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Field versus laboratory experiments to evaluate the fate of azoxystrobin in an amended vineyard soil
Journal of Environmental Management 163 (2015) 78e86
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Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman
Research article
Field versus laboratory experiments to evaluate the fate of azoxystrobin in an amended vineyard soil ndez a, J.M. Marín-Benito a, M.S. Andrades b, M.J. Sa nchez-Martín a, E. Herrero-Herna M.S. Rodríguez-Cruz a, * a b
Institute of Natural Resources and Agrobiology of Salamanca (IRNASA-CSIC), Cordel de Merinas 40-52, 37008 Salamanca, Spain ~ o, Spain Department of Agriculture and Food, University of La Rioja, Madre de Dios 51, 26006 Logron
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 February 2015 Received in revised form 29 July 2015 Accepted 12 August 2015 Available online 22 August 2015
This study reports the effect that adding spent mushroom substrate (SMS) to a representative vineyard soil from La Rioja region (Spain) has on the behaviour of azoxystrobin in two different environmental scenarios. Field dissipation experiments were conducted on experimental plots amended at rates of 50 and 150 t ha1, and similar dissipation experiments were simultaneously conducted in the laboratory to identify differences under controlled conditions. Azoxystrobin dissipation followed biphasic kinetics in both scenarios, although the initial dissipation phase was much faster in the field than in the laboratory experiments, and the half-life (DT50) values obtained in the two experiments were 0.34e46.3 days and 89.2e148 days, respectively. Fungicide residues in the soil profile increased in the SMS amended soil and they were much higher in the top two layers (0e20 cm) than in deeper layers. The persistence of fungicide in the soil profile is consistent with changes in azoxystrobin adsorption by unamended and amended soils over time. Changes in the dehydrogenase activity (DHA) of soils under different treatments assayed in the field and in the laboratory indicated that SMS and the fungicide had a stimulatory effect on soil DHA. The results reveal that the laboratory studies usually reported in the literature to explain the fate of pesticides in amended soils are insufficient to explain azoxystrobin behaviour under real conditions. Field studies are necessary to set up efficient applications of SMS and fungicide, with a view to preventing the possible risk of water contamination. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Azoxystrobin Dissipation Field and laboratory experiments Adsorption Soil dehydrogenase activity
1. Introduction Crop protection is an integral part of modern agriculture, with pesticide application being a major component. However, the presence of pesticides in soils and waters has increased in recent ndez et al., 2013; Ko €ck-Schulmeyer et al., years (Herrero-Herna et al., 2015), and this practice has become exten2014; Masia sively and hotly debated. Pesticide residues in the soil may be taken up by plants, degraded into other chemical forms, or washed into surface and ground waters. The leaching of pesticides into groundwater is a cause of concern, as this is one of the major sources of drinking water in many locations. A positive step toward reducing pesticide leaching, and hence the risk of groundwater contamination by pesticide residues, involves enhancing the retention and degradation of pesticides in
* Corresponding author. E-mail address: [email protected] (M.S. Rodríguez-Cruz). http://dx.doi.org/10.1016/j.jenvman.2015.08.010 0301-4797/© 2015 Elsevier Ltd. All rights reserved.
soils and this can be achieved by increasing soil organic matter (OM) content. As it is known these processes depend on the soil OM content for the non-ionic hydrophobic pesticides (Marín-Benito et al., 2012a,b). Nowadays, a common way of increasing the OM content involves soil amendment with organic residues (Moreno-Casco and Moral-Herrero, 2008). This soil amendment is used to increase OM content in Mediterranean agricultural soils, and there has recently been an increase in interest in assessing the application of these organic residues as a strategy for enhancing the retention and degradation of pesticides in soils in order to avoid their leaching into groundwater. There are numerous studies researching the effect of organic amendments on the fate of pesticides in soils conducted under laboratory conditions (Fenoll et al., 2011; Martin et al., 2012; pez-Pin ~ eiro et al., 2013), and Rodríguez-Cruz et al., 2012a,b; Lo although these studies provide valuable information on the effect organic amendments have on the behaviour of pesticides in the soil,
ndez et al. / Journal of Environmental Management 163 (2015) 78e86 E. Herrero-Herna
they do not always reflect what actually occurs in the field. Consequently, both field studies and laboratory studies are needed to provide complete information on how pesticides dissipate under natural conditions. In general, there are few studies on dissipation in the field (Papiernik et al., 2007; Chai et al., 2009), with only a handful examining the influence of different organic amendments on ndez pesticide dissipation (Dolaptsoglou et al., 2009; Herrero-Herna et al., 2011a). Very few studies have evaluated the dissipation of pesticides under field and laboratory conditions simultaneously (Ahmad et al., 2003; Potter et al., 2005; Chai et al., 2013). Azoxystrobin is a fungicide with a broad spectrum of systemic activity for the control of fungal crop pathogens (Bartlett et al., 2002). It belongs to the strobilurins group, and it is a chemical that has been approved for use on more than 80 different crops in 72 countries. Azoxystrobin is commonly used as a foliar fungicide, and a high proportion of this fungicide may be deposited in the soil when applied in spray form (Adetutu et al., 2008). Some reports on its persistence and mobility have indicated that it may remain in soils for several months (Bending et al., 2007). However, the presence of azoxystrobin detected in the ground and surface waters in ndez et al., different vine growing areas in Spain (Herrero-Herna 2013), Brazil (Menezes Filho et al., 2010), France (Rabiet et al., 2010), and Germany (Neumann et al., 2003) reveals that certain uncontrolled factors affect its behaviour in soils. There are studies on the degradation of this fungicide in some soils amended with organic residues under laboratory conditions ~ a and Bending, 2013), but the (Ghosh and Singh, 2009a; Sopen dissipation of azoxystrobin under field conditions has scarcely been studied (Gajbhiye et al., 2011), and to our knowledge there are no studies on the dissipation of azoxystrobin in soil amended with spent mushroom substrate (SMS). SMS is the composted organic material remaining after a mushroom crop has been harvested, and it is being generated on farms in increasing quantities (Martín et al., 2009). SMS is used as a soil fertilizer and amendment to increase the OM content of vineyard soils in La Rioja region (Spain). The objective of this research was to study the effect that SMS soil amendment has on the fate of azoxystrobin under two environmental scenarios. Accordingly, field versus laboratory experiments were conducted for comparative purposes on a vineyard soil from La Rioja region, both unamended and amended with SMS at two rates, with the aim being to assess the dissipation, persistence, and mobility of azoxystrobin applied at two doses. In support to explain the fate of the fungicide, changes in adsorption of azoxystrobin by unamended and amended soil from field experimental plots and changes in the dehydrogenase activity of unamended and amended soil, untreated and treated with azoxystrobin in field and in laboratory were evaluated over the time. 2. Materials and methods 2.1. Chemicals and organic amendment An analytical standard of azoxystrobin (Methyl (E)-2-{2-[6-(2cyanophenoxy) pyrimidin-4-yloxy]phenyl}-3-methoxyacrylate) from Dr. Ehrenstorfer, Germany (99.0% purity) and the commercial formulation of azoxystrobin from Ortiva (Syngenta, Switzerland) were used in the laboratory and field experiments, respectively. Azoxystrobin is a fungicide with a water solubility of 6 mg L1 and a log Kow of 2.5 (Tomlin, 2000). All other chemicals used were supplied by SigmaeAldrich Quimica SA (Spain). Spent mushroom substrate was supplied by INTRAVAL Environmental Group TRADEBE S.L. (Spain). Its composition was ndez et al. (2011a) and their physicodescribed by Herrero-Herna chemical characteristics determined as described in this previous work are: pH 7.5, organic carbon (OC) content 27.1% and dissolved
79
organic carbon (DOC) content 1.22%. 2.2. Field experimental design and soil sampling The field experiment was conducted in a vineyard in Sajazarra, La Rioja, Spain (42 350 000 N latitude and 2 570 000 W longitude). The soil used was a sandy clay loam soil classified as Typic Calcixerept. Their characteristics were determined by usual analytical methods (MAPA, 1986) (Table 1). Rainfall and temperature were recorded over the 378 days of experimentation at a weather station close to the study site (4 km east) (Fig. S1 in Supplementary material). An experimental layout of randomized complete blocks (18 plots of 1.50 m 3.90 m) was set up with six treatments (unamended (S) and amended soils (Soil þ SMS) at two rates of SMS treated with two doses of azoxystrobin) and three replicates per ndez et al. (2011a,b). Untreatment as indicated in Herrero-Herna amended and amended soils (0e10 cm) at the rates of 50 or 150 t ha1 (Soil þ SMS50 and Soil þ SMS150) on dry weight basis were prepared on November 2010. Azoxystrobin solutions at two doses (0.25 and 1.25 kg ha1) were applied to the plots from the commercial formulation Ortiva-Syngenta (25% w/v of a.i.). Three more plots one unamended and two amended with 50 or 150 t ha1 of SMS, respectively, did not receive fungicide application (control plots). Soil samples from these untreated plots were collected for assays in the laboratory as indicated below. Five topsoil samples were collected from 0 to 10 cm at 0, 2, 7, 14, 21, 28, 35, 84, 115, 150, 181, 235, 300 and 378 days after treatment to determine fungicide dissipation and five soil cores were collected to a depth of 50 cm after 84, 181 and 378 days of azoxystrobin application to determine mobility of fungicide. Soil samples were ndez et al. (2011a). managed as indicated in Herrero-Herna Topsoil OM content was determined two days after SMS application and OM content at different depths of soil cores taken at 84, 181 and 378 days was also determined by triplicate. Results are included in Table 1 and Fig. 2. 2.3. Laboratory experimental design and soil sampling Soil samples for laboratory experiments were taken from 0 to 10 cm of experimental plots prepared for fungicide dissipation study in field on November 2010. Soils collected from control plots, unamended and SMS-amended at dose of 150 t ha1, and without fungicide applied, were transferred to polypropylene containers. They were transported to the laboratory and treated as previously indicated for field soil samples. Solutions of fungicide were prepared in sterile UHQ water and volumes of 10 mL of suitable concentrations to give similar concentrations to those applied in the field experiment (0.25 and 1.25 kg ha1) were added to 500 g fresh weight of soils to determine fungicide dissipation. Unamended and amended soils were then incubated in containers at 20 ± 2 C in the dark during the experiment. The initial moisture content of the soils was adjusted to 40% of their maximum water holding capacity, and it was kept constant during the entire period of the experiment by adding sterile UHQ water when necessary. A sterilized soil sample (z300 g) was also prepared as controls to check the chemical degradation of azoxystrobin as indicated by Marín-Benito et al. (2012b). Soil samples for microbiological control were prepared by adding only sterile UHQ water. Soil samples were taken at day 0 for fungicide analysis and thereafter repeatedly at different time intervals (up to 378 days). 2.4. Fungicide extraction and analysis Samples of moist soil (5 g) were taken by duplicate from each
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Table 1 Selected properties of the soil used in this experiment and initial organic matter (OM) content of unamended and SMS-amended soil. Depth (cm)
Soil texture
pH
OM (%)
N (%)
CaCO3 (%)
Sand (%)
Silt (%)
Clay (%)
0e10
Sandy e e Sandy Sandy Sandy Sandy
7.7 e e 7.8 7.8 7.8 7.9
2.36 6.40a 15.4b 2.22 2.08 1.88 1.44
0.13 e e 0.13 0.13 0.12 0.09
29.5 e e 29.2 29.6 30.5 43.6
60.7 e e 62.6 64.1 63.6 63.3
20.2 e e 18.3 21.4 23.0 15.7
17.1 e e 19.1 14.5 13.4 21.0
10e20 20e30 30e40 40e50
loam loam loam clay loam
S - LD FOMC
80 60 40 20 0 0
100
200
300
100
S+SMS50 - LD FOMC
80 60 40 20 0
400
80
DFOP
60 40 20 0 0
100
200
300
400
Azoxystrobin (% applied)
Azoxystrobin (% applied)
S - HD
100
S+SMS150 - LD FOMC
80 60 40 20 0
0
100
Time (days) 100
Azoxystrobin (% applied)
100
200 300 Time (days)
400
100 S+SMS50 - HD
80
0
Azoxystrobin (% applied)
Azoxystrobin (% applied)
Soil amended at 50 t ha1 of SMS. Soil amended at 150 t ha1 of SMS.
