Ash and biochar mixed biomixtures to degrade co-applied atrazine and fipronil in bio-augmented biobeds

ABSTRACT Retention and degradation of co-applied atrazine and fipronil was studied in laboratory constructed bio-augmented biobeds containing rice straw-compost (BM) and compost replaced with 10% rice husk ash [RHABM(10%)] or 1% wheat straw biochar [WBCBM(1%)] biomixtures. Atrazine (5000 µg, 50% WP) and fipronil (5000 µg, 0.3 G) were co-applied in 100 mL water. After 30 days, 7.31, 4.65, and 11.21 µg g−1 atrazine and 21.59, 7.41, and 37.20 µg g−1 fipronil remained undegraded in the top 3–4 cm of BM, RHABM(10%), WBCBM(1%) biobeds, respectively, suggesting that degradation of both pesticides significantly varied (p < 0.05) among the three biobeds. After 60 days 94.5%–98.3% of initially applied atrazine and 80.3%–86.9% fipronil degradation was observed. Hydroxyatrazine and sulphone were detected as the metabolites of atrazine and fipronil, respectively. Out of 5000 µg of initially applied pesticides, BM, RHABM(10%), and WBCBM(1%) biobeds accounted for 95.2%, 93.1%, and 95.2% of atrazine + hydroxyatrazine and 62.8%, 67.0%, and 71.5% of fipronil + sulphone degradation, respectively. Maximum amounts of recovered atrazine (68.8%–81.1%) and fipronil (89.8%–94.6%) were retained in the 0–5 cm sections of the biobeds. No significant effect of co-applied atrazine + fipronil was evident on the microbial parameters. This study suggested that RHA/WBC-mixed rice straw-compost biomixtures can be exploited for the detoxification of these pesticides in bio-purification systems.


Introduction
Safe handling and proper disposal of pesticide waste generated during spray operation is essential for environmental and human safety.Washing leftover pesticide and spray equipment, due to their poisonous nature, poses greater risks of pesticide migration and contamination to water bodies.Indian farmers are less aware and educated about safe disposal of pesticide waste.Therefore, studies related to the use of bio-purification (biobeds) systems developed using low-cost locally available resources to address the point source of pollution are important.Biobedmediated on-farm disposal of pesticide waste greatly reduces environmental load and toxicity of pesticides [1].Traditionally, biobeds contain a biomixture of soil, peat/compost, and lignocellulosic biomass [2].Substituting compost/peat with ash and biochar to enhance pesticide retention and enhanced degradation is an innovative approach.De Wilde et al. [3] and Mukherjee et al. [4,5] recommended digestate and biocharbased biomixtures to enhance pesticide retention and degradation potential.Mukherjee et al. [4] reported that addition of digestate and biochar to biomixtures enhanced bentazone, boscalid, and pyrimethanil sorption.Degradation studies suggested that a mixture of digestate (5%) and biochar (5%) gave optimal results with respect to mineralisation and simultaneous sorption for all three pesticides [5].Thus, these biomixtures can be promising substrate for a novel biobed setup.Reports suggest that ashes produced from burning of sugarcane bagasse, mustard plant, rice husk, rice straw, and wheat straw have considerable capacity to adsorb 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA), diuron, and pretilachlor [6][7][8][9][10][11]. Further, ashes enhanced degradation of sulfosulfuron and pretilachlor in soils and water [12,13]; therefore, can be used to enhance pesticide retention in biomixtures as they are available locally at no cost.
Few studies exist related to the use of bio-purification systems for pesticide waste disposal under Indian tropical environment [14,15].Atrazine, a pre-and postemergent systemic herbicide for monocot and dicot weeds, and fipronil, a broadspectrum systemic insecticide for sucking insect pests, are used in sugarcane plantation in India.Both pesticides are persistent in nature with half-life (t1/2) of 60 to 70 for atrazine [16] and 132 days for fipronil [17].Hydroxyatrazine and fipronil sulphone, major metabolites of atrazine and fipronil, too are highly persistent with t 1/2 of 121 days [18] and 188 days [17], respectively.Sulphone is a metabolite of toxicological relevance; therefore, it is included in the residue definition for fipronil.Recently, authors have reported that rice husk ash (RHA), sugarcane bagasse ash (SBA), and wheat straw biochar (WBC)-mixed rice straw-compost biomixtures act as alternative to the traditionally used biomixtures for these two pesticides [19,20].Compared to control (biomixture without RHA and SBA), ashes (10%) significantly enhanced atrazine adsorption; while, WBC increased adsorption of both atrazine and fipronil [20].Degradation study suggested that RHA/SBA (10%) and WBC (1%) did not show any effect on the degradation of individually applied atrazine or fipronil; however, WBC (5%) slowed atrazine (200-300%) and fipronil (40-60%) degradation.Bioaugmentation with atrazine/fipronil degrading microbial cultures enhanced degradation of both pesticides and effect on atrazine was significant (p -0.05) [14].Based on the observations of sorption and degradation studies, 10% RHA and 1% WBC-mixed biomixtures were identified as the best biomixtures for optimum retention and degradation of individually applied atrazine or fipronil.However, adsorption and degradation dynamics change when pesticides are co-applied.This can be attributed to the competition for adsorption sites and combined toxicity of pesticides to microbial community.Therefore, the present study evaluated comparative retention and degradation of co-applied atrazine and fipronil, two sugarcane pesticides, in ricestraw-compost (BM), 10% RHA and 1% WBC mixed biomixtures in laboratory constructed biobeds augmented with atrazine and fipronil degrading microbes.Effect of co-applied pesticides on microbial parameters was monitored.

