Research Article Received: 13 March 2014
Revised: 13 June 2014
Accepted article published: 5 August 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/ps.3876
Establishment of multiple pesticide biodegradation capacities from pesticide-primed materials in on-farm biopurification system microcosms treating complex pesticide-contaminated wastewater Kristel Sniegowski and Dirk Springael* Abstract BACKGROUND: On-farm biopurification systems (BPSs) treat pesticide-containing wastewater at farms by biodegradation and sorption processes. The inclusion of pesticide-primed material carrying a pesticide-degrading microbial community is beneficial for improving biodegradation, but no data exist for treating wastewater containing multiple pesticides, as often occurs on farms. In a microcosm set-up, an examination was carried out to determine whether multiple pesticide degradation activities could be simultaneously established in the matrix of a BPS by the simultaneous inclusion of different, appropriate pesticide-primed materials. The microcosms were fed with a mixture of pesticides including the fungicide metalaxyl and the herbicides bentazon, isoproturon, linuron and metamitron, and pesticide-degrading activities were monitored over time. RESULTS: The strategy immediately provided the microcosms with a multiple pesticide degradation/mineralisation capacity, which improved during feeding of the pesticide mixture. Not only did the degradation of the parent compound improve but also that of the produced metabolites and compound mineralisation. The time to achieve maximum degradation/mineralisation capacity depended on the pesticide degradation capacity of the pesticide-primed materials. CONCLUSIONS: The data obtained show that the addition of pesticide-primed materials into the matrix of a BPS as an approach to improve biodegradation can be extended to the treatment of pesticide mixtures. © 2014 Society of Chemical Industry Supporting information may be found in the online version of this article. Keywords: on-farm biopurification system; pesticide mixtures; biodegradation; pesticide primed materials; bioaugmentation
1
INTRODUCTION
Direct pesticide losses during the handling of pesticides in farmyards contribute largely to contamination of surface water.1 Treatment of pesticide-contaminated wastewater by means of on-farm biopurification systems (BPSs) can significantly reduce pesticide losses.1 Different variants of BPSs exist, such as the biobed, the biofilter and Phytobac®, but all of them operate as a biofilter that contains a matrix of organic waste materials, designated as biomix, in which removal of pesticides in the wastewater occurs by sorption and biodegradation.1,2 Previously, bioaugmentation of the biomix with pesticide-primed materials improved the pesticide degradation capacity of lab-scale BPSs for over 1 year, even while enduring successive stress conditions.3 Pesticide-primed materials are materials that have been treated on a long-term basis with specific pesticides, resulting in the proliferation of microbial populations that have adapted to degrade and often metabolise the compound. Such materials include long-term pesticide-treated soils or biomix materials from BPSs in operation.4,5 Target improvements include the introduction Pest Manag Sci (2014)
of missing catabolic phenotypes, the acceleration of pesticide degradation activities and the avoidance of production of toxic metabolites.5,6 The method has been suggested as an alternative to bioaugmentation with axenic pesticide-mineralising pure cultures, and has been successfully demonstrated using linuron as a model pesticide and a linuron-treated soil as a model pesticide-primed soil.5 However, in reality, different types of pesticide are being used on farms, often simultaneously, and as such will be present as mixtures in the contaminated water. Although several reports suggest the removal of multiple pesticide compounds in BPSs,7,8 no data yet exist on the feasibility of bioaugmentation with pesticide-primed materials to improve multiple pesticide degradation in BPSs. Such a strategy would
∗
Correspondence to: Dirk Springael, Division of Soil and Water Management, KU Leuven, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium. E-mail: [email protected] Division of Soil and Water Management, KU Leuven, Heverlee, Belgium
www.soci.org
© 2014 Society of Chemical Industry
www.soci.org involve adding a mixture of different pesticide-primed materials with catabolic activity towards different pesticides. In the present study, an investigation was carried out to determine whether inoculation of a BPS with pesticide-primed materials targeting simultaneously the degradation of different pesticide compounds would result in the successful transfer and establishment of the respective pesticide degradation capacities in the biomix of the BPS. Use was made of BPS microcosms (BMs) containing a simulated biomix matrix, as described previously.5 The BMs were irrigated with artificially contaminated water containing a mixture of the fungicide metalaxyl and the herbicides bentazon, isoproturon, linuron and metamitron or with water without pesticides. The material used for bioaugmentation was a mix of pesticide-primed materials that had been treated for several years with the pesticides applied in the mixture. As a control set-up, BMs were included that instead of the primed material mix contained a soil without history of pesticide contamination. The pesticide degradation and mineralisation capacities of the BMs were monitored, and related to the pesticide-degrading/mineralisation capacities of the pesticide-primed materials used for bioaugmentation.
2
EXPERIMENTAL METHODS
2.1 Pesticides used Linuron [3-(3,4-dichlorophenyl)-1-methoxy-1-methyl urea] (purity 99.5%) and isoproturon [(3-(4-isopropylphenyl)-1,1-dimethyl urea) (purity 99.0%) were purchased from Sigma-Aldrich (Belgium). Metalaxyl [methyl-N-(2,6-dimethylphenyl)-N-(2-methoxyacetyl)-D, L-alaninate] (purity 95.5%) was obtained from Syngenta (Switzerland), and bentazon (3-isopropyl-2,1,3-benzothiadiazine-4-on-2,2dioxide) (purity 98.8%) from BASF (Belgium). Metamitron [4-amino -3-methyl-6-phenyl-1,2,4-triazin-5(4H)-one] (purity 95%) was donated by Agrichem B.V. (The Netherlands). [Phenyl-U-14 C] linuron (16.93 mCi mmol−1 ), [phenyl-U-14 C] isoproturon (12.02 mCi mmol−1 ), [carbonyl-14 C] bentazon (46.70 mCi mmol−1 ) and [phenyl -U-14 C] metamitron (14.11 mCi mmol−1 ) were obtained from Izotop (Hungary). 2.2 BPS microcosm set-ups BPS microcosms (BMs) were set up as described.5 The biomix contained 25 vol% cut straw (±0.5 cm2 ), 35 vol% peat, 25 vol% coconut husk chips (±0.5 cm2 ), 5 vol% cow manure (crushed dry pellets) and 10 vol% of a pesticide-primed material mix or a soil without pesticide application history. An overview of the used soils/pesticide-primed materials and their origin is given in Table 1. The soil without pesticide treatment history, designated as soil C, was collected from a construction site without any known history of pesticide treatment, as described.5 The pesticide-primed material mix consisted of soil L, soil I + M, soil B and biomix MX. Soil L originated from a potato field in Halen, Belgium, and has been described previously.5 The field had been treated yearly with linuron until the time of sampling. Soil I + M originated from an agricultural field treated with both isoproturon and metamitron. Metamitron had been used until the time of sampling, while the last isoproturon treatment was applied 4 years before sampling. Soil B originated from an agricultural field that had been treated with bentazon for several years until the time of sampling. Soils L, I + M and B were sampled from the upper 20 cm of the field 1 month before initiating the experiment. The soils were stored at 4 ∘ C in the dark before use. Biomix MX was sampled from an
wileyonlinelibrary.com/journal/ps
K Sniegowski, D Springael
Table 1. Overview of the origins of the materials that were included in the biomix of BMs in this study Designation Soil C Soil L Soil I + M Soil B Biomix MX
Origin St-Truiden (construction site) Halen (potato field) Tielt-Winge (cereals, beet field) Leefdaal (corn field) Chastre (BPS)
Pesticide(s) applied No pesticides Linuron Isoproturon, metamitron Bentazon Metalaxyl
on-farm BPS that had processed pesticide-contaminated water containing metalaxyl for 4 years until the time of sampling and was stored at 4 ∘ C before use. The pesticide-primed material mix was prepared by mixing 2.5 vol% of each pesticide-primed material. The individual pesticide-primed materials as well as the mixture were tested for their ability to degrade or mineralise the corresponding pesticide(s) during a maximum incubation period of 60 days. Table 2 shows an overview of the BM experimental set-ups. Each set-up included three replicate BMs. The BMs were incubated at 20 ∘ C and supplied for 10 weeks with either 19.1 L m−3 day−1 of sterile tap water (microcosm set-ups C− and S− ) or sterile tap water containing a mixture of the five pesticides at concentrations of 50 mg L−1 each (microcosm set-ups C+ and S+ ). Weekly, on Monday, Wednesday and Friday, the solution (7 mL on each of the indicated days) was manually spread dropwise over the surface of the biomix using a 10 mL pipette. Samples for assessing the pesticide degradation capacity of the BMs in batch assays were taken from the upper layer of the biomix of the BMs at weeks 0, 2, 4, 6, 8 and 10. For each set-up, each of the three replicates were sampled. Pesticide mineralisation assays were performed with samples taken at weeks 0 and 10. 2.3 Batch pesticide degradation assay Batch pesticide degradation assays were performed in sterile 100 mL Erlenmeyers containing 200 mg (wet weight) of either soil C, pesticide-primed material mix or biomix taken from the mixed upper layer (1 cm) of the BMs and 50 mL of MMO medium (MMN supplied with NH4 + , pH 5.8)9 containing a mixture of linuron, metamitron, isoproturon, metalaxyl and bentazon at a concentration of 20 mg L−1 each. In the case of the BM set-ups, each replicate BM was assessed once at each time point. In addition, the pesticide degradation capacity of each of the pesticide-primed materials was tested individually in identical batch degradation experiments, except that the medium contained only one pesticide. Each pesticide-primed material was tested for degradation of each of the pesticides that had been applied on that material. Abiotic removal was assessed a single time in a batch experiment with samples taken from set-up S− supplied with MMO adjusted to pH 2 with HCl (1 mM) to inhibit biotic activity, and was found to be negligible. For each series, a control without microcosm material was included to act as an additional abiotic control. The Erlenmeyers were incubated on a horizontal shaker (125 rpm) at 20 ∘ C in the dark. Frequently, 700 𝜇L samples were taken from the aqueous phase and, after centrifugation at 12 000 rpm for 5 min, analysed by reverse-phase HPLC (LaChrom; Merck Hitachi). The HPLC system was equipped with a Platinum EPS C18 (7.5 × 4.6 mm) guard column, a Platinum EPS C18 150 × 4.6 mm end-capped column packed with silica beads (3 μm) and a UV-vis detector set at 208 nm. The compounds were separated by gradient elution with
© 2014 Society of Chemical Industry
Pest Manag Sci (2014)
Mixed pesticide pollution treated in a biopurification system
www.soci.org
Table 2. Overview of the BM set-ups examined in this study Relevant added material Microcosm set-up C− C+ S− S+
Non-pesticide-primed soil (soil C) + + − −
Treatment
Pesticide-primed material mixa
Pesticide-contaminated waterb
− − + +
− + − +
a Mix of soil L, soil I + M, soil B and biomix MX (2.5 vol% each) b Sterile tap water with linuron, isoproturon, metamitron, bentazon, and metalaxyl (50 mg L−1
CH3 CN/H2 O (0.005% H3 PO4 ) (0 min: 78/22; 10.5 min: 45/55; 13 min: 60/40; 15 min: 78/22) and a flow rate of 1.0 mL min−1 . The ratio of the concentration in the soil slurry (C) and the concentration in the medium without soil inoculum (C 0 ) were used to present the data graphically as a function of time. When unidentified pesticide degradation products were observed, the corresponding peak surface (C) was expressed with reference to the maximum peak surface (C max ) recorded in the HPLC chromatograms. The lag phase, defined as the period between initiation of the degradation assay and the start of degradation, was calculated as the intersection of the X-axis with the linear regression line between two successive points of the degradation curve where the amount of linuron removed was the largest. The slope of the linear regression line of maximum degradation determined the maximum degradation rate, as described.10 Averages of the lag times and maximum degradation rates of triplicate BM samples were analysed by ANOVA for significance analysis (P < 0.05). 2.4 Pesticide mineralisation assay 14 C-Pesticide mineralisation assays were performed as described.5 From each replicate BM, four samples were taken in order to assess the capacity for mineralisation of either linuron, isoproturon, metamitron or bentazon. Mineralisation of metalaxyl was not assessed because 14 C-metalaxyl was not commercially available. Microcosm biomix samples (200 mg wet weight) were supplied with 5 mL of MMO containing a mixture of the target pesticide as 14 C-labelled and unlabelled compounds, as well as each of the other four pesticides as unlabelled compounds. Unlabelled pesticides were added at concentrations of 20 mg L−1 . For each target compound, final radioactivity added to the assay was 238 Bq mL−1 , which corresponded to 0.092 mg L−1 (2.58 MBq mg−1 ) of 14 C-metamitron, 0.11 mg L−1 (2.16 MBq mg−1 ) of 14 C-isoproturon, 0.095 mg L−1 (2.52 MBq mg−1 ) of 14 C-linuron or 0.33 mg L−1 (7.13 MBq mg−1 ) of 14 C-bentazon. The pesticide mineralisation capacity of the pesticide-primed material mixture and soil C was tested identically for each of the pesticides, while that of the individual pesticide-primed materials was assessed only for the pesticide with which they had been treated. The vials were incubated at 20 ∘ C on a rotary shaker at 150 rpm, and the amount 14 CO2 produced was measured as described by Sniegowski5 and expressed as the percentage of the initial amount of 14 C-pesticide added. Cumulative mineralisation curves were established on the basis of a control without inoculation of the soil sample. The lag time and mineralisation rate were calculated as for the degradation experiments. Averages of the lag times and mineralisation rates of triplicate BM samples were analysed by ANOVA for significance analysis (P < 0.05). Pest Manag Sci (2014)
Non-contaminated water
3
+ − + −
each).
