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Evaluation of soil biological activity after a diesel fuel spill
Science of the Total Environment 407 (2009) 4056–4061
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Science of the Total Environment j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s c i t o t e n v
Evaluation of soil biological activity after a diesel fuel spill A. Serrano a, M. Tejada b,⁎, M. Gallego a, J.L. Gonzalez c a b c
Department of Analytical Chemistry, Campus of Rabanales, University of Cordoba, E-14071 Cordoba, Spain Department of Crystallography, Mineralogy and AgroChemistry, Crta de Utrera Km1, University of Seville, E-41013, Seville, Spain Department of AgroChemistry and Pedology, Campus of Rabanales, University of Cordoba, E-14071 Cordoba, Spain
a r t i c l e
i n f o
Article history: Received 17 November 2008 Received in revised form 25 February 2009 Accepted 9 March 2009 Available online 23 April 2009 Keywords: Diesel fuel spill Aromatic and aliphatic hydrocarbons Soil biological activities Germination index
a b s t r a c t Diesel fuel contamination in soils may be toxic to soil microorganisms and plants and acts as a source of groundwater contamination. The objective of this study was to evaluate the soil biological activity and phytotoxicity to garden cress (Lepidium sativum L.) in a soil polluted with diesel fuel. For this, a diesel fuel spill was simulated on agricultural soil at dose 1 l m− 2. During the experiment (400 days) the soil was not covered in vegetation and no agricultural tasks were carried out. A stress period of 18 days following the spill led to a decrease in soil biological activity, reflected by the soil microbial biomass and soil enzymatic activities, after which it increased again. The n-C17/Pristine and n-C18/Phytane ratios were correlated negatively and significantly with the dehydrogenase, arylsulphatase, protease, phosphatase and urease activities and with the soil microbial biomass during the course of the experiment. The β-glucosidase activity indicated no significant connection with the parameters related with the evolution of hydrocarbons in the soil. Finally, the germination activity of the soil was seen to recover 200 days after the spill. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The uncontrolled spillage of fuels can lead to the deterioration of the environmental quality, especially as regards the negative influence on the soil's properties and water as result of their intrinsic chemical stability, resistance to different types of degradation and high toxicity to living microorganisms (Alexander, 1999; Andreoni et al., 2004; Eibes et al., 2006). The spilling of petroleum by-products (mainly aliphatic hydrocarbon) produces an important deterioration of soil physical properties (Luthy et al., 1997) and a large variations in soil microbial diversity (Saadoun, 2002; Maliszewska-Kordybach and Smreczak, 2003; Gianfreda et al., 2005), with a noticeable increase in the presence of microorganisms that degrade hydrocarbons (Aislabie et al., 2004). This fact is reflected by the variations observed in the levels of different soil enzymatic activities (Baran et al., 2004; Tejada et al., 2008). Soil microorganisms, being in intimate contact with the soil's environment, are very sensitive to any ecosystem perturbation, and are therefore considered to be the best indicators of soil pollution (Andreoni et al., 2004) because: (i) they are a measure of the soil microbial activity and therefore they are strictly related to the nutrient cycles and transformations, (ii) they may rapidly respond to the changes caused by both natural and
⁎ Corresponding author. Tel.: +34 954486469; fax: +34 954486436. E-mail address: [email protected] (M. Tejada). 0048-9697/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2009.03.017
anthropogenic factors and (iii) they are easy to measure. For these reasons, soil enzymatic activities may be considered early and sensitive indicators for measuring the degree of soil degradation in both natural and agro-ecosystems, being thus well suited in assessing the impact of pollution on the soil quality (Baran et al., 2004; Labud et al., 2007). Aliphatic hydrocarbon analysis can be used to fingerprint spilled oils and to provide additional information on the source of hydrocarbon contamination and the extent of degradation of the spilled oil (Wang et al., 1999, Zhu et al., 2005). Comparing biodegradation indicators such as n-C17/pristine and n-C18/phytane throughout a period of time provides a measure of the effect of microbial degradation on the loss of hydrocarbons at the spill site (Wang et al., 1999). These indicators are used since pristine and phytane degradation will generally be considered negligible when n-alkanes are still available as substrates. This resistance to degradation is though to result from the greater complexity of the molecular structures of branched compounds (viz. pristine and phytane) as compared with linear alkanes (Pond et al., 2002; Hejazi and Husain, 2004a,b). This experiment formed part of a research project concerning the natural attenuation of the effect of contamination in diesel-contaminated agricultural soils. Such contamination can be caused by an accidental spillage, given that these fuels are transported by road through agricultural areas, or by leakages from fuel storage tanks located on agricultural land. For this purpose, a diesel fuel spill from a tank truck was simulated, creating contamination levels in the soil of 1 l m− 2. The natural attenuation of the volatile fraction (mainly
A. Serrano et al. / Science of the Total Environment 407 (2009) 4056–4061 Table 1 Properties of the studied agricultural soil at different depths. Depth (cm)
Sand (g kg− 1)
Silt (g kg− 1)
Clay (g kg− 1)
pH
Organic-C (g kg− 1)
C.E.C (cmolc(+) kg− 1)
0–10 10–20 20–30
303 315 342
417 384 305
290 301 353
7.6 7.7 7.7
13.3 11.6 11.1
25.4 25.8 26.5
benzene derivatives) of diesel fuel was monitored in a previous experiment, in which it was concluded that these compounds start to disperse instantly, mainly through volatilization, disappearing completely 50 days after the spill (Serrano et al., 2006). Immediately after the spill, between days 0 and 18, evaporation was the main cause in the drastic decrease of the pollutants in the soil (Serrano et al., 2006). From 18 days onwards, the soil microorganisms started to use the aliphatic hydrocarbons as source of carbon and energy as corroborated the degradation ratios (C17/Pristine and C18/ Phytane); by day 50 after spiking, biodegradation by soil microorganisms played an important role in the removal of the soil pollutants. The present experiment, therefore, focused on changes in the levels of enzymatic activities to determine the possibility of using these values as indicators of quality in agricultural soils contaminated by spills of hydrocarbons. 2. Materials and methods 2.1. Site and experimental layout The study was conducted from April 2004 to May 2005 in Córdoba (Andalusia, Spain). The soil of the field experiment is a Vertic Chromoxerert (Soil Survey Staff, 2003) and its general properties (0–30 cm) are shown in Table 1. This study site enjoyed Mediterranean climatic conditions: temperatures of 0–40 °C, average annual rainfall of 620 mm and 3000 h of annual sunshine. Soil pH was determined in distilled water with a glass electrode (soil: H2O ratio 1:2.5). The particle size distribution was determined densitometrically (Benítez et al., 1998). Total C was determined by oxidising the soil organic matter with K2Cr2O7 in sulphuric acid (96%) for 30 min and measuring the Cr(III) concentration formed (Sims and Haby, 1971). Total N was determined by the Kjeldahl method (MAPA, 1994). The cation exchange capacity was determined saturating the soil with 1 M sodium acetate and 1 M ammonium acetate (MAPA, 1994). 2.2. Soil spiking and sampling A 16 m2 plot of land located in an area with a b2% slope was used in the study. The area was cleared of stones and plant residues, after which it was fenced to keep animals off. The soil was spiked using ground spray application equipment loaded with a diesel fuel (medium light) to a final concentration of 1 l m− 2. Diesel fuel
