Meinan Zhen ,Benru Song ,Xiaomei Liu ,Radhika Chandankere ,Jingchun Tang ,*

1 Key Laboratory of Pollution Processes and Environmental Criteria(Ministry of Education),Tianjin Engineering Center of Environmental Diagnosis and Contamination Remediation,College of Environmental Science and Engineering,Nankai University,Tianjin 300071,China

2 Department of Building,Civil and Environmental Engineering,Concordia University,Montreal,H3G 1M8,Canada

Keywords:Organophosphorus pesticides Biochar NH3NO4 Remediation Greenhouse gas Microbial community

A B S T R A C T Organophosphorus pesticides(OPPs)are a setoftoxic persistentorganic pollutants(POPs)presentin the environment.Recently,biochar-mediated bioremediation has exhibited many advantages over conventional methods for the remediation of pesticide-contaminated soil.In the present study,biochar and nitrogen fertilizer(NH4NO3)were employed to remediate OPP-contaminated soil and the greenhouse gas(GHG)emission during 90 days of incubation was investigated.After thermal desorption treatment,the content of organophosphorus pesticides reduced from 175.61 μg·kg-1 to 62.68 μg·kg-1.The addition of NH4NO3 in the following bioremediation led to larger reduction(34.35%)of the pesticide concentration than that of biochar(31.90%)for the contaminated soils with thermal desorption treatment,while the simultaneous addition of biochar and NH4NO3 led to the largest reduction of pesticide concentration(11.07%)for the soil without thermal desorption treatment.The addition of biochar and NH4NO3 only slightly increased the emission rate of GHGs from the soil without thermal treatment,but remarkably increased the emission rate of GHGs from the soil after thermal treatment.In most cases,the addition of NH4NO3 is more effective than biochar to promote the degradation of pesticide,but also exhibited higher GHG emission.The microbial community analysis suggests that the enhanced degradation of pesticide is mainly owing to the increased activity of microorganism.

1.Introduction

Modern agricultural practices use large amounts of pesticides that in turn has increased environmental pollution,leading to serious health concerns.These pesticides are toxic and can accumulate easily into non-target organisms including humans[1–3].Organophosphorus pesticides(OPPs)are a set of persistent organic pollutants(POPs)that are toxic,persistent in the environment,and can be biomagnified through the food chain[4].OPPs are commonly used in China for agriculture and the chronic application of pesticide has resulted in some serious environmental problems[5].Many researchers have extensively studied the impact of OPPs on the soil system near various pesticide producing industries[6,7].Additionally,high concentrations of OPP residues in soils can be volatilized and subsequently enter into the vegetation or animals through the air.As a result,vegetation and terrestrial animals around pesticide-producing industries may be at risk[8].These results support the general concern towards OPP-mediated contamination into the terrestrial ecosystems.For example,it has been reported that methyl parathion inhibits the cholinesterase enzyme in the human brain,causes a genotoxic effect in mammalian somatic cells in culture medium and increases the abnormalities in chromosomal structure[9,10].

There are a number of sites contaminated by OPPs in areas designated for commercial development in China[11].It is very important to develop effective methods to remediate these sites.Of the wide range of remediation technologies used for the treatment of pesticide contaminated sites,thermal desorption is one of the most commonly employed methods[7].According to the specific physical and chemical properties of the targeted volatile organic pollutants,an external heat source is used to heat the soil and promote the volatilization of pollutants.After heating treatment,a recovery unit is used to capture the volatile organic matter[12].It is important to note that the central goal of remediation is to restore the ecological function of the contaminated soil[13].However,the toxicity of pesticide in soils after thermal desorption treatment is still very high and requires further treatment before it is safe for use[14–16].