Azoxystrobin (% applied)
a b
loam
DFOP
60 40 20 0 0
100
Time (days)
200 300 Time (days)
400
100
200 300 Time (days)
400
100 S+SMS150 - HD
80
DFOP
60 40 20 0 0
100
200
300
400
Time (days)
Fig. 1. Dissipation of azoxystrobin in unamended and SMS-amended soil (50 or 150 t ha1) after the application of fungicide at low (LD) and high (HD) doses under field conditions. Error bars represent the standard deviation of the mean value (n ¼ 2).
plot (field experiment) or from each container (laboratory experiment) and extracted with 10 mL of methanol in glass tubes. The samples were shaken at 20 C for 24 h and then centrifuged at 5045 g for 15 min. After an appropriate volume (6 mL) was taken, evaporated and redissolved in 1 mL of methanol-UHQ water (1:1 v/ ndez et al. v) in an HPLC glass vial as indicated by Herrero-Herna (2011a) for analysis of azoxystrobin and their metabolites. The mean recovery values of azoxystrobin from spiked soils at concentration of 0.125 mg kg1 and performing the extraction procedure as described above were 101 ± 2.0%, 74.3 ± 3.6% and 71.7 ± 2.6% for unamended and SMS-amended soils (50 and 150 t ha1), respectively. The concentrations of azoxystrobin were corrected for recovery values. The analysis of azoxystrobin and their possible degradation products was performed by HPLC using a Waters chromatograph (Waters Assoc., Milford, MA, USA) attached to a ZQ mass spectrometer detector (MS). A Waters Symmetry C18 column (75 4.6 mm I.D., 3.5 mm) was used and the mobile phase was 70:30 methanol/water in a 0.1% formic acid solution. The flow rate of the mobile phase was 0.30 mL min1 and the sample injection volume was 20 mL. Detection was carried out by HPLC/MS by monitoring the positive molecular ion (m/z) 404.0 for azoxystrobin. The retention time of the fungicide was 5.6 min. Quantitative analysis was performed using the peak area obtained from the total ion chromatogram in SIM mode. External calibration curves were performed from 0.05 to 2.0 mg L1 using standard solutions. The limit of detection (LOD) and limit of quantification (LOQ) were 0.09
and 0.16 mg L1, respectively. In each analysis, a possible azoxystrobin metabolite (azoxystrobin acid), with ion (m/z) [M]þ of 390, was monitored during the dissipation experiment. 2.5. Adsorption experiments Azoxystrobin adsorption isotherms by unamended and amended soil samples taken from control plots (without fungicide) after 2, 84, 181 and 378 days of SMS application were obtained using the batch equilibrium technique. Duplicate soil samples (<2 mm) of 1 g were equilibrated with 5 mL of fungicide solution in UHQ water at concentrations of 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mg L1. Treatment of suspensions was performed as previously described by Herrerondez et al. (2011a). Herna 2.6. Dehydrogenase activity measurements Soil dehydrogenase activity (DHA), expressed as mg TPF g1 dry soil, was determined following the Tabatabai's method (Tabatabai, 1994) at 0, 28, 84, 181 and 378 days after fungicide application in samples from field experiments and in samples from laboratory experiments. 2.7. Data analysis The dissipation kinetics for the fungicide were fitted to a single first-order (SFO) kinetic model, a first-order multicompartment
ndez et al. / Journal of Environmental Management 163 (2015) 78e86 E. Herrero-Herna
Azoxystrobin (μg g-1 dry soil)
Azoxystrobin (μg g-1 dry soil) 0.0
0.1
0.2
0.3
0.4
0.5
0.6
81
0.0
0.7
0.5
1.0
1.5
2.0
2.5
0
3
OM total (μg g-1 ) 6 9
12
0 0-10
0-10
10-20
10-20
Low Dose-84 days
30-40
20-30
Depth (cm)
20-30
Depth (cm)
Depth (cm)
10
High Dose-84 days
20 30
30-40 Soil Soil+SMS50 Soil+SMS150
40-50
Soil Soil+SMS50 Soil+SMS150
40-50
Soil Soil+SMS50 Soil+SMS150
40 50
0.0
Azoxystrobin (μg g-1 dry soil) 0.1 0.2 0.3 0.4 0.5 0.6
0.0
0.7
Azoxystrobin (μg g-1 dry soil) 0.5 1.0 1.5 2.0
0
2.5
3
OM total (μg g-1) 6 9
12
0
0-10
0-10
10-20
10-20
Low Dose-181 days
20-30 High Dose-181 days
20 30
30-40
30-40 Soil Soil+SMS50 Soil+SMS150
40-50
Soil Soil+SMS50 Soil+SMS150
40-50
0.1
0.2
0.3
0.4
0.5
0.6
Soil Soil+SMS50 Soil+SMS150
40 50
Azoxystrobin (μg g-1 dry soil)
Azoxystrobin (μg g-1 dry soil) 0.0
Depth (cm)
20-30
Depth (cm)
Depth (cm)
10
0.7
0.0
0-10
0-10
10-20
10-20
0.5
1.0
1.5
2.0
0
2.5
3
OM total (μg g-1 ) 6 9
12
0
Low Dose-378 days
High Dose-378 days
20 30
30-40
30-40 40-50
20-30
Depth (cm)
20-30
Depth (cm)
Depth (cm)
10
Soil Soil+SMS50 Soil+SMS150
40-50
Soil Soil+SMS50 Soil+SMS150
40
Soil Soil+SMS50 Soil+SMS150
50
Fig. 2. Distribution of azoxystrobin and organic matter content in unamended and SMS-amended soil profile at 84, 181 and 378 days after application of fungicide at low and high doses. Error bars represent the standard deviation of the mean value (n ¼ 6).
(FOMC) model or the double first order in parallel (DFOP) model. FOCUS work group guidance recommendations were followed (FOCUS, 2006) to select the best kinetic model according to the goodness of fit. Values for the time to 50% dissipation, or DT50 values, were used to characterize the decay curves and they were estimated using the Excel Solver add-in package (FOCUS, 2006). The adsorption data for the fungicide were fitted to the linearized form of the Freundlich equation (Rodríguez-Cruz et al., 2012a). DHA data were submitted to an analysis of variance (ANOVA) to evaluate differences at each sampling time and at each treatment. Correlations between adsorption parameters and characteristics of the soils were determined by Pearson's correlation coefficient (r). IBM SPSS (version 22; USA) statistical software was used. 3. Results and discussion 3.1. Azoxystrobin dissipation in field experiments Rainfall and temperature evolution over time (378 days) are included in Fig. S1 in Supplementary material. The average air temperature was 11.3 C, with min and max temperatures of 8.2
and 25.9 C. Rainfall > 10 mm was recorded 4, 49, 52, 54, 190, 217, 220, 224, 235, 307, 339 and 369 days after fungicide application, with the maximum rainfall of 18 mm being registered 235 days after fungicide application. The cumulative precipitation during the experiment was 448 mm. Note should be taken of a cumulative precipitation of 27.2 mm during the first week after fungicide application. Azoxystrobin dissipation under field conditions was determined in unamended and amended plots (0e10 cm). The amount found in the samples taken immediately after its application was considered as a reference for the fungicide applied. Soil samples were analyzed before the fungicide was applied, and no azoxystrobin was detected in any of the soil samples. Under field conditions, azoxystrobin dissipation followed biphasic FOMC and DFOP models, with an initially very rapid dissipation rate followed by very slow and prolonged dissipation in the second phase (Fig. 1). A similar bi-phasic model for dissipation under experimental field conditions has also been reported for other compounds, although azoxystrobin dissipation was much ndez faster than that reported for other fungicides (Herrero-Herna et al., 2011a). The first phase was <7 days after fungicide
ndez et al. / Journal of Environmental Management 163 (2015) 78e86 E. Herrero-Herna
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application, and dissipation varied in the 65e75% range (unamended soil or soil þ SMS50) and in the 45e65% range (soil þ SMS150) when applied at both doses. Azoxystrobin dissipation was influenced more by the SMS amount in the soil than by the dose of fungicide applied. The dissipation kinetics of azoxystrobin applied at low dose (0.25 kg ha1) fitted the FOMC model best in the unamended sample and soil þ SMS, (lower c2 error values) whereas the dissipation kinetics of the fungicide applied at high dose (1.25 kg ha1) fitted the DFOP model best (Table 2). The DT50 values obtained ranged from 0.34 to 0.72 days and from 1.93 to 46.3 days for azoxystrobin applied at low and high dose, respectively, in unamended and amended soils. The highest rate of azoxystrobin dissipation occurred in unamended soil or soil þ SMS50 when applied at low and high dose. The dissipation rate decreased when fungicide was applied in soil þ SMS150. The DT50 values obtained under these conditions increased 1.4e2.3 times (low dose) and 22e24 times (high dose) in this amended soil relative to the DT50 values of azoxystrobin obtained under other conditions. Azoxystrobin has been classified as a non-persistent-tomoderately persistent compound with DT50 values of 14e62 days (PMRA, 2007) or of 3e39 days (EC, 1998) obtained in different soils after field studies, and DT50 values of 5.4e10.4 days obtained in a soil after its application at recommended and double dose (Gajbhiye et al., 2011). The kinetic parameters obtained in this paper indicated that the dissipation rate of azoxystrobin was only lower in amended soils at a high SMS rate than that reported in previous field studies for unamended soil. The differences in the overall dissipation kinetics of azoxystrobin in the field were small in unamended soil and soil þ SMS50, and the influence of soil amendment at a high rate was observed especially during the second phase of the dissipation kinetics. The concentrations of fungicide decreased continuously during this slower second phase, although amounts of fungicide were detectable in the topsoil until the last sampling at 378 days after application in all treatments, and higher residues of azoxystrobin were found in amended soils (5e9%) than in unamended soil (<3%). The influence of the fungicide dose was observed during the second phase of dissipation, with a higher persistence of azoxystrobin when applied at the highest dose, and with the highest SMS rate (Fig. 1). This slower second-phase dissipation might be due to a fraction of strongly adsorbed pesticide that would be less accessible to dissipation processes, as found for other