Biobed setup and degradation studies
Based on the results obtained in the previous adsorption [19] and degradation studies [20], three biomixtures [BM, RHABM(10%) and WBCBM(1%)] were used for biobed setup.Biobeds were installed in laboratory and were constructed in 5 L capacity aspirator bottles (28 cm height × 28 cm diameter) fitted with a tap (Supplementary Figure A).The bottom of the container was filled with 1200 g of gravels of average size of 2 × 2.5 × 0.7 cm.Above the gravel, 885 g (oven dry weight) of biomixture [BM/ RHABM(10%)/WBCBM(1%)] was packed upto to 15 cm height.Biobeds were supplemented with sterile water to provide moisture content of 60% water holding capacity (WHC).Biobed were kept undisturbed for 10 days at room temperature (20 ± 5°C) for conditioning.Then, biobeds were fortified with 100 mL aqueous solution of atrazine (Atrataf, 50% WP) and fipronil (Regent, 0.3 G) formulations at 50 μg mL −1 concentration.Simultaneously, they were inoculated with atrazine degrading enrichment culture [24] and fipronil degrading bacteria, Bacillus megaterium [25].Biobeds were covered with a plastic sheet having 10 pinholes for gaseous exchange and incubated at room temperature.After 30 days, biomixture samples from all biobeds were taken using core sampler, 3-4 cm from the surface at 5 spots.Biomixture samples were analysed for pesticide residues and microbial activity.After 60 days, the experiment was discontinued, biobeds were dismantled, and biomixture from each biobed was divided horizontally into three sections of 5 cm each.The biomixture samples from each section were analysed for pesticide residues and microbial activity.

Microbial activity
Microbial biomass carbon (MBC) content was estimated by chloroform fumigation extraction method [26], dehydrogenase activity by monitoring the rate of production of triphenyl formazan (TPF) from triphenyl tetrazolium chloride [27] and fluorescein diacetate hydrolysis activity (FDA) assay by method reported by Green et al. [28].

Pesticide extraction and analysis
Pesticide residues from biomixtures were extracted using the dipping and shaking method reported by Kumari et al. [20].Briefly, biomixture samples (1 g, in triplicate from each biobed) were taken in 100 mL Erlenmeyer flasks, supplemented with 2 mL water and extracted using 20 mL ethyl acetate by shaking (1 h) on a rotary shaking machine.After shaking, 1 mL supernatant was evaporated at room temperature and reconstituted in 1 mL acetonitrile.Samples were analysed for atrazine and fipronil and their metabolites using liquid chromatography -mass spectroscopy (LC-MS/MS).
Samples were analysed for atrazine, fipronil, and their metabolites using Shimadzu LC-MS/MS (Model: LC-MS/MS-8030) as reported previously by Kumari et al. [20].The LC was equipped with C-18 column (100 mm length, 3 mm i.d.3.5 µm film thickness) and was attached with Triple Quadrupole mass detector.Multiple reaction monitoring (MRM) optimisation for identification of quantifier and qualifier ion transitions of atrazine, fipronil, and metabolites were done under electron spray ionisation (ESI) in positive/ negative mode (Supplementary Table B).Mobile phase was 1:1 mixture of A (5 mM ammonium formate in water: methanol, 80:20) and B (5 mM ammonium formate in water: methanol, 10:90).Argon was used as collisionally activated dissociation gas while, nitrogen gas at flow rate of 3 and 15 mL min −1 was used for nebulising and drying, respectively.The Desorption Line temperature and Heat Block temperature were maintained at 120 °C and 300 °C, respectively.Identification and quantification of pesticide/ metabolite was done using calibration curve of quantifier ion transitions (Supplementary Table B; Figures B, C).The limit of detection (LOD) of atrazine/metabolites was found to be 0.003-0.01µg mL −1 and limit of quantification (LOQ) was found to be 0.01-0.04µg mL −1 .The LOD of fipronil/metabolites was found to be 0.01-0.05µg mL −1 and LOQ was found to be 0.03-0.08µg mL −1 .

