
Fig. 1 Soil enzyme activities of the samples from four different sites in Daqing. a Urease activity; b protease activity; c invertase activity; d dehydrogenase activity; e catalase and f polyphenol oxidase
123
Risk assessment of petroleum-contaminated soil Table 2 Pearson’s correlation coefficients between soil physicochemical properties and their enzyme activities pH
EC
SOM
TN
C/N
TP
EC
0.988**
OM
0.817
0.870
1.000
TN
-0.886
-0.857
-0.492
1.000
0.882
-0.749
1.000
0.019
0.815
-0.229
C/N
0.968*
AN
AP
AK
1.000
0.953*
TP
-0.463
-0.442
AN
-0.965*
-0.920*
-0.749
0.825
-0.972*
0.353
1.000
AP
-0.892
-0.860
-0.497
1.000**
-0.759
0.805
0.837
AK
-0.835
-0.867
-0.606
0.915*
-0.682
0.776
0.680
0.930*
-0.169
-0.828
-0.654
-0.737
0.996**
-0.150
-0.949*
-0.701
-0.644
SC
0.905*
TPH
0.946*
0.948* 0.939*
0.981**
-0.650
0.915*
-0.691
1.000 1.000 0.907*
1.000
Ure
-0.895
-0.894
-0.580
0.981**
-0.753
0.797
0.790
0.978*
0.975*
Prot
-0.975*
-0.932*
-0.738
0.860
-0.966*
0.412
0.998**
0.871
0.722
Inver
-0.994**
-0.979*
-0.845
0.836
-0.990**
0.367
0.979*
0.844
0.771
DHA
-0.977*
-0.942*
-0.676
0.949*
-0.914*
0.591
0.961*
0.955*
0.837
CAT PPO
0.966* 0.978*
0.931* 0.941*
0.805 0.679
-0.787 -0.943*
0.989** 0.919*
-0.799 -0.950*
-0.666 -0.827
SC
TPH
Ure
Prot
Invert
SC
1.000
TPH
0.945*
-0.286 -0.579 DHA
-0.725
-0.705
1.000
Prot
-0.827
-0.939*
0.828
Inver
-0.918*
-0.974*
0.839
0.982*
DHA CAT
-0.795 0.869
-0.875 0.974*
0.925 -0.765
0.977* -0.990*
0.960* -0.986*
1.000 -0.942
0.880
-0.918
-0.980*
-0.962*
0.947
0.796
CAT
PPO
1.000
Ure
PPO
-0.995** -0.965*
1.000 1.000 1.000 -1.000**
1.000
Marked correlations are significant at: *P \ 0.05; **P \ 0.01 EC electrical conductivity, OM soil organic matter, TN total nitrogen, C/N soil organic carbon and total nitrogen ratio, TP total phosphorous, AN available nitrogen, AP available phosphorous, AK available potassium, SC salt content, TPH pseudo-total petroleum hydrocarbons content, Ure urease, Prot protease, Inver invertase, DHA dehydrogenase, CAT catalase, PPO polyphenol oxidase
less (P \ 0.05) in S0 soils compared to soils collected at the other 3 sites, while the CA % decreased significantly (P \ 0.05) in S0 soils (Table 3).
Discussion In the present study, by taking petroleum-contaminated soil from Daqing region in Northeast China, the physicochemical properties, enzyme activities and genoticixity to Vicia faba were investigated. Enzyme activities are commonly used as indicators of soil health because they play an important role in nutrient cycling (Gianfreda et al. 2005), and can be sensitive indicators of pollution with hydrocarbons, heavy metals or pesticides (Bayer et al. 1982; Dick 1997; Top et al. 1999, Margesin et al. 2000a, Baran et al. 2004, Yao et al. 2012). Protease is the enzyme that catalyses protein hydrolysis to
peptides and aminoacids (Alef and Nannipieri 1995), and urease catalyses the hydrolysis of urea to carbon dioxide and ammonium, and it is widely distributed in microorganisms, plants and animals (Nannipieri et al. 2002), these enzymes are both involved in the N-cycle. Invertase is known to be a very stable and persistent enzyme and its association with soil components is well documented (Kiss et al. 1978; Sannino and Gianfreda 2001), it is involved in process of C-cycle in the soil. Invertase can facilitate the hydrolysis process of the decomposition of organic matter, which suggests their important roles in increasing the soluble nutrients in soil. The present study showed suppressive trends of these enzyme activities, and the inhibition is probably due to changes in the molecular structure of the enzyme caused by the inhibitors as illustrated by former studies (Verrhiest et al. 2002; Kolesnikov et al. 2007; Wyszkowska and Wyszkowski 2010). For petroleum hydrocarbons, it is assumed that they may denature the