RESULTS
3.1 Pesticide degradation capacity of the pesticide-primed materials and mixture The pesticide-primed materials used for bioaugmenation were individually tested for their ability to degrade or mineralise the pesticide(s) with which they had been treated, during an incubation period of 60 days maximum. Soil C and the pesticide material mix were assessed for degradation/mineralisation of all tested pesticides. When degradation and/or mineralisation occurred, lag times were observed that differed significantly between the different pesticides and materials (see supporting information Fig. S1). This lag time was previously related to the size of the pesticide-degrading population and hence to the pesticide degrading capacity.11 An overview of the pesticide degradation/mineralisation potential of the tested materials, expressed as lag time, is shown in Table 3. Most of the individual materials showed the expected degradation/mineralisation potential, i.e. soil L showed degradation and mineralisation of linuron, soil I + M showed degradation and mineralisation of metamitron and biomix MX showed degradation of metalaxyl. However,
Table 3. Overview of the pesticide degradation capacity of pesticide-primed materials, expressed as lag time observed in the batch degradation and mineralisation assays
Material Soil C
Soil L Soil I + M Soil B Biomix MX Pesticide-primed material mix
Pesticide tested
Degradation Mineralisation lag time (days) lag time (days)
Linuron Metamitron Isoproturon Bentazon Metalaxyl Linuron Isoproturon Metamitron Bentazon Metalaxyl Linuron
27.0(±3.6) 37.3(±7.4)a >60 >60 >60 18.8(±1.7) >60 3.5(±4.9)a >60 9.3(±1.7)a 38.0(±10.6)
>60 >60 >60 >60 NDb 16.9(±1.6) >60 5.5(±4.2) >60 NDb 27.6(±3.1)
Metamitron Isoproturon Bentazon Metalaxyl
33.9(±12.8)a >60 >60 9.3(±1.7)
25.4(±3.7) >60 >60 NDb
a A metabolite accumulated during the degradation assays. b ND: not done.
© 2014 Society of Chemical Industry
wileyonlinelibrary.com/journal/ps
www.soci.org 1.2
(A) % 14C-linuron mineralised
C/C0 linuron
0.8 0.6 0.4 0.2
0
10
20
30 40 Time (days)
50
0.6 0.4 0.2 0
20 10 0
10
20
30 40 Time (days)
50
C/Cmaxmetamitron metbolite
0.8 0.6 0.4 0.2
10
20
30 40 Time (days)
Set-up C+
50
60
Set-up S+
10
20
30 40 50 Time (days)
60
60
80
60
70
(D)
50 40 30 20 10
0
10
20
30 40 Time (days)
50
(F)
1.2
1.0
0
0
60
0
60
(E)
1.2
C/C0 metalaxyl
30
% 14C-metamitron mineralised
C/C0 metamitron
0.8
0.0
40
60
1.0
0.0
50
(C)
1.2
(B)
60
1.0
0.0
K Sniegowski, D Springael
1.0 0.8 0.6 0.4 0.2 0.0
0
10
20
30 40 Time (days)
50
60
Pesticide-primed material mix
Figure 1. Kinetics of linuron degradation (A), linuron mineralisation (B), metamitron degradation (C), metamitron mineralisation (D) and metalaxyl degradation (E), recorded with samples taken from BMs of set-up C+ , set-up S+ and the pesticide-primed material mix. The values are averages of three replicates with indicated standard deviations. Similar observations were made for set-ups C− and S− in A, C and E, the Y-axis presents the fraction of the pesticide concentration in reference to the concentration in the abiotic control (C0 ). In B, D and F, the Y-axis presents the cumulative amount of 14 C in the produced 14 CO2 over the amount of 14 C initially added as 14 C-pesticide.
isoproturon was not degraded/mineralised in soil I + M, and bentazon underwent neither process in soil B. In soil I + M, apparently only the phenyl group of metamitron was degraded, as the 14 C-labelled metamitron used was labelled in the phenyl ring and 14 CO2 was produced in the mineralisation assay, while a metabolite appeared in the degradation assay. A metabolite also appeared during degradation of metalaxyl (see supporting information Fig. S1, panel F). In both cases, the metabolite accumulated over time and was not degraded within the 60 days of monitoring. Unexpectedly, the non-primed soil C also degraded (but did not mineralise) linuron and metamitron, but with lag times (27.0 ± 3.6 days and 37.3 ± 7.4 days respectively) that were significantly larger (P = 0.017 and 0.002 respectively), and with a significantly lower degradation rate (zero order) than those observed with the pesticide-primed soil L and soil I + M.
wileyonlinelibrary.com/journal/ps
As with soil I + M, a metabolite accumulated from metamitron in soil C. Soil C did not degrade/mineralise metalaxyl, isoproturon or bentazon. Results for the pesticide-primed material mix are given in Fig. 1 and in supporting information Fig. S1. Degradation and mineralisation of linuron and metamitron were observed as well as degradation of metalaxyl, showing that the pesticide degradation capacities of the individual soils were retained after they were mixed, although differences in lag time were noted between the materials when individually tested and tested as a mixture, especially for metamitron. In the case of metamitron and metalaxyl, a metabolite accumulated (data not shown). As with the individual materials, no degradation and/or mineralisation of isoproturon and bentazon were observed in the applied time period with the mixture.
© 2014 Society of Chemical Industry
Pest Manag Sci (2014)
Mixed pesticide pollution treated in a biopurification system
www.soci.org
1.2
(A)
1.0 0.8 0.6 0.4 0.2 0.0
0
detected, but at a very low rate (0.3 ± 0.04% day−1 ) (Fig. 1D). As in set-ups S− and S+ , degradation of metamitron resulted in the production of an unidentified metabolite, but in contrast to set-ups S− and S+ , this compound was not transient and accumulated (data not shown). In addition, as degradation of metamitron was not completed after 60 days, the amount of produced metabolite remained limited. In set-ups C− and C+ , no initial degradation and/or mineralisation capacity for isoproturon, metalaxyl and bentazon was observed (Fig. 1D and data not shown). 3.3 Effect of pesticide supply on the pesticide degradation/mineralisation capacity of the BMs As illustrated in Fig. 4A, set-up S+ showed a gradually decreasing lag time for linuron degradation from 31 ± 5.9 days at week 0 to 3.3 ± 0.7 days at week 10, indicating an improvement in the linuron degradation capacity as a response to the mixed pesticide supply. The dynamics of linuron mineralisation capacity largely followed that of the degradation capacity, with an average lag time of 2.0 ± 0.2 days at week 10 (Fig. 5A). Also, metamitron degradation and mineralisation capacity improved with time in set-up S+ , as lag times decreased from 31.6 ± 19.8 days at week 0 to 1.91 ± 0.43 days at week 10 for metamitron degradation, and from 9.4 ± 0.76 days at week 0 to 1.1 ± 0.28 days at week 10 for metamitron mineralisation. The metalaxyl degradation capacity in set-up S+ also improved, as shown by the lag times decreasing from 10.5 ± 0.69 days at week 0 to 0.51 ± 0.59 days at week 10 (Fig. 4E). For both linuron and metamitron degradation and mineralisation, the required period of pesticide supply to shorten the lag time to its minimum differed between the replicates and apparently depended on the initial lag time (as recorded at week 0). At week 10, however, standard deviations in lag times and rates were strongly reduced. Interestingly, in addition to metamitron, for metalaxyl also an improvement in the capacity to degrade respective observed metabolites was recorded (Figs 2A and 3A). Moreover, for metalaxyl, until week 8, degradation of the metabolite resulted in the accumulation of a second degradation product, without further degradation (Fig. 3C). However, at week 10, the second metabolite also started to degrade (Fig. 3C). Although set-up S− did not receive pesticides and no enhanced degradation capacity was observed with the degradation assays, some improvement in the linuron mineralisation capacity occurred, as indicated by the reduced lag time (P = 0.043) and increased mineralisation rate (P = 0.44) at week 10 compared with week 0 (Fig. 5A). C/Cmax metamitron metabolite
C/Cmax metamitron metabolite
3.2 Initial pesticide degradation and mineralisation capacity in BMs Before initiating pesticide application, for both set-ups S− and S+ , degradation and mineralisation potentials for linuron and for metamitron were recorded (data not shown and Fig. 1). Linuron and metamitron were degraded and mineralised with large differences in lag times between replicates. Degradation and mineralisation of linuron showed a lag time of 25–35 days (Fig. 1A) and 40–48 days (Fig. 1B) respectively. Metamitron started to degrade and mineralise after lag times of 9–45 days (Fig. 1C) and 7.5–14 days (Fig. 1D) respectively. For linuron, the observed degradation kinetics recorded with the S− and S+ microcosm samples was similar to the kinetics observed with the pesticide-primed material mix, indicating that the additional substrata in the microcosm biomix had no direct effect on the degradation capacity of the primed material mix. However, mixing the primed material with the other biomix substrata appeared to have a negative effect on the linuron mineralisation capacity, as the lag times were longer and the mineralisation rates lower for the microcosm samples than those observed for the primed material mix (Figs 1A and B). This was not the case for metamitron degradation and mineralisation, where the lag time for set-ups S− and S+ was not significantly different from that recorded with the primed material mix (P = 0.14 and 0.09 respectively) (Figs 1C and D). As observed with soil I + M and the primed material mix, an unidentified metabolite appeared during degradation of metamitron in the degradation assay (see Figs 2A and B, week 0). In addition to metamitron and linuron, set-ups S− and S+ directly showed the capacity to degrade metalaxyl (Fig. 1E). Metalaxyl degradation occurred after a relative short lag time of 9.8 days, with data showing small standard deviations, and followed degradation kinetics similar to that observed for the primed material mix. As observed with the initial pesticide-primed material, degradation of metalaxyl resulted in the accumulation of a metabolite (see Figs 3A and B, week 0). Neither set-up S− nor set-up S+ showed the ability to degrade or mineralise isoproturon or bentazon (data not shown). Samples taken from BMs inoculated with soil C (set-ups C− and + C ) also degraded linuron and metamitron. No clear lag time was observed, but degradation occurred at a much lower rate than those recorded for set-ups S− and S+ and clearly showed zero-order kinetics (Figs 1A and C). Moreover, in contrast to set-ups S− and S+ , set-ups C− and C+ showed no linuron mineralisation capacity (Fig. 1B). Some metamitron mineralisation activity was
10
20
30
40
50
60
70
1.2 (B) 1.0 0.8 0.6 0.4 0.2 0.0
0
10
20
Time (days) week 0
week 4
week 6
30 40 50 Time (days)
week 8
60
70
week 10
Figure 2. Formation and degradation of the metamitron metabolite, observed during degradation of metamitron for samples taken from BMs of set-up S+ (A) and set-up S− (B) after different periods of feeding with the pesticide cocktail. Because of the large variability between BM replicates, and for the sake of clarity of the figure, only data obtained for one replicate are presented. The arrow indicates the evolution of the lag time associated with degradation of the metabolite during the incubation period. The Y-axis presents the fraction of the peak surface associated with the metabolite (C) in reference to the maximum peak surface (C max ) observed in the HPLC chromatograms obtained with the samples from the batch degradation assays.