Statistical analysis
Degradation data was subjected to statistical analysis (one-way ANOVA) using IBM SPSS Statistics for Windows, Version 21.0, Armonk, NY.
The present study suggested that (i) pesticides and their metabolites were mainly restricted in the surface 0-5 cm sections and (ii) atrazine and hydroxyatrazine degraded at faster rate than fipronil and sulphone.Biobeds were highly effective in degrading atrazine + hydroxyatrazine as only 4.80%, 6.93%, and 4.84% of initially applied atrazine remained undegraded after 60 days in BM, RHABM(10%), and WBCBM(1%) biobeds, respectively.Thus, enrichment culture was highly effective in degrading atrazine to hydroxyatrazine and hydroxyatrazine to CO 2 , NH 3 , and H 2 O via the formation of cyanuric acid and biuret [24,29].Findings of this study are very encouraging as hydroxyatrazine is highly persistent in nature with a half-life of 121 days [18].Persistence of hydroxyatrazine is attributed to its poor water solubility of 5.9 mg L −1 and higher sorption to soil organic carbon.Fipronil was relatively more persistent than atrazine in all biobeds (Figure 2) and 80.29%, 84.53%, and 86.92% of fipronil or 62.8%, 67.0%, and 71.5% of fipronil + sulphone degradation was observed in BM, RHABM(10%), and WBCBM(1%) biobeds, respectively.,Findings of this study suggesting that B. megaterium was capable of degrading sulphone formed from fipronil degradation in the bio-augmented biomixtures and will help in addressing toxicity-related issues with fipronil.Degradation of co-applied atrazine and fipronil in biobeds was relatively slower than the degradation observed with individual pesticide [20].When applied alone, atrazine showed 100% degradation in BM, RHABM(10%) and WBCBM(1%) biomixtures in 25 days while ~90% fipronil degradation was observed after 37 day.Thus, slow degradation of co-applied atrazine and fipronil, which is the scenario under real condition, can be attributed to (i) low temperature as the biobed experiment was performed at room temperature, which fluctuated between 15°C and 25°C and (ii) coapplication of both pesticides might have affected microbial community structure.This is the first report on pesticide degradation in the ash-mixed biomixtures and our results suggested that they provide an alternate to traditional biomixtures.Biochar-mixed biomixtures used by previous researchers indicated inhibition of pesticide degradation [5,30].Biochar content used in these studies was relatively higher (>5%) than used in the present study and our results suggested no significant inhibition of atrazine degradation while 1% WBC enhanced fipronil degradation.
After 60 days, MBC content in the 0-5 cm section of BM (2884.77µg C g −1 ).RHABM (10%) (2885.88µg C g −1 ), and WBCBM(1%) (2604.86 µg C g −1 ) biobeds was slightly higher than that observed at 30 th days, but the difference was non-significant.The dehydrogenase activity (µg TPF released g −1 biomixture d −1 ) in different sections greatly varied and was significantly less in the top section (43.11-52.9)than the middle (96.16-114.54)and bottom (96.97-116) sections and was less than that observed on the 30 th day.Atrazine/ fipronil metabolites probably significantly decreased dehydrogenase activity.Dehydrogenase activity in the 5-10 and 10-15 cm sections was nearly double of the activity in the top section, where concentration of atrazine and fipronil/metabolites was notably less than that detected in the 0-5 cm section.The FDA activity, compared to activity on 30 th day, significantly increased on 60 th day in all biobeds.No difference in FDA activity among different section was observed and it varied from 21.68 to 25.67 µg, 19.53 to 25.28 µg, and 21.86 to 26.02 µg fluorescein released g −1 2 h −1 in BM, RHABM(10%), and WBCBM(1%) biobeds, respectively.Results suggested that there was no variation in MBC content and enzymes estimated among different biobeds.Preconditioning of biobeds probably built their tolerance to the negative effects of pesticides/metabolites as it significantly increased microbial activity and diversity [31,32].There are mixed reports about the role of microbial parameters, viz., MBC, phenoloxidase activity, respiration, and FDA activity and pesticide degradation in biomixtures.Castillo and Torstensson [2] reported that pesticide degradation positively correlated with phenoloxidase and respiration activity, while Karanasios et al. [33] observed no correlation as effect varied with the nature of pesticide [34].

Conclusion
Innovative biomixtures prepared using locally available materials have shown enhanced adsorption capabilities and can play a crucial role in the success of bio-purification systems.Rice husk ash (RHA, 10%) and wheat straw biochar (WBC, 1%) mixed rice strawcompost (BM) biomixtures were used for atrazine and fipronil degradation in laboratory setups of biobeds.Biobeds were bio-augmented with atrazine degrading enrichment culture and fipronil degrading B. megaterium.The study concluded that all three biomixtures were effective in retaining co-applied atrazine and fipronil (5000 µg each) in the surface layer.Compared to fipronil (80.29% to 86.92%), atrazine (96.91% to 98.61%) degraded easily.Besides the parent compounds, toxicologically relevant and persistent metabolites of atrazine (hydroxyatrazine) and fipronil (sulphone) were also degraded.This study suggested that biomixtures made of low cost and easily available materials and bioaugmented with pesticide degrading microbial cultures have potential use in biopurification system(s) for successful pesticide degradation.

Table 2 .
Atrazine and fipronil and their metabolites recovered from the rice straw-compost (BM), rice husk ash [RHABM(10%)] and wheat straw biochar [WBCBM(1%)] biomixture in constructed biobed setup after 60 days.Figures in the parenthesis are percent amount Values followed by different letters within a column are significantly different at 5% level based on the Duncan's multiple range test performed using SPSS. *