hydrophobic pesticides in our previous studies (Herrerondez et al., 2011a; Marín-Benito et al., 2012b). Herna The rapid decrease in azoxystrobin concentration observed in the topsoil may be attributed to the movement, biodegradation, photodegradation and bound residue formation of the fungicide over time (EC, 1998; Bartlett et al., 2002; Adetutu et al., 2008). Likewise, this rapid decrease in the fungicide concentration observed here might be favoured by the rainfall episodes recorded in the first week after its application on the experimental plots (Fig. S1 in Supplementary material). The dissipation of pesticides in the field may be affected by a variety of factors, such as soil texture, varying moisture content, varying soil temperature, and agricultural practices such as ploughing, fertilization and the use of organic amendments. Some of these factors have a positive or negative impact on the interactions between soil microorganisms and pesticides, and here they may affect azoxystrobin dissipation (Adetutu et al., 2008). The dissipation study was carried out simultaneously with a study of the distribution of azoxystrobin through the soil profile. Azoxystrobin concentrations were determined at different soil depths (0e50 cm) at 84, 181 and 378 days after fungicide application on the unamended and SMS-amended plots to check for its possible mobility through the soil. The results obtained are included in Fig. 2, together with the OM contents determined at the different soil depths. The leaching of azoxystrobin to deeper soil layers was observed, but the amount of fungicide residues was much higher in the top two layers (0e10 cm and 10e20 cm) than in deeper layers. Similar results were reported by Ghosh and Singh (2009b) when studying the leaching of azoxystrobin in intact and packed soil columns under laboratory conditions. Spliid et al. (2006) also reported that azoxystrobin was immobilized in the top layer (0e10 cm) of a biobed according to its high estimated Koc value. The amounts of fungicide found through the soil profile were higher on the plots treated with a high dose, and also higher in the soils amended with SMS (50 and 150 t ha1) than in the unamended one (Fig. 2). SMS application increased the soil OM content in terms of depth, and the OM enhanced the persistence of azoxystrobin at different soil depths. In fact, a significant correlation was found between azoxystrobin amounts and the OM content determined in the unamended and amended soil profiles when azoxystrobin was applied at low dose (r ¼ 0.796e0.972, p < 0.001), and at high dose (r ¼ 0.839e0.881, p < 0.001). The residual amounts
Table 2 Dissipation of azoxystrobin applied at low and high doses in unamended and SMS-amended soils under field and laboratory conditions. DT50 values were obtained from the kinetics fitting to a single first-order (SFO), Gustafson and Holden (FOMC) and double first-order in parallel (DFOP) models. Samples
Field: low dose Sa S þ SMS50b S þ SMS150c Field: high dose S S þ SMS50 S þ SMS150 Laboratory: low dose S S þ SMS150 Laboratory: high dose S S þ SMS150 a b c
SFO
FOMC
DFOP
DT50 (days)
r2
c2
1.71 2.24 142
0.941 0.938 0.613
55.1 55.0 42.1
0.52 0.34 0.71
0.994 0.994 0.986
9.9 8.6 9.2
7.92 143 191
0.863 0.672 0.836
50.5 36.7 18.8
0.92 1.41 16.8
0.991 0.978 0.899
10.8 10.7 15.6
113 148
0.985 0.991
7.0 4.5
89.2 148
0.995 0.991
4.1 4.6
163 176
0.979 0.964
6.5 8.0
138 138
0.983 0.971
6.1 7.5
Unamended soil. Soil amended at 50 t ha1 of SMS. Soil amended at 150 t ha1 of SMS.
DT50 (days)
r2
c2
r2
c2
2.14 1.61 2.61
0.974 0.990 0.972
22.1 11.2 14.2
2.12 1.93 46.3
0.994 0.993 0.974
10.2 6.1 8.7
68.8 139
0.992 0.991
5.8 5.2
154 155
0.979 0.965
8.8 13.2
DT50 (days)
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83
of the fungicide in the topsoil amended with 50 t ha1 were 1.45e1.95 times (low dose) and 1.82e2.27 times (high dose) higher than in the unamended soil. When the soil was amended with 150 t ha1 the amount of fungicide found was 1.64e2.30 times (low dose) and 2.02e2.34 times (high dose) higher than that found in the unamended soil at the three sampling times (84, 181 and 378 days). The relationship between the amounts of azoxystrobin found in the amended soil and unamended soil was >1 in most of the soil layers at the three times after fungicide application, and these amounts decreased with time for all the soil treatments. The results obtained show an initial increase of fungicide retention on the surface due to the SMS applied, although the fungicide's mobility at deeper soil layers was also recorded in unamended and amended soils over time. Azoxystrobin has a low-to-moderate mobility in soils (EC, 1998; PMRA, 2007), although its high potential for leaching to groundwater under certain climatic and soil conditions has been reported. Using the Groundwater Ubiquity Score (GUS) assessment method, azoxystrobin is classified as a borderline leacher (GUS index ¼ 2.1). Ghosh and Singh (2009b) determined that preferential flow through the macropores of intact soil columns reduced the retention of azoxystrobin in the soil profile, and may thus increase the risk of groundwater contamination. Azoxystrobin concentrations were detected at deeper soil layers (50 cm) through to the end of the study. They decreased from 0.003e0.061 mg g1 at 84 days to 0.002 and 0.007 mg g1 at 378 days after fungicide application. This may be due to the immobilization or mineralization of the fungicide over time, as no degradation products were identified.
in laboratory versus field experiments; the DT50 values of azoxystrobin applied at low dose increased more than 170 times in unamended soil, and more than 200 times in amended soil. These increases were lower for azoxystrobin applied at high dose (65 and three times, respectively). This finding was also observed for other pesticides (PPDB, 2014), and it was explained by processes affecting azoxystrobin under field conditions that may not occur under laboratory conditions. The persistence of azoxystrobin in soils under laboratory conditions could be affected simply by soil characteristics, such as soil pH (Bending et al., 2006), soil OM content, and the addition of ~ a and Bending, amendments (Ghosh and Singh, 2009a; Sopen 2013), and not by soil management practice or different environmental processes that are mainly responsible for leaching the fungicide through the soil (Bending et al., 2007). The azoxystrobin dissipation kinetics carried out in sterilized soils in the laboratory (Fig. 3) indicated that the fungicide was mainly degraded by the soil microorganisms, which was much slower in sterilized soils. An amount of azoxystrobin > 40% and >50% remained in the sterilized soil after 378 days when it was applied at low or high dose. The degradation of azoxystrobin in sterilized soils may be also due to other abiotic factors, but photodegradation did not occur, as the soils were kept in the dark during incubation. No degradation products were detected during the laboratory experiments. In previous works, the azoxystrobin acid metabolite was the major degradation product formed during the dissipation of this fungicide in soils (Ghosh and Singh, 2009a,b; Manna et al., 2013).
3.2. Azoxystrobin dissipation in laboratory experiments
3.3. Adsorption of azoxystrobin by unamended and amended soils
Under laboratory conditions, the dissipation rate of azoxystrobin was lower than that under field conditions, and the dissipation kinetics fitted the SFO and FOMC models better (Fig. 3, Table 2). Previous reports in the literature fitted FOMC and DFOP or SFO kinetic models to describe the degradation of azoxystrobin in different substrates (compost-based biomixtures and soil) (Karanasios et al., 2010) or in unamended and manure- or compostamended soils (Ghosh and Singh, 2009a), respectively. Fungicide dissipation in laboratory studies was also more rapid in unamended than in SMS-amended soil, and DT50 values increased from 89.2 days to 148 days when azoxystrobin was applied at low dose to the unamended soil and soil þ SMS150 (Table 2), and no differences were seen in DT50 values (138 days) when azoxystrobin was applied at high dose in unamended and amended soils. These values were higher than those reported in other works, which ranged from 7 to 90 days (USEPA, 1997; Karanasios et al., 2010), although azoxystrobin was also reported to be very persistent in soils in other papers with DT50 values higher than those found here (Kalinin et al., 2002; Bending et al., 2006, 2007). The influence of SMS in soils for increasing the DT50 values of the fungicide was greater than for other organic amendments, such as biomixtures or soil amended with these materials, because some of these materials were readily capable of degrading fungicides and recorded a shorter half-life of azoxystrobin compared with the values reported in the soil (Ghosh and Singh, 2009a; Karanasios et al., 2010; Marinozzi et al., 2013). However, the differences in the dissipation kinetics of azoxystrobin applied at high dose were not seen in the unamended soil and soil þ SMS150 under laboratory conditions. The concentrations of fungicide decreased continuously, although amounts between 15 and 20% were detected in the topsoil in the last sampling at 378 days after application in all treatments. A significantly lower azoxystrobin dissipation rate was observed
The adsorption of azoxystrobin by unamended and SMSamended soils was studied as support for explaining the behaviour of fungicide in the field and/or laboratory over time. Soils were taken from experimental control plots (untreated with fungicide) after 2, 84, 181 and 378 days, and adsorption isotherms were obtained under laboratory conditions (Fig. S2 in Supplementary material). These adsorption isotherms were fitted to the Freundlich equation (r2 > 0.97, p < 0.01), and the Kf and nf parameters were determined (Table 3). The nf values were, in general, <1 in the unamended soil and soil þ SMS50 and >1 in soil amended with SMS at a higher rate (150 t ha1), providing L-type and S-type isotherms of the azoxystrobin by the soils. These isotherms correspond to a decrease or an increase in adsorption, respectively, with the increase in fungicide concentration in solution. The results indicate an increase in adsorption through the addition of SMS to the soil, with the Kf values of azoxystrobin in soil þ SMS150 increasing by factors of 34.3, 16.5, 5.9 and 4.6 after 2, 84, 181 and 378 days of SMS application, compared to the unamended soil. The influence of the soil OM content on the adsorption of azoxystrobin and other fungicides has also been et al., 2011). On the other hand, reported previously (Kodesova changes in the adsorption of azoxystrobin over time were found in the amended soils, but not in the unamended soil; these changes in the amended soil indicated Kf values decreased from 28.6 to 5.57 (Soil þ SMS50) and from 234 to 30.1 (Soil þ SMS150) over time. These changes in adsorption coefficients corresponded to changes in the soil OM contents shown in Fig. 2. In fact, a significant correlation was obtained between the Kf values of azoxystrobin for the unamended and amended soils collected over time and the OM content of these soils (r ¼ 0.93, p < 0.001). A decrease has also been reported in the adsorption of other fungicides in SMS-soils over 12 months of soil incubation in the laboratory (Marín-Benito et al.,
ndez et al. / Journal of Environmental Management 163 (2015) 78e86 E. Herrero-Herna
S - LD FOMC
80 60 40 20
80 60 40 20 0
0
100
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100 S - HD FOMC
80
0
Azoxystrobin (% applied)
Azoxystrobin (% applied)
S+SMS150 - LD SFO
60 40 20 0
100
200 300 Time (days)
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80 60 40 20 0
0
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0
100
200 300 Time (days)
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80 60 40 20 0
400
0
Azoxystrobin (% applied)
0
100
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100
Azoxystrobin (% applied)
Azoxystrobin (% applied)
84
100
200 300 Time (days)
400
100 SS - HD
80 60 40 20 0 0
100
200 300 Time (days)
400
Fig. 3. Dissipation of azoxystrobin in unamended, SMS-amended soil (150 t ha1) and sterilized soil after the application of fungicide at low (LD) and high (HD) doses under laboratory conditions. Error bars represent the standard deviation of the mean value (n ¼ 2), they were smaller than the symbol.