123
J. Ma et al.
Fig. 2 Microphotograph of the fava bean root tip aberrant cells induced by the petroleum-contaminated soil at different stages of mitosis. a Micronucleated cell in interphase; b MN of small size in interphase; c MN in prophase; d anaphase with chromosome loss and
unoriented chromosome; e disturbed anaphase with unoriented chromosomes; f chromosome break; g anaphase with chromosome loss; h chromosome bridge and late-separating chromosomes in telophase. Scale in figure 10 lm
Table 3 Genotoxic effects on Vicia faba tip cells of the soil at different sites
activity, which was followed by a subsequent increase in enzymatic activity, possibly due to adaptation of the indigenous microorganisms. In some cases, a stimulatory activity was observed (Margesin et al. 2000b). Polyphenol oxidase plays an important role in the process that convert the aromatic organic compounds into humus in soil and it is an important measure of the soil microflora’s capacity to degrade potentially recalcitrant organic ones (Gianfreda et al. 2005). Therefore, the presence of petroleum hydrocarbons can result in an activated in polyphenol oxidase activity. The influence of PAHs on the enzymatic activity depends on a significant level on soil properties like total organic carbon content and pH (Baran et al. 2004). Organic C is the main constituent of the soil organic matter, and as such it may represent a source of enzyme production but also a substrate for enzyme degradation (Gianfreda et al. 2005). Gianfreda et al. (2005) showed that the measured organic C contents can be mostly attributed to the presence of organic wastes, rather than to organic biomass, in the soils polluted by petroleum and PAHs. These results could indicate that the high organic C content does not necessarily reflect corresponding increases of enzyme activities. In a general way, soil enzyme activities and soil pH are significantly correlated, and positive, negative or no correlations have been reported (Kang and Freeman 1999; Acosta-Martinez and Tabatabai 2000; Canet et al. 2000, Gianfreda et al. 2005). In our situation, dehydrogenase, polyphenol oxidase, catalase
Station
Mitotic index (MI %)
Micronucleus rate (MN %)
Chromosome aberration rate (CA %)
S0
14.61 ± 2.10c
6.11 ± 1.16a
6.34 ± 0.96a
S1
12.22 ± 1.67c
7.44 ± 1.02b
8.15 ± 0.87b
S2
b
9.40 ± 0.92
c
10.39 ± 1.01c
11.74 ± 1.39
d
13.84 ± 0.96d
S3
7.24 ± 1.40
a
3.60 ± 1.39
The used data mean and standard deviation presentation The small letters in the same vertical line show the statistical significance between different at level of P \ 0.05 (Tukey HSD test)
entire protein structure (Shen and Sali 2006). Dehydrogenase, which is also involved in process of C-cycle in the soil, is almost exclusively intracellular to the majority of organisms and used as an indicator of the oxidative potential of a soil. Contamination with PAHs and alkanes inhibited the dehydrogenase activity were reported in several soils (Menharaj et al. 2000; Ihra et al. 2003; Gianfreda et al. 2005, Wyszkowska and Wyszkowski 2010). A similar tendency was observed in our study. The influence of PAHs on soil enzyme depends mainly on the amount and type of pollutants introduced into the environment (Boopathy 2000). Claassens et al. (2006) showed that the addition of the petroleum hydrocarbons initially resulted in a decrease in the potential enzyme
123
Risk assessment of petroleum-contaminated soil
