Journal of Hazardous Materials 273 (2014) 207–214
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Behavior of oxyfluorfen in soils amended with different sources of organic matter. Effects on soil biology Isidoro Gómez a , Bruno Rodríguez-Morgado b , Juan Parrado b , Carlos García c , Teresa Hernández c , Manuel Tejada a,∗ a Grupo de investigación “Edafología ambiental”, Departamento de Cristalografía, Mineralogía y Química Agrícola, E.T.S.I.A. Universidad de Sevilla, Crta de Utrera km. 1, 41013 Sevilla, Spain b Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Sevilla, C/Prof. García González 2, 41012 Sevilla, Spain c Departamento de Conservación de Suelos y Agua y Manejo de Residuos Orgánicos, Centro de Edafología y Biología Aplicada del Segura, CEBAS-CSIC, PO Box 4195, 30080 Murcia, Spain
h i g h l i g h t s • Oxyfluorfen caused a negative effect on soil biological properties. • The application of organic wastes decreased the toxic action of oxyfluorfen. • The low molecular weight protein of wastes increased the degradation of herbicide.
a r t i c l e
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Article history: Received 15 October 2013 Received in revised form 19 February 2014 Accepted 17 March 2014 Available online 1 April 2014 Keywords: Oxyfluorfen Biofertilizer/biostimulant Municipal solid waste Sheep manure Soil enzymatic activities Soil microbial community
a b s t r a c t We performed a laboratory study on the effect of oxyfluorfen at a rate of 4 l ha−1 on biological properties of a soil amended with four organic wastes (two biostimulants/biofertilizers, obtained from rice bran, RB1 and RB2; municipal solid waste, MSW; and sheep manure, SM). Soil was mixed with SM at a rate of 1%, MSW at a rate of 0.52%, RB1 at a rate of 0.39% and RB2 at a rate of 0.30%, in order to apply the same amount of organic matter to the soil. The enzymatic activities and microbial community in the soil were determined during the incubation times. The application of RB1 and RB2 to soil without oxyfluorfen increased the enzymatic activities and biodiversity, peaking at day 10 of the incubation period. This stimulation was higher in the soil amended with RB2 than in that amended with RB1. In SM and CF-amended soils, the stimulation of enzymatic activities and soil biodiversity increased during the experiment. The application of herbicide in organic-amended soils decreased the inhibition of soil enzymatic activities and soil biodiversity. Possibly the low molecular weight protein content easily assimilated by soil microorganisms and the higher fat content in the biostimulants/biofertilizers are responsible for the lower inhibition of these soil biological properties. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The increase in the levels of pesticide residues in soils and ground water is a current environmental problem. Chemicals that are highly soluble in water, minimally adsorbed by soil particles and not readily degradable, are easily leached by infiltrating water and hence, are likely to be found in ground water [1–3]. Several bioremediation strategies for reducing the presence of pesticides in soil from which they can reach groundwater have been
∗ Corresponding author. Tel.: +34 95448646; fax: +34 954486436. E-mail address: [email protected] (M. Tejada). http://dx.doi.org/10.1016/j.jhazmat.2014.03.051 0304-3894/© 2014 Elsevier B.V. All rights reserved.
proposed. One such is remediation by enhancing the microbial population capable of specifically degrading the target compounds [4]. This strategy has been approached by adding organic matter of different origins. The application of organic matter to the soils causes an increase in their biological activity accelerating the degradation of the pesticide in the soil. Furthermore, the pesticide adsorption capacity of humic substances reduces the xenobiotic levels in the soil solution and consequently decreases the toxic effects of the pesticide [4–7]. However, as these organic products to activate soil microorganisms, the organic compounds need to be degraded into simpler easily assimilated forms, requiting a great expenditure of energy by soil microorganisms. Moreover, this degradation depends both
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Table 1 Characteristics of the experimental soil (mean ± S.E.). Data are the means of three samples. pH (H2 O) Coarse sand (g kg−1 ) Fine sand (g kg−1 ) Silt (g kg−1 ) Clay (g kg−1 ) Total N (g kg−1 ) Organic matter (g kg−1 )
7.9 486 130 123 260 0.93 17
± ± ± ± ± ± ±
Table 3 Molecular weight distribution of (mean ± S.E.) of the organic matter studied. Data are the means of three samples. Rows followed by the same letter(s) are not significantly different (p > 0.05).
0.2 49 25 29 35 0.08 1
on soil factors and the chemical composition of the organic material applied to soil [4,7,8]. In recent years the use of hydrolysate organic biofertilizers/biostimulants (BS) obtained from different organic materials by hydrolysis reactions has been increasing [9–12]. These BS, generally comprising peptides, amino acids, polysaccharides, humic acids, phytohormones, etc., are directly absorbed by soil microorganisms and plants which thus have to devote less energy to the absorption process. Therefore, not only does the application of these BS to the soil lead to an increased content of organic matter and macro- and micro-nutrients, it also leads to a significant activation of the soil’s microbial community. As a result, the development of new BS has become the focus of interest in research. Biological and biochemically mediated processes in soils are of the utmost importance to ecosystem function. Many studies have shown that biological parameters have been used to assess soil quality and health as affected by agricultural practices [4,13]. Also, the number of physiological groups of bacteria has also proved to be useful for measuring structural changes in soil due to several anthropogenic factors [14,15]. Therefore, the changes in soil microbial community could be of help in evaluating the impact of herbicides on soils. The objective of this study was to investigate under laboratory conditions the behavior of oxyfluorfen in soils amended with different organic wastes and its influence on soil enzymatic activities biological and soil biodiversity.
Molecular weight (Da)
SM (%)
>10,000 10,000–5000 5000–1000 1000–300 0.05). SM pH (1:2.5) Organic matter (g kg−1 ) Total N (g kg−1 ) Fat (g kg−1 ) P (g kg−1 ) K (g kg−1 ) Ca (g kg−1 ) Mg (g kg−1 ) Fe (mg kg−1 ) Cu (mg kg−1 ) Mn (mg kg−1 ) Zn (mg kg−1 ) Cd (mg kg−1 ) Pb (mg kg−1 ) Ni (mg kg−1 ) Nd: Not determined.
7.8a ± 225a ± 16.8a ± Nd 13.8b ± 19.7b ± 16.8b ± 10.3b ± 1345b ± 6.9b ± 86.4c ± 70.1c ± 1.2a ± 1.4a ± 1.9a ±
MSW 0.2 13 1.7 1.9 2.5 2.1 1.9 106 1.8 8.2 8.3 0.3 0.5 0.4
6.7a ± 475b ± 18.6a ± Nd 11.6b ± 10.1b ± 133c ± 68.3c ± 835a ± 3.9a ± 17.1a ± 40.8b ± 1.6a ± 0.8a ± 1.1a ±
RB1 0.3 22 2.1 1.4 1.7 18 5.9 77 1.2 2.6 4.1 0.5 0.2 0.4
7.9a 548c 17.9a 2.8a 8.1a 2.2a 0.74a 1.3a 273a 3.4a 29.1ab 18.4a 1.9a 1.1a 1.8a
RB2 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.2 25 1.1 0.4 1.7 0.4 0.12 0.2 35 0.9 1.2 2.6 0.3 0.3 0.2
7.8a 553c 18.4a 11.2b 8.6a 2.5a 0.88a 1.4a 288a 3.0a 33.6b 21.1a 1.6a 1.2a 1.7a
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.1 18 2.0 1.4 1.9 0.3 0.17 0.2 29 0.6 2.3 1.8 0.7 0.4 0.4
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insoluble molecules, and the supernatant was passed through a 0.2 m filter and loaded into a 0.1 ml loop connected to an ÄKTApurifier system. The column was equilibrated, and eluted with 0.25 M Tris–HCl buffer (pH 7.0) in isocratic mode, at a flow-rate of 0.5 ml min−1 , and proteins/peptides were detected at 280 and 215 nm with a GE Healthcare UV900 module coupled to the column elution. For MSW and SM, the proteins were extracted at pH = 9, temperature = 55 ◦ C and time = 3 h. Subsequently, the distribution of proteins was also determined by size-exclusion chromatography 2.2. Incubation procedure Five hundred grams of dried and 2 mm-sieved soil were preincubated at 25 ◦ C for 7 days at 30–40% of their water-holding capacity according to Tejada [3], prior to the treatments. Two days after applying oxyfluorfen to soil, organic wastes were also applied to the soil. Soil samples were mixed with SM at a rate of 1% or MSW at a rate of 0.52%, or RB1 at a rate of 0.39% or RB2 at a rate of 0.30% applying to the soil the same amount of organic matter with each organic waste. Both BS were liquid and were solubilized in distilled water before the application. An unamended soil was used as control. Distilled water was added to each soil to bring it to 60% of its water-holding capacity. The incubation treatments are detailed as follows: 1. C, control soil, soil non-organic amended and without oxyfluorfen; 2. H, soil with oxyfluorfen and non-organic amended; 3. SM, soil without oxyfluorfen and amended with SM; 4. MSW, soil without oxyfluorfen and amended with MSW; 5. RB1, soil without oxyfluorfen and amended with RB1; 6. RB2, soil without oxyfluorfen and amended with RB2; 7. SM + H, soil with oxyfluorfen and amended with SM; 8. MSW + H, soil with oxyfluorfen and amended with MSW; 9. RB1 + H, soil with oxyfluorfen and amended with RB1; 10. RB2 + H, soil with oxyfluorfen and amended with RB2. Triplicate treatments were kept in semi-closed microcosms at 25 ± 1 ◦ C for 120 days. 2.3. Soil analysis The activity levels of four soil enzymes for each treatment were measured at days 5, 10, 20, 40, 80 and 120 during the incubation period. Dehydrogenase activity was measured as the reduction of 2-p-iodo-3-nitrophenyl 5-phenyl tetrazolium chloride to iodonitrophenyl formazan [22]. Urease activity was determined by the buffered method of Kandeler and Gerber [23], using urea as substrate. The -glucosidase activity was determined using pnitrophenyl--d-glucopyranoside as substrate [24]. Phosphatase activity was measured using p-nitrophenyl phosphate as substrate [25]. Phospholipids were extracted at days 5, 10, 20 and 120 during the incubation period for each treatment, (three replicates per treatment) using a chloroform–methanol extraction based on Bligh and Dyer [26]. They were fractioned and quantified using the procedure described by Frostegard et al. [27] and Bardgett et al. [28]. Twenty-six separated fatty acid methyl esters were identified using gas chromatography and a flame ionization detector. The phospholipids were transformed by alkaline methanolysis into fatty acid methyl esters (FAMEs), which were quantified with a gas chromatograph (GC/FID, AutoSystem XL Gas Chromatograph, Varian Saturno 2000) fitted with a 50-m capillary column, using helium as the carrier gas. The injector temperature was 260 ◦ C, the flame ionization detector temperature was 280 ◦ C, and the initial temperature was 70 ◦ C (for 2 min); it was increased to 160 ◦ C at 30 ◦ C min−1 and then to 280 ◦ C at 3 ◦ C min−1 .