Pest Manag Sci (2014)
© 2014 Society of Chemical Industry
wileyonlinelibrary.com/journal/ps
1.2
K Sniegowski, D Springael
C/Cmax metalaxyl metabolite 1
C/Cmax metalaxyl metabolite 1
www.soci.org
A
1.0 0.8 0.6 0.4 0.2 0.0 10
C/Cmax metalaxyl metabolite 2
0
20 30 40 Time (days) 1.2
50
60
1.2
B
1.0 0.8 0.6 0.4 0.2 0.0
0
10
20
30
40
50
60
Time (days)
C
1.0 0.8 0.6 0.4
week 0 week 4 week 6 week 10
0.2 0.0 0
10
20
30
40
50
60
Time (days) Figure 3. Formation and degradation of the first (A, B) and second (C) metabolites of metalaxyl degradation, recorded with samples taken from BMs of set-up S+ (A, C) and set-up S− (B) after different periods of pesticide cocktail supply (set-up S+ ) or non-contaminated water (set-up S− ). Because of the large variability between BM replicates, and for the sake of clarity of the figure, only data from one replicate are presented. The arrow indicates the evolution of the lag time during the incubation period. The Y-axis presents the fraction of the peak surface associated with the metabolite (C) in reference to the maximum peak surface (C max ) observed in the HPLC chromatograms obtained with the samples from the batch degradation assays.
Nevertheless, lag times were up to 6 times lower with samples of the S+ set-up compared with those of the S− set-up. No significant change in the lag time of metamitron degradation (P = 0.782, data not shown) or metamitron mineralisation (P = 0.189) (Fig. 5C) was observed for set-up S− . Also, the capacity to degrade the metamitron metabolite (Fig. 2B) did not significantly improve over time for set-up S− . This indicates that metamitron degradation/mineralisation improvement in set-up S+ was due to the feeding of metamitron. For metalaxyl, the degradation lag time recorded for set-up S− decreased from 9.8 to 5.8 days, which is a much lower extent compared with set-up S+ (data not shown). Moreover, samples from set-up S− never showed degradation of the first metalaxyl metabolite, even after 10 weeks of incubation (Fig. 3B). In the course of the incubation period, neither set-up S+ nor set-up S− developed the capacity to degrade/mineralise isoproturon or bentazon (data not shown). The capacity to degrade and mineralise linuron also improved in set-up C+ as a response to the mixed pesticide supply (Figs 4B and 5B). However, the capacity to degrade and mineralise linuron at day 10 was less for set-up C+ compared with set-up S+ . In the degradation assays this was only indicated by a lower degradation rate, which was 5.6 ± 0.024% day−1 for set-up C+ compared with 16.4 ± 0.021% day−1 for set-up S+ at week 10 (P = 0.0060). Owing to the linear degradation rate, the calculated lag time did not change significantly. On the other hand, in the mineralisation assays, reduced mineralisation capacity was indicated in particular by a significantly longer lag time, i.e. 6.8 ± 0.1 days for
wileyonlinelibrary.com/journal/ps
set-up C+ compared with 2.0 ± 0.2 days for set-up S+ (P = 0.011). Also, the capacity to degrade metamitron in BMs of set-up C+ improved during the period of pesticide supply, with apparent zero-order kinetics changing into first-order degradation kinetics from week 0, indicating that a population with metamitron metabolic abilities started to proliferate (Fig. 4D). The recorded average lag time and metamitron degradation rate for set-up C+ at week 10 were 25.8 ± 15.6 days and 7.2 ± 0.03% day−1 respectively, which were still significantly longer (P = 0.025) and lower (P = 0.0027), respectively, than those recorded for set-up S+ at week 10. Also, metamitron mineralisation capacity improved during the pesticide cocktail supply for C+ (Fig. 5D). However, the lag times recorded for set-up C+ were significantly longer (6.3 ± 3.2 days) compared with those recorded for set-up S+ (1.1 ± 0.3 days) (P = 0.049). Furthermore, as the capacity to degrade metamitron improved over time in set-up C+ , so did the capacity to degrade the observed metabolite (data not shown), and set-up C+ samples taken at week 10 degraded the metabolite at a rate similar to samples of set-up S− (data not shown). No change in linuron or metamitron degradation (data not shown) and metamitron mineralisation kinetics (Fig. 5D) nor in degradation of the metamitron metabolite (data not shown) were observed for set-up C− samples during the 10 weeks of water supply. Moreover, no change in the metalaxyl degradation kinetics was observed with samples taken from set-ups C− and C+ (data not shown). In addition, as in set-ups S+ and S− , no apparent proliferation of isoproturon and bentazon microorganisms occurred in C+ and C− during the 10 weeks
© 2014 Society of Chemical Industry
Pest Manag Sci (2014)
Mixed pesticide pollution treated in a biopurification system 1.2
www.soci.org
A
1.0
0.8
C/C0 linuron
C/C0 linuron
1.0
0.6 0.4 0.2 0.0
B
1.2
0.8 0.6 0.4 0.2
0
10
20
30
40
50
0.0
60
0
10
20 30 40 Time (days)
Time (days) 1.2
1.2
C C/C0 metamitron
C/C0 metamitron
60
D
1.0
1.0 0.8 0.6 0.4
0.8 0.6 0.4 0.2
0.2 0.0
50
0
10
20
30
40
0.0 30
50
40
40
50
Time (days)
Time (days) E
1.2
C/C0 metalaxyl
1.0 0.8 0.6 0.4 0.2 0.0
Week 0
0
2
4
6 8 10 Time (days)
Week 4
12
14
Week 6
16
Week 8
Week 10
Figure 4. Pesticide degradation kinetics recorded with samples taken from BMs of set-up S+ for linuron (A), metamitron (C) and metalaxyl (E) and set-up C+ for linuron (B) and metamitron (D), taken at different weeks after starting the supply of the pesticide cocktail. The data for linuron and metalaxyl are average values of triplicate BM samples with indicated standard deviations. Because of the large variability between BM replicates, and for the sake of clarity of the figure, for metamitron the data of only one BM are given. At week 8, no data were obtained for set-up C+ at week 8. The arrow indicates the evolution of the lag time over the incubation period. The Y-axis presents the fraction of the pesticide concentration in reference to the concentration in the abiotic control (C 0 ).
of feeding with the pesticide cocktail or non-contaminated water, respectively, as no changes in lag times of isoproturon and bentazon degradation and mineralisation occurred (data not shown).
4
DISCUSSION
Previous bioaugmentation experiments with a single pesticide-primed soil showed promising results for enhancing the degradation and mineralisation capacity of a pesticide in BPSs.5 As, in farmyards, different pesticides are used, and often simultaneously, the present study attempted to coenhance the degradation capacity of a BPS for different pesticides simultaneously. Pest Manag Sci (2014)
Therefore, the pesticide degradation/mineralisation capacities of lab-scale BPS microcosms inoculated with four appropriate pesticide-primed materials were compared with those of microcosms inoculated with a soil without a history of pesticide treatment. Prior to inoculation, a pesticide-primed material mix with samples originating from appropriately treated agricultural fields (isoproturon, metamitron, linuron and bentazon) or BPS (metalaxyl) was prepared. Degradation/mineralisation assays confirmed the corresponding degradation capacity of the pesticide-primed materials for linuron, metamitron and metalaxyl, but not for isoproturon and bentazon. Apparently, the soils treated with isoproturon and bentazon, in spite of being long-term
© 2014 Society of Chemical Industry
wileyonlinelibrary.com/journal/ps
70
A
70 60 50 40 30 20 10 0
K Sniegowski, D Springael
% 14C-linuron mineralised
% 14C-linuron mineralised
www.soci.org
0
10
20
30
40
50
60 50 40 30 20 10 0
60
B
0
10
20 30 40 Time (days)
50
60
10
20
50
60
60
% 14C-metamitron mineralised
% 14C-metamitron mineralised
Time (days) C
50 40 30 20 10 0
0
10
20
30
40
50
60
60
D
50 40 30 20 10 0
0
30
40
Time (days)
Time (days)
Set-up, S-, week 0 Set-up, S+, week 0 Set-up, S-, week 10 Set-up, S+, week 10
Set-up, C-, week 0 Set-up, C+, week 0 Set-up, C-, week 10 Set-up, C+, week 10
Figure 5. Pesticide mineralisation kinetics recorded with samples taken from BMs of set-ups S− and S+ for linuron (A) and metamitron (C) and from BMs of set-ups C− and C+ for linuron (B) and metamitron (D). The samples were taken at week 0 and at 10 weeks after starting the supply of the pesticide mixture. The data are average values of triplicate BM samples with indicated standard deviations. The Y-axis presents the cumulative amount of 14 C produced as CO2 over the total amount of 14 C initially added as 14 C-pesticide. The metamitron mineralisation kinetics of C− and C+ at week 0 was very similar, and corresponding symbols are therefore difficult to distinguish from each other in the graph.