activity and soil pH are significantly correlated (P \ 0.05), whereas protease (P \ 0.05), invertase (P \ 0.01) activity and soil pH are negatively correlated. These results suggest that enzymes commonly present as an extracellular enzymes that combine inorganic or organic soil colloids, and some enzyme activities are pH dependent (Gianfreda et al. 2005). Soil enzymes activities are usually correlated with TN contents or C/N (Deng and Tabatabai 1997; Acosta-Martinez and Tabatabai 2000; Dodor and Tabatabai 2002, Taylor et al. 2002). Our survey also showed the positively correlated relationship between urase, dehydrogenase activity and TN contents (P \ 0.05), and negatively correlated relationship between polyphenol oxidase activity and TN contents (P \ 0.05). Previous studies consistently showed that among the hydrocarbons, the PAHs, present in crude oil, are some of the most dangerous environmental contaminants due to their toxic, carcinogenic, and mutagenic effect (Aina et al. 2006). The cytotoxicity levels of a bioindicator can be determined by the increase or decrease in the MI (Linnainmaa et al. 1978; Romansik et al. 2007). Our findings are in agreement with the earlier studies (Leme et al. 2008), the interaction of TPH with the proteins essential for cell cycle progression may be the cause of inhibition of MI. Findings of the present study reflecting the MI of Vicia can be considered as a reliable method to determine the presence of cytotoxic agents in the environment, and thus can be considered as a sensitive test to estimate petroleum pollution levels. The present data showed a significant MN number in the roots exposed to the petroleum-contaminated soils and MN presented in these root cells showed different sizes. The MN may result from acentric fragments (aneugenic agent) or whole chromosomes (clastogenic agent) that were not incorporated to the main nucleus during the cell cycle (Fenech 2000). Petroleum, which composed of a complex mixture of hydrocarbons, can have both clastogenic and aneugenic actions. Our results confirmed these two actions, and coincide with the previous findings (Kirsch-Volders et al. 2002; Aina et al. 2006; Leme et al. 2008). The types of mitotic chromosomal abnormalities induced by the petroleum hydrocarbons in Vicia root cells can be grouped into two classes. The first class comprises those which indicate that the mutagens are either spindle inhibitors or clastogens, represented by chromosome lagging and unoriented chromosomes at anaphase cells and micronuclei. The second comprises chromosomal adherence, chromosomal bridges and late-separating chromosomes. Chromosomal adherence, which usually leads to chromosomal bridges and, thereby, chromosomal breaks (Fiskesjo¨ and Levan 1993; Marcano et al. 2004; Leme et al. 2008), is a common sign of genotoxicity and may cause irreversible effects on the cell, triggering the cell death
process. In our study this distortion phenomenon was observed, the presence of this abnormality can explain of chromosomal bridges and breaks in these roots.
Conclusion The data from this study provides useful information on the impact of TPHs on the soil ecosystem in Daqing region in Northeastern China. The soil enzymatic activities changed significantly in petroleum-contaminated soil compared to control soil. Moreover, positive and negative correlations between soil properties, TPHs and soil enzymatic activities were observed. Petroleum-contaminated samples indicated serious cytotoxicity and genotoxicity to plants, including decreased index level of MI, increased frequency of MN and CA. The present study reveals that combination of enzymatic activities and genotoxicity test via Vicia faba can be used as an important indicator for assessing the risk of TPH on soil ecosystem. Acknowledgments We thank Dr. Richard S Halbrook and Dr. JiDong Gu for their invaluable assistance in reviewing the manuscript. This study was supported by the National Natural Science Foundation of China (Grant No. 31170479), Programs for Science and Technology Development of Heilongjiang Province, China (Grant No. GC12B304), and Aid program for Science and Technology Innovative Research Team in Higher Educational Institutions of Heilongjiang Province (2010TD10) and Harbin Normal University (KJTD2011-2). Conflict of interest of interest.