According to Beesley et al.[17],biochar derived from renewable biomass appears very suitable for the bioremediation of contaminated soils.Biochar plays a positive role in carbon sequestration,reduction of greenhouse gas emissions and improvement of soil fertility[18–20].Meanwhile,the presence of biochar in contaminated soil not only enhances the sorption of different pollutants but also affects their bioavailability[21,22].Numerous studies have demonstrated the beneficial effects of biochar for pesticide sorption,compared to soil organic matter[23–25].Moreover,previous studies have demonstrated that the application of biochar and nitrogen fertilizer remarkably affects the emissions of greenhouse gases(GHG),including CO2,CH4and N2O from forest soil[27].Both the remediation performance and the emission of GHG are closely related to the activity of microorganisms.Therefore,it's very necessary to systematically investigate the effect of biochar on the bioavailability and toxicological effect of pesticides,the activity of microorganisms as well as its subsequent impacton the emission of greenhouse gas(GHG),to comprehensively understand the remediation performance of biochar to guide the practical application.

In the present study,biochar and NH4NO3were applied to the OPP contaminated soil to enhance bioremediation,to regulate the greenhouse gas emission and to reduce the toxicity of the contaminated soils.The concentrations of six representative OPPs in the contaminated soil after different treatments were analyzed.At the same time,the emission rate of greenhouse gases,including CO2,CH4and N2O was determined during the incubation process.Furthermore,the toxicity of pesticides in the treated soil was assayed by mortality and massloss of earthworm using Eisenia fetida as model animal.

2.Materials and Methods

2.1.Chemicals and reagents

Methamidophos,phorate,isocarbophos,terbufos,malathion,parathion and methyl parathion(purity＞98%)were purchased from the Ministry of Agriculture Environmental Monitoring Station(Tianjin,China).All other chemical reagents were of analytical grade and were used without further purification.

2.2.Preparation of biochar

Biochar was produced by the pyrolysis of rice husk.The rice husk(RH)used in this work was collected from Nanjing,Jiangsu Province,China and washed with deionized water 4 times to remove dirt and then dried in the oven at 60°C.The dried RH was loosely placed in a muffle furnace for 3 h at a temperature of 700°C under oxygenlimited condition[26].The biochar was washed by water and ethanol sufficiently to remove free metal ions and then ground to pass through a 0.50 mm sieve.The samples were then washed with deionized water for several times to remove impurities,oven-dried at 80°C,and sealed in a glass container before use.The pH and electrical conductivity of biochar were determined to be 7.94(±0.05)and 102(±2) μS·cm-1,respectively,according to the method in International Biochar Initiative(IBI)protocols[27].The surface area of the resulting biochar was determined to be 285.5 m2·g-1.The C,H,O and N contents were 46.48%,1.09%,1.87%and 0.39%,respectively.The Si content was 4.17 g·kg-1and the P,S,Ca,Mg,K,Zn and Mn contents were 683.17,58.90,303.13,121.93,598.80,2.60 and 24.90 mg·kg-1,respectively.

2.3.Site description and soil preparation

All soil samples were taken from an abandoned OPP producing factory located in the Beichen district of Tianjin,China.Soil samples were collected from the surface to a depth of 200 mm.The 0.3 m of surface soil was drilled with an auger of 0.10 m in diameter,then a PVC ground sleeve was placed into the borehole to prevent contamination resulting from topsoil material.A clean core was retrieved for every 0.1 m by removing the soil from the flanks and the top and bottom of the core with a knife.Each soil sample was stored separately in a firmly closed plastic box.All the soil samples were air dried for 7 days and properly homogenized and ground to pass through a 2 mm sieve.Finally,all the samples were stored in glass bottles at 4°C prior to further analyses.The soil contained approximately 22%of sand,55%of silt,23%of clay and 2.52%of organic carbon(OC).The physicochemical properties of the soils were determined and listed in Table 1.