treated, had not developed corresponding pesticide degradation capacities. The last treatment of soil I + M was also done 4 years before sampling of the soil, and the long-term lack of treatment might have affected isoproturon-degrading populations that could have developed during the field treatment. As such, supplementing the biomix with the pesticide-primed material mix was expected to result directly in the establishment in the microcosms of degradation/mineralisation capacity for linuron, metamitron and metalaxyl but not for isoproturon and bentazon. This was indeed the case, confirming the success of this bioaugmentation strategy and extending its feasibility towards BPS-based treatment of multiple-pesticide-contaminated wastewaters. Moreover, as only small volumes (i.e. 2.5 vol%) of inoculum material was used for each pesticide-primed matrix material, the present study confirms that such small volumes harbour sufficient capacity for successful application. Previously, a soil vol% of up to 0.5 was sufficient for successful establishment of linuron-degrading capacity in a BPS biomix matrix.12 Ten weeks of supplying the five pesticides simultaneously further enhanced the linuron, metamitron and metalaxyl biodegradation capacities of the microcosms inoculated with the pesticide-primed material mix, showing that the introduced pesticide degradation capacity could easily proliferate in the biomix when present in the added material. This was consistent
wileyonlinelibrary.com/journal/ps
with previous observations with linuron-treated BM experimental set-ups bioaugmented with only linuron-primed soil5 and with metalaxyl-treated BPS mesocosm systems bioaugmented with metalaxyl-primed BPS material.4 The observed decrease in lag time and hence capacity can be related to a proliferation of the corresponding pesticide degraders. The observation that the increase in size of the pesticide-degrading/mineralising capacity was greater in pesticide-treated BMs compared with BMs without pesticide supply suggests that proliferation was at least partially at the expense of the applied pesticides, and hence that the corresponding pesticides were also degraded in the BMs themselves. It should be noted, however, that samples were taken only from the upper layer of the microcosms, and the degradation/mineralisation capacity might not have evolved in the same way for the entire volume of the microcosm. Often some increase in pesticide mineralisation/degradation capacity was also found in BMs treated with water without pesticides. This has been observed before and could be due to the growth of pesticide-degrading populations at the expense of the fresh organic material (straw) added to the system, or to the improvement in moisture content.3 The time needed to reach maximum degradation/mineralisation capacity in the BPS microcosms could be clearly associated with the initial recorded degradation/mineralisation lag time. The
© 2014 Society of Chemical Industry
Pest Manag Sci (2014)
Mixed pesticide pollution treated in a biopurification system larger the initial lag time, and hence the lower the initial degradation/mineralisation capacity, the more time was required to reach maximum degradation/mineralisation capacity. Moreover, initially, a large variability in degradation capacity between microcosms within a set-up was observed for linuron and metamitron. This variability disappeared after the 10 weeks of pesticide supply. These observations are similar to these previously observed in BPS microcosms5 for linuron degradation and in soil microcosms in other studies using other pesticides13 and are explained by the initial spatial heterogeneity of low numbers of pesticide degraders in the pesticide-primed mix.13 Growth of corresponding pesticide-degrading populations in the biomix would then lead to decreased heterogeneity and less variation in the outcome of the degradation/mineralisation assays. Interestingly, as degradation of metamitron and metalaxyl improved in set-up S+ , so did the degradation capacity of their metabolites. In the case of metalaxyl degradation, the first metabolite was converted to a second metabolite, which initially did not further degrade. However, after 10 weeks of pesticide supply, the second metabolite was also degraded, indicating the proliferation of a metabolite-degrading population in the matrix. This was not observed for set-up S− , suggesting again that the metabolites were also degraded in the BMs themselves. As the time needed to enhance the degradation of the metabolite was longer than for metalaxyl, it can be suggested that the degradation of the metabolite(s) is performed by (a) microbial population(s) that is/are different from those that degrade metalaxyl. The nature of the metamitron and metalaxyl metabolites is currently unknown. Metalaxyl is a phenylamide fungicide consisting of a diphenyl ring containing a complex secondary amide structure. Pathways for metalaxyl degradation are not well known. Several metabolites of metalaxyl have previously been identified.14,15 The metabolite detected in the present study is likely the primary degradation product designated as CGA 6286 (see supporting information Fig. S2).15 As compound CGA 6286 is toxic and has potential to leach to groundwater, EU directive 2002/64/EG imposes special measures to lower the risk for leaching when metalaxyl is applied in vulnerable areas.16 In one study, 50% of metalaxyl could still be found as product CGA 6286 in soil 10 weeks after application of the fungicide.17 Metamitron is a triazinone herbicide consisting of a triazine moiety substituted with an amino group, a methyl group and a phenyl ring. Two metabolic pathways for metamitron degradation have been proposed. In the first, degradation is initiated with the attack of the phenyl ring by a dioxygenase, which is then cleaved and mineralised, leading to 3-methyl-4-amino-1,2,4-triazin-5(4H)one.18 The second alternative pathway involves an initial amidohydrolase cleavage and degradation of the triazin ring, producing benzoylformate, which is further mineralised by the mandelate pathway.19 As the metamitron mineralisation curves were in accordance with the metamitron degradation curves, and as the 14 C label in the 14 C-labelled metamitron used as a substrate in the mineralisation tests is within the phenyl moiety, it is suggested that the metabolite from metamitron that was detected by UV absorption in the HPLC measurements is the triazin moiety of metamitron, and hence that degradation of metamitron in the S+ set-up occurs by initial attack of the phenyl ring. The mechanism behind the observed establishment and proliferation of the pesticide-degrading capacities is unknown and could either be by establishment and proliferation of added microbial populations or through horizontal gene transfer of the appropriate catabolic genes from organisms residing in the added materials towards microbial residents in the other materials used.20 – 23 In a previous Pest Manag Sci (2014)
www.soci.org study, the use of molecular tools and appropriate BM set-ups suggested that bacteria belonging to the genus Variovorax and originating from the linuron-primed soil could be linked to linuron degradation.5 Bentazon- and isoproturon-degrading capacities did not develop in the S set-ups, even after 12 weeks of feeding the pesticides to the microbiota in set-up S+ . Apparently, organisms that could degrade these compounds were absent in the corresponding primed material (as suggested by their inability to degrade/mineralise these compounds) or were not able to proliferate under the set-up conditions. The capacity to degrade/mineralise linuron and metamitron (including degradation of its metabolite) also improved in microcosms inoculated with the non-primed soil C (i.e. in set-up C+ ) upon continuous feed with the pesticide mix. Apparently, the soil contained microbiota, probably at initial low densities, that proliferated under the feed conditions. The proliferation of a linuron-degrading/mineralising microbial population upon feeding with the pesticide mixture was not unexpected as this was observed previously with soil C in other BM set-ups when fed with linuron.5 However, based on the lag times, the degradation and mineralisation capacities for both compounds was always much lower than those in microcosms bioaugmented with the pesticide-primed soils (i.e. in set-up S+ ). In addition, the capacity to degrade metalaxyl never developed in BMs of set-up S+ within the experimental time period. These observations show that the addition of pesticide-primed materials is beneficial for activation of BPS, as their inclusion in the biomix matrix resulted in a more rapid establishment and greater abundance of pesticide-degrading capacities than without the addition of primed materials.
ACKNOWLEDGEMENTS This research was supported by the IWT-Vlaanderen Agricultural Research project LBO 040272 and by the KBBE-EU project BACSIN Contract 211684.
SUPPORTING INFORMATION Supporting information may be found in the online version of this article.
REFERENCES 1 De Wilde T, Spanoghe P, Debaer C, Ryckeboer J, Springael D and Jaeken P, Overview of on-farm bioremediation systems to reduce the occurrence of point source contamination. Pest Manag Sci 63:111–128 (2007). 2 Karanasios E, Tsiropoulos NG and Karpouzas DG, On-farm biopurification systems for the depuration of pesticide wastewaters: recent biotechnological advances and future perspectives. Biodegradation 23:787–802 (2012). 3 Sniegowski K, Bers K, Ryckeboer J, Jaeken P, Spanoghe P and Springael D, Robust linuron degradation in on-farm biopurification systems exposed to sequential environmental changes. Appl Environ Microbiol 77:6614–6621 (2011). 4 De Wilde T, Spanoghe P, Sniegowski K, Ryckeboer J, Jaeken P and Springael D, Transport and degradation of metalaxyl and isoproturon in biopurification columns inoculated with pesticide-primed material. Chemosphere 78:56–60 (2010). 5 Sniegowski K, Bers K, Van Goethem K, Ryckeboer J, Jaeken P, Spanoghe P et al., Improvement of pesticide mineralization in on-farm biopurification systems by bioaugmentation with pesticide primed soil. FEMS Microbiol Ecol 76:64–73 (2011). 6 Dejonghe W, Boon N, Seghers D, Top EM and Verstraete W, Bioaugmentation of soils by increasing microbial richness: missing links. Environ Microbiol 3:649–657 (2001).
© 2014 Society of Chemical Industry
wileyonlinelibrary.com/journal/ps
www.soci.org 7 Pigeon O, De Vleeschouwer C, Cors F, Weickmans B, Huyghebaert B, Planchon V et al., Biofilters to treat the pesticides wastes from spraying applications: results after 4 years of study. Comm Agric Appl Biol Sci 71:9–19 (2006). 8 Pussemier L, De Vleeschouwer C and Debongnie P, Self-made biofilters for on-farm clean-up of pesticides wastes. Outlooks Pest Manag 15:60–63 (2004). 9 Dejonghe W, Berteloot E, Goris J, Boon N, Crul K, Maertens S et al., Synergistic degradation of linuron by a bacterial consortium and isolation of a single linuron-degrading Variovorax strain. Appl Environ Microbiol 69:1532–1541 (2003). 10 Broos K, Mertens J and Smolders E, Toxicity of heavy metals in soil assessed with various soil microbial and plant growth assays: as comparative study. Environ Toxicol Chem 24:634–640 (2005). 11 Sniegowski K, Mertens J, Diels J, Smolders E and Springael D, Inverse modeling of pesticide degradation and pesticide-degrading population size dynamics in a bioremediation system: parameterizing the Monod model. Chemosphere 75:726–731 (2009). 12 Sniegowski K, Bers K, Ryckeboer J, Jaeken P, Spanoghe P and Springael D, Minimal pesticide-primed soil inoculum density to secure maximum pesticide degradation efficiency in on-farm biopurification systems. Chemosphere 88:1114–1118 (2012). 13 Cullington JE and Walker A, Rapid biodegradation of diuron and other phenylurea herbicides by a soil bacterium. Soil Biol Biochem 31:677–686 (1999). 14 Pesaro M, Nicollier G, Zeyer J and Widmer F, Impact of soil drying– rewetting stress on microbial communities and activities and on degradation of two crop protection products. Appl Environ Microbiol 70:2577–2587 (2004). 15 Droby S and Coffey MD, Biodegradation process and the nature of metabolism of metalaxyl in soil. Ann Appl Biol 118:543–553 (1991).
wileyonlinelibrary.com/journal/ps
K Sniegowski, D Springael 16 Directive 2002/64/EG of the Commission of 15 July 2002, Alteration of Directive 91/414/EEG of the Council. [Online]. European Community, Publ. E.-1 (2002). Available: http://eur-lex.europa.eu/LexUriServ/ LexUriServ.do?uri=OJ:L:2002:189:0027:0032:NL:PDF [4 March 2014]. 17 Sukul P and Spiteller M, Metalaxyl: Persistence, degradation, metabolism, and analytical methods. Rev Environ Contam Toxicol 164:1–26 (2000). 18 Parekh NR, Walker A, Roberts SL and Welch SJ, Rapid degradation of the triazinone herbicide metamitron by a Rhodococcus sp. isolated from treated soil. J Appl Bacteriol 77:467–475 (1994). 19 Engelhardt G, Ziegler W, Wollnofer PR, Jarczyk HJ and Oehlmann L, Degradation of the triazinone herbicide metamitron by Arthrobacter spec. DSM 20369. J Agric Food Chem 30:278–282 (1982). 20 DiGiovanni GD, Neilson JW, Pepper IL and Sinclair NA, Gene transfer of Alcaligenes eutrophus JMP134 plasmid pJP4 to indigenous soil recipients. Appl Environ Microbiol 62:2521–2526 (1996). 21 Devers M, Henry S, Hartmann A and Martin-Laurent F, Horizontal gene transfer of atrazine-degrading genes (atz) from Agrobacterium tumefaciens St96-4 pADP1: Tn5 to bacteria of maize-cultivated soil. Pest Manag Sci 61:870–880 (2005). 22 Hirkala DLM and Germida JJ, Field and soil microcosm studies on the survival and conjugation of a Pseudomonas putida strain bearing a recombinant plasmid, pADPTel. Can J Microbiol 50:595–604 (2004). 23 Dunon V, Sniegowski K, Bers K, Lavigne R, Smalla K and Springael D, High prevalence of IncP-1 plasmids and IS1071 insertion sequences in on-farm biopurification systems and other pesticide polluted environments. FEMS Microbiol Ecol 86:415–431 (2013).