2012a). These findings clearly indicate the influence of the organic amendment on azoxystrobin adsorption by soil, and likewise the influence in the persistence and/or dissipation of the fungicide. According to the DT50 values, azoxystrobin increased from 1.4 to 22 times in soil þ SMS150 in relation to unamended soil after the application under field conditions of a low or high dose of the fungicide, respectively. Under laboratory conditions, this effect was noted only when fungicide was applied at low dose. Azoxystrobin adsorption involves rapid dissipation in amended soils, and might limit initial fungicide leaching, although the results reveal the presence of fungicide in the soil profile over time after 378 days (Fig. 2), and this might indicate that the fungicide adsorption prevented its degradation. However the OM content decreased from SMS amended soils over time, which should favour the natural reversibility of compound and its natural degradation.
Table 3 Freundlich adsorption parameters of azoxystrobin by unamended and amended soils in field with SMS at 50 and 150 t ha1 after 2, 84, 181 and 378 days. Adsorption parameters
Time after SMS application (days) T2
T84
T181
T378
6.83 ± 0.56 0.77 ± 0.04 0.99
6.86 ± 0.22 0.79 ± 0.03 0.99
7.33 ± 0.50 0.89 ± 0.03 0.99
6.46 ± 0.30 1.04 ± 0.02 0.98
28.6 ± 1.76 1.06 ± 0.03 0.99
11.2 ± 0.51 0.89 ± 0.01 0.97
8.25 ± 0.37 0.86 ± 0.02 0.98
5.57 ± 0.31 0.77 ± 0.03 0.98
234 ± 23.8 1.32 ± 0.10 0.99
113 ± 0.38 1.48 ± 0.06 0.97
43.1 ± 4.07 1.13 ± 0.05 0.99
30.1 ± 0.97 0.98 ± 0.03 0.99
a
S Kf ± SDd nf ± SD r2 S þ SMS50b Kf ± SD nf ± SD r2 S þ SMS150c Kf ± SD nf ± SD r2 a b c d
Unamended soil. Soil amended at 50 t ha1 of SMS. Soil amended at 150 t ha1 of SMS. SD ¼ standard deviation of replicates (n ¼ 2).
3.4. Dehydrogenase activity of soils in field and laboratory experiments DHA results are included in Fig. 4 for unamended and SMSamended soils both untreated (controls) and treated with azoxystrobin under field conditions and under laboratory controlled conditions over different time periods. Overall, both the sampling time and the soils used, as well as their interactions, had a significant effect on DHA (p < 0.0001). The DHA mean values were higher in amended soils than in unamended ones under field or laboratory conditions (LSD ¼ 74.00 and LSD ¼ 36.58, respectively, p < 0.0004), as reported in the literature (Chen et al., 2001). The higher DHA values in amended soils compared to unamended ones is attributed to the greater OM content provided by SMS which contains available carbon that may stimulate soil microbial community, and to the presence of new active microbial populations introduced with the amendment. Under field conditions, DHA mean values were higher in soils treated with azoxystrobin at two doses than in untreated soils (unamended and amended) (LSD ¼ 27.73, p ¼ 0.0001), revealing the stimulatory effect of the fungicide on soil DHA. In SMSamended soils, the increase in DHA after azoxystrobin application was smaller than in the unamended soil. This effect has also been reported for other fungicides (Chen et al., 2001), with the authors describing the ability fungicides have to both inhibit and stimulate certain groups of microorganisms in soils. Although DHA was higher in most of the soils treated with azoxystrobin, no clear differences were found with fungicide dose as reported for tebucondez et al. (2011a) despite some authors nazole by Herrero-Herna have reported an increase in the toxic effect of pesticides for soil bacteria with the highest doses of compound (Xie et al., 2004). Likewise the DHA mean values increased significantly over time in unamended and amended soils (Fig. 4) under field conditions and DHA values were higher in all the soils at the end of the experiment (p values between 0.0002 and 0.0612) especially in unamended soil.
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Fig. 4. Soil dehydrogenase activity for unamended and SMS-amended soil (150 t ha1), untreated or treated with a low (LD) or high (HD) dose of azoxystrobin at different sampling times under field (A) and laboratory (B) conditions. Bars represent the standard deviation of the mean (n ¼ 3). Treatments with the same letter are not significantly different from each other.
Under laboratory conditions DHA values were higher at the beginning of the experiment in unamended soils, untreated or treated with azoxystrobin. A similar effect was observed by Chen et al. (2001), being explained by the initial mixing and wetting of soils prior to the stabilization of microbial activity. A significant DHA decrease was subsequently recorded over time in all the soils (treated and untreated) (p values between 0.001 and 0.003). In SMS-amended soils, the decrease in DHA after azoxystrobin application was smaller than in the unamended soil. A DHA ~ a and decrease in soils treated with biochar was observed by Sopen Bending (2013) following azoxystrobin application. These authors believed that a decrease in DHA induced by azoxystrobin in treated soils might reflect an impact on fungal biomass, with the direct inhibition of fungi, or indirect changes reflecting altered competitive interactions between microbes in the presence of the fungicide (Bending et al., 2007). Furthermore, Karanasios et al. (2010) observed a decrease in microbial respiration over time in the degradation assays of pesticides in biomixtures in the laboratory. Changes may occur in the physico-chemical properties of soil, with the loss of soil active microbial biomass at the end of the fungicide incubation time in containers under laboratory controlled conditions. Therefore, differences in the management of residual soil amounts in both experimental procedures may explain the much higher persistence of fungicide under laboratory rather than field conditions.
4. Conclusions The results obtained in this work revealed that azoxystrobin dissipation was faster in unamended soils than in amended ones in the two environmental scenarios. However a significantly lower azoxystrobin dissipation rate was observed in laboratory versus field experiments and it was explained by processes affecting azoxystrobin that may not occur under laboratory conditions. The adsorption and mobility of fungicide in the soil were affected by SMS application, enhancing its rapid dissipation under field conditions and increasing its persistence in the amended soil profile. Likewise, the increase in DHA over time under field conditions is consistent with the influence of microbial activity, and also supports the greater dissipation of azoxystrobin under these conditions than under laboratory conditions. Our research highlights the importance of examining the dissipation of azoxystrobin under field situations in order to obtain a realistic assessment of dissipation rates in agricultural practices involving the joint use of SMS and azoxystrobin. This would help to better predict its behaviour in the soil environment, with the purpose being to avoid the risk of water being contaminated by this compound. A long-term assessment of the effect of SMS amendment has on the fate of azoxystrobin in vineyard soils is required to take preventive measures against the groundwater contamination derived from its intensive use in agriculture.