2.4.Experimental design

Contaminated soil(150 g)was placed in a 250 ml flask,to which different doses of biochar and NH4NO3were added and then mixed thoroughly.The mixture was incubated at 25°C for a certain period.Initial contaminated soils with thermal desorption and without thermal treatment were named A0and B0,respectively.Eight treatments were tested,including(1)A:contaminated soil after thermal desorption treatment,(2)A+N:NH4NO3loadings(150 mg·kg-1and 300 mg·kg-1)were added to sample A,(3)A+C:biochar loadings(1 wt%,3 wt%and 6 wt%with respect to the mass of soil)were added to sample A,(4)A+C+N:both NH4NO3(300 mg·kg-1)and biochar(6 wt%with respect to the mass of soil)were added to sample A,(5)B:initial soil contaminated by pesticides,(6)B+N:NH4NO3loadings(150 mg·kg-1and 300 mg·kg-1)were added to sample B,(7)B+C:biochar loadings(1 wt%,3 wt%and 6 wt%with respect to the mass of soil)were added to sample B,and(8)B+C+N:both NH4NO3(300 mg·kg-1)and biochar(6 wt%with respect to the mass of soil)were added to sample B.The incubation units were weighed daily to determine the evaporation amount of water,in order to estimate the water content in soil sample.The relative water content(RWC)in all the treatments was maintained at approximately 40%during the treatment process by adding deionized water.Each treatment was carried out in triplicate and incubated for 3 months.

2.5.Analysis of physical and chemical properties

2.5.1.Conductivity of salinity and pH

Glass containers with soil samples were removed from the refrigerator and stored at room temperature in the dark for at least 12 h before processing.In brief,5 g freeze drying soil(1:5 soil:deionized water,w/w)was added to water.The conductivity and pH of the soil suspension were measured by a conductivity meter(Mettler Toledo,FiveEasy)and a PB-10 pHmeter,respectively(Sartorius,Gottingen,Germany).The solution mixture was centrifuged at 3000 r·min-1for 15 min and supernatant was collected.Hydrogen peroxide(6%)was added to the supernatant as an oxidant and then freeze-dried to weigh residual and calculate the total dissolved salts[28].All samples were analyzed in triplicate.

2.5.2.GHG flux measurements

The GHG flux measurements of soils were carried out by a Greenhouse Gas Analyzer G2508(Picarro Inc.,Santa Clara,CA,USA)daily between 8 a.m.and 8 p.m.over a 90-day period[27,29].The measurement was conducted according to the process described in a previous study[27].Briefly,the soil sample was weighted and placed in the static closed chamber which is connected to the analyzer for 30 min.During this process,the concentration of CO2,CH4and N2Owas measured every 2 s.The emission rate of CO2,CH4and N2O was determined from the accumulation rate of greenhouse gas concentration in the chamber.The emission rate is calculated according the following equation:

where F[μg·(kg soil)-1·h-1]is the emission rate of GHG.V(1.25 ×10-3m3)is the total volume of the system,including the chamber and pipeline.Ciand Cf(μl·L-1)represent the initial and final concentrationof the CO2,CH4and N2O,respectively.T(K)and P(kPa)are the absolute temperature and pressure in the chamber during the measurement process.T0(273.15 K)and P0(101.325 kPa)are the absolute temperature and pressure under standard state.Δt(h)is the measurement time and ms(g)is the mass of soils ample.k(1.96×103,0.71×103and 1.96 × 103μg·m-3for CO2,CH4and N2O,respectively)is the coefficient which converts ppm into mass concentration(μg·m-3)under ideal state.

Table 1Physicochemical properties of the soil samples after 90 days'treatments

2.5.3.Extraction and analysis of OPPs

Samples were collected at different days and stored at-20°C for further analyses.OPPs were extracted from each sample using Soxhletextraction by using 5 g of dried soil samples with 100 ml extraction solvent(1:1,hexane:acetone,v/v)for 24 h.Individual standard solutions of pesticide were prepared using methanol:water(8:2 v/v)+5 mmol·L-1of ammonium acetate as solvent with a concentration of 200 mg·L-1.The chromatographic analysis was performed on a HPLC(Waters 1525,USA),equipped with a quaternary pump,an automatic injector and a temperature-controlled column compartment connected to a Quattro micro API triple quadrupole mass spectrometer,equipped with a Z-spray electrospray ionization source,from Micromass(Manchester,UK).The chromatographic separation was performed with an Atlantis dC18(2.1 mm × 150 mm,5 μm)column from Waters.The mobile phase components used were(A)methanol:water(1:9)+5 mmol·L-1ammonium acetate and(B)methanol:water(9:1)+5 mmol·L-1ammonium acetate with a constant flow rate of0.3 ml·min-1.The injection volume was 20 μl and the temperature of the column compartment was set to 30°C[30].