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To estimate the various proportions of the main taxa in the samples according to the PLFAs, the biomarkers i15:0, a15:0, i16:0, 16:17c, 17:0, i17:0, cy17:0, 18:19c, and cy19:0 were used to represent bacterial biomass (bacPLFA) [28,29] and 18:26 (fungPLFA) was taken to indicate fungal biomass [30]. The ratio of bacPLFA to fungPLFA (bacPLFA/fungPLFA) represents the ratio between bacterial and fungal biomass [28]. The Gram+ specific fatty acids i15:0, a15:0, i16:0, and i17:0 and the Gram− specific fatty acids cy17:0, 18:19c, and cy19:0 were taken as a measure of the ratio of the Gram+ and Gram− bacterial biomass (Gram+ :Gram− ). All results are given in nmol g−1 . For each treatment and each incubation time, 20 g of soil were taken. Soil samples were stored in sealed polyethylene bags at 4 ◦ C for 15 d, prior to analysis of the enzymatic activities [7], and at −20 ◦ C prior to phospholipid analysis [31]. 2.4. Oxyfluorfen determination in soil The extraction of oxyfluorfen from soil was realized using the Anastassiades et al. [32] method. Oxyfluorfen was extracted with a mixture of triphenyl phosphate and acetonitrile. Once the supernatant had been shaken and centrifuged, magnesium sulfate was added to it and it was, stirred and centrifuged again. The supernatant was concentrated and the dried residue was recomposed with 1 ml of cyclohexane:ethyl acetate (9:1). Oxyfluorfen was determined using a tandem mass spectrometer and electron impact, where the chromatographic conditions were as follows: carrier gas: He at 1 ml min−1 , initial temperature inlet: 70 ◦ C for 0.50 min, 310 ◦ C to 100 ◦ C min−1 for 10 min, column: 30 m × 0.25 mm ID, initial temperature of the column oven: 70 ◦ C for 3.5 min., 180 ◦ C to 35 ◦ C min−1 , 300 ◦ C at 10 ◦ C min−1 for 5 min; temperature detector: trap to 250 ◦ C; manifold 60 ◦ C; xferline to 280 ◦ C; injection volume: 5 l. 2.5. Statistical analysis Data were submitted to two-way ANOVA with treatment and sampling time as factors followed by Tukey’s significant difference as a post hoc test, considering a significance level of p < 0.05 throughout the study. The ANOVA was performed using the using the Statgraphics Plus 2.1 software package. For the ANOVA, triplicate data were used for each treatment and each day of incubation. 3. Results 3.1. Characterization of organic wastes Molecular weight distribution showed significant differences (p < 0.05) in the organic compounds studied (Table 3). In this respect, RB1 and RB2 did not differ significantly in terms of protein weight distribution, with a high content of small molecular weight proteins. MSW and SM, however, have a higher content of high molecular weight protein., MSW presented a higher content of larger molecular protein than SM. 3.2. Evolution of soil biological properties Statistical analysis indicated a significant (p < 0.05) stimulation of the dehydrogenase activity during the first days after the application of both BS, peaking 10 days after the beginning of the experiment (Table 4). In this respect, and compared to the control, the soil dehydrogenase activity increased significantly (p < 0.05) by 87.8% and 92.5% in the RB1 and RB2 treatments, respectively. After the first 10 days, the dehydrogenase activity began to decline gradually. At the end of the experimental period BS treatments had very similar values to the control soil. In SM and CF-amended soils,
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Table 4 Evolution of dehydrogenase and urease activities in soils amended with the organic matter studied and with oxyfluorfen during the experimental period. Data are expressed as mean values ± standard error. Columns followed by the same letter(s) are not significantly different (p > 0.05). INTF: 2-p-iodo-3-nitrophenyl formazan. Dehydrogenase activity (g INTF g−1 h−1 ) 5
10
40
80
120
0.4 0.3 0.7 0.4 1.4 1.6 0.4 0.3 1.2 1.5
2.6b 1.2a 4.2b 3.8b 21.4d 34.6e 3.4b 2.9b 12.6d 19.3d
± ± ± ± ± ± ± ± ± ±
0.5 0.4 0.5 0.8 1.8 2.6 0.6 0.4 0.9 1.2
2.5b 1.2a 5.1b 4.3b 9.6cd 12.8d 4.4b 3.3b 5.6b 6.3b
± ± ± ± ± ± ± ± ± ±
0.6 0.2 0.9 0.6 1.1 1.5 0.4 0.6 0.6 0.5
2.5b 1.3a 6.4b 5.4b 4.3b 6.6b 5.7b 4.0b 3.7b 4.1b
± ± ± ± ± ± ± ± ± ±
0.4 0.3 0.6 0.7 1.0 0.9 0.8 0.6 0.3 0.4
2.4b 1.2a 7.7c 6.5b 2.8b 3.5b 6.2b 4.8b 2.4b 2.4b
± ± ± ± ± ± ± ± ± ±
0.3 0.2 0.6 0.4 0.9 0.4 0.7 0.6 0.3 0.2
2.3b 1.2a 10.1c 7.5c 2.2b 2.5b 7.7c 5.6b 2.3b 2.4b
± ± ± ± ± ± ± ± ± ±
0.5 0.3 0.8 0.7 0.3 0.6 0.6 0.6 0.4 0.5
Urease activity (g NH4 + g−1 h−1 ) C 1.9b ± 0.4 H 1.1a ± 0.2 SM 2.2b ± 0.3 MSW 2.1b ± 0.2 RB1 2.0b ± 0.3 RB2 2.0b ± 0.4 2.0b ± 0.4 SM + H 2.0b ± 0.2 MSW + H RB1 + H 1.0a ± 0.2 1.0a ± 0.3 RB2 + H
2.0b 1.1a 2.8b 2.6b 1.9b 2.0b 2.6b 2.4b 0.9a 1.0a
± ± ± ± ± ± ± ± ± ±
0.2 0.3 0.4 0.3 0.3 0.2 0.4 0.4 0.1 0.2
1.8b 0.9a 4.1bc 3.6b 2.1b 2.1b 3.4b 2.9b 1.0a 0.9a
± ± ± ± ± ± ± ± ± ±
0.2 0.1 0.5 0.3 0.3 0.4 0.3 0.5 0.2 0.2
1.9b 1.0a 5.6c 4.8bc 2.0b 1.9b 4.0bc 3.4b 0.9a 1.0a
± ± ± ± ± ± ± ± ± ±
0.3 0.2 0.4 0.6 0.3 0.2 0.5 0.4 0.2 0.3
1.8b 0.9a 6.8c 5.4c 1.9b 2.0b 4.9bc 4.0bc 1.0a 0.9a
± ± ± ± ± ± ± ± ± ±
0.2 0.2 0.8 0.5 0.2 0.2 0.6 0.8 0.2 0.2
1.9b 1.0a 8.0d 6.6c 2.0b 1.9b 5.8c 4.6bc 0.9a 1.0a
± ± ± ± ± ± ± ± ± ±
0.4 0.3 1.1 0.9 0.3 0.3 0.6 0.7 0.1 0.2
C H SM MSW RB1 RB2 SM + H MSW + H RB1 + H RB2 + H
2.7b 1.3a 3.3b 3.2b 12.6 15.9 2.6b 2.4b 8.7cd 10.1d
± ± ± ± ± ± ± ± ± ±
20
the dehydrogenase activity was stimulated progressively over the whole experimental period. At the end of the experimental period and compared to the control soil, the dehydrogenase activity significantly (p < 0.05) increased 68% and 76.2% in soils amended with MSW and SM, respectively. The application of oxyfluorfen in soil showed a significant decrease in dehydrogenase activity (Table 4). At the end of the incubation period and compared with the control treatment, dehydrogenase activity decreased significantly (p < 0.05) by 47.8%. The application of both BS in oxyfluorfen soils caused a minor decrease in dehydrogenase activity (Table 4). In this respect, 10 days after applying the herbicide in the soil, and compared to RB1 treatment, the dehydrogenase activity in the RB1 + H treatment decreased (41.1%). Compared to RB2 treatment, the dehydrogenase activity in the RB2 + H treatment decreased 44.2%. At day 20 of the incubation period, this decrease in soil dehydrogenase activity was diminished progressively over the experimental period, noting that in soils amended with both BS, this activity showed values similar to those of soil amended without oxyfluorfen. The application of SM and MSW in soils with oxyfluorfen caused a decrease in the enzyme inhibition. However, this inhibition was lower than that caused by both BS. Furthermore, this inhibition was significantly (p < 0.05) higher in MSW + H treatment than in the SM + H treatment (2.3%). Unlike the dehydrogenase activity, soil urease activity was not stimulated after the application of the two BS studied. However in SM and CF-amended soils, the dehydrogenase activity was progressively stimulated over the whole experimental period (Table 4). Similar to the dehydrogenase activity, when oxyfluorfen was applied to the unamended soil, there was a significant (p < 0.05) decrease in this enzyme activity throughout the experimental period. These same results were also observed when the different organic wastes studied where applied to the soil with the herbicide. Similar to the dehydrogenase activity, the -glucosidase activity was also stimulated in organically amended soils. However this stimulation depended on the organic matter applied to the soil (Table 4). The -glucosidase activity was significantly (p < 0.05) stimulated during the first days after the application of both BS, peaking 10 days after the start of the experiment (Table 4). In this respect, and compared to the control, the soil -glucosidase
activity significantly (p < 0.05) increased by 81.2% and 85.7% in the RB1 and RB2 treatments, respectively. After the first 10 days, the -glucosidase activity began to decline gradually. In SM and CF-amended soils, the dehydrogenase activity was progressively stimulated over the whole experimental period. At the end of the experimental period and compared to the control soil, the glucosidase activity increased significantly (p < 0.05) by 87.2% and 92.6% in soils amended with MSW and SM, respectively. The response of -glucosidase activity to the application of oxyfluorfen in soil was very similar to other enzyme activities studied (Table 5). An inhibition of this enzyme in the soils with oxyfluorfen was observed. Moreover, the application of organic wastes to the soil decreased the inhibition of this enzymatic activity. However this inhibition depended on the organic matter applied to the soil. In the case of both BS, and 10 days after the start the experiment, this decrease was higher in RB1 + H treatment than in that of the RB2 + H treatment. Thirty days after beginning the incubation period, this decrease in soil -glucosidase activity diminished progressively over the experimental period, noting that in soils amended with both BS, this activity showed similar values to those of the amended soil without oxyfluorfen. The application of SM and MSW in soils with oxyfluorfen also caused a decrease in the enzyme inhibition. Again, this inhibition was lower than that caused by both BS. The soil phosphatase activity was also significantly (p < 0.05) stimulated after the application of organic wastes to the soil (Table 5). Compared to both BS, this stimulation was different in MSW and SM-amended soils. Similar to the dehydrogenase and -glucosidase activities, the soil phosphatase activity showed a higher stimulation in soil amended with both BS 10 days after beginning the experiment. In this respect, and compared to the C treatment, soil phosphatase activity significantly increased by 88.6% and 91.1% in the RB1 and RB2 treatments, respectively. This stimulation decreased as the experimental period progressed. In SM and CF-amended soils, the phosphatase activity was progressively stimulated over the whole experimental period. At the end of the experimental period and compared to the control soil, the dehydrogenase activity increased significantly (p < 0.05) by 71.4 and 80.2% in soils amended with MSW and SM, respectively. Furthermore, applying oxyfluorfen to the soil inhibited this enzymatic activity during the experimental period (Table 5).