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consists of a complex mixture of aliphatic (80–90%) and aromatic (20–10%) hydrocarbons. Four sampling areas (A, B, C and D), 2 × 2 m each, were established in the plot. A, B and C areas were contaminated in order to obtain an adequate number of representative samples that did not overlap during the studied period. Three sub-samples per area were used in each analysis (n = 9). Cores (sub-samples), 2.5 cm in diameter, were taken from 0–30 cm soil depth in the spiked plot. A parallel study was carried out in the non-contaminated area (D). Approximately 3 g of soil from each area were placed in pre-weighed 20 ml glass vials, to which aqueous saline solution and ethyl acetate (containing fluorobenzene as internal standard) were added, and immediately sealed. Samples were placed in a portable freezer to transfer them to the laboratory, where they were weighed and stored at 4 °C until analysis (1 day). In this way, evaporation losses of the volatile aromatic hydrocarbons (VAHs) in the sampling process were minimised (Couch et al., 2000). Adding saline solution and transporting the samples at 4 °C, VAHs degradation losses were avoided (Hewitt, 1997). Several of the VAHs are suspected carcinogens and some safety cautions must be taken with them. Inside the laboratory, all handling of the solutions and samples should be performed in a ventilated hood and wearing latex gloves to avoid inhalation or skin contact. For the same reason, soil sample handling in the field study should also be performed wearing special latex gloves, goggles and masks for these compounds. Two different methods were used to study the diesel fuel spill; volatile aromatic hydrocarbons (benzene derivatives) were determined using a headspace-gas chromatography-mass spectrometry method (Serrano et al., 2006) while the aliphatic hydrocarbons (from C11 to C27, including pristine and phytane) were determined using a continuous flow system that included microwave-assisted and liquid– liquid extraction units; the organic extract was finally injected into a gas chromatograph-mass spectrometer (Serrano and Gallego, 2006). The evolution of VAHs, TPHs, and the n-C17/Pri and n-C18/Phy ratios were determined 0, 4, 8, 18, 26, 50, 100, 200, 300 and 400 days after the diesel fuel contamination. 2.3. Determination of soil biochemical parameters Soil samples were collected 0, 4, 8, 18, 26, 50, 100, 200, 300 and 400 days after the diesel fuel contamination. Soils from plots were thoroughly homogenised and stored (fresh samples) at 4 °C for analysis. Soil microbial biomass was determined using the CHCl3 fumigation– extraction method (Vance et al., 1987). The levels of six enzymatic activities in the soil were measured. Dehydrogenase activity was measured by reduction of 2-p-iodo-3-nitrophenyl 5-phenyl tetrazolium chloride to iodonitrophenyl formazan (García et al., 1993). Urease activity was determined by the buffered method of Kandeler and Gerber (1988), using urea as substrate. Protease activity (BBA protease) was measured using N-α-benzoyl-L-argininamide as substrate (Nannipieri et al., 1980). Phosphatase activity was measured using p-nitrophenyl phosphate as substrate (Tabatabai and Bremner, 1969). β-glucosidase
Table 2 Evolution of VAH, TPH, (mg kg− 1soil), and n-C17/Pri and n-C18/Phy ratios and standard errors. Experimental period (Days) 0 VAH TPH n-C17/ Pri ratio n-C18/ Phy ratio
1
4
8
13
18
26
39
50
100
347.4 ± 22.1 104.9 ± 9.4 46.3 ± 3.2 24.6 ± 2.1 18.2 ± 1.6 11.8 ± 1.5 5.9 ± 0.5 1.1 ± 0.3 0.03 ± 0.01 2784 ± 136 2066 ± 106 1230 ± 96 958 ± 46 788 ± 40 549 ± 35 435 ± 16 243 ± 20 154 ± 19 129 ± 11 1.45 ± 0.16 1.47 ± 0.11 1.50 ± 0.13 1.56 ± 0.14 1.56 ± 0.12 1.54 ± 0.15 1.48 ± 0.13 1.37 ± 0.13 1.36 ± 0.16 1.33 ± 0.18 1.36 ± 0.11
1.40 ± 0.13
1.48 ± 0.15 1.54 ± 0.10 1.53 ± 0.11
1.54 ± 0.12 1.48 ± 0.17 1.40 ± 0.14
1.40 ± 0.14
200
300
76 ± 13 1.16 ± 0.13
69 ± 9 38 ± 10 1.13 ± 0.08 0.85 ± 0.06
1.37 ± 0.12 1.21 ± 0.09 1.17 ± 0.11
400
0.91 ± 0.04
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Fig. 1. Evolution of soil microbial biomass and dehydrogenase, urease and BBA-protease activities in soils contaminated with diesel fuel. INTF: 2-p-iodo-3-nitrophenyl. NS, ⁎, ⁎⁎, ⁎⁎⁎: non-significant or significant at p b 0.05, 0.01 or 0.001, respectively.
activity was determined using p-nitrophenyl-β-D-glucopyranoside as substrate (Masciandaro et al., 1994). Arylsulfatase activity was determined using p-nitrophenylsulphate as substrate (Tabatabai and Bremner, 1970). 2.4. Phytotoxicity test Germination experiments (in triplicate) were carried out in Petri dishes using soil samples taken 1, 4, 8, 13, 18, 26, 39, 50, 100, 200, 300 and 400 days after the diesel fuel contamination. Thirty grams of the corresponding soil samples were introduced into dishes and moistened, while distilled water on filter paper was used as control in other dishes. Ten seeds of garden cress (Lepidium sativum L.) were then placed on the dishes, which were placed in a germination chamber and maintained at 28 °C in darkness. The seeds were supplied by the Botanical Garden of Córdoba (Spain). The germination index was calculated according to the method described by Zucconi et al. (1981) and modified by Potenz et al. (1985). The germination index is calculated by:
G:I: =
a×b × 100 a1 × b1
where a is the average of the germinated seeds, b is the average length of each root, a1 is average of the germinated seeds in the control of the experiment, and a2 is the average length of each root in the control of the experiment. 2.5. Statistical analysis Analysis of variance (ANOVA) was performed for all variables and parameters, considering all the data collected and using the Statgraphics v. 5.0 software package (Statistical Graphics Corporation, 1991). The means were separated by Tukey's test, considering a significance level of p b 0.05 throughout the study. For the ANOVA analysis, triplicate data for each treatment were used. To compare the correlations between the parameters studied, a correlation matrix was calculated; the significance of the correlation
Fig. 2. Evolution of β-glucosidase, phosphatase and arylsulphatase activities in soils contaminated with diesel fuel. PNP: p-nitrophenol; PNF: p-nitrophenyl. NS, ⁎, ⁎⁎: nonsignificant or significant at p b 0.05 or 0.01, respectively.