2.5.4.Analysis of the microbial community

In this section,the soil after different treatments was sampled at certain times.Total microbial DNA was extracted from each soil sample using a DNA MiniPrep?kit(Zymo Research,USA)following the protocol provided by the manufacturer.Agarose gel electrophoresis and 16S rDNA were used to analyze the bacterial communities[31].The primer set targeting the V3 region of bacterial 16S rDNA consisted of GC-338f(5′-GC clamp-CAC GGG GGG ACT CCT ACG GGA GGC AGC AG-3′)(GC clamp=CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG G)and 518r(5′-ATT ACC GCG GCT GCT GG-3′)[32].PCR amplicons were loaded with loading dye into 8%polyacrylamide gels(37.5:1 acrylamide:bisacrylamide)with a denaturing gradient from 40%to 60%(100%denaturant consisted of 7 mol·L-1urea and 40%form amide,v/v).Conditions for PCR were set according to the manufacturer:initial denaturation at94°C for 2 min,30 cycles of denaturing at 94 °C for 20 s,annealing at 55 °C for 1 min,and extension at 72 °C for 2 min.The DGGE analysis was carried out on a Universal Mutation Detection System D-code(BioRad,CA,USA)at 150 V and 60°C for 4 h.Gels were then stained with ethidium bromide,and visualized with an UV transilluminator(ATTO orporation,Japan).Dendrogram and intensity analysis of DGGE banding patterns were performed using Quantity One 4.6(Bio-Rad Laboratories,CA,USA).The extracted DNA was sentto Beijing Genomics Institute(Wuhan,China)for pyrosequencing.Calculation of the pair-wise similarities was based on the Dice correlation coefficient and dendrograms were created using the algorithm of unweighted pair-group method with arithmetic averages(UPGMA).

2.5.5.Toxicity analysis

Earthworms(E.fetida)were purchased from the College of Animal Sciences,Zhejiang University,China.Earthworm cultivation was carried out as with slight modification as reported earlier[33].Briefly,E.fetida was acclimatized in the mixture of manure at(20 ±1)°C for 2 weeks.Mature earthworms with mass between 300 and 600 mg were selected and starved for 24 h before adding into the artificial soil.The artificial soil was prepared with the following composition:70%industrial sand,20%kaolin,and 10%sphagnum peat moss with the pH adjusted to 6.0±0.5 using CaCO3(OECD 1984;ISO 11268-1 1993).All the experiments were carried out in triplicate and each replicate had ten depurated earthworms.The water content in soil was measured every day and 35%of water-holding capacity was maintained by adding distilled water as needed[34].Mortality and mass of earthworms were recorded after 7 days.

3.Results and Discussion

3.1.Physical and chemical properties of soil

Thermal desorption treatment and the addition of biochar and NH4NO3both could influence the conductivity,salinity and pH of soil[35].The pH value is a very important factor for soil,which not only affects the dissolution and oxidation reduction of minerals,but also affects the activity of living organisms[36].The thermal desorption treatment slightly increased the pH of soil,while the addition of NH4NO3buffered the soil from weakly alkaline to neutral due to the alkalinity of biochar(Table 1).This is in accordance with previous report[27].The thermal desorption treatment decreased the total salt content from 0.81%to 0.79%.The addition of biochar to soil A decreased the total salt content from 0.79%to 0.61%,while the addition of NH4NO3increased the total salt content from 0.79%to 0.82%.Thermal desorption treatment had obvious effect on the electrical conductivity of the soil sample.For soil A the addition of biochar reduced the conductivity from 723.26 to 664.93 μS·cm-1,while the addition of NH4NO3increased the conductivity to 787.63 μS·cm-1.Itwas inferred that the improved soil physical and chemical properties are related to the activity of microorganisms in soil[39].