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Table 5 Evolution of -glucosidase and phosphatase activities in soils amended with the organic matter studied and with oxyfluorfen during the experimental period. Data are expressed as mean values ± S.E. Columns followed by the same letter(s) are not significantly different (p > 0.05). PNP: p-nitrophenol. -glucosidase activity (mmol PNP g−1 h−1 ) 5 C H SM MSW RB1 RB2 SM + H MSW + H RB1 + H RB2 + H
10 1.4b 0.5a 2.0b 1.9b 3.8c 5.2c 1.8b 1.7b 2.3b 3.1c
± ± ± ± ± ± ± ± ± ±
0.3 0.1 0.4 0.3 0.9 1.4 0.2 0.2 0.2 0.3
Phosphatase activity (mol PNP g−1 h−1 ) C 3.6b ± 1.2 H 1.9a ± 0.4 SM 4.3b ± 0.9 MSW 4.0b ± 1.1 RB1 19.4d ± 2.1 RB2 21.9d ± 2.8 3.7b ± 1.4 SM + H 3.7b ± 1.3 MSW + H RB1 + H 10.4c ± 1.2 13.6c ± 1.8 RB2 + H
20
40
80
120
1.3b 0.6a 3.0c 2.7b 6.9d 9.1e 2.4b 2.0b 4.8c 6.0cd
± ± ± ± ± ± ± ± ± ±
0.2 0.2 0.5 0.2 0.9 1.3 0.3 0.3 0.5 0.8
1.2b 0.7a 5.1c 3.9c 4.7c 5.8c 3.0c 2.5b 3.0c 4.1c
± ± ± ± ± ± ± ± ± ±
0.3 0.1 0.9 0.6 0.4 0.9 0.4 0.4 0.4 0.6
1.2b 0.6a 7.1d 5.0c 2.9b 3.2c 4.1c 3.2c 2.1b 3.0c
± ± ± ± ± ± ± ± ± ±
0.3 0.2 1.2 0.5 0.5 0.5 0.6 0.4 0.4 0.5
1.1b 0.5a 9.7e 6.3d 1.3b 1.3b 5.2c 4.3c 1.2b 1.3b
± ± ± ± ± ± ± ± ± ±
0.1 0.1 1.7 1.0 0.2 0.2 0.6 0.4 0.3 0.2
1.0b 0.6a 13.6f 7.8d 1.2b 1.1b 6.6d 5.0c 1.1b 1.1b
± ± ± ± ± ± ± ± ± ±
0.2 0.1 2.2 1.4 0.2 0.2 1.1 0.4 0.2 0.1
3.5b 2.0a 5.9b 5.1b 30.6d 39.4e 4.6b 4.2b 15.6 20.9
± ± ± ± ± ± ± ± ± ±
1.1 0.5 1.3 1.5 3.3 3.0 1.0 1.4 1.5 2.2
3.5b 1.8a 7.0c 6.3b 25.6d 30.9de 6.0b 5.1b 13.6c 17.6
± ± ± ± ± ± ± ± ± ±
1.4 0.3 1.1 0.9 2.5 3.4 1.6 1.5 2.0 1.9
3.4b 1.5a 8.8c 7.6c 8.1c 10.9c 7.8c 5.9c 6.1c 7.2c
± ± ± ± ± ± ± ± ± ±
1.1 0.3 1.4 1.0 1.9 1.3 1.5 1.4 1.6 1.3
3.3b 1.6a 11.0c 9.0c 3.9b 3.8b 8.4c 6.6c 3.5b 3.6b
± ± ± ± ± ± ± ± ± ±
1.1 0.4 1.3 1.2 1.1 1.4 1.5 1.3 1.1 1.2
3.2b 1.8a 16.2d 11.2c 3.0b 3.1b 9.6c 7.8c 3.1b 3.3b
± ± ± ± ± ± ± ± ± ±
1.4 0.5 1.4 1.1 1.1 0.9 1.2 1.5 1.2 1.3
Similar to the enzymes studied, when applied organic wastes were applied to the soil with oxyfluorfen the inhibition of phosphatase activity decreased. This decrease was lower in the RB2 + H treatment, followed by RB1 + H, SM + H and MSW + H treatments, respectively. The application of the organic waste increased the soil bacteria and fungi population (Table 6). Similar to the results of the enzymatic activities, this stimulation depended on the organic matter applied to the soil. Again, and in the BS treatments, the highest populations of bacteria and fungi were found after 10 days of incubation. These populations, however, decreased over the experimental period. At the end of the experiment, the bacteria population was similar to that observed in the control treatment. The bacGram+ /Gram− and bacPLFA/fungPLFA rates increased in BS- treatments, also indicating the variability in the biodiversity of these soils during the first days of the experiment (Table 6). At the end of the experimental period, the rates showed similar values to those obtained in the control treatment. In the MSW and SM-amended soils, the stimulation occurred progressively over the experimental period and at the end of the experiment, the bacteria population was higher in SM than in MSW treatment. When the herbicide was applied to the soil, the total bacterial population decreased significantly (p < 0.05) while the fungal population decreased slightly. However, and with respect to the control treatment, no significant (p > 0.05) differences were found in bacGram+ /Gram− and bacPLFA/fungPLFA rates (Table 6). Compared with contaminated soils, the application of organic matter to soils with oxyfluorfen caused a minor decrease in the bacteria and fungi populations. This decrease was higher when the BS applied to the soil was RB2. 3.3. Evolution of oxyfluorfen in soil The application of organic matter to soil with herbicide, decreased the soil oxyfluorfen concentration (Fig. 1). However, this decrease depended on the organic matter type applied to the soil. Compared to the MSW and SM treatments, the application of both BS in oxyfluorfen contaminated soils caused a higher decrease in dehydrogenase activity. The values suggest a more rapid degradation of the herbicide when the RB2 was applied to the soil with herbicide. In this respect, 10 days after applying the herbicide to the
soil, and compared to the RB1 + H treatment the oxyfluorfen significantly (p < 0.05) decreased with the RB2 + H treatment (25%). At the end of the experimental period, the concentration of herbicide in soil was significantly (p < 0.05) lower in the soils with oxyfluorfen and amended with both BS than that found in the control soil. For MSW + H and SM + H treatments, the evolution of oxyfluorfen indicated that the degradation of the herbicide occurred more slowly and gradually during the experiment. The degradation of the oxyfluorfen in soil was higher in SM + H treatment than for MSW + treatment. 4. Discussion Oxyfluorfen caused a toxic effect on soil enzymatic activity and soil diversity. These results are in agreement with those obtained by García-Orenes et al. [33] and Nadiger et al. [34], who found an important toxic effect of this herbicide on agricultural soil microorganisms. Moreover, our results show a high persistence of oxyfluorfen in soil. These results are in agreement with those obtained by other authors. Baruah and Mishra [35] indicate that oxyfluorfen has a long persistence in soil (half-life of 72 to 160 days) in natural conditions. Also, Ying and Williams [36] found that this herbicide persisted in soil for 119 days. The application of diverse organic wastes caused an increase in soil microbial activity. When SM and MSW were applied to the soil, the stimulation of soil microorganisms increased progressively during the experimental period. These results are in agreement with those obtained by other authors when applying organic matter of very different chemical nature such as urban waste composts, vermicomposts, green manure, poultry manure etc. [37–39] to soil. Soil microorganisms were also stimulated when BS, RB1 or RB2 were applied to the soil. These results are in agreement with those obtained by Parrado et al. [9], García-Martínez et al. [10,11] and Tejada et al. [4,12] who indicate that the incorporation of different BS, obtained from condensed distillers soluble wheat, enzymatic carob germ, rice bran extract and sewage sludge, stimulates microbial activity in soil. However, the evolution of the soil microbial stimulation was differed, depending on the type of organic waste applied. This might be a consequence of the different protein size distribution obtained for different organic wastes. Our results indicate that protein size
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Table 6 Evolution of bacterial Gram+ , bacterial Gram− , total bacterial, fungal PLFAs (nmol g−1 ), Gram+ /Gram− and bacteria/fungi during the experimental period. Data are the means of three samples. Columns (mean ± S.E.) followed by the same letter(s) are not significantly different (p > 0.05). bacGram+ C (5 d) C (10 d) C (20 d) C (120 d) H (5 d) H (10 d) H (20 d) H (120 d) SM (5 d) SM (10 d) SM (20 d) SM (120 d) MSW (5 d) MSW (10 d) MSW (20 d) MSW (120 d) RB1 (5 d) RB1 (10 d) RB1 (20 d) RB1 (120 d) RB2 (5 d) RB2 (10 d) RB2 (20 d) RB2 (120 d) SM + H (5 d) SM + H (10 d) SM + H (20 d) SM + H (120 d) MSW + H (5 d) MSW + H (10 d) MSW + H (20 d) MSW + H (120 d) RB1 + H (5 d) RB1 + H (10 d) RB1 + H (20 d) RB1 + H (120 d) RB2 + H (5 d) RB2 + H (10 d) RB2 + H (20 d) RB2 + H (120 d)