© 2014 Society of Chemical Industry
Pest Manag Sci (2014)
Revised: 13 June 2014
Accepted article published: 5 August 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/ps.3876
Establishment of multiple pesticide biodegradation capacities from pesticide-primed materials in on-farm biopurification system microcosms treating complex pesticide-contaminated wastewater Kristel Sniegowski and Dirk Springael* Abstract BACKGROUND: On-farm biopurification systems (BPSs) treat pesticide-containing wastewater at farms by biodegradation and sorption processes. The inclusion of pesticide-primed material carrying a pesticide-degrading microbial community is beneficial for improving biodegradation, but no data exist for treating wastewater containing multiple pesticides, as often occurs on farms. In a microcosm set-up, an examination was carried out to determine whether multiple pesticide degradation activities could be simultaneously established in the matrix of a BPS by the simultaneous inclusion of different, appropriate pesticide-primed materials. The microcosms were fed with a mixture of pesticides including the fungicide metalaxyl and the herbicides bentazon, isoproturon, linuron and metamitron, and pesticide-degrading activities were monitored over time. RESULTS: The strategy immediately provided the microcosms with a multiple pesticide degradation/mineralisation capacity, which improved during feeding of the pesticide mixture. Not only did the degradation of the parent compound improve but also that of the produced metabolites and compound mineralisation. The time to achieve maximum degradation/mineralisation capacity depended on the pesticide degradation capacity of the pesticide-primed materials. CONCLUSIONS: The data obtained show that the addition of pesticide-primed materials into the matrix of a BPS as an approach to improve biodegradation can be extended to the treatment of pesticide mixtures. © 2014 Society of Chemical Industry Supporting information may be found in the online version of this article. Keywords: on-farm biopurification system; pesticide mixtures; biodegradation; pesticide primed materials; bioaugmentation
1
INTRODUCTION
Direct pesticide losses during the handling of pesticides in farmyards contribute largely to contamination of surface water.1 Treatment of pesticide-contaminated wastewater by means of on-farm biopurification systems (BPSs) can significantly reduce pesticide losses.1 Different variants of BPSs exist, such as the biobed, the biofilter and Phytobac®, but all of them operate as a biofilter that contains a matrix of organic waste materials, designated as biomix, in which removal of pesticides in the wastewater occurs by sorption and biodegradation.1,2 Previously, bioaugmentation of the biomix with pesticide-primed materials improved the pesticide degradation capacity of lab-scale BPSs for over 1 year, even while enduring successive stress conditions.3 Pesticide-primed materials are materials that have been treated on a long-term basis with specific pesticides, resulting in the proliferation of microbial populations that have adapted to degrade and often metabolise the compound. Such materials include long-term pesticide-treated soils or biomix materials from BPSs in operation.4,5 Target improvements include the introduction Pest Manag Sci (2014)
of missing catabolic phenotypes, the acceleration of pesticide degradation activities and the avoidance of production of toxic metabolites.5,6 The method has been suggested as an alternative to bioaugmentation with axenic pesticide-mineralising pure cultures, and has been successfully demonstrated using linuron as a model pesticide and a linuron-treated soil as a model pesticide-primed soil.5 However, in reality, different types of pesticide are being used on farms, often simultaneously, and as such will be present as mixtures in the contaminated water. Although several reports suggest the removal of multiple pesticide compounds in BPSs,7,8 no data yet exist on the feasibility of bioaugmentation with pesticide-primed materials to improve multiple pesticide degradation in BPSs. Such a strategy would
∗
Correspondence to: Dirk Springael, Division of Soil and Water Management, KU Leuven, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium. E-mail: [email protected] Division of Soil and Water Management, KU Leuven, Heverlee, Belgium
www.soci.org
© 2014 Society of Chemical Industry
www.soci.org involve adding a mixture of different pesticide-primed materials with catabolic activity towards different pesticides. In the present study, an investigation was carried out to determine whether inoculation of a BPS with pesticide-primed materials targeting simultaneously the degradation of different pesticide compounds would result in the successful transfer and establishment of the respective pesticide degradation capacities in the biomix of the BPS. Use was made of BPS microcosms (BMs) containing a simulated biomix matrix, as described previously.5 The BMs were irrigated with artificially contaminated water containing a mixture of the fungicide metalaxyl and the herbicides bentazon, isoproturon, linuron and metamitron or with water without pesticides. The material used for bioaugmentation was a mix of pesticide-primed materials that had been treated for several years with the pesticides applied in the mixture. As a control set-up, BMs were included that instead of the primed material mix contained a soil without history of pesticide contamination. The pesticide degradation and mineralisation capacities of the BMs were monitored, and related to the pesticide-degrading/mineralisation capacities of the pesticide-primed materials used for bioaugmentation.
2
EXPERIMENTAL METHODS
2.1 Pesticides used Linuron [3-(3,4-dichlorophenyl)-1-methoxy-1-methyl urea] (purity 99.5%) and isoproturon [(3-(4-isopropylphenyl)-1,1-dimethyl urea) (purity 99.0%) were purchased from Sigma-Aldrich (Belgium). Metalaxyl [methyl-N-(2,6-dimethylphenyl)-N-(2-methoxyacetyl)-D, L-alaninate] (purity 95.5%) was obtained from Syngenta (Switzerland), and bentazon (3-isopropyl-2,1,3-benzothiadiazine-4-on-2,2dioxide) (purity 98.8%) from BASF (Belgium). Metamitron [4-amino -3-methyl-6-phenyl-1,2,4-triazin-5(4H)-one] (purity 95%) was donated by Agrichem B.V. (The Netherlands). [Phenyl-U-14 C] linuron (16.93 mCi mmol−1 ), [phenyl-U-14 C] isoproturon (12.02 mCi mmol−1 ), [carbonyl-14 C] bentazon (46.70 mCi mmol−1 ) and [phenyl -U-14 C] metamitron (14.11 mCi mmol−1 ) were obtained from Izotop (Hungary). 2.2 BPS microcosm set-ups BPS microcosms (BMs) were set up as described.5 The biomix contained 25 vol% cut straw (±0.5 cm2 ), 35 vol% peat, 25 vol% coconut husk chips (±0.5 cm2 ), 5 vol% cow manure (crushed dry pellets) and 10 vol% of a pesticide-primed material mix or a soil without pesticide application history. An overview of the used soils/pesticide-primed materials and their origin is given in Table 1. The soil without pesticide treatment history, designated as soil C, was collected from a construction site without any known history of pesticide treatment, as described.5 The pesticide-primed material mix consisted of soil L, soil I + M, soil B and biomix MX. Soil L originated from a potato field in Halen, Belgium, and has been described previously.5 The field had been treated yearly with linuron until the time of sampling. Soil I + M originated from an agricultural field treated with both isoproturon and metamitron. Metamitron had been used until the time of sampling, while the last isoproturon treatment was applied 4 years before sampling. Soil B originated from an agricultural field that had been treated with bentazon for several years until the time of sampling. Soils L, I + M and B were sampled from the upper 20 cm of the field 1 month before initiating the experiment. The soils were stored at 4 ∘ C in the dark before use. Biomix MX was sampled from an
wileyonlinelibrary.com/journal/ps
K Sniegowski, D Springael
Table 1. Overview of the origins of the materials that were included in the biomix of BMs in this study Designation Soil C Soil L Soil I + M Soil B Biomix MX
Origin St-Truiden (construction site) Halen (potato field) Tielt-Winge (cereals, beet field) Leefdaal (corn field) Chastre (BPS)
Pesticide(s) applied No pesticides Linuron Isoproturon, metamitron Bentazon Metalaxyl
on-farm BPS that had processed pesticide-contaminated water containing metalaxyl for 4 years until the time of sampling and was stored at 4 ∘ C before use. The pesticide-primed material mix was prepared by mixing 2.5 vol% of each pesticide-primed material. The individual pesticide-primed materials as well as the mixture were tested for their ability to degrade or mineralise the corresponding pesticide(s) during a maximum incubation period of 60 days. Table 2 shows an overview of the BM experimental set-ups. Each set-up included three replicate BMs. The BMs were incubated at 20 ∘ C and supplied for 10 weeks with either 19.1 L m−3 day−1 of sterile tap water (microcosm set-ups C− and S− ) or sterile tap water containing a mixture of the five pesticides at concentrations of 50 mg L−1 each (microcosm set-ups C+ and S+ ). Weekly, on Monday, Wednesday and Friday, the solution (7 mL on each of the indicated days) was manually spread dropwise over the surface of the biomix using a 10 mL pipette. Samples for assessing the pesticide degradation capacity of the BMs in batch assays were taken from the upper layer of the biomix of the BMs at weeks 0, 2, 4, 6, 8 and 10. For each set-up, each of the three replicates were sampled. Pesticide mineralisation assays were performed with samples taken at weeks 0 and 10. 2.3 Batch pesticide degradation assay Batch pesticide degradation assays were performed in sterile 100 mL Erlenmeyers containing 200 mg (wet weight) of either soil C, pesticide-primed material mix or biomix taken from the mixed upper layer (1 cm) of the BMs and 50 mL of MMO medium (MMN supplied with NH4 + , pH 5.8)9 containing a mixture of linuron, metamitron, isoproturon, metalaxyl and bentazon at a concentration of 20 mg L−1 each. In the case of the BM set-ups, each replicate BM was assessed once at each time point. In addition, the pesticide degradation capacity of each of the pesticide-primed materials was tested individually in identical batch degradation experiments, except that the medium contained only one pesticide. Each pesticide-primed material was tested for degradation of each of the pesticides that had been applied on that material. Abiotic removal was assessed a single time in a batch experiment with samples taken from set-up S− supplied with MMO adjusted to pH 2 with HCl (1 mM) to inhibit biotic activity, and was found to be negligible. For each series, a control without microcosm material was included to act as an additional abiotic control. The Erlenmeyers were incubated on a horizontal shaker (125 rpm) at 20 ∘ C in the dark. Frequently, 700 𝜇L samples were taken from the aqueous phase and, after centrifugation at 12 000 rpm for 5 min, analysed by reverse-phase HPLC (LaChrom; Merck Hitachi). The HPLC system was equipped with a Platinum EPS C18 (7.5 × 4.6 mm) guard column, a Platinum EPS C18 150 × 4.6 mm end-capped column packed with silica beads (3 μm) and a UV-vis detector set at 208 nm. The compounds were separated by gradient elution with
© 2014 Society of Chemical Industry
Pest Manag Sci (2014)
Mixed pesticide pollution treated in a biopurification system
www.soci.org
Table 2. Overview of the BM set-ups examined in this study Relevant added material Microcosm set-up C− C+ S− S+
Non-pesticide-primed soil (soil C) + + − −
Treatment
Pesticide-primed material mixa
Pesticide-contaminated waterb
− − + +
− + − +
a Mix of soil L, soil I + M, soil B and biomix MX (2.5 vol% each) b Sterile tap water with linuron, isoproturon, metamitron, bentazon, and metalaxyl (50 mg L−1
CH3 CN/H2 O (0.005% H3 PO4 ) (0 min: 78/22; 10.5 min: 45/55; 13 min: 60/40; 15 min: 78/22) and a flow rate of 1.0 mL min−1 . The ratio of the concentration in the soil slurry (C) and the concentration in the medium without soil inoculum (C 0 ) were used to present the data graphically as a function of time. When unidentified pesticide degradation products were observed, the corresponding peak surface (C) was expressed with reference to the maximum peak surface (C max ) recorded in the HPLC chromatograms. The lag phase, defined as the period between initiation of the degradation assay and the start of degradation, was calculated as the intersection of the X-axis with the linear regression line between two successive points of the degradation curve where the amount of linuron removed was the largest. The slope of the linear regression line of maximum degradation determined the maximum degradation rate, as described.10 Averages of the lag times and maximum degradation rates of triplicate BM samples were analysed by ANOVA for significance analysis (P < 0.05). 2.4 Pesticide mineralisation assay 14 C-Pesticide mineralisation assays were performed as described.5 From each replicate BM, four samples were taken in order to assess the capacity for mineralisation of either linuron, isoproturon, metamitron or bentazon. Mineralisation of metalaxyl was not assessed because 14 C-metalaxyl was not commercially available. Microcosm biomix samples (200 mg wet weight) were supplied with 5 mL of MMO containing a mixture of the target pesticide as 14 C-labelled and unlabelled compounds, as well as each of the other four pesticides as unlabelled compounds. Unlabelled pesticides were added at concentrations of 20 mg L−1 . For each target compound, final radioactivity added to the assay was 238 Bq mL−1 , which corresponded to 0.092 mg L−1 (2.58 MBq mg−1 ) of 14 C-metamitron, 0.11 mg L−1 (2.16 MBq mg−1 ) of 14 C-isoproturon, 0.095 mg L−1 (2.52 MBq mg−1 ) of 14 C-linuron or 0.33 mg L−1 (7.13 MBq mg−1 ) of 14 C-bentazon. The pesticide mineralisation capacity of the pesticide-primed material mixture and soil C was tested identically for each of the pesticides, while that of the individual pesticide-primed materials was assessed only for the pesticide with which they had been treated. The vials were incubated at 20 ∘ C on a rotary shaker at 150 rpm, and the amount 14 CO2 produced was measured as described by Sniegowski5 and expressed as the percentage of the initial amount of 14 C-pesticide added. Cumulative mineralisation curves were established on the basis of a control without inoculation of the soil sample. The lag time and mineralisation rate were calculated as for the degradation experiments. Averages of the lag times and mineralisation rates of triplicate BM samples were analysed by ANOVA for significance analysis (P < 0.05). Pest Manag Sci (2014)
Non-contaminated water
3
+ − + −
each).