3.2.Changes in concentration of organophosphorus pesticides

Previous studies showed that the use of biochar has complex influences on the transportation and degradation of different pesticides in soil.For instance,Felipe et al.reported that the use of biochar significantly promotes the degradation of iprodione[38].In contrast,Tang et al.reported that the employment of biochar is adverse to the degradation of difenoconazole in soil[39].Therefore,it's very important to investigate the impact of biochar on the microbial degradation of different pesticides in soil,in order to comprehensively evaluate the feasibility of using biochar in soil.As shown in Fig.1(a),thermal desorption treatment reduced the total concentration of OPPs from 175.61 to 62.68 μg·kg-1.It was observed that for soil A the addition of biochar and NH4NO3reduces the pesticide concentration by 31.90%and 34.35%,respectively.Furthermore,the simultaneous addition of biochar and NH4NO3only led to slightly higher reduction(36.01%)of pesticide concentration,suggesting that there is an antagonistic effect between biochar and NH4NO3for the bioremediation of soil with relatively low concentration of pesticides.Among the tested pesticides,the degradation rates of different kinds of pesticides decrease in the following order:methyl parathion＞parathion＞isocarbophos＞malathion＞terbufos＞methamidophos.For soilB,the separate addition of biochar or NH4NO3could only reduce the pesticide concentration slightly(8.22%and 6.44%,respectively).In contrast,the simultaneous addition of biochar and NH4NO3led to more remarkable reduction of pesticide concentration(11.07%),suggesting that there is a synergistic effect between biochar and NH4NO3for the bioremediation of soil with high concentration of pesticides.The different phenomena observed with soil A and soil B were mainly attributed to the different activities of microorganisms in different soil samples.In soil A with relatively high concentration of pesticide,the activity of microorganisms can be stimulated either by biochar or NH4NO3.In contrast,only simultaneous application of biochar and NH4NO3could stimulate the highest activity of microorganisms in soil B owing to its high toxicity to microorganisms.

Fig.1.(a)Concentration of OPPs in soil samples after 90 days' treatment.(b)Degradation rate of parathion.Condition:the weight percent of biochar is6 wt%with respect to soil and the concentration of NH4NO3 is 300 mg·kg-1.

Since the concentration of parathion is the highest in all the soil samples,we further investigated the degradation rate using parathion as a representative pesticide.As shown in Fig.1(b),the degradation rate of parathion increased with the treatment days,and the highest degradation rate occurred from 30 days to 60 days.This may be due to the fact that the microorganisms in soils have the fastest growth rate during this period[41].The lower removal capacity of biochar or NH4NO3in soil B,compared with soil A,was attributed to the higher toxicity of soil B which inhibits the growth of microorganisms[41].The thermal desorption treatment remarkably decreased the concentration of OPPs,thus obviously alleviating their inhibition to the activity of microorganisms and then promoting the degradation of OPPs.

3.3.Greenhouse gas emission during remediation process

During the remediation process,the emission rates of CO2,CH4and N2O were measured.As shown in Fig.2(a),the addition of biochar to soil A increased the emission of CO2,but the increment is limited.For all treatments,the measured CO2emission rates were continuously increasing from the first day to the 45th day of incubation.After 45 days of incubation,the CO2emission rates decreased slightly.When the loading of biochar is 6 wt%with respect to the mass of soil,the maximal CO2emission rate increased from 1801 to 2366 μg CO2·(kg soil)-1·h-1(approximate 31.37%).When 300 mg·kg-1of NH3NO4was added to soil A,the maximal CO2emission rate increased from 1801 to 5097 μg CO2·(kg soil)-1·h-1(approaching 1.8 times).In contrast,the simultaneous addition of biochar and NH3NO4could not further increase the emission of CO2,compared with only adding NH3NO4.Meanwhile,in view of that the dose of NH3NO4is remarkably lower than that of biochar,these results suggest that the addition of NH3NO4is more effective in enhancing microbial growth in soil than biochar.The CO2emission rate from soil B(Fig.2(b))is significantly higher than that from soil A during the initial several days of incubation,because the thermal treatment may eradicate most of indigenous microorganisms.In contrast,the CO2emission rate increases slightly with the incubation time for all the treatments.The maximal CO2emission rate[3300 μg CO2·(kg soil)-1·h-1]obtained with soil B is obviously lower than that[5097 μg CO2·(kg soil)-1·h-1]obtained with soil A,indicating the respiration intensity in soil B is obviously lower than that in soil A.The addition of NH3NO4to soil B leads to the largest increment(23.3%)of CO2emission rate.These results suggest that the addition of NH3NO4and biochar,especially the former could help to recover the activity of microorganisms and then result in the improvement of CO2emission rate,which is in accordance with previous reports[29].