13.5b 13.0b 13.3b 13.4b 8.9a 9.0a 9.2a 8.9a 13.8b 15.9b 18.2b 20.4c 13.4b 15.0b 16.1b 18.0c 28.9c 39.6d 22.8c 13.7b 34.4 45.6e 19.9c 13.6b 12.9b 14.1b 15.0b 16.6c 12.9b 13.8b 14.6b 15.9c 19.2c 24.3c 19.5c 13.2b 21.4c 33.1d 20.6c 13.4b
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.4 1.3 1.3 1.4 1.1 1.2 0.9 0.7 1.4 1.1 1.6 1.5 1.1 1.4 1.2 1.3 2.1 2.6 1.5 1.5 2.4 3.1 1.6 1.1 1.3 1.4 1.3 1.2 1.4 1.5 1.3 1.3 1.7 2.2 1.4 1.1 1.8 2.6 2.1 1.3
bacGram− 1.5b 1.4b 1.5b 1.5b 1.0a 0.8a 0.9a 1.0a 1.7b 2.5b 4.1c 5.2c 1.6b 2.0b 3.0b 4.4c 2.8b 3.5c 2.3b 1.5b 2.8b 3.5c 2.3b 1.5b 1.6b 2.0b 3.3c 4.3c 1.5b 1.8b 2.6b 3.6c 1.4b 2.4b 1.8b 1.5b 1.6b 2.7b 2.0b 1.6b
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.3 0.4 0.3 0.3 0.2 0.2 0.2 0.3 0.3 0.4 0.7 0.5 0.3 0.5 0.5 0.9 0.6 0.6 0.7 0.4 0.6 0.6 0.7 0.4 0.6 0.9 0.6 1.1 0.8 0.6 1.1 1.2 0.3 0.7 0.5 0.3 0.3 0.4 0.4 0.2
Total bacterial PLFA 15.0b 14.4b 14.8b 14.9b 9.9a 9.8a 10.1a 9.9a 15.5b 18.4b 22.3c 25.6c 15.0b 17.0b 19.1b 22.4c 31.7d 43.1d 25.1c 15.2b 37.2d 49.1d 22.2c 15.1b 14.5b 16.1b 18.3b 20.9c 14.4b 15.6b 17.2b 19.5c 20.6c 26.7c 21.3c 14.7b 23.0c 35.8d 22.6c 15.0b
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.5 1.4 1.4 1.5 1.2 1.2 1.1 1.2 1.4 1.9 1.7 2.0 1.6 1.8 1.1 1.9 2.7 2.1 2.0 1.7 1.9 2.7 2.0 1.9 1.1 1.9 1.6 1.8 1.3 1.5 1.4 1.6 1.4 1.5 1.5 1.2 2.1 3.0 1.8 1.4
distribution differs greatly for each organic waste. While BS have a higher content of low molecular weight proteins, SM and MSW have a higher content of high molecular weight proteins. Tejada et al. [12] found that after applying the BS obtained from sewage sludge to the soil, the soil microbial activity showed a greater increase when the BS applied contained a higher proportion of
Fungal PLFA 0.9b 0.9b 1.0b 0.9b 0.5a 0.5a 0.6a 0.6a 1.0b 1.6b 2.8c 4.1d 1.0b 1.3b 2.0c 3.0c 1.5b 1.9c 1.3b 1.0b 1.7b 2.6c 1.5b 1.0b 0.9b 1.3b 1.9c 2.9c 0.9b 1.1b 1.5b 2.4c 1.3b 1.6b 1.2b 1.0b 1.5b 1.9c 1.3b 1.0b
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.1 0.1 0.2 0.1 0.1 0.1 0.2 0.1 0.1 0.3 0.5 0.4 0.2 0.2 0.4 0.4 0.3 0.3 0.2 0.1 0.4 0.4 0.3 0.2 0.1 0.3 0.3 0.5 0.1 0.2 0.4 0.5 0.2 0.2 0.2 0.3 0.3 0.4 0.2 0.2
bacGram+ /bacGram− 9.0b 9.3b 8.9b 8.9b 8.9b 11.3b 10.2b 8.9b 9.9b 6.4a 4.4a 3.9a 8.4b 7.5b 5.4a 4.1a 10.3b 11.3b 9.9b 9.1b 12.3c 13.0c 8.7b 9.1b 8.1b 7.1b 4.5a 3.9a 8.6b 7.7a 5.6a 4.4a 13.7c 10.1b 10.8b 8.8b 13.4c 12.3c 10.3b 8.4b
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.1 1.0 1.1 1.1 1.1 1.3 1.3 1.2 1.1 1.4 1.0 1.1 1.0 1.2 0.9 1.0 1.8 1.6 1.6 1.4 1.5 1.8 1.2 1.0 1.1 1.4 1.3 0.8 1.0 1.1 1.1 1.0 1.1 1.4 1.1 1.0 1.2 1.3 1.1 1.0
bacPLFA/fungPLFA 16.7b 16.0b 14.8b 16.5b 19.8c 19.6c 16.8b 16.5b 15.0b 11.5b 8.0a 6.2a 15.0b 13.1b 9.6a 7.5a 21.1c 22.7c 19.3c 15.2b 21.9c 18.9c 14.8b 15.1b 16.1b 12.4b 9.6a 7.2a 16.0b 14.2b 11.5b 8.1a 15.8b 16.7b 17.8b 14.7b 15.3b 18.8c 17.4b 15.0 b
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.3 1.5 1.4 1.4 1.5 1.4 1.4 1.3 1.2 1.7 1.4 1.4 1.4 1.6 1.4 1.3 1.6 1.3 1.1 1.2 1.4 1.6 1.3 1.1 1.0 1.1 0.9 0.9 0.9 1.1 1.0 0.9 1.0 1.1 0.9 1.0 1.4 1.7 1.5 1.3
low molecular weight proteins. The decrease in protein molecular size indicates that the N is in a form that is more readily available for the soil microorganisms. This, in turn, promotes an increased proliferation of soil microorganisms [40]. This greater assimilation of low molecular weight proteins might be responsible for the fact that the soil urease activity
Fig. 1. Evolution of oxyfluorfen (mean ± S.E.) in soils during the experimental period. Data are the means of three samples. Columns (mean ± S.E.) followed by the same letter(s) are not significantly different (p > 0.05).
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exhibits no significant stimulation after the RB1 and RB2 application. Since soil microorganisms can obtain this easily available N without any energy expenditure, microorganisms do not need to excrete any enzyme to obtain it. These results also agree with those obtained by García-Martínez et al. [10,11] and Tejada et al. [12], who found that after applying different protein hydrolysates to the soil, no stimulation was observed in the soil urease activity due to the fact that these chemical compounds were rich in low molecular weight proteins. In SM- and MSW-amended soils, the urease activity was progressively stimulated during the experimental period. The higher molecular weight proteins are also a source of N for microorganisms. However, as this N is not easily assimilated by microorganisms, they first need to conduct a mineralization process, thus expending energy. Therefore, they excrete enzymes to degrade these higher molecular weight proteins. Therefore, the stimulation of soil microorganisms was higher in SM than in MSWamended soils. Finally, the RB1 and RB2 stimulation in soils differed. The main difference between the two BS is the fat content. Possibly the higher fat content of RB2 caused a higher soil microbial stimulation in order to degrade this fat and to obtain nutrients and energy from it. With respect to the soil microbial diversity, Marschner et al. [41] found that the degradation of organic matter requires enzymes that are produced by a limited number of microbial species and may increase the competitive ability of microorganisms. It is very probable that this is the reason why the microbial biodiversity of soils amended with both BS increased during the first days of the experimental period. Once these easily degradable organic complexes have been metabolized, the structure of the soil microbial population is reestablished again. These authors also note the availability of substrate as a reason for finding differences in the structure of the soil bacterial community. Perhaps due to the high number of higher molecular weight proteins, the degradation of SM and MSW is slower than both BS. This degradation involves the continuous excretion of enzymes into the medium of the microorganisms in order to obtain nutrients. Therefore, our results suggest that a continuous evolution of soil microbial diversity occurred during the experiment. When the organic wastes were added to soils with oxyfluorfen, a lower inhibition of the enzymatic activities under study occurred. The toxic effect of the herbicide in soil was considerably reduced. This degradation was faster in the contaminated soil amended with RB1 and RB2 than with SM and MSW, probably due to the greater stimulation of soil microorganisms in the soil treated with both BS. Coinciding with the results obtained by Tejada et al. [4] applying different BS constituted by a high number of lower molecular weight proteins obtained from soluble condensed distillers wheat, enzymatic carob germ and rice bran extract to a soil contaminated by herbicide MCPA, BS stimulate soil microorganisms and therefore favor and accelerate degradation of the xenobiotic compound in the soil. In line with what was previously commented earlier, the higher content of low molecular weight proteins in BS easily assimilated by microorganisms may be responsible for this fact.
5. Conclusions It can be concluded that the oxyfluorfen herbicide caused a negative effect on soil enzymatic activities and microbial diversity. The application of organic wastes decreased the toxic action of oxyfluorfen on soil biological properties. However, this effect depended on the different size distribution of the proteins of these organic wastes. The higher assimilation by soil microorganisms of low molecular weight proteins might be responsible for the greater degradation of oxyfluorfen in soil.