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Fig. 3. Dose (concentration x time) response curve between VAH, TPH, n-C17/Pri and n-C18/Phy ratios and soil microbial biomass-C.
coefficients is shown by using ⁎, ⁎⁎, and ⁎⁎⁎ to indicate the 95%, 99% and 99.9% probability levels, respectively. In order to establishing relationships between the soil evolution of each biochemical variable with the VAHs and TPHs contents and the values of n-C17/Pri and n-C18/Phy, linear regression analyses were performed. 3. Results 3.1. Hydrocarbons and soil biological parameters Table 2 shows the soil contents of the total volatile aromatic hydrocarbons (VAHs), and total aliphatic hydrocarbons (TPHs), and
the C17-Pristine (C17-Pri) and C18-Phytane (C18-Phy) ratios. The fraction corresponding to VAHs practically disappeared 50 days after spiking, while TPHs lasted longer, falling throughout the 400 days of the experiment, after which only about 1% of the original levels remained. This fall was more pronounced in the days following the application since after 50 days only 6% of the original concentration remained. The C17/Pri and C18/Phy ratios tended to increase until 13– 18 days after the spill, coinciding with the predominance of the volatilization (Serrano et al., 2006), and then decreased until the end of the test, coinciding with the predominance of the biodegradation. Soil microbial biomass and enzymatic activities were inhibited during the first 50 days following spiking (Figs. 1 and 2). By 50 days post-contamination, the soil microbial biomass and dehydrogenase,
Table 3 Correlation matrix of parameters studied in soil contaminated by diesel fuel. Dehydrogenase Urease activity activity
BBA-protease activity
β-glucosidase activity
Microbial 0.979 (+)(⁎⁎⁎) 0.316(+) 0.822 (+)(⁎⁎⁎) 0.428(−) biomass Dehydrogenase 0.323(+) 0.776(+)(⁎⁎⁎) 0.440(−) activity Urease activity 0.580(+)(⁎) 0.931(+) (⁎⁎⁎) BBA-protease 0.101(−) activity β-glucosidase activity Phosphatase activity Arylsulphatase activity n-C18/Phy ratio n-C17/Pri ratio TPH (+), (−) positive or negative correlation. (⁎) p b 0.05; (⁎⁎) p b 0.01; (⁎⁎⁎) p b 0.001.
Phosphatase activity
Arylsulphatase n-C18/Phy ratio n-C17/Pri ratio TPH activity
VAH
0.586(+)(⁎)
0.593(+)(⁎)
0.744(−)(⁎⁎)
0.711(−)(⁎⁎)
0.111(+)
0.311(+)
0.573(+)(⁎)
0.554(+)(⁎)
0.776(−)(⁎⁎)
0.721(−)(⁎⁎)
0.075(+)
0.254(+)
0.758(+)(⁎⁎) 0.786(+)(⁎⁎) 0.655(−)(⁎) 0.800(+)(⁎⁎⁎) 0.931(+)(⁎⁎⁎) 0.729(−)(⁎⁎)
0.741(−)(⁎⁎) 0.703(−)(⁎⁎)
0.150(−) 0.099(+)
0.042(+) 0.288(+)
0.271(+)
0.020(+)
0.355(−)
0.296(−)
0.857(+)(⁎⁎⁎) 0.796(−)(⁎⁎)
0.821(−)(⁎⁎)
0.266(+)
0.101(+)
0.766(−)(⁎⁎)
0.743(−)(⁎⁎)
0.543(−)(⁎)
0.399(−)
0.418(+)
0.243(+) 0.977(−)(⁎⁎⁎) 0.756(−)(⁎⁎) 0.731(−)(⁎⁎) 0.890(+)(⁎⁎⁎)
0.067(+)
0.130(+)
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urease, BBA-protease, β-glucosidase, phosphatase and arylsulphatase activities had decreased by 59.2%, 72.5%, 37.4%, 41.3%, 68.6%, 36.1% and 58.3%, respectively, compared with the control soil. From 50 days onwards, such enzymatic activity increased significantly until the end of the experimental period, increasing by 16.1%, 75.8%, 71.9%, 34.8%, 65%, 31.8% and 42.3%, respectively, with respect to the unpolluted soil. The increase in soil biological parameters coincide with the disappearance of soil VAHs and the biodegradation of the soil n-C17/Pri and n-C18/Phy ratios. Fig. 3 shows the dose (concentration x time) response curve between VAH, TPH, n-C17/Pri and n-C18/Phy ratios and soil microbial biomass-C. The results indicate that VAH contents negatively influence the soil microbial biomass development. Although the soil VAH decreases, microbial biomass continues decreasing, possibly because soil microorganisms had not recovered at this time. After the disappearance of VAH, the microbial biomass started to increase, reaching final values that were higher than those observed before applying the pollutant to soil. It is possible that TPH, pristine and phytane significantly affect this increase in soil microbial biomass. The correlation matrix of the different chemical and biochemical parameters determined in the polluted soil indicated that the enzymatic activity showing the lowest number of significant correlations was β-glucosidase; while phosphatase, BBA-protease and arylsulphatase activities displayed the greatest number of correlations followed, by the dehydrogenase and urease activities (Table 3). The nC18/Phy and, especially, the n-C17/Pri ratios were negatively correlated with the biochemical parameters examined. From this, it is concluded that this relation can be used as an indicator of the soil biochemical quality in the case of diesel fuel. Table 4 shows the multiple correlations between the soil microbial biomass and the different enzymatic activities (as dependent variables), and the soil VAH and TPH contents and the values of nC17/Pri and n-C18/Phy (as independent variables). For soil microbial biomass and of the dehydrogenase and urease enzymatic activities, a better fit is obtained using only the n-C17/Pri and n-C18/Phy ratios, whereas for BBA-protease, phosphatase and arylsulphatase activities the best fit is obtained taking four independent variables into consideration, namely the above mentioned relations and the soil VAH and TPH contents.
Table 4 Multiple correlations between variables. SMB = 633.4 − 158.1 C18/Phy − 174.9 C17/Pri − 4.54 × 10− 3 TPH + 4.31 × 10− 7 VAH SMB = 628.4 − 758.0 C18/Phy + 436.8 C17/Pri DHA = 24.79 + 4.614 C18/Phy17.93 C17/Pri + 5.6 × 10− 4 TPH +1.3 × 10− 5 VAH DHA = 24.03 + 22.86 C18/Phy + 10.57 C17/Pri URA = 3.229 + 2.859 C18/Phy − 3.929 C17/Pri − 6.4 × 10− 6TPH + 1.0 × 10− 7VAH URA = 3.233 + 2.791 C18/Phy − 3.865 C17/Pri BBA = 0.617 + 0.604 C18/Phy −0.921 C17/Pri + 2.86 × 10− 5 TPH + 3.61 × 10− 7 VAH BBA = 0.585 − 0.363 C18/Phy + 0.087 C17/Pri GLUA = 12.55 + 7.32 C18/Phy − 8.75 C17/Pri + 1.7 × 10− 4 TPH + 1.9 × 10− 6VAH GLUA = 12.43 + 1.423 C18/Phy + 2.537 C17/Pri PHOA = 10.98 + 8.44 C18/Phy − 11.61 C17/Pri + 2.9 × 10− 4 TPH + 2.3 × 10− 6VAH PHOA = 10.67 + 0.651 C18/Phy + 3.440 C17/Pri ASA = 15.81 + 14.28 C18/Phy − 22.03 C17/Pri + 4.8 × 10− 4 TPH + 1.0 × 10− 5 VAH ASA = 15.18 + 8.47 C18/Phy + 1.57 C17/Pri
p b 0.05; r2 = 75.75 p b 0.01; r2 = 64.96 p b 0.05; r2 = 71.9 p b 0.01; r2 = 60.69 p b 0.05; r2 = 66.52 p b 0.01; r2 = 66.50 p b 0.05; r2 = 76.36 p b 0.05; r2 = 53.33 p b 0.01; r2 = 84.22 p b 0.01; r2 = 62.31 p b 0.01; r2 = 83.69 p b 0.01; r2 = 67.65 p b 0.01; r2 = 83.09 p b 0.05; r2 = 58.89
SMB: Soil microbial biomass; DHA: dehydrogenase activity; URA: urease activity; BBA: BBA-protease activity; GLUA: β-glucosidase activity; PHOA: phosphatase activity; ASA: arylsulphatase activity.