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Acknowledgements This work was funded by the Spanish Ministry of Science and Innovation (Project AGL2007-61674/AGR). E.H.H. and J.M.M.B. thank CSIC for their JAE contracts. We would like to thank L.F. Lorenzo, J.M. Ordax and A. Gonzalez for their technical assistance and CVNE winery, CTICH and INTRAVAL S.L. from La Rioja, Spain, for their collaborations. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jenvman.2015.08.010. References Adetutu, E.M., Ball, A.S., Osborn, A.M., 2008. Azoxystrobin and soil interactions: degradation and impact on soil bacterial and fungal communities. J. Appl. Microbiol. 105, 1777e1790. Ahmad, R., James, T.K., Rahman, A., Holland, P.T., 2003. Dissipation of the herbicide clopyralid in an allophanic soil: laboratory and field studies. J. Environ. Sci. Health B 38, 683e695. Bartlett, D.W., Clough, J.M., Godwin, J.R., Hall, A.A., Hamer, M., Parr-Dobrzanski, B., 2002. Review: the strobilurin fungicides. Pest Manag. Sci. 58, 649e662. Bending, G.D., Lincoln, S.D., Edmondson, R.N., 2006. Spatial variation in the degradation rate of the pesticides isoproturon, azoxystrobin and diflufenican in soil and its relationship with chemical and microbial properties. Environ. Pollut. 139, 279e287. Bending, G.D., Rodríguez-Cruz, M.S., Lincoln, D.S., 2007. Fungicide impacts on microbial communities in soils with contrasting management histories. Chemosphere 69, 82e88. Chai, L.-K., Mohd-Tahir, N., Hansen, H.C.B., 2009. Dissipation of acephate, chlorpyrifos, cypermethrin and their metabolites in a humid-tropical vegetable production system. Pest Manag. Sci. 65, 189e196. Chai, L.-K., Wong, M.-H., Hansen, H.C.B., 2013. Degradation of chlorpyrifos in humid tropical soils. J. Environ. Manag. 125, 28e32. Chen, S.-K., Edwards, C.A., Subler, S., 2001. Effects of the fungicide benomyl, captan and chlorothalonil on soil microbial activity and nitrogen dynamics in laboratory incubations. Soil Biol. Biochem. 33, 1971e1980. Dolaptsoglou, C., Karpouzas, D., Menkissoglu-Spiroudi, U., Eleftherohorinos, I., Voudrias, E.A., 2009. Influence of different organic amendments on the leaching and dissipation of terbuthylazine in a column and a field study. J. Environ. Qual. 38, 782e791. EC (European Commision. Directorate General for Agriculture. DG VI-B.II-1), 1998. Review Report for the Active Substance Azoxystrobin, 7581/VI/97-Final. p. 17. Fenoll, J., Ruiz, E., Flores, P., Vela, N., Hellín, P., Navarro, S., 2011. Use of farming and agro-industrial wastes as versatile barriers in reducing pesticide leaching through soil columns. J. Hazard. Mater. 187, 206e212. FOCUS, 2006. Guidance Document on Estimating Persistence and Degradation Kinetics from Environmental Fate Studies on Pesticides in EU Registration. Report of the FOCUS Work Group on Degradation Kinetics; EC Document Reference Sanco/10058/2005 version 2.0. Gajbhiye, V.T., Gupta, S., Mukherjee, I., Singh, S.B., Singh, N., Dureja, P., Kumar, Y., 2011. Persistence of azoxystrobin in/on grapes and soil in different grapes growing areas of India. Bull. Environ. Contam. Toxicol. 86, 90e94. Ghosh, R.K., Singh, N., 2009a. Effect of organic manure on sorption and degradation of azoxystrobin in soil. J. Agric. Food Chem. 57, 632e636. Ghosh, R.K., Singh, N., 2009b. Leaching behaviour of azoxystrobin and metabolites in soil columns. Pest Manag. Sci. 65, 1009e1014. nchez-Martín, M.J., Herrero-Hern andez, E., Andrades, M.S., Marín-Benito, J.M., Sa Rodríguez-Cruz, M.S., 2011a. Field-scale dissipation of tebuconazole in a vineyard soil amended with spent mushroom substrate and its potential environmental impact. Ecotoxicol. Environ. Saf. 74, 1480e1488. Herrero-Hern andez, E., Andrades, M.S., Rodríguez-Cruz, M.S., S anchez-Martín, M.J., 2011b. Effect of spent mushroom substrate applied to vineyard soil on the behaviour of copper-based fungicide residues. J. Environ. Manag. 92, 1849e1857. Herrero-Hern andez, E., Andrades, M.S., Alvarez-Martín, A., Pose-Juan, E., RodríguezCruz, M.S., S anchez-Martín, M.J., 2013. Occurrence of pesticides and some of their degradation products in waters in a Spanish wine region. J. Hydrol. 486, 234e245. Kalinin, V.A., Bykov, K.V., Osman, A.G., 2002. Effects of azoxystrobin on soil microorganisms under laboratory conditions. In: The BCPC Conference: Pest and Diseases, Vol. 1 and 2. Proceeding of an International Conference Held at Brighton, UK, pp. 279e284. Karanasios, E., Tsiropoulos, N.G., Karpouzas, D.G., Ehaliotis, C., 2010. Degradation and adsorption of pesticides in compost-based biomixtures as potential substrates for biobeds in Southern Europe. J. Agric. Food Chem. 58, 9147e9156.
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Research article
Field versus laboratory experiments to evaluate the fate of azoxystrobin in an amended vineyard soil ndez a, J.M. Marín-Benito a, M.S. Andrades b, M.J. Sa nchez-Martín a, E. Herrero-Herna M.S. Rodríguez-Cruz a, * a b
Institute of Natural Resources and Agrobiology of Salamanca (IRNASA-CSIC), Cordel de Merinas 40-52, 37008 Salamanca, Spain ~ o, Spain Department of Agriculture and Food, University of La Rioja, Madre de Dios 51, 26006 Logron
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 February 2015 Received in revised form 29 July 2015 Accepted 12 August 2015 Available online 22 August 2015
This study reports the effect that adding spent mushroom substrate (SMS) to a representative vineyard soil from La Rioja region (Spain) has on the behaviour of azoxystrobin in two different environmental scenarios. Field dissipation experiments were conducted on experimental plots amended at rates of 50 and 150 t ha1, and similar dissipation experiments were simultaneously conducted in the laboratory to identify differences under controlled conditions. Azoxystrobin dissipation followed biphasic kinetics in both scenarios, although the initial dissipation phase was much faster in the field than in the laboratory experiments, and the half-life (DT50) values obtained in the two experiments were 0.34e46.3 days and 89.2e148 days, respectively. Fungicide residues in the soil profile increased in the SMS amended soil and they were much higher in the top two layers (0e20 cm) than in deeper layers. The persistence of fungicide in the soil profile is consistent with changes in azoxystrobin adsorption by unamended and amended soils over time. Changes in the dehydrogenase activity (DHA) of soils under different treatments assayed in the field and in the laboratory indicated that SMS and the fungicide had a stimulatory effect on soil DHA. The results reveal that the laboratory studies usually reported in the literature to explain the fate of pesticides in amended soils are insufficient to explain azoxystrobin behaviour under real conditions. Field studies are necessary to set up efficient applications of SMS and fungicide, with a view to preventing the possible risk of water contamination. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Azoxystrobin Dissipation Field and laboratory experiments Adsorption Soil dehydrogenase activity
1. Introduction Crop protection is an integral part of modern agriculture, with pesticide application being a major component. However, the presence of pesticides in soils and waters has increased in recent ndez et al., 2013; Ko €ck-Schulmeyer et al., years (Herrero-Herna et al., 2015), and this practice has become exten2014; Masia sively and hotly debated. Pesticide residues in the soil may be taken up by plants, degraded into other chemical forms, or washed into surface and ground waters. The leaching of pesticides into groundwater is a cause of concern, as this is one of the major sources of drinking water in many locations. A positive step toward reducing pesticide leaching, and hence the risk of groundwater contamination by pesticide residues, involves enhancing the retention and degradation of pesticides in
* Corresponding author. E-mail address: [email protected] (M.S. Rodríguez-Cruz). http://dx.doi.org/10.1016/j.jenvman.2015.08.010 0301-4797/© 2015 Elsevier Ltd. All rights reserved.
soils and this can be achieved by increasing soil organic matter (OM) content. As it is known these processes depend on the soil OM content for the non-ionic hydrophobic pesticides (Marín-Benito et al., 2012a,b). Nowadays, a common way of increasing the OM content involves soil amendment with organic residues (Moreno-Casco and Moral-Herrero, 2008). This soil amendment is used to increase OM content in Mediterranean agricultural soils, and there has recently been an increase in interest in assessing the application of these organic residues as a strategy for enhancing the retention and degradation of pesticides in soils in order to avoid their leaching into groundwater. There are numerous studies researching the effect of organic amendments on the fate of pesticides in soils conducted under laboratory conditions (Fenoll et al., 2011; Martin et al., 2012; pez-Pin ~ eiro et al., 2013), and Rodríguez-Cruz et al., 2012a,b; Lo although these studies provide valuable information on the effect organic amendments have on the behaviour of pesticides in the soil,
ndez et al. / Journal of Environmental Management 163 (2015) 78e86 E. Herrero-Herna
they do not always reflect what actually occurs in the field. Consequently, both field studies and laboratory studies are needed to provide complete information on how pesticides dissipate under natural conditions. In general, there are few studies on dissipation in the field (Papiernik et al., 2007; Chai et al., 2009), with only a handful examining the influence of different organic amendments on ndez pesticide dissipation (Dolaptsoglou et al., 2009; Herrero-Herna et al., 2011a). Very few studies have evaluated the dissipation of pesticides under field and laboratory conditions simultaneously (Ahmad et al., 2003; Potter et al., 2005; Chai et al., 2013). Azoxystrobin is a fungicide with a broad spectrum of systemic activity for the control of fungal crop pathogens (Bartlett et al., 2002). It belongs to the strobilurins group, and it is a chemical that has been approved for use on more than 80 different crops in 72 countries. Azoxystrobin is commonly used as a foliar fungicide, and a high proportion of this fungicide may be deposited in the soil when applied in spray form (Adetutu et al., 2008). Some reports on its persistence and mobility have indicated that it may remain in soils for several months (Bending et al., 2007). However, the presence of azoxystrobin detected in the ground and surface waters in ndez et al., different vine growing areas in Spain (Herrero-Herna 2013), Brazil (Menezes Filho et al., 2010), France (Rabiet et al., 2010), and Germany (Neumann et al., 2003) reveals that certain uncontrolled factors affect its behaviour in soils. There are studies on the degradation of this fungicide in some soils amended with organic residues under laboratory conditions ~ a and Bending, 2013), but the (Ghosh and Singh, 2009a; Sopen dissipation of azoxystrobin under field conditions has scarcely been studied (Gajbhiye et al., 2011), and to our knowledge there are no studies on the dissipation of azoxystrobin in soil amended with spent