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Acknowledgments This work was supported by the Ministry of Science and Innovation (Spain), Plan Nacional I+D CTM2011-2993001, CTM2011-29930-03 and by Proyecto Excelencia, Junta de Andalucía P11-RNM-7887. References [1] M.J. Carrizosa, W.C. Koskinen, M.C. Hermosin, J. Cornejo, Dicambaadsorption–desorption on organoclays, Appl. Clay Sci. 18 (5–6) (2001) 223–231. [2] N. Singh, H. Kloeppel, W. Klein, Movement of metolachlor and terbuthylazine in core and packed soil columnsm, Chemosphere 47 (4) (2002) 409–415. [3] M. Tejada, Evolution of soil biological properties after addition of glyphosate, diflufenican and glyphosate + diflufenican herbicides, Chemosphere 76 (3) (2009) 365–373. [4] M. Tejada, A. García-Martínez, I. Gómez, J. Parrado, Application of MCPA herbicide on soils amended with bioestimulants: short-time effects on soil biological properties, Chemosphere 80 (9) (2010) 1088–1094. ˜ Compost and vermicompost of olive cake to [5] L. Delgado-Moreno, A. 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Behavior of oxyfluorfen in soils amended with different sources of organic matter. Effects on soil biology Isidoro Gómez a , Bruno Rodríguez-Morgado b , Juan Parrado b , Carlos García c , Teresa Hernández c , Manuel Tejada a,∗ a Grupo de investigación “Edafología ambiental”, Departamento de Cristalografía, Mineralogía y Química Agrícola, E.T.S.I.A. Universidad de Sevilla, Crta de Utrera km. 1, 41013 Sevilla, Spain b Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Sevilla, C/Prof. García González 2, 41012 Sevilla, Spain c Departamento de Conservación de Suelos y Agua y Manejo de Residuos Orgánicos, Centro de Edafología y Biología Aplicada del Segura, CEBAS-CSIC, PO Box 4195, 30080 Murcia, Spain
h i g h l i g h t s • Oxyfluorfen caused a negative effect on soil biological properties. • The application of organic wastes decreased the toxic action of oxyfluorfen. • The low molecular weight protein of wastes increased the degradation of herbicide.
a r t i c l e
i n f o
Article history: Received 15 October 2013 Received in revised form 19 February 2014 Accepted 17 March 2014 Available online 1 April 2014 Keywords: Oxyfluorfen Biofertilizer/biostimulant Municipal solid waste Sheep manure Soil enzymatic activities Soil microbial community
a b s t r a c t We performed a laboratory study on the effect of oxyfluorfen at a rate of 4 l ha−1 on biological properties of a soil amended with four organic wastes (two biostimulants/biofertilizers, obtained from rice bran, RB1 and RB2; municipal solid waste, MSW; and sheep manure, SM). Soil was mixed with SM at a rate of 1%, MSW at a rate of 0.52%, RB1 at a rate of 0.39% and RB2 at a rate of 0.30%, in order to apply the same amount of organic matter to the soil. The enzymatic activities and microbial community in the soil were determined during the incubation times. The application of RB1 and RB2 to soil without oxyfluorfen increased the enzymatic activities and biodiversity, peaking at day 10 of the incubation period. This stimulation was higher in the soil amended with RB2 than in that amended with RB1. In SM and CF-amended soils, the stimulation of enzymatic activities and soil biodiversity increased during the experiment. The application of herbicide in organic-amended soils decreased the inhibition of soil enzymatic activities and soil biodiversity. Possibly the low molecular weight protein content easily assimilated by soil microorganisms and the higher fat content in the biostimulants/biofertilizers are responsible for the lower inhibition of these soil biological properties. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The increase in the levels of pesticide residues in soils and ground water is a current environmental problem. Chemicals that are highly soluble in water, minimally adsorbed by soil particles and not readily degradable, are easily leached by infiltrating water and hence, are likely to be found in ground water [1–3]. Several bioremediation strategies for reducing the presence of pesticides in soil from which they can reach groundwater have been
∗ Corresponding author. Tel.: +34 95448646; fax: +34 954486436. E-mail address: [email protected] (M. Tejada). http://dx.doi.org/10.1016/j.jhazmat.2014.03.051 0304-3894/© 2014 Elsevier B.V. All rights reserved.
proposed. One such is remediation by enhancing the microbial population capable of specifically degrading the target compounds [4]. This strategy has been approached by adding organic matter of different origins. The application of organic matter to the soils causes an increase in their biological activity accelerating the degradation of the pesticide in the soil. Furthermore, the pesticide adsorption capacity of humic substances reduces the xenobiotic levels in the soil solution and consequently decreases the toxic effects of the pesticide [4–7]. However, as these organic products to activate soil microorganisms, the organic compounds need to be degraded into simpler easily assimilated forms, requiting a great expenditure of energy by soil microorganisms. Moreover, this degradation depends both
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Table 1 Characteristics of the experimental soil (mean ± S.E.). Data are the means of three samples. pH (H2 O) Coarse sand (g kg−1 ) Fine sand (g kg−1 ) Silt (g kg−1 ) Clay (g kg−1 ) Total N (g kg−1 ) Organic matter (g kg−1 )
7.9 486 130 123 260 0.93 17
± ± ± ± ± ± ±
Table 3 Molecular weight distribution of (mean ± S.E.) of the organic matter studied. Data are the means of three samples. Rows followed by the same letter(s) are not significantly different (p > 0.05).
0.2 49 25 29 35 0.08 1
on soil factors and the chemical composition of the organic material applied to soil [4,7,8]. In recent years the use of hydrolysate organic biofertilizers/biostimulants (BS) obtained from different organic materials by hydrolysis reactions has been increasing [9–12]. These BS, generally comprising peptides, amino acids, polysaccharides, humic acids, phytohormones, etc., are directly absorbed by soil microorganisms and plants which thus have to devote less energy to the absorption process. Therefore, not only does the application of these BS to the soil lead to an increased content of organic matter and macro- and micro-nutrients, it also leads to a significant activation of the soil’s microbial community. As a result, the development of new BS has become the focus of interest in research. Biological and biochemically mediated processes in soils are of the utmost importance to ecosystem function. Many studies have shown that biological parameters have been used to assess soil quality and health as affected by agricultural practices [4,13]. Also, the number of physiological groups of bacteria has also proved to be useful for measuring structural changes in soil due to several anthropogenic factors [14,15]. Therefore, the changes in soil microbial community could be of help in evaluating the impact of herbicides on soils. The objective of this study was to investigate under laboratory conditions the behavior of oxyfluorfen in soils amended with different organic wastes and its influence on soil enzymatic activities biological and soil biodiversity.
Molecular weight (Da)
SM (%)
>10,000 10,000–5000 5000–1000 1000–300 0.05). SM pH (1:2.5) Organic matter (g kg−1 ) Total N (g kg−1 ) Fat (g kg−1 ) P (g kg−1 ) K (g kg−1 ) Ca (g kg−1 ) Mg (g kg−1 ) Fe (mg kg−1 ) Cu (mg kg−1 ) Mn (mg kg−1 ) Zn (mg kg−1 ) Cd (mg kg−1 ) Pb (mg kg−1 ) Ni (mg kg−1 ) Nd: Not determined.
7.8a ± 225a ± 16.8a ± Nd 13.8b ± 19.7b ± 16.8b ± 10.3b ± 1345b ± 6.9b ± 86.4c ± 70.1c ± 1.2a ± 1.4a ± 1.9a ±
MSW 0.2 13 1.7 1.9 2.5 2.1 1.9 106 1.8 8.2 8.3 0.3 0.5 0.4
6.7a ± 475b ± 18.6a ± Nd 11.6b ± 10.1b ± 133c ± 68.3c ± 835a ± 3.9a ± 17.1a ± 40.8b ± 1.6a ± 0.8a ± 1.1a ±
RB1 0.3 22 2.1 1.4 1.7 18 5.9 77 1.2 2.6 4.1 0.5 0.2 0.4
7.9a 548c 17.9a 2.8a 8.1a 2.2a 0.74a 1.3a 273a 3.4a 29.1ab 18.4a 1.9a 1.1a 1.8a
RB2 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.2 25 1.1 0.4 1.7 0.4 0.12 0.2 35 0.9 1.2 2.6 0.3 0.3 0.2
7.8a 553c 18.4a 11.2b 8.6a 2.5a 0.88a 1.4a 288a 3.0a 33.6b 21.1a 1.6a 1.2a 1.7a
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.1 18 2.0 1.4 1.9 0.3 0.17 0.2 29 0.6 2.3 1.8 0.7 0.4 0.4
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insoluble molecules, and the supernatant was passed through a 0.2 m filter and loaded into a 0.1 ml loop connected to an ÄKTApurifier system. The column was equilibrated, and eluted with 0.25 M Tris–HCl buffer (pH 7.0) in isocratic mode, at a flow-rate of 0.5 ml min−1 , and proteins/peptides were detected at 280 and 215 nm with a GE Healthcare UV900 module coupled to the column elution. For MSW and SM, the proteins were extracted at pH = 9, temperature = 55 ◦ C and time = 3 h. Subsequently, the distribution of proteins was also determined by size-exclusion chromatography 2.2. Incubation procedure Five hundred grams of dried and 2 mm-sieved soil were preincubated at 25 ◦ C for 7 days at 30–40% of their water-holding capacity according to Tejada [3], prior to the treatments. Two days after applying oxyfluorfen to soil, organic wastes were also applied to the soil. Soil samples were mixed with SM at a rate of 1% or MSW at a rate of 0.52%, or RB1 at a rate of 0.39% or RB2 at a rate of 0.30% applying to the soil the same amount of organic matter with each organic waste. Both BS were liquid and were solubilized in distilled water before the application. An unamended soil was used as control. Distilled water was added to each soil to bring it to 60% of its water-holding capacity. The incubation treatments are detailed as follows: 1. C, control soil, soil non-organic amended and without oxyfluorfen; 2. H, soil with oxyfluorfen and non-organic amended; 3. SM, soil without oxyfluorfen and amended with SM; 4. MSW, soil without oxyfluorfen and amended with MSW; 5. RB1, soil without oxyfluorfen and amended with RB1; 6. RB2, soil without oxyfluorfen and amended with RB2; 7. SM + H, soil with oxyfluorfen and amended with SM; 8. MSW + H, soil with oxyfluorfen and amended with MSW; 9. RB1 + H, soil with oxyfluorfen and amended with RB1; 10. RB2 + H, soil with oxyfluorfen and amended with RB2. Triplicate treatments were kept in semi-closed microcosms at 25 ± 1 ◦ C for 120 days. 2.3. Soil analysis The activity levels of four soil enzymes for each treatment were measured at days 5, 10, 20, 40, 80 and 120 during the incubation period. Dehydrogenase activity was measured as the reduction of 2-p-iodo-3-nitrophenyl 5-phenyl tetrazolium chloride to iodonitrophenyl formazan [22]. Urease activity was determined by the buffered method of Kandeler and Gerber [23], using urea as substrate. The -glucosidase activity was determined using pnitrophenyl--d-glucopyranoside as substrate [24]. Phosphatase activity was measured using p-nitrophenyl phosphate as substrate [25]. Phospholipids were extracted at days 5, 10, 20 and 120 during the incubation period for each treatment, (three replicates per treatment) using a chloroform–methanol extraction based on Bligh and Dyer [26]. They were fractioned and quantified using the procedure described by Frostegard et al. [27] and Bardgett et al. [28]. Twenty-six separated fatty acid methyl esters were identified using gas chromatography and a flame ionization detector. The phospholipids were transformed by alkaline methanolysis into fatty acid methyl esters (FAMEs), which were quantified with a gas chromatograph (GC/FID, AutoSystem XL Gas Chromatograph, Varian Saturno 2000) fitted with a 50-m capillary column, using helium as the carrier gas. The injector temperature was 260 ◦ C, the flame ionization detector temperature was 280 ◦ C, and the initial temperature was 70 ◦ C (for 2 min); it was increased to 160 ◦ C at 30 ◦ C min−1 and then to 280 ◦ C at 3 ◦ C min−1 .