The authors declare that they have no conflict
References Achuba FI, Peretiemo-Clarke BO (2008) Effect of spent engine oil on soil catalase and dehydrogenase activities. Int Agrophys 22:1–4 Acosta-Martinez V, Tabatabai MA (2000) Enzyme activities in a limed agricultural soil. Biol Fertl Soils 31:85–91 Aina R, Palin L, Citterio S (2006) Molecular evidence for benzo[a]pyrene and naphthalene genotoxicity in Trifolium repens L. Chemosphere 65:666–673 Alef K, Nannipieri P (1995) Methods in applied soil microbiology and biochemistry. Academic Press, London Alkorta I, Aizpurua A, Riga P, Albizu I, Ame´zaga I, Garbisu C (2003) Soil enzyme activities as biological indicators of soil health. Rev Environ Health 18:65–73 Baran S, Bielin´ska JE, Oleszczuk P (2004) Enzymatic activity in an airfield soil polluted with polycyclic aromatic hydrocarbons. Geoderma 118:221–232 Bayer H, Mitterer M, Schinner F (1982) Der einfluss von insektiziden auf mikrobiogene prozesse in Ah-materialien eines landwirtschaftlich genutzten bodens. Pedobiologia 23:311–319 Be´kaert C, Rast C, Ferrier V, Bispo A, Jourdain MJ, Vasseur P (1999) Use of in vitro (Ames and Mutatox tests) and in vivo (Amphibian Micronucleus test) assays to assess the genotoxicity of leachates from a contaminated soil. Org Geochem 30:953–962 Boopathy R (2000) Factors limiting bioremediation technologies. Bioresource Technol 74:64–67
123
J. Ma et al. Canet R, Albiach R, Pomares F (2000) Indexes of biological activity as tools for diagnosing soil fertility in organic farming. In: Garcia C, Herna´ndez T (eds) Research and perspectives of soil enzymology in Spain. Cebas-CSIC, Murcia, pp 27–39 Claassens S, Van Rensburg L, Riedel KJ, Bezuidenhout JJ, Jansen Van Rensburg PJ (2006) Evaluation of the efficiency of various commercial products for the bioremediation of hydrocarbon contaminated soil. Environmentalist 26:51–62 Coleman JE (1992) Zinc proteins: enzymes, storage proteins, transcription factors, and replication proteins. Annu Rev Biochem 61:897–946 Cotelle S, Masfaraud JF, Fe´rard JF (1999) Assessment of the genotoxicity of contaminated soil with the Allium/Vicia-micronucleus and the Tradescantia-micronucleus assays. Mutat Res Fund Mol M 426:167–171 Deng SP, Tabatabai MA (1997) Effect of tillage and residue management on enzyme activities in soils: III phosphatases and arylsulfatase. Biol Fertl Soils 24:141–146 Dick RP (1997) Soil enzyme activities as integrative indicators of soil health. In: Panhurst CE, Double BM, Gupta VV (eds) Biological indicators of soil health. Commonwealth Agricultural Bureaux International, Oxford, pp 121–157 Dodor DE, Tabatabai MA (2002) Effects of cropping system and microbial biomass on arylamidase activity in soils. Biol Fertl Soils 35:253–261 Fenech M (2000) The in vitro micronucleus technique. Muta Res Fund Mol M 455:81–95 Fiskesjo¨ G, Levan A (1993) Evaluation of the first ten MEIC chemicals in the Allium test. ATLA 21:139–149 Gestel KV, Mergaert J, Swings J, Coosemans J, Ryckeboer J (2003) Bioremediation of diesel oil-contaminated soil by composting with biowaste. Environ Pollut 125:361–368 Gianfreda L, Rao MA, Piotrowska A, Palumbo G, Colombo C (2005) Soil enzyme activities as affected by anthropogenic alterations: intensive agricultural practices and organic pollution. Sci Total Environ 341:265–279 Gil-Sotres F, Trasar-Cepeda C, Leiros MC, Seoane S (2005) Different approaches to evaluating soil quality using biochemical properties. Soil Biol Biochem 37:877–887 Gogoi BK, Dutta NN, Goswami P, Krishna Mohan TR (2003) A case study of bioremediation of petroleum-hydrocarbon contaminated soil at a crude oil spill site. Adv Environ Res 7:767–782 Grant WF (1999) Higher plants assays for the detection of chromosomal aberrations and gene mutations: a brief historical background on their use for screening environmental chemicals. Mutat