Fig. 4. Germination index of Lepidium sativum seeds in diesel oil polluted soil.
3.2. Phytotoxicity test The germination index (GI) gives an idea of the effect of soil contamination on both seed germination and root growth. For unpolluted soil, the GI remained constant at a value of 100%. However, during the first 50 days of the experiment, the soil contaminated with diesel oil showed the lowest GI values (Fig. 4). At 4 days of the experiment, GI decreased by 84.9% with respect to the control soil. This inhibition was less 50 days into the experiment (47.9% compared with the unpolluted soil). From 100 days, the GI increased, reaching similar values to those obtained with unpolluted soil. 4. Discussion The decrease in soil microbial biomass, soil enzymatic activities and the germination index in soils polluted with diesel oil indicates that this polycyclic aromatic hydrocarbon has a toxic effect on soil microorganisms and plants, results which are in agreement with those of Adam and Duncan (2002), Siddiqui and Adams (2002) and Labud et al. (2007). The inhibitory effect of the diesel fuel on soil enzymatic activities is probably due to the suppression of the microbial populations involved in the C, N, P and S cycles (Andreoni et al., 2004); and/or to the fact that it may cover both organic-mineral and cell surfaces, thus hindering the interaction between enzyme active sites and soluble substrates with an adverse effect on enzyme activity expression (Kiss et al., 1998). The negative effect of diesel fuel on the germination index may be attributed to their inherent toxicity and/or to the perturbations they cause in soil and plants due to their hydrophobic properties (Adam and Duncan, 2002; Ogboghodo et al., 2004). Hydrocarbons may coat roots, preventing or reducing gas and water exchange and nutrient absorption; they may also enter the seeds and alter the metabolic reactions and/or kill the embryo by direct, acute toxicity; after penetrating the plant tissues, hydrocarbons damage cell membranes and reduce the metabolic transport and respiration rate (Adam and Duncan, 2002; Labud et al., 2007). But, a more likely reason for the inhibitory effect of diesel fuel on germination is its physical water repellent property. The film of diesel around the seeds may act as a physical barrier, preventing or reducing both water and oxygen from entering the seeds. This would inhibit the germination response (Adam and Duncan, 2002). However this inhibitory effect on the soil biological properties and germination index lasted 50 days after the diesel spill. From this date until the end of the experimental period, the soil biological properties and germination index increased. These results coincide with the data obtained for soil VAHs, suggesting that the inhibitory effect of diesel on the soil biological properties and germination index is a
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consequence of the VAH content in the soil. These results agree with those of Siddiqui and Adams (2002) and Labud et al. (2007). After the disappearance of VAHs from the soil, the TPH contents, pristine and phytane did not exercise any toxic effect on the soil's biological properties or germination index, suggesting that TPHs, pristine and phytane acts as newly available substrates for the microorganisms and the plant, since these hydrocarbon compounds represent a new source of carbon for the soil, consequently, a new source of energy and nutrients for the plant and microorganisms (Siddiqui and Adams, 2002). According to Blagodatsky et al. (2000) and Tejada and González (2007), soil microbial biomass responds rapidly to the addition of readily available C. The increase in soil enzymatic activities suggests the availability of a high quantity of biodegradable substrates (Tejada and González, 2007). For this reason, the soil biological properties and the germination index were greater than in the non-polluted soil at the end of the experimental period. Obviously, the duration of these new substrates in the soil will depend on the quantity of the spilled diesel fuel. The statistical analyses illustrated the behaviour of each soil biological parameter measured according to the soil hydrocarbon contents. This is also very important because it helps understand the behaviour of the microorganisms involved in the different nutrient cycles. 5. Conclusions We conclude that the soil biological properties and germination index can be useful tools for assessing the effect of diesel fuel on soil. The VAHs had a negative effect on soil biological properties and the germination index. After the disappearance of the VAHs from the soil, the contents of TPHs, pristine and phytane did not exercise any toxic effect on the soil biological properties and germination index, suggesting that TPHs, pristine and phytane act as new substrates available for the microorganisms and the plant, since these hydrocarbon compounds represent a new source of carbon to the soil and, as a consequence, a new source of energy and nutrients for both plants and microorganisms. Acknowledgement The authors would like to thank Spain's DGICyT for its financial support awarded in the form of Grant CTQ2004-02798. References Adam G, Duncan H. Influence of diesel fuel on seed germination. Environ Poll 2002;120:363–70. Aislabie JM, Balks MR, Foght JM, Waterhouse EJ. Hydrocarbon spills on Antarctic soils: effects and management. Environ Sci Technol 2004;38:1265–74. Alexander M. Biodegradation and bioremediation. San Diego: Academic Press, Inc.; 1999. Andreoni V, Cavalca L, Rao MA, Nocerino G, Bernasconi S, Dell'Amico E, Colombo M, Gianfreda L. Bacterial communities and enzyme activities of PAHs polluted soils. Chemosphere 2004;57:401–12. Baran S, Bielinska JE, Oleszczuk P. Enzymatic activity in an airfield soil polluted with polycyclic aromatic hydrocarbons. Geoderma 2004;118:221–32. Benítez C, Bellido E, González JL, Medina M. Influence of pedological and climatic factors on nitrogen mineralization in soils treated with pig slurry compost. Biores Technol 1998;63:147–50. Blagodatsky S, Heinemeyer O, Richter J. Estimating the active and total soil microbial biomass by kinetic respiration analysis. Biol Fertil Soils 2000;32:73–81. Couch MW, Schmidt CJ, Wasdo SC. A comparison of sampling techniques for VOCs in soil. Adv Environ Res 2000;4:97-102. Eibes G, Cajthaml T, Moreira MT, Feijoo G, Lema JM. Enzymatic degradation of anthracene, dibenzothiophene and pyrene by manganese peroxidase in media containing acetone. Chemosphere 2006;64:408–14.