209
To estimate the various proportions of the main taxa in the samples according to the PLFAs, the biomarkers i15:0, a15:0, i16:0, 16:17c, 17:0, i17:0, cy17:0, 18:19c, and cy19:0 were used to represent bacterial biomass (bacPLFA) [28,29] and 18:26 (fungPLFA) was taken to indicate fungal biomass [30]. The ratio of bacPLFA to fungPLFA (bacPLFA/fungPLFA) represents the ratio between bacterial and fungal biomass [28]. The Gram+ specific fatty acids i15:0, a15:0, i16:0, and i17:0 and the Gram− specific fatty acids cy17:0, 18:19c, and cy19:0 were taken as a measure of the ratio of the Gram+ and Gram− bacterial biomass (Gram+ :Gram− ). All results are given in nmol g−1 . For each treatment and each incubation time, 20 g of soil were taken. Soil samples were stored in sealed polyethylene bags at 4 ◦ C for 15 d, prior to analysis of the enzymatic activities [7], and at −20 ◦ C prior to phospholipid analysis [31]. 2.4. Oxyfluorfen determination in soil The extraction of oxyfluorfen from soil was realized using the Anastassiades et al. [32] method. Oxyfluorfen was extracted with a mixture of triphenyl phosphate and acetonitrile. Once the supernatant had been shaken and centrifuged, magnesium sulfate was added to it and it was, stirred and centrifuged again. The supernatant was concentrated and the dried residue was recomposed with 1 ml of cyclohexane:ethyl acetate (9:1). Oxyfluorfen was determined using a tandem mass spectrometer and electron impact, where the chromatographic conditions were as follows: carrier gas: He at 1 ml min−1 , initial temperature inlet: 70 ◦ C for 0.50 min, 310 ◦ C to 100 ◦ C min−1 for 10 min, column: 30 m × 0.25 mm ID, initial temperature of the column oven: 70 ◦ C for 3.5 min., 180 ◦ C to 35 ◦ C min−1 , 300 ◦ C at 10 ◦ C min−1 for 5 min; temperature detector: trap to 250 ◦ C; manifold 60 ◦ C; xferline to 280 ◦ C; injection volume: 5 l. 2.5. Statistical analysis Data were submitted to two-way ANOVA with treatment and sampling time as factors followed by Tukey’s significant difference as a post hoc test, considering a significance level of p < 0.05 throughout the study. The ANOVA was performed using the using the Statgraphics Plus 2.1 software package. For the ANOVA, triplicate data were used for each treatment and each day of incubation. 3. Results 3.1. Characterization of organic wastes Molecular weight distribution showed significant differences (p < 0.05) in the organic compounds studied (Table 3). In this respect, RB1 and RB2 did not differ significantly in terms of protein weight distribution, with a high content of small molecular weight proteins. MSW and SM, however, have a higher content of high molecular weight protein., MSW presented a higher content of larger molecular protein than SM. 3.2. Evolution of soil biological properties Statistical analysis indicated a significant (p < 0.05) stimulation of the dehydrogenase activity during the first days after the application of both BS, peaking 10 days after the beginning of the experiment (Table 4). In this respect, and compared to the control, the soil dehydrogenase activity increased significantly (p < 0.05) by 87.8% and 92.5% in the RB1 and RB2 treatments, respectively. After the first 10 days, the dehydrogenase activity began to decline gradually. At the end of the experimental period BS treatments had very similar values to the control soil. In SM and CF-amended soils,
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Table 4 Evolution of dehydrogenase and urease activities in soils amended with the organic matter studied and with oxyfluorfen during the experimental period. Data are expressed as mean values ± standard error. Columns followed by the same letter(s) are not significantly different (p > 0.05). INTF: 2-p-iodo-3-nitrophenyl formazan. Dehydrogenase activity (g INTF g−1 h−1 ) 5
10
40
80
120
0.4 0.3 0.7 0.4 1.4 1.6 0.4 0.3 1.2 1.5
2.6b 1.2a 4.2b 3.8b 21.4d 34.6e 3.4b 2.9b 12.6d 19.3d
± ± ± ± ± ± ± ± ± ±
0.5 0.4 0.5 0.8 1.8 2.6 0.6 0.4 0.9 1.2
2.5b 1.2a 5.1b 4.3b 9.6cd 12.8d 4.4b 3.3b 5.6b 6.3b
± ± ± ± ± ± ± ± ± ±
0.6 0.2 0.9 0.6 1.1 1.5 0.4 0.6 0.6 0.5
2.5b 1.3a 6.4b 5.4b 4.3b 6.6b 5.7b 4.0b 3.7b 4.1b
± ± ± ± ± ± ± ± ± ±
0.4 0.3 0.6 0.7 1.0 0.9 0.8 0.6 0.3 0.4
2.4b 1.2a 7.7c 6.5b 2.8b 3.5b 6.2b 4.8b 2.4b 2.4b
± ± ± ± ± ± ± ± ± ±
0.3 0.2 0.6 0.4 0.9 0.4 0.7 0.6 0.3 0.2
2.3b 1.2a 10.1c 7.5c 2.2b 2.5b 7.7c 5.6b 2.3b 2.4b
± ± ± ± ± ± ± ± ± ±
0.5 0.3 0.8 0.7 0.3 0.6 0.6 0.6 0.4 0.5
Urease activity (g NH4 + g−1 h−1 ) C 1.9b ± 0.4 H 1.1a ± 0.2 SM 2.2b ± 0.3 MSW 2.1b ± 0.2 RB1 2.0b ± 0.3 RB2 2.0b ± 0.4 2.0b ± 0.4 SM + H 2.0b ± 0.2 MSW + H RB1 + H 1.0a ± 0.2 1.0a ± 0.3 RB2 + H
2.0b 1.1a 2.8b 2.6b 1.9b 2.0b 2.6b 2.4b 0.9a 1.0a
± ± ± ± ± ± ± ± ± ±
0.2 0.3 0.4 0.3 0.3 0.2 0.4 0.4 0.1 0.2
1.8b 0.9a 4.1bc 3.6b 2.1b 2.1b 3.4b 2.9b 1.0a 0.9a
± ± ± ± ± ± ± ± ± ±
0.2 0.1 0.5 0.3 0.3 0.4 0.3 0.5 0.2 0.2
1.9b 1.0a 5.6c 4.8bc 2.0b 1.9b 4.0bc 3.4b 0.9a 1.0a
± ± ± ± ± ± ± ± ± ±
0.3 0.2 0.4 0.6 0.3 0.2 0.5 0.4 0.2 0.3
1.8b 0.9a 6.8c 5.4c 1.9b 2.0b 4.9bc 4.0bc 1.0a 0.9a
± ± ± ± ± ± ± ± ± ±
0.2 0.2 0.8 0.5 0.2 0.2 0.6 0.8 0.2 0.2
1.9b 1.0a 8.0d 6.6c 2.0b 1.9b 5.8c 4.6bc 0.9a 1.0a
± ± ± ± ± ± ± ± ± ±
0.4 0.3 1.1 0.9 0.3 0.3 0.6 0.7 0.1 0.2
C H SM MSW RB1 RB2 SM + H MSW + H RB1 + H RB2 + H
2.7b 1.3a 3.3b 3.2b 12.6 15.9 2.6b 2.4b 8.7cd 10.1d
± ± ± ± ± ± ± ± ± ±
20
the dehydrogenase activity was stimulated progressively over the whole experimental period. At the end of the experimental period and compared to the control soil, the dehydrogenase activity significantly (p < 0.05) increased 68% and 76.2% in soils amended with MSW and SM, respectively. The application of oxyfluorfen in soil showed a significant decrease in dehydrogenase activity (Table 4). At the end of the incubation period and compared with the control treatment, dehydrogenase activity decreased significantly (p < 0.05) by 47.8%. The application of both BS in oxyfluorfen soils caused a minor decrease in dehydrogenase activity (Table 4). In this respect, 10 days after applying the herbicide in the soil, and compared to RB1 treatment, the dehydrogenase activity in the RB1 + H treatment decreased (41.1%). Compared to RB2 treatment, the dehydrogenase activity in the RB2 + H treatment decreased 44.2%. At day 20 of the incubation period, this decrease in soil dehydrogenase activity was diminished progressively over the experimental period, noting that in soils amended with both BS, this activity showed values similar to those of soil amended without oxyfluorfen. The application of SM and MSW in soils with oxyfluorfen caused a decrease in the enzyme inhibition. However, this inhibition was lower than that caused by both BS. Furthermore, this inhibition was significantly (p < 0.05) higher in MSW + H treatment than in the SM + H treatment (2.3%). Unlike the dehydrogenase activity, soil urease activity was not stimulated after the application of the two BS studied. However in SM and CF-amended soils, the dehydrogenase activity was progressively stimulated over the whole experimental period (Table 4). Similar to the dehydrogenase activity, when oxyfluorfen was applied to the unamended soil, there was a significant (p < 0.05) decrease in this enzyme activity throughout the experimental period. These same results were also observed when the different organic wastes studied where applied to the soil with the herbicide. Similar to the dehydrogenase activity, the -glucosidase activity was also stimulated in organically amended soils. However this stimulation depended on the organic matter applied to the soil (Table 4). The -glucosidase activity was significantly (p < 0.05) stimulated during the first days after the application of both BS, peaking 10 days after the start of the experiment (Table 4). In this respect, and compared to the control, the soil -glucosidase
activity significantly (p < 0.05) increased by 81.2% and 85.7% in the RB1 and RB2 treatments, respectively. After the first 10 days, the -glucosidase activity began to decline gradually. In SM and CF-amended soils, the dehydrogenase activity was progressively stimulated over the whole experimental period. At the end of the experimental period and compared to the control soil, the glucosidase activity increased significantly (p < 0.05) by 87.2% and 92.6% in soils amended with MSW and SM, respectively. The response of -glucosidase activity to the application of oxyfluorfen in soil was very similar to other enzyme activities studied (Table 5). An inhibition of this enzyme in the soils with oxyfluorfen was observed. Moreover, the application of organic wastes to the soil decreased the inhibition of this enzymatic activity. However this inhibition depended on the organic matter applied to the soil. In the case of both BS, and 10 days after the start the experiment, this decrease was higher in RB1 + H treatment than in that of the RB2 + H treatment. Thirty days after beginning the incubation period, this decrease in soil -glucosidase activity diminished progressively over the experimental period, noting that in soils amended with both BS, this activity showed similar values to those of the amended soil without oxyfluorfen. The application of SM and MSW in soils with oxyfluorfen also caused a decrease in the enzyme inhibition. Again, this inhibition was lower than that caused by both BS. The soil phosphatase activity was also significantly (p < 0.05) stimulated after the application of organic wastes to the soil (Table 5). Compared to both BS, this stimulation was different in MSW and SM-amended soils. Similar to the dehydrogenase and -glucosidase activities, the soil phosphatase activity showed a higher stimulation in soil amended with both BS 10 days after beginning the experiment. In this respect, and compared to the C treatment, soil phosphatase activity significantly increased by 88.6% and 91.1% in the RB1 and RB2 treatments, respectively. This stimulation decreased as the experimental period progressed. In SM and CF-amended soils, the phosphatase activity was progressively stimulated over the whole experimental period. At the end of the experimental period and compared to the control soil, the dehydrogenase activity increased significantly (p < 0.05) by 71.4 and 80.2% in soils amended with MSW and SM, respectively. Furthermore, applying oxyfluorfen to the soil inhibited this enzymatic activity during the experimental period (Table 5).