Res Fund Mol M 426:107–112 Guo D, Ma J, Li R, Guo G (2010) Genotoxicity effect of nitrobenzene on soybean (Glycine max) root tip cells. J Hazard Mater 178:1030–1034 Howcroft CF, Gravato C, Amorim MJB, Novais SC, Soares AMVM, Guilhermino L (2011) Biochemical characterization of cholinesterases in Enchytraeus albidus and assessment of in vivo and in vitro effects of different soil properties, copper and phenmedipham. Ecotoxicology 20(1):119–130 Ihra N, Slet J, Petersell V (2003) Effect of heavy metals and PAH on soil assessed via dehydrogenase assay. Environ Int 28:779–782 Justino CI, Pereira R, Freitas AC, Rocha-Santos TA, Panteleitchouk TS, Duarte AC (2012) Olive oil mill wastewaters before and after treatment: a critical review from the ecotoxicological point of view. Ecotoxicology 21(2):615–629 Kang H, Freeman C (1999) Phosphatase and arylsulphatase activities in wetland soils: annual variation and controlling factors. Soil Biol Biochem 31:449–454 Kiss S, Dragan-Bularda M, Radulescu D (1978) Soil polysaccharidases: activity and agricultural importance. In: Burns RG (ed) Soil enzymes. Academic Press, London, pp 117–147
123
Kolesnikov SI, Tatosyan ML, Aznaur’yan DK (2007) Change in enzymatic activity of common chernozem polluted with crude oil and its products in model experiments. Russ Agric Sci 33:318–320 Krisch-Volders M, Vanhauwaert A, De Boeck M, Decordier I (2002) Importance of detecting numerical versus structural chromosome aberrations. Mutat Res Fund Mol M 504:137–148 Ladd JN, Brisbane PG, Butler JHA, Amato M (1976) Studies on soil fumigation. 3. Effects on enzyme-activities, bacterial numbers and extractable ninhydrin reactive compounds. Soil Biol Biochem 8:255–260 Lah B, Vidic T, Glasencnik E, Cepeljnik T, Gorjanc G, MarinsekLogar R (2008) Genotoxicity evaluation of water soil leachates by Ames test, comet assay, and preliminary Tradescantia micronucleus assay. Environ Monit Assess 139:107–118 Leme DM, de Angelis DF, Marin-Morales MA (2008) Action mechanisms of petroleum hydrocarbons present in waters impacted by an oil spill on the genetic material of Allium cepa root cells. Aquat Toxicol 88:214–219 Linnainmaa K, Meretoja T, Sorsa M, Vainto H (1978) Cytogenetic effects of styrene and styrene oxide. Mutat Res Gen Tox En 58:277–286 Ma T-H, Xu Z, Chengen Xu, McConnell H, Valtierra Rabago E, Adriana Arreola G, Zhang H (1995) The improved Allium/Vicia root tip micronucleus assay for clastogenicity of environmental pollutants. Mutat Res Gen Tox En 334:185–195 Maliszewska-Kordybach B, Klimkowicz-Pawlas A, Smreczak B, Janusauskaite D (2007) Ecotoxic effect of phenanthrene on nitrifying bacteria in soils of different propertie. J Environ Qual 36:1635–1645 Marcano L, Carruyo I, Del Campo A, Montiel X (2004) Cytoxicity and mode of action of maleic hydrazide in root tips of Allium cepa L. Environ Res 94:221–226 Marcato-Romain CE, Guiresse M, Cecchi M, Cotelle S, Pinelli E (2009) New direct contact approach to evaluate soil genotoxicity using the Vicia faba micronucleus test. Chemosphere 77:345–350 Margesin R, Walder G, Schinner F (2000a) The impact of hydrocarbon remediation (diesel oil and polycyclic aromatic hydrocarbons) on enzyme activities and microbial properties of soil. Acta Biotechnol 20:313–333 Margesin R, Zimmerbauer A, Schinner F (2000b) Monitoring of bioremediation by soil biological activities. Chemosphere 40:339–346 Megharaj M, Singleton I, McClure NC, Naidu R (2000) Influence of petroleum hydrocarbon contamination on microalgae and microbial activities in a long-term contaminated soil. Arch Environ Con Tox 38:439–445 Methods of Soil Analysis. (1996) In: Sparks DL (ed) Part 3 chemical methods. SSA book series, vol 5. Soil Science Society of America and American Society of Agronomy, Madison Nannipieri PB, Kandler E, Ruggiero P (2002) Enzyme activity and microbial and biochemical