mushroom substrate (SMS). SMS is the composted organic material remaining after a mushroom crop has been harvested, and it is being generated on farms in increasing quantities (Martín et al., 2009). SMS is used as a soil fertilizer and amendment to increase the OM content of vineyard soils in La Rioja region (Spain). The objective of this research was to study the effect that SMS soil amendment has on the fate of azoxystrobin under two environmental scenarios. Accordingly, field versus laboratory experiments were conducted for comparative purposes on a vineyard soil from La Rioja region, both unamended and amended with SMS at two rates, with the aim being to assess the dissipation, persistence, and mobility of azoxystrobin applied at two doses. In support to explain the fate of the fungicide, changes in adsorption of azoxystrobin by unamended and amended soil from field experimental plots and changes in the dehydrogenase activity of unamended and amended soil, untreated and treated with azoxystrobin in field and in laboratory were evaluated over the time. 2. Materials and methods 2.1. Chemicals and organic amendment An analytical standard of azoxystrobin (Methyl (E)-2-{2-[6-(2cyanophenoxy) pyrimidin-4-yloxy]phenyl}-3-methoxyacrylate) from Dr. Ehrenstorfer, Germany (99.0% purity) and the commercial formulation of azoxystrobin from Ortiva (Syngenta, Switzerland) were used in the laboratory and field experiments, respectively. Azoxystrobin is a fungicide with a water solubility of 6 mg L1 and a log Kow of 2.5 (Tomlin, 2000). All other chemicals used were supplied by SigmaeAldrich Quimica SA (Spain). Spent mushroom substrate was supplied by INTRAVAL Environmental Group TRADEBE S.L. (Spain). Its composition was ndez et al. (2011a) and their physicodescribed by Herrero-Herna chemical characteristics determined as described in this previous work are: pH 7.5, organic carbon (OC) content 27.1% and dissolved
79
organic carbon (DOC) content 1.22%. 2.2. Field experimental design and soil sampling The field experiment was conducted in a vineyard in Sajazarra, La Rioja, Spain (42 350 000 N latitude and 2 570 000 W longitude). The soil used was a sandy clay loam soil classified as Typic Calcixerept. Their characteristics were determined by usual analytical methods (MAPA, 1986) (Table 1). Rainfall and temperature were recorded over the 378 days of experimentation at a weather station close to the study site (4 km east) (Fig. S1 in Supplementary material). An experimental layout of randomized complete blocks (18 plots of 1.50 m 3.90 m) was set up with six treatments (unamended (S) and amended soils (Soil þ SMS) at two rates of SMS treated with two doses of azoxystrobin) and three replicates per ndez et al. (2011a,b). Untreatment as indicated in Herrero-Herna amended and amended soils (0e10 cm) at the rates of 50 or 150 t ha1 (Soil þ SMS50 and Soil þ SMS150) on dry weight basis were prepared on November 2010. Azoxystrobin solutions at two doses (0.25 and 1.25 kg ha1) were applied to the plots from the commercial formulation Ortiva-Syngenta (25% w/v of a.i.). Three more plots one unamended and two amended with 50 or 150 t ha1 of SMS, respectively, did not receive fungicide application (control plots). Soil samples from these untreated plots were collected for assays in the laboratory as indicated below. Five topsoil samples were collected from 0 to 10 cm at 0, 2, 7, 14, 21, 28, 35, 84, 115, 150, 181, 235, 300 and 378 days after treatment to determine fungicide dissipation and five soil cores were collected to a depth of 50 cm after 84, 181 and 378 days of azoxystrobin application to determine mobility of fungicide. Soil samples were ndez et al. (2011a). managed as indicated in Herrero-Herna Topsoil OM content was determined two days after SMS application and OM content at different depths of soil cores taken at 84, 181 and 378 days was also determined by triplicate. Results are included in Table 1 and Fig. 2. 2.3. Laboratory experimental design and soil sampling Soil samples for laboratory experiments were taken from 0 to 10 cm of experimental plots prepared for fungicide dissipation study in field on November 2010. Soils collected from control plots, unamended and SMS-amended at dose of 150 t ha1, and without fungicide applied, were transferred to polypropylene containers. They were transported to the laboratory and treated as previously indicated for field soil samples. Solutions of fungicide were prepared in sterile UHQ water and volumes of 10 mL of suitable concentrations to give similar concentrations to those applied in the field experiment (0.25 and 1.25 kg ha1) were added to 500 g fresh weight of soils to determine fungicide dissipation. Unamended and amended soils were then incubated in containers at 20 ± 2 C in the dark during the experiment. The initial moisture content of the soils was adjusted to 40% of their maximum water holding capacity, and it was kept constant during the entire period of the experiment by adding sterile UHQ water when necessary. A sterilized soil sample (z300 g) was also prepared as controls to check the chemical degradation of azoxystrobin as indicated by Marín-Benito et al. (2012b). Soil samples for microbiological control were prepared by adding only sterile UHQ water. Soil samples were taken at day 0 for fungicide analysis and thereafter repeatedly at different time intervals (up to 378 days). 2.4. Fungicide extraction and analysis Samples of moist soil (5 g) were taken by duplicate from each
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Table 1 Selected properties of the soil used in this experiment and initial organic matter (OM) content of unamended and SMS-amended soil. Depth (cm)
Soil texture
pH
OM (%)
N (%)
CaCO3 (%)
Sand (%)
Silt (%)
Clay (%)
0e10
Sandy e e Sandy Sandy Sandy Sandy
7.7 e e 7.8 7.8 7.8 7.9
2.36 6.40a 15.4b 2.22 2.08 1.88 1.44
0.13 e e 0.13 0.13 0.12 0.09
29.5 e e 29.2 29.6 30.5 43.6
60.7 e e 62.6 64.1 63.6 63.3
20.2 e e 18.3 21.4 23.0 15.7
17.1 e e 19.1 14.5 13.4 21.0
10e20 20e30 30e40 40e50
loam loam loam clay loam
S - LD FOMC
80 60 40 20 0 0
100
200
300
100
S+SMS50 - LD FOMC
80 60 40 20 0
400
80
DFOP
60 40 20 0 0
100
200
300
400
Azoxystrobin (% applied)
Azoxystrobin (% applied)
S - HD
100
S+SMS150 - LD FOMC
80 60 40 20 0
0
100
Time (days) 100
Azoxystrobin (% applied)
100
200 300 Time (days)
400
100 S+SMS50 - HD
80
0
Azoxystrobin (% applied)
Azoxystrobin (% applied)
Soil amended at 50 t ha1 of SMS. Soil amended at 150 t ha1 of SMS.
Azoxystrobin (% applied)
a b
loam
DFOP
60 40 20 0 0
100
Time (days)
200 300 Time (days)
400
100
200 300 Time (days)
400
100 S+SMS150 - HD
80
DFOP
60 40 20 0 0
100
200
300
400
Time (days)
Fig. 1. Dissipation of azoxystrobin in unamended and SMS-amended soil (50 or 150 t ha1) after the application of fungicide at low (LD) and high (HD) doses under field conditions. Error bars represent the standard deviation of the mean value (n ¼ 2).
plot (field experiment) or from each container (laboratory experiment) and extracted with 10 mL of methanol in glass tubes. The samples were shaken at 20 C for 24 h and then centrifuged at 5045 g for 15 min. After an appropriate volume (6 mL) was taken, evaporated and redissolved in 1 mL of methanol-UHQ water (1:1 v/ ndez et al. v) in an HPLC glass vial as indicated by Herrero-Herna (2011a) for analysis of azoxystrobin and their metabolites. The mean recovery values of azoxystrobin from spiked soils at concentration of 0.125 mg kg1 and performing the extraction procedure as described above were 101 ± 2.0%, 74.3 ± 3.6% and 71.7 ± 2.6% for unamended and SMS-amended soils (50 and 150 t ha1), respectively. The concentrations of azoxystrobin were corrected for recovery values. The analysis of azoxystrobin and their possible degradation products was performed by HPLC using a Waters chromatograph (Waters Assoc., Milford, MA, USA) attached to a ZQ mass spectrometer detector (MS). A Waters Symmetry C18 column (75 4.6 mm I.D., 3.5 mm) was used and the mobile phase was 70:30 methanol/water in a 0.1% formic acid solution. The flow rate of the mobile phase was 0.30 mL min1 and the sample injection volume was 20 mL. Detection was carried out by HPLC/MS by monitoring the positive molecular ion (m/z) 404.0 for azoxystrobin. The retention time of the fungicide was 5.6 min. Quantitative analysis was performed using the peak area obtained from the total ion chromatogram in SIM mode. External calibration curves were performed from 0.05 to 2.0 mg L1 using standard solutions. The limit of detection (LOD) and limit of quantification (LOQ) were 0.09
and 0.16 mg L1, respectively. In each analysis, a possible azoxystrobin metabolite (azoxystrobin acid), with ion (m/z) [M]þ of 390, was monitored during the dissipation experiment. 2.5. Adsorption experiments Azoxystrobin adsorption isotherms by unamended and amended soil samples taken from control plots (without fungicide) after 2, 84, 181 and 378 days of SMS application were obtained using the batch equilibrium technique. Duplicate soil samples (<2 mm) of 1 g were equilibrated with 5 mL of fungicide solution in UHQ water at concentrations of 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mg L1. Treatment of suspensions was performed as previously described by Herrerondez et al. (2011a). Herna 2.6. Dehydrogenase activity measurements Soil dehydrogenase activity (DHA), expressed as mg TPF g1 dry soil, was determined following the Tabatabai's method (Tabatabai, 1994) at 0, 28, 84, 181 and 378 days after fungicide application in samples from field experiments and in samples from laboratory experiments. 2.7. Data analysis The dissipation kinetics for the fungicide were fitted to a single first-order (SFO) kinetic model, a first-order multicompartment
ndez et al. / Journal of Environmental Management 163 (2015) 78e86 E. Herrero-Herna
Azoxystrobin (μg g-1 dry soil)
Azoxystrobin (μg g-1 dry soil) 0.0
0.1
0.2
0.3
0.4
0.5
0.6
81
0.0
0.7
0.5
1.0
1.5
2.0
2.5
0
3
OM total (μg g-1 ) 6 9
12
0 0-10
0-10
10-20
10-20
Low Dose-84 days
30-40
20-30
Depth (cm)
20-30
Depth (cm)
Depth (cm)
10
High Dose-84 days
20 30
30-40 Soil Soil+SMS50 Soil+SMS150
40-50
Soil Soil+SMS50 Soil+SMS150
40-50
Soil Soil+SMS50 Soil+SMS150
40 50
0.0
Azoxystrobin (μg g-1 dry soil) 0.1 0.2 0.3 0.4 0.5 0.6
0.0
0.7
Azoxystrobin (μg g-1 dry soil) 0.5 1.0 1.5 2.0
0
2.5
3
OM total (μg g-1) 6 9
12
0
0-10
0-10
10-20
10-20
Low Dose-181 days
20-30 High Dose-181 days
20 30
30-40
30-40 Soil Soil+SMS50 Soil+SMS150
40-50
Soil Soil+SMS50 Soil+SMS150
40-50
0.1
0.2
0.3
0.4
0.5
0.6
Soil Soil+SMS50 Soil+SMS150
40 50
Azoxystrobin (μg g-1 dry soil)
Azoxystrobin (μg g-1 dry soil) 0.0
Depth (cm)
20-30
Depth (cm)
Depth (cm)
10
0.7
0.0
0-10
0-10
10-20
10-20
0.5
1.0
1.5
2.0
0
2.5
3
OM total (μg g-1 ) 6 9
12
0
Low Dose-378 days
High Dose-378 days
20 30
30-40
30-40 40-50
20-30
Depth (cm)
20-30
Depth (cm)
Depth (cm)
10
Soil Soil+SMS50 Soil+SMS150
40-50
Soil Soil+SMS50 Soil+SMS150
40
Soil Soil+SMS50 Soil+SMS150
50
Fig. 2. Distribution of azoxystrobin and organic matter content in unamended and SMS-amended soil profile at 84, 181 and 378 days after application of fungicide at low and high doses. Error bars represent the standard deviation of the mean value (n ¼ 6).