CH4is generally the main end carbon-product under anaerobic conditions and the emission rate of CH4is closely related to the total activity of soil microbes and the quantity of methanogens[40].As exhibited in Fig.2(c)and(d),the emission rates of CH4increased slightly during the first 7 days of incubation when biochar or NH3NO4was added to soil A,and then decrease in the following days of incubation.The separate addition of biochar and NH3NO4to soil A increased the maximal CH4emission rate by 18.0%and 26.2%,respectively.The simultaneous addition of biochar and NH3NO4to soil A increased the maximal CH4emission rate by 27.8%.In the subsequent period,the CH4emission rate deceased gradually to a level approaching the control group.In contrast,the CH4emission rate in soil B changed slightly with time and the addition of either biochar or NH3NO4had little influence on the emission of CH4.

As shown in Fig.2(e)and(f),the N2O emission rate in soil A increased slightly until the 30th day of incubation and then decreased gradually for most of treatments,except for treatment A+C+N

where the maximal N2Oemission rate occurred at45 days of incubation.The addition of biochar to soil A only increased N2O emission slightly,while the addition of NH3NO4increased the maximal N2O emission rate by 1.96 times.When biochar and NH3NO4were simultaneously applied to soil A,the maximal N2O emission rate increased by 2.71 times.The maximal N2O emission rate obtained with soil A[0.27 μg N2O·(kg soil)-1·h-1]was obviously lower than that obtained with soil B[0.27 μg N2O·(kg soil)-1·h-1].For soil B,the N2O emission rate is almost unchanged when biochar is added.In contrast,the addition of NH3NO4increased the maximal N2O emission rate by 1.69 times.The N2O emission rate is mainly related to soil nitrifying bacteria and denitrifying bacteria[42].Since adding biochar could improve the activity of microorganisms in soil[43],biochar can affect the emission of N2O by influencing the soil microbial activity.

Fig.2.Greenhouse gas emission from soil samples with thermal treatment(a,c and e)and without thermal treatment(b,d and f)during the 90 days'treatment.In this section,different loadings of NH4NO3(150 mg·kg-1 and 300 mg·kg-1)and biochar(1 wt%,3 wt%and 6 wt%with respect to soil)were used.

3.4.Microbial quantity and community analysis

Fig.3.DGGE analysis results of OPP bioremediation process and cluster analysis of microbial community of soil sample with thermal desorption.Control:A0 soil before remediation.Sets I,II,III and IV correspond to treatments A,A+C,A+N and A+C+N,respectively.(b)Cluster analysis by DGGE.(c)Phylogenetic tree analysis based on the 16S rDNA sequences.A1–A13 represent the 16S rDNA sequences corresponding to the selected bands in the DGGE profile.

The microbial community structure and diversity from different samples were investigated by PCR-DGGE[44].Based on PCR-DGGE analyses of soil with thermal desorption treatment(Fig.3(a)),more dominant microbial species were found in set III compared to other sets.The dominant bacteria in soil B0were Pseudomonas sp.,Salinicola sp.,Chromohalobacter sp.and Halomonas sp.,while the dominant bacteria in soil A0were Sphingomonas sp.and Pseudomonas sp.The species of microorganisms in soil A0were obviously lower than that in soil B0,indicating that the thermal desorption treatment eradicated some indigenous microorganisms.For soil A,the dominant bacterial in soil samples were Pseudomonas sp.and Azotobacter sp.after the addition of NH4NO3.After the addition of biochar,the dominant bacterial in soil samples were Sanguibacter inulinus.After simultaneous addition of both biochar and NH4NO3the dominant bacterial in soil samples were similar to that from soil samples with only NH4NO3.Among these soil bacteria,Azotobacter sp.was dominant due to its ability to fix nitrogen(Fig.3(c)).Previous studies have reported the role of Azotobacter sp.in the reduction of N2to ammonia[45].These results indicate that NH4NO3plays important roles in the proliferation of the complex microbial community[46].For soil B,the species of microorganisms from the initial soil are similar with those from soil after the addition of either biochar or NH4NO3(Fig.4(a)and(b)).However,the samples with the addition of biochar,NH4NO3or both were clustered in one group,respectively.This indicates that the different treatments have certain effects on the microbial community structure,though a slight change in the dominant bacteria was observed.