RESULTS
3.1 Pesticide degradation capacity of the pesticide-primed materials and mixture The pesticide-primed materials used for bioaugmenation were individually tested for their ability to degrade or mineralise the pesticide(s) with which they had been treated, during an incubation period of 60 days maximum. Soil C and the pesticide material mix were assessed for degradation/mineralisation of all tested pesticides. When degradation and/or mineralisation occurred, lag times were observed that differed significantly between the different pesticides and materials (see supporting information Fig. S1). This lag time was previously related to the size of the pesticide-degrading population and hence to the pesticide degrading capacity.11 An overview of the pesticide degradation/mineralisation potential of the tested materials, expressed as lag time, is shown in Table 3. Most of the individual materials showed the expected degradation/mineralisation potential, i.e. soil L showed degradation and mineralisation of linuron, soil I + M showed degradation and mineralisation of metamitron and biomix MX showed degradation of metalaxyl. However,
Table 3. Overview of the pesticide degradation capacity of pesticide-primed materials, expressed as lag time observed in the batch degradation and mineralisation assays
Material Soil C
Soil L Soil I + M Soil B Biomix MX Pesticide-primed material mix
Pesticide tested
Degradation Mineralisation lag time (days) lag time (days)
Linuron Metamitron Isoproturon Bentazon Metalaxyl Linuron Isoproturon Metamitron Bentazon Metalaxyl Linuron
27.0(±3.6) 37.3(±7.4)a >60 >60 >60 18.8(±1.7) >60 3.5(±4.9)a >60 9.3(±1.7)a 38.0(±10.6)
>60 >60 >60 >60 NDb 16.9(±1.6) >60 5.5(±4.2) >60 NDb 27.6(±3.1)
Metamitron Isoproturon Bentazon Metalaxyl
33.9(±12.8)a >60 >60 9.3(±1.7)
25.4(±3.7) >60 >60 NDb
a A metabolite accumulated during the degradation assays. b ND: not done.
© 2014 Society of Chemical Industry
wileyonlinelibrary.com/journal/ps
www.soci.org 1.2
(A) % 14C-linuron mineralised
C/C0 linuron
0.8 0.6 0.4 0.2
0
10
20
30 40 Time (days)
50
0.6 0.4 0.2 0
20 10 0
10
20
30 40 Time (days)
50
C/Cmaxmetamitron metbolite
0.8 0.6 0.4 0.2
10
20
30 40 Time (days)
Set-up C+
50
60
Set-up S+
10
20
30 40 50 Time (days)
60
60
80
60
70
(D)
50 40 30 20 10
0
10
20
30 40 Time (days)
50
(F)
1.2
1.0
0
0
60
0
60
(E)
1.2
C/C0 metalaxyl
30
% 14C-metamitron mineralised
C/C0 metamitron
0.8
0.0
40
60
1.0
0.0
50
(C)
1.2
(B)
60
1.0
0.0
K Sniegowski, D Springael
1.0 0.8 0.6 0.4 0.2 0.0
0
10
20
30 40 Time (days)
50
60
Pesticide-primed material mix
Figure 1. Kinetics of linuron degradation (A), linuron mineralisation (B), metamitron degradation (C), metamitron mineralisation (D) and metalaxyl degradation (E), recorded with samples taken from BMs of set-up C+ , set-up S+ and the pesticide-primed material mix. The values are averages of three replicates with indicated standard deviations. Similar observations were made for set-ups C− and S− in A, C and E, the Y-axis presents the fraction of the pesticide concentration in reference to the concentration in the abiotic control (C0 ). In B, D and F, the Y-axis presents the cumulative amount of 14 C in the produced 14 CO2 over the amount of 14 C initially added as 14 C-pesticide.
isoproturon was not degraded/mineralised in soil I + M, and bentazon underwent neither process in soil B. In soil I + M, apparently only the phenyl group of metamitron was degraded, as the 14 C-labelled metamitron used was labelled in the phenyl ring and 14 CO2 was produced in the mineralisation assay, while a metabolite appeared in the degradation assay. A metabolite also appeared during degradation of metalaxyl (see supporting information Fig. S1, panel F). In both cases, the metabolite accumulated over time and was not degraded within the 60 days of monitoring. Unexpectedly, the non-primed soil C also degraded (but did not mineralise) linuron and metamitron, but with lag times (27.0 ± 3.6 days and 37.3 ± 7.4 days respectively) that were significantly larger (P = 0.017 and 0.002 respectively), and with a significantly lower degradation rate (zero order) than those observed with the pesticide-primed soil L and soil I + M.
wileyonlinelibrary.com/journal/ps
As with soil I + M, a metabolite accumulated from metamitron in soil C. Soil C did not degrade/mineralise metalaxyl, isoproturon or bentazon. Results for the pesticide-primed material mix are given in Fig. 1 and in supporting information Fig. S1. Degradation and mineralisation of linuron and metamitron were observed as well as degradation of metalaxyl, showing that the pesticide degradation capacities of the individual soils were retained after they were mixed, although differences in lag time were noted between the materials when individually tested and tested as a mixture, especially for metamitron. In the case of metamitron and metalaxyl, a metabolite accumulated (data not shown). As with the individual materials, no degradation and/or mineralisation of isoproturon and bentazon were observed in the applied time period with the mixture.
© 2014 Society of Chemical Industry
Pest Manag Sci (2014)
Mixed pesticide pollution treated in a biopurification system
www.soci.org
1.2
(A)
1.0 0.8 0.6 0.4 0.2 0.0
0
detected, but at a very low rate (0.3 ± 0.04% day−1 ) (Fig. 1D). As in set-ups S− and S+ , degradation of metamitron resulted in the production of an unidentified metabolite, but in contrast to set-ups S− and S+ , this compound was not transient and accumulated (data not shown). In addition, as degradation of metamitron was not completed after 60 days, the amount of produced metabolite remained limited. In set-ups C− and C+ , no initial degradation and/or mineralisation capacity for isoproturon, metalaxyl and bentazon was observed (Fig. 1D and data not shown). 3.3 Effect of pesticide supply on the pesticide degradation/mineralisation capacity of the BMs As illustrated in Fig. 4A, set-up S+ showed a gradually decreasing lag time for linuron degradation from 31 ± 5.9 days at week 0 to 3.3 ± 0.7 days at week 10, indicating an improvement in the linuron degradation capacity as a response to the mixed pesticide supply. The dynamics of linuron mineralisation capacity largely followed that of the degradation capacity, with an average lag time of 2.0 ± 0.2 days at week 10 (Fig. 5A). Also, metamitron degradation and mineralisation capacity improved with time in set-up S+ , as lag times decreased from 31.6 ± 19.8 days at week 0 to 1.91 ± 0.43 days at week 10 for metamitron degradation, and from 9.4 ± 0.76 days at week 0 to 1.1 ± 0.28 days at week 10 for metamitron mineralisation. The metalaxyl degradation capacity in set-up S+ also improved, as shown by the lag times decreasing from 10.5 ± 0.69 days at week 0 to 0.51 ± 0.59 days at week 10 (Fig. 4E). For both linuron and metamitron degradation and mineralisation, the required period of pesticide supply to shorten the lag time to its minimum differed between the replicates and apparently depended on the initial lag time (as recorded at week 0). At week 10, however, standard deviations in lag times and rates were strongly reduced. Interestingly, in addition to metamitron, for metalaxyl also an improvement in the capacity to degrade respective observed metabolites was recorded (Figs 2A and 3A). Moreover, for metalaxyl, until week 8, degradation of the metabolite resulted in the accumulation of a second degradation product, without further degradation (Fig. 3C). However, at week 10, the second metabolite also started to degrade (Fig. 3C). Although set-up S− did not receive pesticides and no enhanced degradation capacity was observed with the degradation assays, some improvement in the linuron mineralisation capacity occurred, as indicated by the reduced lag time (P = 0.043) and increased mineralisation rate (P = 0.44) at week 10 compared with week 0 (Fig. 5A). C/Cmax metamitron metabolite
C/Cmax metamitron metabolite
3.2 Initial pesticide degradation and mineralisation capacity in BMs Before initiating pesticide application, for both set-ups S− and S+ , degradation and mineralisation potentials for linuron and for metamitron were recorded (data not shown and Fig. 1). Linuron and metamitron were degraded and mineralised with large differences in lag times between replicates. Degradation and mineralisation of linuron showed a lag time of 25–35 days (Fig. 1A) and 40–48 days (Fig. 1B) respectively. Metamitron started to degrade and mineralise after lag times of 9–45 days (Fig. 1C) and 7.5–14 days (Fig. 1D) respectively. For linuron, the observed degradation kinetics recorded with the S− and S+ microcosm samples was similar to the kinetics observed with the pesticide-primed material mix, indicating that the additional substrata in the microcosm biomix had no direct effect on the degradation capacity of the primed material mix. However, mixing the primed material with the other biomix substrata appeared to have a negative effect on the linuron mineralisation capacity, as the lag times were longer and the mineralisation rates lower for the microcosm samples than those observed for the primed material mix (Figs 1A and B). This was not the case for metamitron degradation and mineralisation, where the lag time for set-ups S− and S+ was not significantly different from that recorded with the primed material mix (P = 0.14 and 0.09 respectively) (Figs 1C and D). As observed with soil I + M and the primed material mix, an unidentified metabolite appeared during degradation of metamitron in the degradation assay (see Figs 2A and B, week 0). In addition to metamitron and linuron, set-ups S− and S+ directly showed the capacity to degrade metalaxyl (Fig. 1E). Metalaxyl degradation occurred after a relative short lag time of 9.8 days, with data showing small standard deviations, and followed degradation kinetics similar to that observed for the primed material mix. As observed with the initial pesticide-primed material, degradation of metalaxyl resulted in the accumulation of a metabolite (see Figs 3A and B, week 0). Neither set-up S− nor set-up S+ showed the ability to degrade or mineralise isoproturon or bentazon (data not shown). Samples taken from BMs inoculated with soil C (set-ups C− and + C ) also degraded linuron and metamitron. No clear lag time was observed, but degradation occurred at a much lower rate than those recorded for set-ups S− and S+ and clearly showed zero-order kinetics (Figs 1A and C). Moreover, in contrast to set-ups S− and S+ , set-ups C− and C+ showed no linuron mineralisation capacity (Fig. 1B). Some metamitron mineralisation activity was
10
20
30
40
50
60
70
1.2 (B) 1.0 0.8 0.6 0.4 0.2 0.0
0
10
20
Time (days) week 0
week 4
week 6
30 40 50 Time (days)
week 8
60
70
week 10
Figure 2. Formation and degradation of the metamitron metabolite, observed during degradation of metamitron for samples taken from BMs of set-up S+ (A) and set-up S− (B) after different periods of feeding with the pesticide cocktail. Because of the large variability between BM replicates, and for the sake of clarity of the figure, only data obtained for one replicate are presented. The arrow indicates the evolution of the lag time associated with degradation of the metabolite during the incubation period. The Y-axis presents the fraction of the peak surface associated with the metabolite (C) in reference to the maximum peak surface (C max ) observed in the HPLC chromatograms obtained with the samples from the batch degradation assays.