(FOMC) model or the double first order in parallel (DFOP) model. FOCUS work group guidance recommendations were followed (FOCUS, 2006) to select the best kinetic model according to the goodness of fit. Values for the time to 50% dissipation, or DT50 values, were used to characterize the decay curves and they were estimated using the Excel Solver add-in package (FOCUS, 2006). The adsorption data for the fungicide were fitted to the linearized form of the Freundlich equation (Rodríguez-Cruz et al., 2012a). DHA data were submitted to an analysis of variance (ANOVA) to evaluate differences at each sampling time and at each treatment. Correlations between adsorption parameters and characteristics of the soils were determined by Pearson's correlation coefficient (r). IBM SPSS (version 22; USA) statistical software was used. 3. Results and discussion 3.1. Azoxystrobin dissipation in field experiments Rainfall and temperature evolution over time (378 days) are included in Fig. S1 in Supplementary material. The average air temperature was 11.3 C, with min and max temperatures of 8.2
and 25.9 C. Rainfall > 10 mm was recorded 4, 49, 52, 54, 190, 217, 220, 224, 235, 307, 339 and 369 days after fungicide application, with the maximum rainfall of 18 mm being registered 235 days after fungicide application. The cumulative precipitation during the experiment was 448 mm. Note should be taken of a cumulative precipitation of 27.2 mm during the first week after fungicide application. Azoxystrobin dissipation under field conditions was determined in unamended and amended plots (0e10 cm). The amount found in the samples taken immediately after its application was considered as a reference for the fungicide applied. Soil samples were analyzed before the fungicide was applied, and no azoxystrobin was detected in any of the soil samples. Under field conditions, azoxystrobin dissipation followed biphasic FOMC and DFOP models, with an initially very rapid dissipation rate followed by very slow and prolonged dissipation in the second phase (Fig. 1). A similar bi-phasic model for dissipation under experimental field conditions has also been reported for other compounds, although azoxystrobin dissipation was much ndez faster than that reported for other fungicides (Herrero-Herna et al., 2011a). The first phase was <7 days after fungicide
ndez et al. / Journal of Environmental Management 163 (2015) 78e86 E. Herrero-Herna
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application, and dissipation varied in the 65e75% range (unamended soil or soil þ SMS50) and in the 45e65% range (soil þ SMS150) when applied at both doses. Azoxystrobin dissipation was influenced more by the SMS amount in the soil than by the dose of fungicide applied. The dissipation kinetics of azoxystrobin applied at low dose (0.25 kg ha1) fitted the FOMC model best in the unamended sample and soil þ SMS, (lower c2 error values) whereas the dissipation kinetics of the fungicide applied at high dose (1.25 kg ha1) fitted the DFOP model best (Table 2). The DT50 values obtained ranged from 0.34 to 0.72 days and from 1.93 to 46.3 days for azoxystrobin applied at low and high dose, respectively, in unamended and amended soils. The highest rate of azoxystrobin dissipation occurred in unamended soil or soil þ SMS50 when applied at low and high dose. The dissipation rate decreased when fungicide was applied in soil þ SMS150. The DT50 values obtained under these conditions increased 1.4e2.3 times (low dose) and 22e24 times (high dose) in this amended soil relative to the DT50 values of azoxystrobin obtained under other conditions. Azoxystrobin has been classified as a non-persistent-tomoderately persistent compound with DT50 values of 14e62 days (PMRA, 2007) or of 3e39 days (EC, 1998) obtained in different soils after field studies, and DT50 values of 5.4e10.4 days obtained in a soil after its application at recommended and double dose (Gajbhiye et al., 2011). The kinetic parameters obtained in this paper indicated that the dissipation rate of azoxystrobin was only lower in amended soils at a high SMS rate than that reported in previous field studies for unamended soil. The differences in the overall dissipation kinetics of azoxystrobin in the field were small in unamended soil and soil þ SMS50, and the influence of soil amendment at a high rate was observed especially during the second phase of the dissipation kinetics. The concentrations of fungicide decreased continuously during this slower second phase, although amounts of fungicide were detectable in the topsoil until the last sampling at 378 days after application in all treatments, and higher residues of azoxystrobin were found in amended soils (5e9%) than in unamended soil (<3%). The influence of the fungicide dose was observed during the second phase of dissipation, with a higher persistence of azoxystrobin when applied at the highest dose, and with the highest SMS rate (Fig. 1). This slower second-phase dissipation might be due to a fraction of strongly adsorbed pesticide that would be less accessible to dissipation processes, as found for other
hydrophobic pesticides in our previous studies (Herrerondez et al., 2011a; Marín-Benito et al., 2012b). Herna The rapid decrease in azoxystrobin concentration observed in the topsoil may be attributed to the movement, biodegradation, photodegradation and bound residue formation of the fungicide over time (EC, 1998; Bartlett et al., 2002; Adetutu et al., 2008). Likewise, this rapid decrease in the fungicide concentration observed here might be favoured by the rainfall episodes recorded in the first week after its application on the experimental plots (Fig. S1 in Supplementary material). The dissipation of pesticides in the field may be affected by a variety of factors, such as soil texture, varying moisture content, varying soil temperature, and agricultural practices such as ploughing, fertilization and the use of organic amendments. Some of these factors have a positive or negative impact on the interactions between soil microorganisms and pesticides, and here they may affect azoxystrobin dissipation (Adetutu et al., 2008). The dissipation study was carried out simultaneously with a study of the distribution of azoxystrobin through the soil profile. Azoxystrobin concentrations were determined at different soil depths (0e50 cm) at 84, 181 and 378 days after fungicide application on the unamended and SMS-amended plots to check for its possible mobility through the soil. The results obtained are included in Fig. 2, together with the OM contents determined at the different soil depths. The leaching of azoxystrobin to deeper soil layers was observed, but the amount of fungicide residues was much higher in the top two layers (0e10 cm and 10e20 cm) than in deeper layers. Similar results were reported by Ghosh and Singh (2009b) when studying the leaching of azoxystrobin in intact and packed soil columns under laboratory conditions. Spliid et al. (2006) also reported that azoxystrobin was immobilized in the top layer (0e10 cm) of a biobed according to its high estimated Koc value. The amounts of fungicide found through the soil profile were higher on the plots treated with a high dose, and also higher in the soils amended with SMS (50 and 150 t ha1) than in the unamended one (Fig. 2). SMS application increased the soil OM content in terms of depth, and the OM enhanced the persistence of azoxystrobin at different soil depths. In fact, a significant correlation was found between azoxystrobin amounts and the OM content determined in the unamended and amended soil profiles when azoxystrobin was applied at low dose (r ¼ 0.796e0.972, p < 0.001), and at high dose (r ¼ 0.839e0.881, p < 0.001). The residual amounts
Table 2 Dissipation of azoxystrobin applied at low and high doses in unamended and SMS-amended soils under field and laboratory conditions. DT50 values were obtained from the kinetics fitting to a single first-order (SFO), Gustafson and Holden (FOMC) and double first-order in parallel (DFOP) models. Samples
Field: low dose Sa S þ SMS50b S þ SMS150c Field: high dose S S þ SMS50 S þ SMS150 Laboratory: low dose S S þ SMS150 Laboratory: high dose S S þ SMS150 a b c
SFO
FOMC
DFOP
DT50 (days)
r2
c2
1.71 2.24 142
0.941 0.938 0.613
55.1 55.0 42.1
0.52 0.34 0.71
0.994 0.994 0.986
9.9 8.6 9.2
7.92 143 191
0.863 0.672 0.836
50.5 36.7 18.8
0.92 1.41 16.8
0.991 0.978 0.899
10.8 10.7 15.6
113 148
0.985 0.991
7.0 4.5
89.2 148
0.995 0.991
4.1 4.6
163 176
0.979 0.964
6.5 8.0
138 138
0.983 0.971
6.1 7.5
Unamended soil. Soil amended at 50 t ha1 of SMS. Soil amended at 150 t ha1 of SMS.
DT50 (days)
r2
c2
r2
c2
2.14 1.61 2.61
0.974 0.990 0.972
22.1 11.2 14.2
2.12 1.93 46.3
0.994 0.993 0.974
10.2 6.1 8.7
68.8 139
0.992 0.991
5.8 5.2
154 155
0.979 0.965
8.8 13.2
DT50 (days)
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83
of the fungicide in the topsoil amended with 50 t ha1 were 1.45e1.95 times (low dose) and 1.82e2.27 times (high dose) higher than in the unamended soil. When the soil was amended with 150 t ha1 the amount of fungicide found was 1.64e2.30 times (low dose) and 2.02e2.34 times (high dose) higher than that found in the unamended soil at the three sampling times (84, 181 and 378 days). The relationship between the amounts of azoxystrobin found in the amended soil and unamended soil was >1 in most of the soil layers at the three times after fungicide application, and these amounts decreased with time for all the soil treatments. The results obtained show an initial increase of fungicide retention on the surface due to the SMS applied, although the fungicide's mobility at deeper soil layers was also recorded in unamended and amended soils over time. Azoxystrobin has a low-to-moderate mobility in soils (EC, 1998; PMRA, 2007), although its high potential for leaching to groundwater under certain climatic and soil conditions has been reported. Using the Groundwater Ubiquity Score (GUS) assessment method, azoxystrobin is classified as a borderline leacher (GUS index ¼ 2.1). Ghosh and Singh (2009b) determined that preferential flow through the macropores of intact soil columns reduced the retention of azoxystrobin in the soil profile, and may thus increase the risk of groundwater contamination. Azoxystrobin concentrations were detected at deeper soil layers (50 cm) through to the end of the study. They decreased from 0.003e0.061 mg g1 at 84 days to 0.002 and 0.007 mg g1 at 378 days after fungicide application. This may be due to the immobilization or mineralization of the fungicide over time, as no degradation products were identified.
in laboratory versus field experiments; the DT50 values of azoxystrobin applied at low dose increased more than 170 times in unamended soil, and more than 200 times in amended soil. These increases were lower for azoxystrobin applied at high dose (65 and three times, respectively). This finding was also observed for other pesticides (PPDB, 2014), and it was explained by processes affecting azoxystrobin under field conditions that may not occur under laboratory conditions. The persistence of azoxystrobin in soils under laboratory conditions could be affected simply by soil characteristics, such as soil pH (Bending et al., 2006), soil OM content, and the addition of ~ a and Bending, amendments (Ghosh and Singh, 2009a; Sopen 2013), and not by soil management practice or different environmental processes that are mainly responsible for leaching the fungicide through the soil (Bending et al., 2007). The azoxystrobin dissipation kinetics carried out in sterilized soils in the laboratory (Fig. 3) indicated that the fungicide was mainly degraded by the soil microorganisms, which was much slower in sterilized soils. An amount of azoxystrobin > 40% and >50% remained in the sterilized soil after 378 days when it was applied at low or high dose. The degradation of azoxystrobin in sterilized soils may be also due to other abiotic factors, but photodegradation did not occur, as the soils were kept in the dark during incubation. No degradation products were detected during the laboratory experiments. In previous works, the azoxystrobin acid metabolite was the major degradation product formed during the dissipation of this fungicide in soils (Ghosh and Singh, 2009a,b; Manna et al., 2013).