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Table 5 Evolution of -glucosidase and phosphatase activities in soils amended with the organic matter studied and with oxyfluorfen during the experimental period. Data are expressed as mean values ± S.E. Columns followed by the same letter(s) are not significantly different (p > 0.05). PNP: p-nitrophenol. -glucosidase activity (mmol PNP g−1 h−1 ) 5 C H SM MSW RB1 RB2 SM + H MSW + H RB1 + H RB2 + H
10 1.4b 0.5a 2.0b 1.9b 3.8c 5.2c 1.8b 1.7b 2.3b 3.1c
± ± ± ± ± ± ± ± ± ±
0.3 0.1 0.4 0.3 0.9 1.4 0.2 0.2 0.2 0.3
Phosphatase activity (mol PNP g−1 h−1 ) C 3.6b ± 1.2 H 1.9a ± 0.4 SM 4.3b ± 0.9 MSW 4.0b ± 1.1 RB1 19.4d ± 2.1 RB2 21.9d ± 2.8 3.7b ± 1.4 SM + H 3.7b ± 1.3 MSW + H RB1 + H 10.4c ± 1.2 13.6c ± 1.8 RB2 + H
20
40
80
120
1.3b 0.6a 3.0c 2.7b 6.9d 9.1e 2.4b 2.0b 4.8c 6.0cd
± ± ± ± ± ± ± ± ± ±
0.2 0.2 0.5 0.2 0.9 1.3 0.3 0.3 0.5 0.8
1.2b 0.7a 5.1c 3.9c 4.7c 5.8c 3.0c 2.5b 3.0c 4.1c
± ± ± ± ± ± ± ± ± ±
0.3 0.1 0.9 0.6 0.4 0.9 0.4 0.4 0.4 0.6
1.2b 0.6a 7.1d 5.0c 2.9b 3.2c 4.1c 3.2c 2.1b 3.0c
± ± ± ± ± ± ± ± ± ±
0.3 0.2 1.2 0.5 0.5 0.5 0.6 0.4 0.4 0.5
1.1b 0.5a 9.7e 6.3d 1.3b 1.3b 5.2c 4.3c 1.2b 1.3b
± ± ± ± ± ± ± ± ± ±
0.1 0.1 1.7 1.0 0.2 0.2 0.6 0.4 0.3 0.2
1.0b 0.6a 13.6f 7.8d 1.2b 1.1b 6.6d 5.0c 1.1b 1.1b
± ± ± ± ± ± ± ± ± ±
0.2 0.1 2.2 1.4 0.2 0.2 1.1 0.4 0.2 0.1
3.5b 2.0a 5.9b 5.1b 30.6d 39.4e 4.6b 4.2b 15.6 20.9
± ± ± ± ± ± ± ± ± ±
1.1 0.5 1.3 1.5 3.3 3.0 1.0 1.4 1.5 2.2
3.5b 1.8a 7.0c 6.3b 25.6d 30.9de 6.0b 5.1b 13.6c 17.6
± ± ± ± ± ± ± ± ± ±
1.4 0.3 1.1 0.9 2.5 3.4 1.6 1.5 2.0 1.9
3.4b 1.5a 8.8c 7.6c 8.1c 10.9c 7.8c 5.9c 6.1c 7.2c
± ± ± ± ± ± ± ± ± ±
1.1 0.3 1.4 1.0 1.9 1.3 1.5 1.4 1.6 1.3
3.3b 1.6a 11.0c 9.0c 3.9b 3.8b 8.4c 6.6c 3.5b 3.6b
± ± ± ± ± ± ± ± ± ±
1.1 0.4 1.3 1.2 1.1 1.4 1.5 1.3 1.1 1.2
3.2b 1.8a 16.2d 11.2c 3.0b 3.1b 9.6c 7.8c 3.1b 3.3b
± ± ± ± ± ± ± ± ± ±
1.4 0.5 1.4 1.1 1.1 0.9 1.2 1.5 1.2 1.3
Similar to the enzymes studied, when applied organic wastes were applied to the soil with oxyfluorfen the inhibition of phosphatase activity decreased. This decrease was lower in the RB2 + H treatment, followed by RB1 + H, SM + H and MSW + H treatments, respectively. The application of the organic waste increased the soil bacteria and fungi population (Table 6). Similar to the results of the enzymatic activities, this stimulation depended on the organic matter applied to the soil. Again, and in the BS treatments, the highest populations of bacteria and fungi were found after 10 days of incubation. These populations, however, decreased over the experimental period. At the end of the experiment, the bacteria population was similar to that observed in the control treatment. The bacGram+ /Gram− and bacPLFA/fungPLFA rates increased in BS- treatments, also indicating the variability in the biodiversity of these soils during the first days of the experiment (Table 6). At the end of the experimental period, the rates showed similar values to those obtained in the control treatment. In the MSW and SM-amended soils, the stimulation occurred progressively over the experimental period and at the end of the experiment, the bacteria population was higher in SM than in MSW treatment. When the herbicide was applied to the soil, the total bacterial population decreased significantly (p < 0.05) while the fungal population decreased slightly. However, and with respect to the control treatment, no significant (p > 0.05) differences were found in bacGram+ /Gram− and bacPLFA/fungPLFA rates (Table 6). Compared with contaminated soils, the application of organic matter to soils with oxyfluorfen caused a minor decrease in the bacteria and fungi populations. This decrease was higher when the BS applied to the soil was RB2. 3.3. Evolution of oxyfluorfen in soil The application of organic matter to soil with herbicide, decreased the soil oxyfluorfen concentration (Fig. 1). However, this decrease depended on the organic matter type applied to the soil. Compared to the MSW and SM treatments, the application of both BS in oxyfluorfen contaminated soils caused a higher decrease in dehydrogenase activity. The values suggest a more rapid degradation of the herbicide when the RB2 was applied to the soil with herbicide. In this respect, 10 days after applying the herbicide to the
soil, and compared to the RB1 + H treatment the oxyfluorfen significantly (p < 0.05) decreased with the RB2 + H treatment (25%). At the end of the experimental period, the concentration of herbicide in soil was significantly (p < 0.05) lower in the soils with oxyfluorfen and amended with both BS than that found in the control soil. For MSW + H and SM + H treatments, the evolution of oxyfluorfen indicated that the degradation of the herbicide occurred more slowly and gradually during the experiment. The degradation of the oxyfluorfen in soil was higher in SM + H treatment than for MSW + treatment. 4. Discussion Oxyfluorfen caused a toxic effect on soil enzymatic activity and soil diversity. These results are in agreement with those obtained by García-Orenes et al. [33] and Nadiger et al. [34], who found an important toxic effect of this herbicide on agricultural soil microorganisms. Moreover, our results show a high persistence of oxyfluorfen in soil. These results are in agreement with those obtained by other authors. Baruah and Mishra [35] indicate that oxyfluorfen has a long persistence in soil (half-life of 72 to 160 days) in natural conditions. Also, Ying and Williams [36] found that this herbicide persisted in soil for 119 days. The application of diverse organic wastes caused an increase in soil microbial activity. When SM and MSW were applied to the soil, the stimulation of soil microorganisms increased progressively during the experimental period. These results are in agreement with those obtained by other authors when applying organic matter of very different chemical nature such as urban waste composts, vermicomposts, green manure, poultry manure etc. [37–39] to soil. Soil microorganisms were also stimulated when BS, RB1 or RB2 were applied to the soil. These results are in agreement with those obtained by Parrado et al. [9], García-Martínez et al. [10,11] and Tejada et al. [4,12] who indicate that the incorporation of different BS, obtained from condensed distillers soluble wheat, enzymatic carob germ, rice bran extract and sewage sludge, stimulates microbial activity in soil. However, the evolution of the soil microbial stimulation was differed, depending on the type of organic waste applied. This might be a consequence of the different protein size distribution obtained for different organic wastes. Our results indicate that protein size
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Table 6 Evolution of bacterial Gram+ , bacterial Gram− , total bacterial, fungal PLFAs (nmol g−1 ), Gram+ /Gram− and bacteria/fungi during the experimental period. Data are the means of three samples. Columns (mean ± S.E.) followed by the same letter(s) are not significantly different (p > 0.05). bacGram+ C (5 d) C (10 d) C (20 d) C (120 d) H (5 d) H (10 d) H (20 d) H (120 d) SM (5 d) SM (10 d) SM (20 d) SM (120 d) MSW (5 d) MSW (10 d) MSW (20 d) MSW (120 d) RB1 (5 d) RB1 (10 d) RB1 (20 d) RB1 (120 d) RB2 (5 d) RB2 (10 d) RB2 (20 d) RB2 (120 d) SM + H (5 d) SM + H (10 d) SM + H (20 d) SM + H (120 d) MSW + H (5 d) MSW + H (10 d) MSW + H (20 d) MSW + H (120 d) RB1 + H (5 d) RB1 + H (10 d) RB1 + H (20 d) RB1 + H (120 d) RB2 + H (5 d) RB2 + H (10 d) RB2 + H (20 d) RB2 + H (120 d)