Pest Manag Sci (2014)
© 2014 Society of Chemical Industry
wileyonlinelibrary.com/journal/ps
1.2
K Sniegowski, D Springael
C/Cmax metalaxyl metabolite 1
C/Cmax metalaxyl metabolite 1
www.soci.org
A
1.0 0.8 0.6 0.4 0.2 0.0 10
C/Cmax metalaxyl metabolite 2
0
20 30 40 Time (days) 1.2
50
60
1.2
B
1.0 0.8 0.6 0.4 0.2 0.0
0
10
20
30
40
50
60
Time (days)
C
1.0 0.8 0.6 0.4
week 0 week 4 week 6 week 10
0.2 0.0 0
10
20
30
40
50
60
Time (days) Figure 3. Formation and degradation of the first (A, B) and second (C) metabolites of metalaxyl degradation, recorded with samples taken from BMs of set-up S+ (A, C) and set-up S− (B) after different periods of pesticide cocktail supply (set-up S+ ) or non-contaminated water (set-up S− ). Because of the large variability between BM replicates, and for the sake of clarity of the figure, only data from one replicate are presented. The arrow indicates the evolution of the lag time during the incubation period. The Y-axis presents the fraction of the peak surface associated with the metabolite (C) in reference to the maximum peak surface (C max ) observed in the HPLC chromatograms obtained with the samples from the batch degradation assays.
Nevertheless, lag times were up to 6 times lower with samples of the S+ set-up compared with those of the S− set-up. No significant change in the lag time of metamitron degradation (P = 0.782, data not shown) or metamitron mineralisation (P = 0.189) (Fig. 5C) was observed for set-up S− . Also, the capacity to degrade the metamitron metabolite (Fig. 2B) did not significantly improve over time for set-up S− . This indicates that metamitron degradation/mineralisation improvement in set-up S+ was due to the feeding of metamitron. For metalaxyl, the degradation lag time recorded for set-up S− decreased from 9.8 to 5.8 days, which is a much lower extent compared with set-up S+ (data not shown). Moreover, samples from set-up S− never showed degradation of the first metalaxyl metabolite, even after 10 weeks of incubation (Fig. 3B). In the course of the incubation period, neither set-up S+ nor set-up S− developed the capacity to degrade/mineralise isoproturon or bentazon (data not shown). The capacity to degrade and mineralise linuron also improved in set-up C+ as a response to the mixed pesticide supply (Figs 4B and 5B). However, the capacity to degrade and mineralise linuron at day 10 was less for set-up C+ compared with set-up S+ . In the degradation assays this was only indicated by a lower degradation rate, which was 5.6 ± 0.024% day−1 for set-up C+ compared with 16.4 ± 0.021% day−1 for set-up S+ at week 10 (P = 0.0060). Owing to the linear degradation rate, the calculated lag time did not change significantly. On the other hand, in the mineralisation assays, reduced mineralisation capacity was indicated in particular by a significantly longer lag time, i.e. 6.8 ± 0.1 days for
wileyonlinelibrary.com/journal/ps
set-up C+ compared with 2.0 ± 0.2 days for set-up S+ (P = 0.011). Also, the capacity to degrade metamitron in BMs of set-up C+ improved during the period of pesticide supply, with apparent zero-order kinetics changing into first-order degradation kinetics from week 0, indicating that a population with metamitron metabolic abilities started to proliferate (Fig. 4D). The recorded average lag time and metamitron degradation rate for set-up C+ at week 10 were 25.8 ± 15.6 days and 7.2 ± 0.03% day−1 respectively, which were still significantly longer (P = 0.025) and lower (P = 0.0027), respectively, than those recorded for set-up S+ at week 10. Also, metamitron mineralisation capacity improved during the pesticide cocktail supply for C+ (Fig. 5D). However, the lag times recorded for set-up C+ were significantly longer (6.3 ± 3.2 days) compared with those recorded for set-up S+ (1.1 ± 0.3 days) (P = 0.049). Furthermore, as the capacity to degrade metamitron improved over time in set-up C+ , so did the capacity to degrade the observed metabolite (data not shown), and set-up C+ samples taken at week 10 degraded the metabolite at a rate similar to samples of set-up S− (data not shown). No change in linuron or metamitron degradation (data not shown) and metamitron mineralisation kinetics (Fig. 5D) nor in degradation of the metamitron metabolite (data not shown) were observed for set-up C− samples during the 10 weeks of water supply. Moreover, no change in the metalaxyl degradation kinetics was observed with samples taken from set-ups C− and C+ (data not shown). In addition, as in set-ups S+ and S− , no apparent proliferation of isoproturon and bentazon microorganisms occurred in C+ and C− during the 10 weeks
© 2014 Society of Chemical Industry
Pest Manag Sci (2014)
Mixed pesticide pollution treated in a biopurification system 1.2
www.soci.org
A
1.0
0.8
C/C0 linuron
C/C0 linuron
1.0
0.6 0.4 0.2 0.0
B
1.2
0.8 0.6 0.4 0.2
0
10
20
30
40
50
0.0
60
0
10
20 30 40 Time (days)
Time (days) 1.2
1.2
C C/C0 metamitron
C/C0 metamitron
60
D
1.0
1.0 0.8 0.6 0.4
0.8 0.6 0.4 0.2
0.2 0.0
50
0
10
20
30
40
0.0 30
50
40
40
50
Time (days)
Time (days) E
1.2
C/C0 metalaxyl
1.0 0.8 0.6 0.4 0.2 0.0
Week 0
0
2
4
6 8 10 Time (days)
Week 4
12
14
Week 6
16
Week 8
Week 10
Figure 4. Pesticide degradation kinetics recorded with samples taken from BMs of set-up S+ for linuron (A), metamitron (C) and metalaxyl (E) and set-up C+ for linuron (B) and metamitron (D), taken at different weeks after starting the supply of the pesticide cocktail. The data for linuron and metalaxyl are average values of triplicate BM samples with indicated standard deviations. Because of the large variability between BM replicates, and for the sake of clarity of the figure, for metamitron the data of only one BM are given. At week 8, no data were obtained for set-up C+ at week 8. The arrow indicates the evolution of the lag time over the incubation period. The Y-axis presents the fraction of the pesticide concentration in reference to the concentration in the abiotic control (C 0 ).
of feeding with the pesticide cocktail or non-contaminated water, respectively, as no changes in lag times of isoproturon and bentazon degradation and mineralisation occurred (data not shown).
4
DISCUSSION
Previous bioaugmentation experiments with a single pesticide-primed soil showed promising results for enhancing the degradation and mineralisation capacity of a pesticide in BPSs.5 As, in farmyards, different pesticides are used, and often simultaneously, the present study attempted to coenhance the degradation capacity of a BPS for different pesticides simultaneously. Pest Manag Sci (2014)
Therefore, the pesticide degradation/mineralisation capacities of lab-scale BPS microcosms inoculated with four appropriate pesticide-primed materials were compared with those of microcosms inoculated with a soil without a history of pesticide treatment. Prior to inoculation, a pesticide-primed material mix with samples originating from appropriately treated agricultural fields (isoproturon, metamitron, linuron and bentazon) or BPS (metalaxyl) was prepared. Degradation/mineralisation assays confirmed the corresponding degradation capacity of the pesticide-primed materials for linuron, metamitron and metalaxyl, but not for isoproturon and bentazon. Apparently, the soils treated with isoproturon and bentazon, in spite of being long-term
© 2014 Society of Chemical Industry
wileyonlinelibrary.com/journal/ps
70
A
70 60 50 40 30 20 10 0
K Sniegowski, D Springael
% 14C-linuron mineralised
% 14C-linuron mineralised
www.soci.org
0
10
20
30
40
50
60 50 40 30 20 10 0
60
B
0
10
20 30 40 Time (days)
50
60
10
20
50
60
60
% 14C-metamitron mineralised
% 14C-metamitron mineralised
Time (days) C
50 40 30 20 10 0
0
10
20
30
40
50
60
60
D
50 40 30 20 10 0
0
30
40
Time (days)
Time (days)
Set-up, S-, week 0 Set-up, S+, week 0 Set-up, S-, week 10 Set-up, S+, week 10
Set-up, C-, week 0 Set-up, C+, week 0 Set-up, C-, week 10 Set-up, C+, week 10
Figure 5. Pesticide mineralisation kinetics recorded with samples taken from BMs of set-ups S− and S+ for linuron (A) and metamitron (C) and from BMs of set-ups C− and C+ for linuron (B) and metamitron (D). The samples were taken at week 0 and at 10 weeks after starting the supply of the pesticide mixture. The data are average values of triplicate BM samples with indicated standard deviations. The Y-axis presents the cumulative amount of 14 C produced as CO2 over the total amount of 14 C initially added as 14 C-pesticide. The metamitron mineralisation kinetics of C− and C+ at week 0 was very similar, and corresponding symbols are therefore difficult to distinguish from each other in the graph.