3.2. Azoxystrobin dissipation in laboratory experiments
3.3. Adsorption of azoxystrobin by unamended and amended soils
Under laboratory conditions, the dissipation rate of azoxystrobin was lower than that under field conditions, and the dissipation kinetics fitted the SFO and FOMC models better (Fig. 3, Table 2). Previous reports in the literature fitted FOMC and DFOP or SFO kinetic models to describe the degradation of azoxystrobin in different substrates (compost-based biomixtures and soil) (Karanasios et al., 2010) or in unamended and manure- or compostamended soils (Ghosh and Singh, 2009a), respectively. Fungicide dissipation in laboratory studies was also more rapid in unamended than in SMS-amended soil, and DT50 values increased from 89.2 days to 148 days when azoxystrobin was applied at low dose to the unamended soil and soil þ SMS150 (Table 2), and no differences were seen in DT50 values (138 days) when azoxystrobin was applied at high dose in unamended and amended soils. These values were higher than those reported in other works, which ranged from 7 to 90 days (USEPA, 1997; Karanasios et al., 2010), although azoxystrobin was also reported to be very persistent in soils in other papers with DT50 values higher than those found here (Kalinin et al., 2002; Bending et al., 2006, 2007). The influence of SMS in soils for increasing the DT50 values of the fungicide was greater than for other organic amendments, such as biomixtures or soil amended with these materials, because some of these materials were readily capable of degrading fungicides and recorded a shorter half-life of azoxystrobin compared with the values reported in the soil (Ghosh and Singh, 2009a; Karanasios et al., 2010; Marinozzi et al., 2013). However, the differences in the dissipation kinetics of azoxystrobin applied at high dose were not seen in the unamended soil and soil þ SMS150 under laboratory conditions. The concentrations of fungicide decreased continuously, although amounts between 15 and 20% were detected in the topsoil in the last sampling at 378 days after application in all treatments. A significantly lower azoxystrobin dissipation rate was observed
The adsorption of azoxystrobin by unamended and SMSamended soils was studied as support for explaining the behaviour of fungicide in the field and/or laboratory over time. Soils were taken from experimental control plots (untreated with fungicide) after 2, 84, 181 and 378 days, and adsorption isotherms were obtained under laboratory conditions (Fig. S2 in Supplementary material). These adsorption isotherms were fitted to the Freundlich equation (r2 > 0.97, p < 0.01), and the Kf and nf parameters were determined (Table 3). The nf values were, in general, <1 in the unamended soil and soil þ SMS50 and >1 in soil amended with SMS at a higher rate (150 t ha1), providing L-type and S-type isotherms of the azoxystrobin by the soils. These isotherms correspond to a decrease or an increase in adsorption, respectively, with the increase in fungicide concentration in solution. The results indicate an increase in adsorption through the addition of SMS to the soil, with the Kf values of azoxystrobin in soil þ SMS150 increasing by factors of 34.3, 16.5, 5.9 and 4.6 after 2, 84, 181 and 378 days of SMS application, compared to the unamended soil. The influence of the soil OM content on the adsorption of azoxystrobin and other fungicides has also been et al., 2011). On the other hand, reported previously (Kodesova changes in the adsorption of azoxystrobin over time were found in the amended soils, but not in the unamended soil; these changes in the amended soil indicated Kf values decreased from 28.6 to 5.57 (Soil þ SMS50) and from 234 to 30.1 (Soil þ SMS150) over time. These changes in adsorption coefficients corresponded to changes in the soil OM contents shown in Fig. 2. In fact, a significant correlation was obtained between the Kf values of azoxystrobin for the unamended and amended soils collected over time and the OM content of these soils (r ¼ 0.93, p < 0.001). A decrease has also been reported in the adsorption of other fungicides in SMS-soils over 12 months of soil incubation in the laboratory (Marín-Benito et al.,
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S - LD FOMC
80 60 40 20
80 60 40 20 0
0
100
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100 S - HD FOMC
80
0
Azoxystrobin (% applied)
Azoxystrobin (% applied)
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100
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100
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100
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Azoxystrobin (% applied)
84
100
200 300 Time (days)
400
100 SS - HD
80 60 40 20 0 0
100
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400
Fig. 3. Dissipation of azoxystrobin in unamended, SMS-amended soil (150 t ha1) and sterilized soil after the application of fungicide at low (LD) and high (HD) doses under laboratory conditions. Error bars represent the standard deviation of the mean value (n ¼ 2), they were smaller than the symbol.
2012a). These findings clearly indicate the influence of the organic amendment on azoxystrobin adsorption by soil, and likewise the influence in the persistence and/or dissipation of the fungicide. According to the DT50 values, azoxystrobin increased from 1.4 to 22 times in soil þ SMS150 in relation to unamended soil after the application under field conditions of a low or high dose of the fungicide, respectively. Under laboratory conditions, this effect was noted only when fungicide was applied at low dose. Azoxystrobin adsorption involves rapid dissipation in amended soils, and might limit initial fungicide leaching, although the results reveal the presence of fungicide in the soil profile over time after 378 days (Fig. 2), and this might indicate that the fungicide adsorption prevented its degradation. However the OM content decreased from SMS amended soils over time, which should favour the natural reversibility of compound and its natural degradation.
Table 3 Freundlich adsorption parameters of azoxystrobin by unamended and amended soils in field with SMS at 50 and 150 t ha1 after 2, 84, 181 and 378 days. Adsorption parameters
Time after SMS application (days) T2
T84
T181
T378
6.83 ± 0.56 0.77 ± 0.04 0.99
6.86 ± 0.22 0.79 ± 0.03 0.99
7.33 ± 0.50 0.89 ± 0.03 0.99
6.46 ± 0.30 1.04 ± 0.02 0.98
28.6 ± 1.76 1.06 ± 0.03 0.99
11.2 ± 0.51 0.89 ± 0.01 0.97
8.25 ± 0.37 0.86 ± 0.02 0.98
5.57 ± 0.31 0.77 ± 0.03 0.98
234 ± 23.8 1.32 ± 0.10 0.99
113 ± 0.38 1.48 ± 0.06 0.97
43.1 ± 4.07 1.13 ± 0.05 0.99
30.1 ± 0.97 0.98 ± 0.03 0.99
a
S Kf ± SDd nf ± SD r2 S þ SMS50b Kf ± SD nf ± SD r2 S þ SMS150c Kf ± SD nf ± SD r2 a b c d
Unamended soil. Soil amended at 50 t ha1 of SMS. Soil amended at 150 t ha1 of SMS. SD ¼ standard deviation of replicates (n ¼ 2).
3.4. Dehydrogenase activity of soils in field and laboratory experiments DHA results are included in Fig. 4 for unamended and SMSamended soils both untreated (controls) and treated with azoxystrobin under field conditions and under laboratory controlled conditions over different time periods. Overall, both the sampling time and the soils used, as well as their interactions, had a significant effect on DHA (p < 0.0001). The DHA mean values were higher in amended soils than in unamended ones under field or laboratory conditions (LSD ¼ 74.00 and LSD ¼ 36.58, respectively, p < 0.0004), as reported in the literature (Chen et al., 2001). The higher DHA values in amended soils compared to unamended ones is attributed to the greater OM content provided by SMS which contains available carbon that may stimulate soil microbial community, and to the presence of new active microbial populations introduced with the amendment. Under field conditions, DHA mean values were higher in soils treated with azoxystrobin at two doses than in untreated soils (unamended and amended) (LSD ¼ 27.73, p ¼ 0.0001), revealing the stimulatory effect of the fungicide on soil DHA. In SMSamended soils, the increase in DHA after azoxystrobin application was smaller than in the unamended soil. This effect has also been reported for other fungicides (Chen et al., 2001), with the authors describing the ability fungicides have to both inhibit and stimulate certain groups of microorganisms in soils. Although DHA was higher in most of the soils treated with azoxystrobin, no clear differences were found with fungicide dose as reported for tebucondez et al. (2011a) despite some authors nazole by Herrero-Herna have reported an increase in the toxic effect of pesticides for soil bacteria with the highest doses of compound (Xie et al., 2004). Likewise the DHA mean values increased significantly over time in unamended and amended soils (Fig. 4) under field conditions and DHA values were higher in all the soils at the end of the experiment (p values between 0.0002 and 0.0612) especially in unamended soil.
ndez et al. / Journal of Environmental Management 163 (2015) 78e86 E. Herrero-Herna
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Fig. 4. Soil dehydrogenase activity for unamended and SMS-amended soil (150 t ha1), untreated or treated with a low (LD) or high (HD) dose of azoxystrobin at different sampling times under field (A) and laboratory (B) conditions. Bars represent the standard deviation of the mean (n ¼ 3). Treatments with the same letter are not significantly different from each other.
Under laboratory conditions DHA values were higher at the beginning of the experiment in unamended soils, untreated or treated with azoxystrobin. A similar effect was observed by Chen et al. (2001), being explained by the initial mixing and wetting of soils prior to the stabilization of microbial activity. A significant DHA decrease was subsequently recorded over time in all the soils (treated and untreated) (p values between 0.001 and 0.003). In SMS-amended soils, the decrease in DHA after azoxystrobin application was smaller than in the unamended soil. A DHA ~ a and decrease in soils treated with biochar was observed by Sopen Bending (2013) following azoxystrobin application. These authors believed that a decrease in DHA induced by azoxystrobin in treated soils might reflect an impact on fungal biomass, with the direct inhibition of fungi, or indirect changes reflecting altered competitive interactions between microbes in the presence of the fungicide (Bending et al., 2007). Furthermore, Karanasios et al. (2010) observed a decrease in microbial respiration over time in the degradation assays of pesticides in biomixtures in the laboratory. Changes may occur in the physico-chemical properties of soil, with the loss of soil active microbial biomass at the end of the fungicide incubation time in containers under laboratory controlled conditions. Therefore, differences in the management of residual soil amounts in both experimental procedures may explain the much higher persistence of fungicide under laboratory rather than field conditions.
4. Conclusions The results obtained in this work revealed that azoxystrobin dissipation was faster in unamended soils than in amended ones in the two environmental scenarios. However a significantly lower azoxystrobin dissipation rate was observed in laboratory versus field experiments and it was explained by processes affecting azoxystrobin that may not occur under laboratory conditions. The adsorption and mobility of fungicide in the soil were affected by SMS application, enhancing its rapid dissipation under field conditions and increasing its persistence in the amended soil profile. Likewise, the increase in DHA over time under field conditions is consistent with the influence of microbial activity, and also supports the greater dissipation of azoxystrobin under these conditions than under laboratory conditions. Our research highlights the importance of examining the dissipation of azoxystrobin under field situations in order to obtain a realistic assessment of dissipation rates in agricultural practices involving the joint use of SMS and azoxystrobin. This would help to better predict its behaviour in the soil environment, with the purpose being to avoid the risk of water being contaminated by this compound. A long-term assessment of the effect of SMS amendment has on the fate of azoxystrobin in vineyard soils is required to take preventive measures against the groundwater contamination derived from its intensive use in agriculture.
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