13.5b 13.0b 13.3b 13.4b 8.9a 9.0a 9.2a 8.9a 13.8b 15.9b 18.2b 20.4c 13.4b 15.0b 16.1b 18.0c 28.9c 39.6d 22.8c 13.7b 34.4 45.6e 19.9c 13.6b 12.9b 14.1b 15.0b 16.6c 12.9b 13.8b 14.6b 15.9c 19.2c 24.3c 19.5c 13.2b 21.4c 33.1d 20.6c 13.4b
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.4 1.3 1.3 1.4 1.1 1.2 0.9 0.7 1.4 1.1 1.6 1.5 1.1 1.4 1.2 1.3 2.1 2.6 1.5 1.5 2.4 3.1 1.6 1.1 1.3 1.4 1.3 1.2 1.4 1.5 1.3 1.3 1.7 2.2 1.4 1.1 1.8 2.6 2.1 1.3
bacGram− 1.5b 1.4b 1.5b 1.5b 1.0a 0.8a 0.9a 1.0a 1.7b 2.5b 4.1c 5.2c 1.6b 2.0b 3.0b 4.4c 2.8b 3.5c 2.3b 1.5b 2.8b 3.5c 2.3b 1.5b 1.6b 2.0b 3.3c 4.3c 1.5b 1.8b 2.6b 3.6c 1.4b 2.4b 1.8b 1.5b 1.6b 2.7b 2.0b 1.6b
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.3 0.4 0.3 0.3 0.2 0.2 0.2 0.3 0.3 0.4 0.7 0.5 0.3 0.5 0.5 0.9 0.6 0.6 0.7 0.4 0.6 0.6 0.7 0.4 0.6 0.9 0.6 1.1 0.8 0.6 1.1 1.2 0.3 0.7 0.5 0.3 0.3 0.4 0.4 0.2
Total bacterial PLFA 15.0b 14.4b 14.8b 14.9b 9.9a 9.8a 10.1a 9.9a 15.5b 18.4b 22.3c 25.6c 15.0b 17.0b 19.1b 22.4c 31.7d 43.1d 25.1c 15.2b 37.2d 49.1d 22.2c 15.1b 14.5b 16.1b 18.3b 20.9c 14.4b 15.6b 17.2b 19.5c 20.6c 26.7c 21.3c 14.7b 23.0c 35.8d 22.6c 15.0b
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.5 1.4 1.4 1.5 1.2 1.2 1.1 1.2 1.4 1.9 1.7 2.0 1.6 1.8 1.1 1.9 2.7 2.1 2.0 1.7 1.9 2.7 2.0 1.9 1.1 1.9 1.6 1.8 1.3 1.5 1.4 1.6 1.4 1.5 1.5 1.2 2.1 3.0 1.8 1.4
distribution differs greatly for each organic waste. While BS have a higher content of low molecular weight proteins, SM and MSW have a higher content of high molecular weight proteins. Tejada et al. [12] found that after applying the BS obtained from sewage sludge to the soil, the soil microbial activity showed a greater increase when the BS applied contained a higher proportion of
Fungal PLFA 0.9b 0.9b 1.0b 0.9b 0.5a 0.5a 0.6a 0.6a 1.0b 1.6b 2.8c 4.1d 1.0b 1.3b 2.0c 3.0c 1.5b 1.9c 1.3b 1.0b 1.7b 2.6c 1.5b 1.0b 0.9b 1.3b 1.9c 2.9c 0.9b 1.1b 1.5b 2.4c 1.3b 1.6b 1.2b 1.0b 1.5b 1.9c 1.3b 1.0b
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.1 0.1 0.2 0.1 0.1 0.1 0.2 0.1 0.1 0.3 0.5 0.4 0.2 0.2 0.4 0.4 0.3 0.3 0.2 0.1 0.4 0.4 0.3 0.2 0.1 0.3 0.3 0.5 0.1 0.2 0.4 0.5 0.2 0.2 0.2 0.3 0.3 0.4 0.2 0.2
bacGram+ /bacGram− 9.0b 9.3b 8.9b 8.9b 8.9b 11.3b 10.2b 8.9b 9.9b 6.4a 4.4a 3.9a 8.4b 7.5b 5.4a 4.1a 10.3b 11.3b 9.9b 9.1b 12.3c 13.0c 8.7b 9.1b 8.1b 7.1b 4.5a 3.9a 8.6b 7.7a 5.6a 4.4a 13.7c 10.1b 10.8b 8.8b 13.4c 12.3c 10.3b 8.4b
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.1 1.0 1.1 1.1 1.1 1.3 1.3 1.2 1.1 1.4 1.0 1.1 1.0 1.2 0.9 1.0 1.8 1.6 1.6 1.4 1.5 1.8 1.2 1.0 1.1 1.4 1.3 0.8 1.0 1.1 1.1 1.0 1.1 1.4 1.1 1.0 1.2 1.3 1.1 1.0
bacPLFA/fungPLFA 16.7b 16.0b 14.8b 16.5b 19.8c 19.6c 16.8b 16.5b 15.0b 11.5b 8.0a 6.2a 15.0b 13.1b 9.6a 7.5a 21.1c 22.7c 19.3c 15.2b 21.9c 18.9c 14.8b 15.1b 16.1b 12.4b 9.6a 7.2a 16.0b 14.2b 11.5b 8.1a 15.8b 16.7b 17.8b 14.7b 15.3b 18.8c 17.4b 15.0 b
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.3 1.5 1.4 1.4 1.5 1.4 1.4 1.3 1.2 1.7 1.4 1.4 1.4 1.6 1.4 1.3 1.6 1.3 1.1 1.2 1.4 1.6 1.3 1.1 1.0 1.1 0.9 0.9 0.9 1.1 1.0 0.9 1.0 1.1 0.9 1.0 1.4 1.7 1.5 1.3
low molecular weight proteins. The decrease in protein molecular size indicates that the N is in a form that is more readily available for the soil microorganisms. This, in turn, promotes an increased proliferation of soil microorganisms [40]. This greater assimilation of low molecular weight proteins might be responsible for the fact that the soil urease activity
Fig. 1. Evolution of oxyfluorfen (mean ± S.E.) in soils during the experimental period. Data are the means of three samples. Columns (mean ± S.E.) followed by the same letter(s) are not significantly different (p > 0.05).
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exhibits no significant stimulation after the RB1 and RB2 application. Since soil microorganisms can obtain this easily available N without any energy expenditure, microorganisms do not need to excrete any enzyme to obtain it. These results also agree with those obtained by García-Martínez et al. [10,11] and Tejada et al. [12], who found that after applying different protein hydrolysates to the soil, no stimulation was observed in the soil urease activity due to the fact that these chemical compounds were rich in low molecular weight proteins. In SM- and MSW-amended soils, the urease activity was progressively stimulated during the experimental period. The higher molecular weight proteins are also a source of N for microorganisms. However, as this N is not easily assimilated by microorganisms, they first need to conduct a mineralization process, thus expending energy. Therefore, they excrete enzymes to degrade these higher molecular weight proteins. Therefore, the stimulation of soil microorganisms was higher in SM than in MSWamended soils. Finally, the RB1 and RB2 stimulation in soils differed. The main difference between the two BS is the fat content. Possibly the higher fat content of RB2 caused a higher soil microbial stimulation in order to degrade this fat and to obtain nutrients and energy from it. With respect to the soil microbial diversity, Marschner et al. [41] found that the degradation of organic matter requires enzymes that are produced by a limited number of microbial species and may increase the competitive ability of microorganisms. It is very probable that this is the reason why the microbial biodiversity of soils amended with both BS increased during the first days of the experimental period. Once these easily degradable organic complexes have been metabolized, the structure of the soil microbial population is reestablished again. These authors also note the availability of substrate as a reason for finding differences in the structure of the soil bacterial community. Perhaps due to the high number of higher molecular weight proteins, the degradation of SM and MSW is slower than both BS. This degradation involves the continuous excretion of enzymes into the medium of the microorganisms in order to obtain nutrients. Therefore, our results suggest that a continuous evolution of soil microbial diversity occurred during the experiment. When the organic wastes were added to soils with oxyfluorfen, a lower inhibition of the enzymatic activities under study occurred. The toxic effect of the herbicide in soil was considerably reduced. This degradation was faster in the contaminated soil amended with RB1 and RB2 than with SM and MSW, probably due to the greater stimulation of soil microorganisms in the soil treated with both BS. Coinciding with the results obtained by Tejada et al. [4] applying different BS constituted by a high number of lower molecular weight proteins obtained from soluble condensed distillers wheat, enzymatic carob germ and rice bran extract to a soil contaminated by herbicide MCPA, BS stimulate soil microorganisms and therefore favor and accelerate degradation of the xenobiotic compound in the soil. In line with what was previously commented earlier, the higher content of low molecular weight proteins in BS easily assimilated by microorganisms may be responsible for this fact.
5. Conclusions It can be concluded that the oxyfluorfen herbicide caused a negative effect on soil enzymatic activities and microbial diversity. The application of organic wastes decreased the toxic action of oxyfluorfen on soil biological properties. However, this effect depended on the different size distribution of the proteins of these organic wastes. The higher assimilation by soil microorganisms of low molecular weight proteins might be responsible for the greater degradation of oxyfluorfen in soil.
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