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Extraction of phosphatase, urease, protease, organic carbon, and nitrogen from soil. Soil Sci Soc Am J 1980;44:1011–6. Ogboghodo IA, Iruaga EK, Osemwota IO, Chokor JU. An assessment of the effects of crude oil pollution on soil properties, germination and growth of maize (Zea mays) using two crude types-forcados light and escravos light. Environ Monitor Assess 2004;96: 143–52. Pond KL, Huang Y, Wang Y, Kulpa CF. Hydrogen Isotopic Composition of Individual nalkanes as an intrinsic tracer for bioremediation and source identification of petroleum contamination. Environ Sci Technol 2002;36:724–8. Potenz D, Rieghtti E, Bellottieri A, Girardi F, Antonacci P, Calianno LA, Pergolese G. Evoluzione de la fitotossicitain un terreno trattato con acque reflue di frantoio olear. 2. Applicazione dell test “germinazione dell lepidium sativum” e studio comparativo di alcuni parametri chimici e chimici-fisici. Inquinamento 1985;27:49–55. Saadoun L. Isolation and characterization of bacteria from crude petroleum oil contaminated soil and their potential to degrade diesel fuel. J Basic Microb 2002;42: 420–8. Serrano A, Gallego M. Continuous microwave-assisted extraction coupled on-line with liquid–liquid extraction. Determination of aliphatic hydrocarbons in soil and sediments. J Chromatogr 2006;A 1104:323–30. Serrano A, Gallego M, González JL. Assessment of natural attenuation of volatile aromatic hydrocarbons in agricultural soil contaminated with diesel fuel. Environ Pollut 2006;144:203–9. Siddiqui T, Adams WA. The fate of diesel hydrocarbons in soils and their effect on the germination of perennial ryegrass. Environ Toxicol 2002;17:49–62. Sims JR, Haby VA. Simplified colorimetric determination of soil organic matter. Soil Sci 1971;112:137–41. Soil Survey Staff. Keys to soil taxonomy, ninth ed. U.S. Department of Agriculture: Washington, DC. Statistical Graphics Corporation. 1991. Statgraphics 5.0. Statistical Graphics System. Educational Institution Edition, USA; 2003. p. 105. Tabatabai MA, Bremner JM. Use of p-nitrophenol phosphate in assay of soil phosphatase activity. Soil Biol Biochem 1969;1:301–7. Tabatabai MA, Bremner JM. Arylsulfatase activity of soils. Soil Sci Soc Am Proc 1970;34: 225–9. Tejada M, González JL. Application of different organic wastes on soil properties and wheat yield. Agron J 2007;99:1597–606. Tejada M, González JL, Hernández MT, García C. Application of different organic amendments in a gasoline contaminated soil: effect on soil biological properties. Biores Technol 2008;99:2872–80. Vance ED, Brookes PC, Jenkinson DS. An extraction method for measuring soil microbial biomass C. Soil Biol Biochem 1987;19:703–7. Wang Z, Fingas M, Page DS. Oil spill identification. J Chromatogr 1999;A 843:369–411. Zhu Y, Liu H, Cheng H, Xi Z, Liu X, Xu X. The distribution and source apportionment of aliphatic hydrocarbons in soils from the outskirts of Beijing. Org Geochem 2005;36: 475–83. 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Science of the Total Environment j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s c i t o t e n v
Evaluation of soil biological activity after a diesel fuel spill A. Serrano a, M. Tejada b,⁎, M. Gallego a, J.L. Gonzalez c a b c
Department of Analytical Chemistry, Campus of Rabanales, University of Cordoba, E-14071 Cordoba, Spain Department of Crystallography, Mineralogy and AgroChemistry, Crta de Utrera Km1, University of Seville, E-41013, Seville, Spain Department of AgroChemistry and Pedology, Campus of Rabanales, University of Cordoba, E-14071 Cordoba, Spain
a r t i c l e
i n f o
Article history: Received 17 November 2008 Received in revised form 25 February 2009 Accepted 9 March 2009 Available online 23 April 2009 Keywords: Diesel fuel spill Aromatic and aliphatic hydrocarbons Soil biological activities Germination index
a b s t r a c t Diesel fuel contamination in soils may be toxic to soil microorganisms and plants and acts as a source of groundwater contamination. The objective of this study was to evaluate the soil biological activity and phytotoxicity to garden cress (Lepidium sativum L.) in a soil polluted with diesel fuel. For this, a diesel fuel spill was simulated on agricultural soil at dose 1 l m− 2. During the experiment (400 days) the soil was not covered in vegetation and no agricultural tasks were carried out. A stress period of 18 days following the spill led to a decrease in soil biological activity, reflected by the soil microbial biomass and soil enzymatic activities, after which it increased again. The n-C17/Pristine and n-C18/Phytane ratios were correlated negatively and significantly with the dehydrogenase, arylsulphatase, protease, phosphatase and urease activities and with the soil microbial biomass during the course of the experiment. The β-glucosidase activity indicated no significant connection with the parameters related with the evolution of hydrocarbons in the soil. Finally, the germination activity of the soil was seen to recover 200 days after the spill. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The uncontrolled spillage of fuels can lead to the deterioration of the environmental quality, especially as regards the negative influence on the soil's properties and water as result of their intrinsic chemical stability, resistance to different types of degradation and high toxicity to living microorganisms (Alexander, 1999; Andreoni et al., 2004; Eibes et al., 2006). The spilling of petroleum by-products (mainly aliphatic hydrocarbon) produces an important deterioration of soil physical properties (Luthy et al., 1997) and a large variations in soil microbial diversity (Saadoun, 2002; Maliszewska-Kordybach and Smreczak, 2003; Gianfreda et al., 2005), with a noticeable increase in the presence of microorganisms that degrade hydrocarbons (Aislabie et al., 2004). This fact is reflected by the variations observed in the levels of different soil enzymatic activities (Baran et al., 2004; Tejada et al., 2008). Soil microorganisms, being in intimate contact with the soil's environment, are very sensitive to any ecosystem perturbation, and are therefore considered to be the best indicators of soil pollution (Andreoni et al., 2004) because: (i) they are a measure of the soil microbial activity and therefore they are strictly related to the nutrient cycles and transformations, (ii) they may rapidly respond to the changes caused by both natural and
⁎ Corresponding author. Tel.: +34 954486469; fax: +34 954486436. E-mail address: [email protected] (M. Tejada). 0048-9697/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2009.03.017
anthropogenic factors and (iii) they are easy to measure. For these reasons, soil enzymatic activities may be considered early and sensitive indicators for measuring the degree of soil degradation in both natural and agro-ecosystems, being thus well suited in assessing the impact of pollution on the soil quality (Baran et al., 2004; Labud et al., 2007). Aliphatic hydrocarbon analysis can be used to fingerprint spilled oils and to provide additional information on the source of hydrocarbon contamination and the extent of degradation of the spilled oil (Wang et al., 1999, Zhu et al., 2005). Comparing biodegradation indicators such as n-C17/pristine and n-C18/phytane throughout a period of time provides a measure of the effect of microbial degradation on the loss of hydrocarbons at the spill site (Wang et al., 1999). These indicators are used since pristine and phytane degradation will generally be considered negligible when n-alkanes are still available as substrates. This resistance to degradation is though to result from the greater complexity of the molecular structures of branched compounds (viz. pristine and phytane) as compared with linear alkanes (Pond et al., 2002; Hejazi and Husain, 2004a,b). This experiment formed part of a research project concerning the natural attenuation of the effect of contamination in diesel-contaminated agricultural soils. Such contamination can be caused by an accidental spillage, given that these fuels are transported by road through agricultural areas, or by leakages from fuel storage tanks located on agricultural land. For this purpose, a diesel fuel spill from a tank truck was simulated, creating contamination levels in the soil of 1 l m− 2. The natural attenuation of the volatile fraction (mainly
A. Serrano et al. / Science of the Total Environment 407 (2009) 4056–4061 Table 1 Properties of the studied agricultural soil at different depths. Depth (cm)
Sand (g kg− 1)
Silt (g kg− 1)
Clay (g kg− 1)
pH
Organic-C (g kg− 1)
C.E.C (cmolc(+) kg− 1)
0–10 10–20 20–30
303 315 342