treated, had not developed corresponding pesticide degradation capacities. The last treatment of soil I + M was also done 4 years before sampling of the soil, and the long-term lack of treatment might have affected isoproturon-degrading populations that could have developed during the field treatment. As such, supplementing the biomix with the pesticide-primed material mix was expected to result directly in the establishment in the microcosms of degradation/mineralisation capacity for linuron, metamitron and metalaxyl but not for isoproturon and bentazon. This was indeed the case, confirming the success of this bioaugmentation strategy and extending its feasibility towards BPS-based treatment of multiple-pesticide-contaminated wastewaters. Moreover, as only small volumes (i.e. 2.5 vol%) of inoculum material was used for each pesticide-primed matrix material, the present study confirms that such small volumes harbour sufficient capacity for successful application. Previously, a soil vol% of up to 0.5 was sufficient for successful establishment of linuron-degrading capacity in a BPS biomix matrix.12 Ten weeks of supplying the five pesticides simultaneously further enhanced the linuron, metamitron and metalaxyl biodegradation capacities of the microcosms inoculated with the pesticide-primed material mix, showing that the introduced pesticide degradation capacity could easily proliferate in the biomix when present in the added material. This was consistent
wileyonlinelibrary.com/journal/ps
with previous observations with linuron-treated BM experimental set-ups bioaugmented with only linuron-primed soil5 and with metalaxyl-treated BPS mesocosm systems bioaugmented with metalaxyl-primed BPS material.4 The observed decrease in lag time and hence capacity can be related to a proliferation of the corresponding pesticide degraders. The observation that the increase in size of the pesticide-degrading/mineralising capacity was greater in pesticide-treated BMs compared with BMs without pesticide supply suggests that proliferation was at least partially at the expense of the applied pesticides, and hence that the corresponding pesticides were also degraded in the BMs themselves. It should be noted, however, that samples were taken only from the upper layer of the microcosms, and the degradation/mineralisation capacity might not have evolved in the same way for the entire volume of the microcosm. Often some increase in pesticide mineralisation/degradation capacity was also found in BMs treated with water without pesticides. This has been observed before and could be due to the growth of pesticide-degrading populations at the expense of the fresh organic material (straw) added to the system, or to the improvement in moisture content.3 The time needed to reach maximum degradation/mineralisation capacity in the BPS microcosms could be clearly associated with the initial recorded degradation/mineralisation lag time. The
© 2014 Society of Chemical Industry
Pest Manag Sci (2014)
Mixed pesticide pollution treated in a biopurification system larger the initial lag time, and hence the lower the initial degradation/mineralisation capacity, the more time was required to reach maximum degradation/mineralisation capacity. Moreover, initially, a large variability in degradation capacity between microcosms within a set-up was observed for linuron and metamitron. This variability disappeared after the 10 weeks of pesticide supply. These observations are similar to these previously observed in BPS microcosms5 for linuron degradation and in soil microcosms in other studies using other pesticides13 and are explained by the initial spatial heterogeneity of low numbers of pesticide degraders in the pesticide-primed mix.13 Growth of corresponding pesticide-degrading populations in the biomix would then lead to decreased heterogeneity and less variation in the outcome of the degradation/mineralisation assays. Interestingly, as degradation of metamitron and metalaxyl improved in set-up S+ , so did the degradation capacity of their metabolites. In the case of metalaxyl degradation, the first metabolite was converted to a second metabolite, which initially did not further degrade. However, after 10 weeks of pesticide supply, the second metabolite was also degraded, indicating the proliferation of a metabolite-degrading population in the matrix. This was not observed for set-up S− , suggesting again that the metabolites were also degraded in the BMs themselves. As the time needed to enhance the degradation of the metabolite was longer than for metalaxyl, it can be suggested that the degradation of the metabolite(s) is performed by (a) microbial population(s) that is/are different from those that degrade metalaxyl. The nature of the metamitron and metalaxyl metabolites is currently unknown. Metalaxyl is a phenylamide fungicide consisting of a diphenyl ring containing a complex secondary amide structure. Pathways for metalaxyl degradation are not well known. Several metabolites of metalaxyl have previously been identified.14,15 The metabolite detected in the present study is likely the primary degradation product designated as CGA 6286 (see supporting information Fig. S2).15 As compound CGA 6286 is toxic and has potential to leach to groundwater, EU directive 2002/64/EG imposes special measures to lower the risk for leaching when metalaxyl is applied in vulnerable areas.16 In one study, 50% of metalaxyl could still be found as product CGA 6286 in soil 10 weeks after application of the fungicide.17 Metamitron is a triazinone herbicide consisting of a triazine moiety substituted with an amino group, a methyl group and a phenyl ring. Two metabolic pathways for metamitron degradation have been proposed. In the first, degradation is initiated with the attack of the phenyl ring by a dioxygenase, which is then cleaved and mineralised, leading to 3-methyl-4-amino-1,2,4-triazin-5(4H)one.18 The second alternative pathway involves an initial amidohydrolase cleavage and degradation of the triazin ring, producing benzoylformate, which is further mineralised by the mandelate pathway.19 As the metamitron mineralisation curves were in accordance with the metamitron degradation curves, and as the 14 C label in the 14 C-labelled metamitron used as a substrate in the mineralisation tests is within the phenyl moiety, it is suggested that the metabolite from metamitron that was detected by UV absorption in the HPLC measurements is the triazin moiety of metamitron, and hence that degradation of metamitron in the S+ set-up occurs by initial attack of the phenyl ring. The mechanism behind the observed establishment and proliferation of the pesticide-degrading capacities is unknown and could either be by establishment and proliferation of added microbial populations or through horizontal gene transfer of the appropriate catabolic genes from organisms residing in the added materials towards microbial residents in the other materials used.20 – 23 In a previous Pest Manag Sci (2014)
www.soci.org study, the use of molecular tools and appropriate BM set-ups suggested that bacteria belonging to the genus Variovorax and originating from the linuron-primed soil could be linked to linuron degradation.5 Bentazon- and isoproturon-degrading capacities did not develop in the S set-ups, even after 12 weeks of feeding the pesticides to the microbiota in set-up S+ . Apparently, organisms that could degrade these compounds were absent in the corresponding primed material (as suggested by their inability to degrade/mineralise these compounds) or were not able to proliferate under the set-up conditions. The capacity to degrade/mineralise linuron and metamitron (including degradation of its metabolite) also improved in microcosms inoculated with the non-primed soil C (i.e. in set-up C+ ) upon continuous feed with the pesticide mix. Apparently, the soil contained microbiota, probably at initial low densities, that proliferated under the feed conditions. The proliferation of a linuron-degrading/mineralising microbial population upon feeding with the pesticide mixture was not unexpected as this was observed previously with soil C in other BM set-ups when fed with linuron.5 However, based on the lag times, the degradation and mineralisation capacities for both compounds was always much lower than those in microcosms bioaugmented with the pesticide-primed soils (i.e. in set-up S+ ). In addition, the capacity to degrade metalaxyl never developed in BMs of set-up S+ within the experimental time period. These observations show that the addition of pesticide-primed materials is beneficial for activation of BPS, as their inclusion in the biomix matrix resulted in a more rapid establishment and greater abundance of pesticide-degrading capacities than without the addition of primed materials.
ACKNOWLEDGEMENTS This research was supported by the IWT-Vlaanderen Agricultural Research project LBO 040272 and by the KBBE-EU project BACSIN Contract 211684.
SUPPORTING INFORMATION Supporting information may be found in the online version of this article.
REFERENCES 1 De Wilde T, Spanoghe P, Debaer C, Ryckeboer J, Springael D and Jaeken P, Overview of on-farm bioremediation systems to reduce the occurrence of point source contamination. Pest Manag Sci 63:111–128 (2007). 2 Karanasios E, Tsiropoulos NG and Karpouzas DG, On-farm biopurification systems for the depuration of pesticide wastewaters: recent biotechnological advances and future perspectives. Biodegradation 23:787–802 (2012). 3 Sniegowski K, Bers K, Ryckeboer J, Jaeken P, Spanoghe P and Springael D, Robust linuron degradation in on-farm biopurification systems exposed to sequential environmental changes. Appl Environ Microbiol 77:6614–6621 (2011). 4 De Wilde T, Spanoghe P, Sniegowski K, Ryckeboer J, Jaeken P and Springael D, Transport and degradation of metalaxyl and isoproturon in biopurification columns inoculated with pesticide-primed material. Chemosphere 78:56–60 (2010). 5 Sniegowski K, Bers K, Van Goethem K, Ryckeboer J, Jaeken P, Spanoghe P et al., Improvement of pesticide mineralization in on-farm biopurification systems by bioaugmentation with pesticide primed soil. FEMS Microbiol Ecol 76:64–73 (2011). 6 Dejonghe W, Boon N, Seghers D, Top EM and Verstraete W, Bioaugmentation of soils by increasing microbial richness: missing links. Environ Microbiol 3:649–657 (2001).
© 2014 Society of Chemical Industry
wileyonlinelibrary.com/journal/ps
www.soci.org 7 Pigeon O, De Vleeschouwer C, Cors F, Weickmans B, Huyghebaert B, Planchon V et al., Biofilters to treat the pesticides wastes from spraying applications: results after 4 years of study. Comm Agric Appl Biol Sci 71:9–19 (2006). 8 Pussemier L, De Vleeschouwer C and Debongnie P, Self-made biofilters for on-farm clean-up of pesticides wastes. Outlooks Pest Manag 15:60–63 (2004). 9 Dejonghe W, Berteloot E, Goris J, Boon N, Crul K, Maertens S et al., Synergistic degradation of linuron by a bacterial consortium and isolation of a single linuron-degrading Variovorax strain. Appl Environ Microbiol 69:1532–1541 (2003). 10 Broos K, Mertens J and Smolders E, Toxicity of heavy metals in soil assessed with various soil microbial and plant growth assays: as comparative study. Environ Toxicol Chem 24:634–640 (2005). 11 Sniegowski K, Mertens J, Diels J, Smolders E and Springael D, Inverse modeling of pesticide degradation and pesticide-degrading population size dynamics in a bioremediation system: parameterizing the Monod model. Chemosphere 75:726–731 (2009). 12 Sniegowski K, Bers K, Ryckeboer J, Jaeken P, Spanoghe P and Springael D, Minimal pesticide-primed soil inoculum density to secure maximum pesticide degradation efficiency in on-farm biopurification systems. Chemosphere 88:1114–1118 (2012). 13 Cullington JE and Walker A, Rapid biodegradation of diuron and other phenylurea herbicides by a soil bacterium. Soil Biol Biochem 31:677–686 (1999). 14 Pesaro M, Nicollier G, Zeyer J and Widmer F, Impact of soil drying– rewetting stress on microbial communities and activities and on degradation of two crop protection products. Appl Environ Microbiol 70:2577–2587 (2004). 15 Droby S and Coffey MD, Biodegradation process and the nature of metabolism of metalaxyl in soil. Ann Appl Biol 118:543–553 (1991).
wileyonlinelibrary.com/journal/ps
K Sniegowski, D Springael 16 Directive 2002/64/EG of the Commission of 15 July 2002, Alteration of Directive 91/414/EEG of the Council. [Online]. European Community, Publ. E.-1 (2002). Available: http://eur-lex.europa.eu/LexUriServ/ LexUriServ.do?uri=OJ:L:2002:189:0027:0032:NL:PDF [4 March 2014]. 17 Sukul P and Spiteller M, Metalaxyl: Persistence, degradation, metabolism, and analytical methods. Rev Environ Contam Toxicol 164:1–26 (2000). 18 Parekh NR, Walker A, Roberts SL and Welch SJ, Rapid degradation of the triazinone herbicide metamitron by a Rhodococcus sp. isolated from treated soil. J Appl Bacteriol 77:467–475 (1994). 19 Engelhardt G, Ziegler W, Wollnofer PR, Jarczyk HJ and Oehlmann L, Degradation of the triazinone herbicide metamitron by Arthrobacter spec. DSM 20369. J Agric Food Chem 30:278–282 (1982). 20 DiGiovanni GD, Neilson JW, Pepper IL and Sinclair NA, Gene transfer of Alcaligenes eutrophus JMP134 plasmid pJP4 to indigenous soil recipients. Appl Environ Microbiol 62:2521–2526 (1996). 21 Devers M, Henry S, Hartmann A and Martin-Laurent F, Horizontal gene transfer of atrazine-degrading genes (atz) from Agrobacterium tumefaciens St96-4 pADP1: Tn5 to bacteria of maize-cultivated soil. Pest Manag Sci 61:870–880 (2005). 22 Hirkala DLM and Germida JJ, Field and soil microcosm studies on the survival and conjugation of a Pseudomonas putida strain bearing a recombinant plasmid, pADPTel. Can J Microbiol 50:595–604 (2004). 23 Dunon V, Sniegowski K, Bers K, Lavigne R, Smalla K and Springael D, High prevalence of IncP-1 plasmids and IS1071 insertion sequences in on-farm biopurification systems and other pesticide polluted environments. FEMS Microbiol Ecol 86:415–431 (2013).
© 2014 Society of Chemical Industry
Pest Manag Sci (2014)