Fig.4.DGGE analysis results of OPP bioremediation process and cluster analysis of microbial community of soil sample without thermal desorption.Control:B0 soil before remediation.Sets I,II,III and IV correspond to treatments B,B+C,B+N and B+C+N,respectively.(b)Cluster analysis by DGGE.(c)Phylogenetic tree analysis based on the 16S rDNA sequences.B1–B11 represent the 16S rDNA sequences corresponding to the selected bands in the DGGE profile.

The community structures after different treatments at the phylum level showed obvious differences,indicating that thermal desorption treatment,the application of biochar or NH4NO3and incubation time all influence the activity of microorganisms.The thermal desorption process eradicated most of the microorganisms in the soil.The total microbial abundance increased significantly when biochar and NH4NO3were applied to soil A.The results were consistent with those reported by Wang et al.[47].After adding biochar and NH4NO3,the modified environment probably restored the development of a suitable microbial community.Conversely,for soil B the addition of biochar and NH4NO3had a slight effect on microbial diversity[48],presumably due to the presence of indigenous microbes in the original contaminated soil.

Fig.5.Mortality rate and mass of the earthworms in artificialsoil.Condition:the mass percent of biochar is 6 wt%with respect to soil and the concentration of NH4NO3 is 300 mg·kg-1.CK represent clean soil.

3.5.Toxicity tests

According to Ahmad et al.,elucidating the ecological toxicity is an important aspect during biochar-mediated remediation of the contaminated soil[49].After 7 days of exposure,earthworms were removed from the soil sample,washed in water,and placed on moisture filter paper for one day to be weighted.As shown in Fig.5a,all earthworms were dead in soilB0and soil B after7 days of incubation,which indicates that E.fetida is very sensitive to OPPs in contaminated soils.The mortality rates of earthworm in treatments B+C and B+N were 95 and 89%,respectively,indicating that the addition of NH4NO3is more effective than biochar to reduce the toxicity in soil without thermal desorption treatment.The mortality rate(70%)of earthworm in soil A0is remarkably lower than that(approaching 100%)in soil B0,demonstrating that the thermal desorption treatment could significantly alleviate the toxicity of soil.For soil A the application of biochar and NH4NO3not only increased the mass of earthworms,but also decreased(p＜0.05)the mortality rate of earthworms(Fig.5(a)and(b)),as are in accordance with the decreasing trend of OPP concentration.These results are consistent with previous reports[50].The improved growth of earthworms was likely due to the reduced content of OPP pesticides in soil samples through microbial degradation stimulated by the addition of biochar or NH4NO3.

4.Conclusions

For the contaminated soils with thermal desorption treatment,the addition of NH4NO3led to larger reduction(34.35%)of the pesticide concentration than biochar(31.90%).For the soil without thermal desorption treatment the simultaneous addition of biochar and NH4NO3led to the largest reduction of pesticide concentration(11.07%).The emission rate of GHGs from the soil with out thermal treatment increased slightly when biochar and NH4NO3were added,while the emission rate of GHGs from the soil after thermal treatment increased remarkably when biochar and NH4NO3were added.In most cases,the addition of NH4NO3was not only more effective than biochar to promote the degradation of pesticide,but also exhibited higher GHG emission.Besides,the microbial community analysis suggested that the application of biochar and NH4NO3could obviously increase the activity of microorganism.Therefore,the improved degradation of pesticide concentration ismainly owed to the increased activity of microorganism.