417 384 305
290 301 353
7.6 7.7 7.7
13.3 11.6 11.1
25.4 25.8 26.5
benzene derivatives) of diesel fuel was monitored in a previous experiment, in which it was concluded that these compounds start to disperse instantly, mainly through volatilization, disappearing completely 50 days after the spill (Serrano et al., 2006). Immediately after the spill, between days 0 and 18, evaporation was the main cause in the drastic decrease of the pollutants in the soil (Serrano et al., 2006). From 18 days onwards, the soil microorganisms started to use the aliphatic hydrocarbons as source of carbon and energy as corroborated the degradation ratios (C17/Pristine and C18/ Phytane); by day 50 after spiking, biodegradation by soil microorganisms played an important role in the removal of the soil pollutants. The present experiment, therefore, focused on changes in the levels of enzymatic activities to determine the possibility of using these values as indicators of quality in agricultural soils contaminated by spills of hydrocarbons. 2. Materials and methods 2.1. Site and experimental layout The study was conducted from April 2004 to May 2005 in Córdoba (Andalusia, Spain). The soil of the field experiment is a Vertic Chromoxerert (Soil Survey Staff, 2003) and its general properties (0–30 cm) are shown in Table 1. This study site enjoyed Mediterranean climatic conditions: temperatures of 0–40 °C, average annual rainfall of 620 mm and 3000 h of annual sunshine. Soil pH was determined in distilled water with a glass electrode (soil: H2O ratio 1:2.5). The particle size distribution was determined densitometrically (Benítez et al., 1998). Total C was determined by oxidising the soil organic matter with K2Cr2O7 in sulphuric acid (96%) for 30 min and measuring the Cr(III) concentration formed (Sims and Haby, 1971). Total N was determined by the Kjeldahl method (MAPA, 1994). The cation exchange capacity was determined saturating the soil with 1 M sodium acetate and 1 M ammonium acetate (MAPA, 1994). 2.2. Soil spiking and sampling A 16 m2 plot of land located in an area with a b2% slope was used in the study. The area was cleared of stones and plant residues, after which it was fenced to keep animals off. The soil was spiked using ground spray application equipment loaded with a diesel fuel (medium light) to a final concentration of 1 l m− 2. Diesel fuel
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consists of a complex mixture of aliphatic (80–90%) and aromatic (20–10%) hydrocarbons. Four sampling areas (A, B, C and D), 2 × 2 m each, were established in the plot. A, B and C areas were contaminated in order to obtain an adequate number of representative samples that did not overlap during the studied period. Three sub-samples per area were used in each analysis (n = 9). Cores (sub-samples), 2.5 cm in diameter, were taken from 0–30 cm soil depth in the spiked plot. A parallel study was carried out in the non-contaminated area (D). Approximately 3 g of soil from each area were placed in pre-weighed 20 ml glass vials, to which aqueous saline solution and ethyl acetate (containing fluorobenzene as internal standard) were added, and immediately sealed. Samples were placed in a portable freezer to transfer them to the laboratory, where they were weighed and stored at 4 °C until analysis (1 day). In this way, evaporation losses of the volatile aromatic hydrocarbons (VAHs) in the sampling process were minimised (Couch et al., 2000). Adding saline solution and transporting the samples at 4 °C, VAHs degradation losses were avoided (Hewitt, 1997). Several of the VAHs are suspected carcinogens and some safety cautions must be taken with them. Inside the laboratory, all handling of the solutions and samples should be performed in a ventilated hood and wearing latex gloves to avoid inhalation or skin contact. For the same reason, soil sample handling in the field study should also be performed wearing special latex gloves, goggles and masks for these compounds. Two different methods were used to study the diesel fuel spill; volatile aromatic hydrocarbons (benzene derivatives) were determined using a headspace-gas chromatography-mass spectrometry method (Serrano et al., 2006) while the aliphatic hydrocarbons (from C11 to C27, including pristine and phytane) were determined using a continuous flow system that included microwave-assisted and liquid– liquid extraction units; the organic extract was finally injected into a gas chromatograph-mass spectrometer (Serrano and Gallego, 2006). The evolution of VAHs, TPHs, and the n-C17/Pri and n-C18/Phy ratios were determined 0, 4, 8, 18, 26, 50, 100, 200, 300 and 400 days after the diesel fuel contamination. 2.3. Determination of soil biochemical parameters Soil samples were collected 0, 4, 8, 18, 26, 50, 100, 200, 300 and 400 days after the diesel fuel contamination. Soils from plots were thoroughly homogenised and stored (fresh samples) at 4 °C for analysis. Soil microbial biomass was determined using the CHCl3 fumigation– extraction method (Vance et al., 1987). The levels of six enzymatic activities in the soil were measured. Dehydrogenase activity was measured by reduction of 2-p-iodo-3-nitrophenyl 5-phenyl tetrazolium chloride to iodonitrophenyl formazan (García et al., 1993). Urease activity was determined by the buffered method of Kandeler and Gerber (1988), using urea as substrate. Protease activity (BBA protease) was measured using N-α-benzoyl-L-argininamide as substrate (Nannipieri et al., 1980). Phosphatase activity was measured using p-nitrophenyl phosphate as substrate (Tabatabai and Bremner, 1969). β-glucosidase
Table 2 Evolution of VAH, TPH, (mg kg− 1soil), and n-C17/Pri and n-C18/Phy ratios and standard errors. Experimental period (Days) 0 VAH TPH n-C17/ Pri ratio n-C18/ Phy ratio
1
4
8
13
18
26
39
50
100
347.4 ± 22.1 104.9 ± 9.4 46.3 ± 3.2 24.6 ± 2.1 18.2 ± 1.6 11.8 ± 1.5 5.9 ± 0.5 1.1 ± 0.3 0.03 ± 0.01 2784 ± 136 2066 ± 106 1230 ± 96 958 ± 46 788 ± 40 549 ± 35 435 ± 16 243 ± 20 154 ± 19 129 ± 11 1.45 ± 0.16 1.47 ± 0.11 1.50 ± 0.13 1.56 ± 0.14 1.56 ± 0.12 1.54 ± 0.15 1.48 ± 0.13 1.37 ± 0.13 1.36 ± 0.16 1.33 ± 0.18 1.36 ± 0.11
1.40 ± 0.13
1.48 ± 0.15 1.54 ± 0.10 1.53 ± 0.11
1.54 ± 0.12 1.48 ± 0.17 1.40 ± 0.14
1.40 ± 0.14
200
300
76 ± 13 1.16 ± 0.13
69 ± 9 38 ± 10 1.13 ± 0.08 0.85 ± 0.06
1.37 ± 0.12 1.21 ± 0.09 1.17 ± 0.11
400
0.91 ± 0.04
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Fig. 1. Evolution of soil microbial biomass and dehydrogenase, urease and BBA-protease activities in soils contaminated with diesel fuel. INTF: 2-p-iodo-3-nitrophenyl. NS, ⁎, ⁎⁎, ⁎⁎⁎: non-significant or significant at p b 0.05, 0.01 or 0.001, respectively.
activity was determined using p-nitrophenyl-β-D-glucopyranoside as substrate (Masciandaro et al., 1994). Arylsulfatase activity was determined using p-nitrophenylsulphate as substrate (Tabatabai and Bremner, 1970). 2.4. Phytotoxicity test Germination experiments (in triplicate) were carried out in Petri dishes using soil samples taken 1, 4, 8, 13, 18, 26, 39, 50, 100, 200, 300 and 400 days after the diesel fuel contamination. Thirty grams of the corresponding soil samples were introduced into dishes and moistened, while distilled water on filter paper was used as control in other dishes. Ten seeds of garden cress (Lepidium sativum L.) were then placed on the dishes, which were placed in a germination chamber and maintained at 28 °C in darkness. The seeds were supplied by the Botanical Garden of Córdoba (Spain). The germination index was calculated according to the method described by Zucconi et al. (1981) and modified by Potenz et al. (1985). The germination index is calculated by:
G:I: =
a×b × 100 a1 × b1
where a is the average of the germinated seeds, b is the average length of each root, a1 is average of the germinated seeds in the control of the experiment, and a2 is the average length of each root in the control of the experiment. 2.5. Statistical analysis Analysis of variance (ANOVA) was performed for all variables and parameters, considering all the data collected and using the Statgraphics v. 5.0 software package (Statistical Graphics Corporation, 1991). The means were separated by Tukey's test, considering a significance level of p b 0.05 throughout the study. For the ANOVA analysis, triplicate data for each treatment were used. To compare the correlations between the parameters studied, a correlation matrix was calculated; the significance of the correlation
Fig. 2. Evolution of β-glucosidase, phosphatase and arylsulphatase activities in soils contaminated with diesel fuel. PNP: p-nitrophenol; PNF: p-nitrophenyl. NS, ⁎, ⁎⁎: nonsignificant or significant at p b 0.05 or 0.01, respectively.