processes in soil. In: Burns RG, Dick RP (eds) Enzymes in the environment: activity, ecology and applications. Marcel Dekker, New York Ohshima T, Tamura T, Sato M (2007) Influence of pulsed electric field on various enzyme activities. J Electrostat 65:156–161 Perucci P, Casucci C, Dumontet S (2000) An improved method to evaluate the o-diphenol oxidase activity of soil. Soil Biol Biochem 32:1927–1933 Riehm H (1958) Die ammonium lactates sigsaure-methode zur bestimmung der leichtloeslichen phosphosaure in karbonathaltigen boden. Agrochimica 3:49–65 Romansik EM, Reilly CM, Kass PH, Moore PF, London CA (2007) Mitotic index is predictive for survival for canine cutaneous mast cell tumors. Vet Pathol 44:335–341 Sannino F, Gianfreda L (2001) Pesticide influence on soil enzymatic activities. Chemosphere 45:417–425
Risk assessment of petroleum-contaminated soil Shao Y, Zhang W, Liu Z, Sun Y, Chen D, Wu J, Fu S (2012) Responses of soil microbial and nematode communities to aluminum toxicity in vegetated oil-shale-waste lands. Ecotoxicology 21(8):2132–2142 Shen M, Sali A (2006) Statistical potential for assessment and prediction of protein structures. Protein Sci 15(11):2507–2524 Singh A, Ward OP, Kuhad RC (2005) Feasibility studies for microbial remediation hydrocarbon-contaminated soil. Monit Assess Soil Bioremediation 5:131–153 Stanislaw B, Bielin´ska JE, Oleszczuk P (2004) Enzymatic activity in an airfield soil polluted with polycyclic aromatic hydrocarbons. Geoderma 118:221–232 Tabatabai MA (1982) Soil enzymes. In: Page AL, Millar EM, Keeney DR (eds) Methods of soil analysis. ASA and SSSA, Madison, pp 501–538 Tang X, Baosheng Z, Mikael H, Lianyong F (2010) Forecast of oil reserves and production in Daqing oilfield of China. Energy 35:3097–3102 Taylor JP, Wilson B, Mills MS, Burns RG (2002) Comparison of microbial numbers and enzymatic activities in surface soils and subsoils using various techniques. Soil Biol Biochem 34:387–401 Top EM, Maila MP, Clerinx M, Goris J, De Vos P, Verstraete W (1999) Methane oxidation as method to evaluate the removal of 2,4-D from soil by plasmid-mediated bioaugumentation. FEMS Microbiol Ecol 28:203–213 Trasar-Cepeda C, Leiro´s MC, Seoane S, Gil-Sotres F (2000) Limitations of soil enzymes as indicators of soil pollution. Soil Biol Biochem 32:1867–1875 US EPA Method 3550 Ultrasonic Extraction. http://www.epa.gov/sw846/pdfs/3550c.pdf. Accessed 27 Jan 2014
Verrhiest GJ, Cle´ment B, Volat B, Montuelle B, Perrodin Y (2002) Interactions between a polycyclic aromatic hydrocarbon mixture and the microbial communities in a natural freshwater sediment. Chemosphere 46:187–196 Wang C, Tian Y, Wang X, Geng J, Jiang J, Yu H, Wang C (2010) Lead-contaminated soil induced oxidative stress, defense response and its indicative biomarkers in roots of Vicia faba seedlings. Ecotoxicology 19(6):1130–1139 Wyszkowska J, Kucharski J (2000) Biochemical properties of soil contaminated by petrol. Pol J Environ Stud 9:479–485 Wyszkowska J, Wyszkowski M (2010) Activity of soil dehydrogenases, urease, and acid and alkaline phosphatases in soil polluted with petroleum. J Toxicol Env Health 73:1202–1210 Yao Y, Lu Z, Min H, Gao H, Zhu F (2012) The effect of tetrahydrofuran on the enzymatic activity and microbial community in activated sludge from a sequencing batch reactor. Ecotoxicology 21(1):56–65 Zantua MI, Bremner JM (1975) Preservation of soil samples for assay of urease activity. Soil Biol Biochem 7:297–299 Zhao Z, Zhuang YX, Gu JD (2012a) Abundance, composition and vertical distribution of polycyclic aromatic hydrocarbons in sediments of the Mai Po Inner Deep Bay of Hong Kong. Ecotoxicology 21:1734–1742 Zhao ZY, Chu YL, Gu JD (2012b) Distribution and sources of polycyclic aromatic hydrocarbons in sediments of the Mai Po Inner Deep Bay Ramsar Site in Hong Kong. Ecotoxicology 21:1743–1752
123