A. Serrano et al. / Science of the Total Environment 407 (2009) 4056–4061
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Fig. 3. Dose (concentration x time) response curve between VAH, TPH, n-C17/Pri and n-C18/Phy ratios and soil microbial biomass-C.
coefficients is shown by using ⁎, ⁎⁎, and ⁎⁎⁎ to indicate the 95%, 99% and 99.9% probability levels, respectively. In order to establishing relationships between the soil evolution of each biochemical variable with the VAHs and TPHs contents and the values of n-C17/Pri and n-C18/Phy, linear regression analyses were performed. 3. Results 3.1. Hydrocarbons and soil biological parameters Table 2 shows the soil contents of the total volatile aromatic hydrocarbons (VAHs), and total aliphatic hydrocarbons (TPHs), and
the C17-Pristine (C17-Pri) and C18-Phytane (C18-Phy) ratios. The fraction corresponding to VAHs practically disappeared 50 days after spiking, while TPHs lasted longer, falling throughout the 400 days of the experiment, after which only about 1% of the original levels remained. This fall was more pronounced in the days following the application since after 50 days only 6% of the original concentration remained. The C17/Pri and C18/Phy ratios tended to increase until 13– 18 days after the spill, coinciding with the predominance of the volatilization (Serrano et al., 2006), and then decreased until the end of the test, coinciding with the predominance of the biodegradation. Soil microbial biomass and enzymatic activities were inhibited during the first 50 days following spiking (Figs. 1 and 2). By 50 days post-contamination, the soil microbial biomass and dehydrogenase,
Table 3 Correlation matrix of parameters studied in soil contaminated by diesel fuel. Dehydrogenase Urease activity activity
BBA-protease activity
β-glucosidase activity
Microbial 0.979 (+)(⁎⁎⁎) 0.316(+) 0.822 (+)(⁎⁎⁎) 0.428(−) biomass Dehydrogenase 0.323(+) 0.776(+)(⁎⁎⁎) 0.440(−) activity Urease activity 0.580(+)(⁎) 0.931(+) (⁎⁎⁎) BBA-protease 0.101(−) activity β-glucosidase activity Phosphatase activity Arylsulphatase activity n-C18/Phy ratio n-C17/Pri ratio TPH (+), (−) positive or negative correlation. (⁎) p b 0.05; (⁎⁎) p b 0.01; (⁎⁎⁎) p b 0.001.
Phosphatase activity
Arylsulphatase n-C18/Phy ratio n-C17/Pri ratio TPH activity
VAH
0.586(+)(⁎)
0.593(+)(⁎)
0.744(−)(⁎⁎)
0.711(−)(⁎⁎)
0.111(+)
0.311(+)
0.573(+)(⁎)
0.554(+)(⁎)
0.776(−)(⁎⁎)
0.721(−)(⁎⁎)
0.075(+)
0.254(+)
0.758(+)(⁎⁎) 0.786(+)(⁎⁎) 0.655(−)(⁎) 0.800(+)(⁎⁎⁎) 0.931(+)(⁎⁎⁎) 0.729(−)(⁎⁎)
0.741(−)(⁎⁎) 0.703(−)(⁎⁎)
0.150(−) 0.099(+)
0.042(+) 0.288(+)
0.271(+)
0.020(+)
0.355(−)
0.296(−)
0.857(+)(⁎⁎⁎) 0.796(−)(⁎⁎)
0.821(−)(⁎⁎)
0.266(+)
0.101(+)
0.766(−)(⁎⁎)
0.743(−)(⁎⁎)
0.543(−)(⁎)
0.399(−)
0.418(+)
0.243(+) 0.977(−)(⁎⁎⁎) 0.756(−)(⁎⁎) 0.731(−)(⁎⁎) 0.890(+)(⁎⁎⁎)
0.067(+)
0.130(+)
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urease, BBA-protease, β-glucosidase, phosphatase and arylsulphatase activities had decreased by 59.2%, 72.5%, 37.4%, 41.3%, 68.6%, 36.1% and 58.3%, respectively, compared with the control soil. From 50 days onwards, such enzymatic activity increased significantly until the end of the experimental period, increasing by 16.1%, 75.8%, 71.9%, 34.8%, 65%, 31.8% and 42.3%, respectively, with respect to the unpolluted soil. The increase in soil biological parameters coincide with the disappearance of soil VAHs and the biodegradation of the soil n-C17/Pri and n-C18/Phy ratios. Fig. 3 shows the dose (concentration x time) response curve between VAH, TPH, n-C17/Pri and n-C18/Phy ratios and soil microbial biomass-C. The results indicate that VAH contents negatively influence the soil microbial biomass development. Although the soil VAH decreases, microbial biomass continues decreasing, possibly because soil microorganisms had not recovered at this time. After the disappearance of VAH, the microbial biomass started to increase, reaching final values that were higher than those observed before applying the pollutant to soil. It is possible that TPH, pristine and phytane significantly affect this increase in soil microbial biomass. The correlation matrix of the different chemical and biochemical parameters determined in the polluted soil indicated that the enzymatic activity showing the lowest number of significant correlations was β-glucosidase; while phosphatase, BBA-protease and arylsulphatase activities displayed the greatest number of correlations followed, by the dehydrogenase and urease activities (Table 3). The nC18/Phy and, especially, the n-C17/Pri ratios were negatively correlated with the biochemical parameters examined. From this, it is concluded that this relation can be used as an indicator of the soil biochemical quality in the case of diesel fuel. Table 4 shows the multiple correlations between the soil microbial biomass and the different enzymatic activities (as dependent variables), and the soil VAH and TPH contents and the values of nC17/Pri and n-C18/Phy (as independent variables). For soil microbial biomass and of the dehydrogenase and urease enzymatic activities, a better fit is obtained using only the n-C17/Pri and n-C18/Phy ratios, whereas for BBA-protease, phosphatase and arylsulphatase activities the best fit is obtained taking four independent variables into consideration, namely the above mentioned relations and the soil VAH and TPH contents.
Table 4 Multiple correlations between variables. SMB = 633.4 − 158.1 C18/Phy − 174.9 C17/Pri − 4.54 × 10− 3 TPH + 4.31 × 10− 7 VAH SMB = 628.4 − 758.0 C18/Phy + 436.8 C17/Pri DHA = 24.79 + 4.614 C18/Phy17.93 C17/Pri + 5.6 × 10− 4 TPH +1.3 × 10− 5 VAH DHA = 24.03 + 22.86 C18/Phy + 10.57 C17/Pri URA = 3.229 + 2.859 C18/Phy − 3.929 C17/Pri − 6.4 × 10− 6TPH + 1.0 × 10− 7VAH URA = 3.233 + 2.791 C18/Phy − 3.865 C17/Pri BBA = 0.617 + 0.604 C18/Phy −0.921 C17/Pri + 2.86 × 10− 5 TPH + 3.61 × 10− 7 VAH BBA = 0.585 − 0.363 C18/Phy + 0.087 C17/Pri GLUA = 12.55 + 7.32 C18/Phy − 8.75 C17/Pri + 1.7 × 10− 4 TPH + 1.9 × 10− 6VAH GLUA = 12.43 + 1.423 C18/Phy + 2.537 C17/Pri PHOA = 10.98 + 8.44 C18/Phy − 11.61 C17/Pri + 2.9 × 10− 4 TPH + 2.3 × 10− 6VAH PHOA = 10.67 + 0.651 C18/Phy + 3.440 C17/Pri ASA = 15.81 + 14.28 C18/Phy − 22.03 C17/Pri + 4.8 × 10− 4 TPH + 1.0 × 10− 5 VAH ASA = 15.18 + 8.47 C18/Phy + 1.57 C17/Pri
p b 0.05; r2 = 75.75 p b 0.01; r2 = 64.96 p b 0.05; r2 = 71.9 p b 0.01; r2 = 60.69 p b 0.05; r2 = 66.52 p b 0.01; r2 = 66.50 p b 0.05; r2 = 76.36 p b 0.05; r2 = 53.33 p b 0.01; r2 = 84.22 p b 0.01; r2 = 62.31 p b 0.01; r2 = 83.69 p b 0.01; r2 = 67.65 p b 0.01; r2 = 83.09 p b 0.05; r2 = 58.89
SMB: Soil microbial biomass; DHA: dehydrogenase activity; URA: urease activity; BBA: BBA-protease activity; GLUA: β-glucosidase activity; PHOA: phosphatase activity; ASA: arylsulphatase activity.
Fig. 4. Germination index of Lepidium sativum seeds in diesel oil polluted soil.
3.2. Phytotoxicity test The germination index (GI) gives an idea of the effect of soil contamination on both seed germination and root growth. For unpolluted soil, the GI remained constant at a value of 100%. However, during the first 50 days of the experiment, the soil contaminated with diesel oil showed the lowest GI values (Fig. 4). At 4 days of the experiment, GI decreased by 84.9% with respect to the control soil. This inhibition was less 50 days into the experiment (47.9% compared with the unpolluted soil). From 100 days, the GI increased, reaching similar values to those obtained with unpolluted soil. 4. Discussion The decrease in soil microbial biomass, soil enzymatic activities and the germination index in soils polluted with diesel oil indicates that this polycyclic aromatic hydrocarbon has a toxic effect on soil microorganisms and plants, results which are in agreement with those of Adam and Duncan (2002), Siddiqui and Adams (2002) and Labud et al. (2007). The inhibitory effect of the diesel fuel on soil enzymatic activities is probably due to the suppression of the microbial populations involved in the C, N, P and S cycles (Andreoni et al., 2004); and/or to the fact that it may cover both organic-mineral and cell surfaces, thus hindering the interaction between enzyme active sites and soluble substrates with an adverse effect on enzyme activity expression (Kiss et al., 1998). The negative effect of diesel fuel on the germination index may be attributed to their inherent toxicity and/or to the perturbations they cause in soil and plants due to their hydrophobic properties (Adam and Duncan, 2002; Ogboghodo et al., 2004). Hydrocarbons may coat roots, preventing or reducing gas and water exchange and nutrient absorption; they may also enter the seeds and alter the metabolic reactions and/or kill the embryo by direct, acute toxicity; after penetrating the plant tissues, hydrocarbons damage cell membranes and reduce the metabolic transport and respiration rate (Adam and Duncan, 2002; Labud et al., 2007). But, a more likely reason for the inhibitory effect of diesel fuel on germination is its physical water repellent property. The film of diesel around the seeds may act as a physical barrier, preventing or reducing both water and oxygen from entering the seeds. This would inhibit the germination response (Adam and Duncan, 2002). However this inhibitory effect on the soil biological properties and germination index lasted 50 days after the diesel spill. From this date until the end of the experimental period, the soil biological properties and germination index increased. These results coincide with the data obtained for soil VAHs, suggesting that the inhibitory effect of diesel on the soil biological properties and germination index is a
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consequence of the VAH content in the soil. These results agree with those of Siddiqui and Adams (2002) and Labud et al. (2007). After the disappearance of VAHs from the soil, the TPH contents, pristine and phytane did not exercise any toxic effect on the soil's biological properties or germination index, suggesting that TPHs, pristine and phytane acts as newly available substrates for the microorganisms and the plant, since these hydrocarbon compounds represent a new source of carbon for the soil, consequently, a new source of energy and nutrients for the plant and microorganisms (Siddiqui and Adams, 2002). According to Blagodatsky et al. (2000) and Tejada and González (2007), soil microbial biomass responds rapidly to the addition of readily available C. The increase in soil enzymatic activities suggests the availability of a high quantity of biodegradable substrates (Tejada and González, 2007). For this reason, the soil biological properties and the germination index were greater than in the non-polluted soil at the end of the experimental period. Obviously, the duration of these new substrates in the soil will depend on the quantity of the spilled diesel fuel. The statistical analyses illustrated the behaviour of each soil biological parameter measured according to the soil hydrocarbon contents. This is also very important because it helps understand the behaviour of the microorganisms involved in the different nutrient cycles. 5. Conclusions We conclude that the soil biological properties and germination index can be useful tools for assessing the effect of diesel fuel on soil. The VAHs had a negative effect on soil biological properties and the germination index. After the disappearance of the VAHs from the soil, the contents of TPHs, pristine and phytane did not exercise any toxic effect on the soil biological properties and germination index, suggesting that TPHs, pristine and phytane act as new substrates available for the microorganisms and the plant, since these hydrocarbon compounds represent a new source of carbon to the soil and, as a consequence, a new source of energy and nutrients for both plants and microorganisms. Acknowledgement The authors would like to thank Spain's DGICyT for its financial support awarded in the form of Grant CTQ2004-02798. References Adam G, Duncan H. Influence of diesel fuel on seed germination. Environ Poll 2002;120:363–70. Aislabie JM, Balks MR, Foght JM, Waterhouse EJ. Hydrocarbon spills on Antarctic soils: effects and management. Environ Sci Technol 2004;38:1265–74. Alexander M. Biodegradation and bioremediation. San Diego: Academic Press, Inc.; 1999. Andreoni V, Cavalca L, Rao MA, Nocerino G, Bernasconi S, Dell'Amico E, Colombo M, Gianfreda L. Bacterial communities and enzyme activities of PAHs polluted soils. Chemosphere 2004;57:401–12. Baran S, Bielinska JE, Oleszczuk P. Enzymatic activity in an airfield soil polluted with polycyclic aromatic hydrocarbons. Geoderma 2004;118:221–32. Benítez C, Bellido E, González JL, Medina M. Influence of pedological and climatic factors on nitrogen mineralization in soils treated with pig slurry compost. Biores Technol 1998;63:147–50. Blagodatsky S, Heinemeyer O, Richter J. Estimating the active and total soil microbial biomass by kinetic respiration analysis. Biol Fertil Soils 2000;32:73–81. Couch MW, Schmidt CJ, Wasdo SC. A comparison of sampling techniques for VOCs in soil. Adv Environ Res 2000;4:97-102. Eibes G, Cajthaml T, Moreira MT, Feijoo G, Lema JM. Enzymatic degradation of anthracene, dibenzothiophene and pyrene by manganese peroxidase in media containing acetone. Chemosphere 2006;64:408–14.
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