Journal of Hazardous Materials 273 (2014) 155–164

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Responses of soil microbial community to different concentration of fomesafen Xiaohu Wu 1 , Jun Xu 1 , Fengshou Dong, Xingang Liu, Yongquan Zheng ∗ Institute of Plant Protection, Chinese Academy of Agricultural Sciences, State Key Laboratory for Biology of Plant Diseases and Insect Pests, Yuanmingyuan, West Road No. 2, Haidian District, Beijing 100193, China

h i g h l i g h t s • • • •

100× recommended application rate reduced soil microbial activity. 100× recommended application rate changed soil microbial community composition. 100× recommended application rate changed microbial functional diversity. Nitrogen-fixing gene abundance was reduced by high fomesafen application.

a r t i c l e

i n f o

Article history: Received 13 September 2013 Received in revised form 10 March 2014 Accepted 21 March 2014 Available online 30 March 2014 Keywords: Fomesafen Microbial activity Community structure Functional diversity nifH

a b s t r a c t Fomesafen degrades slowly in soils and has been linked to crop damage. However, the effect of its residues on soil microbial communities is unknown. The goal of this work was to assess the effect of applying three different doses of fomesafen on microbial community structure and functional diversity as measured by phospholipid fatty acid (PLFA) levels, community-level physiological profiles (CLPPs) and real-time PCR. Our results indicate that applying 100 times the recommended dose of fomesafen (T100) adversely affects soil microbial activity and stresses soil microbial communities as reflected by the reduced respiratory quotient (qCO2 , QR ). The PLFA analysis showed that high levels of fomesafen treatment (T100) decreased the total amount of PLFAs and both bacterial (both Gram-positive (GP) bacteria and Gram-negative (GN) bacteria) and fungal biomass but increased the microbial stress level. However, the BIOLOG results are not consistent with our other results. The addition of fomesafen significantly increased the average well color development, substrate utilization, and the functional diversity index (H ). Additionally, the abundance of the nifH (N2 -fixing bacteria) gene was reduced in the presence of high concentrations of fomesafen (T100). Taken together, these results suggest that the addition of fomesafen can alter the microbial community structure and functional diversity of the soil, and these parameters do not recover even after a 90-day incubation period. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Fomesafen (5-[2-chloro-4-(trifluoromethyl) phenoxy]-N[methylsulfonyl]-2- nitrobenzamide) is a diphenyl ether that is

Abbreviations: dw, dry weight; RB , basal respiration; SIR, substrate-induced respiration; MBC, microbial biomass carbon; qCO2 and QR , respiratory quotient; PLFA, phospholipids fatty acid; GP, Gram-positive bacteria; GN, Gram-negative bacteria; CLPPs, community level physiological profiles; AWCD, average well-color development; H, functional diversity index; PCA, principal component analysis; PVC, polyvinyl chloride. ∗ Corresponding author. Tel.: +86 01 62815908; fax: +86 01 62815908. E-mail address: [email protected] (Y. Zheng). 1 The authors Xiaohu Wu and Jun Xu contributed equally to this study. http://dx.doi.org/10.1016/j.jhazmat.2014.03.041 0304-3894/© 2014 Elsevier B.V. All rights reserved.

specifically used for early post-emergent control of broad-leaved weeds among bean and soybean crops. This herbicide acts by inhibiting protoporphyrinogen oxidase, which is a key enzyme in the porphyrin biosynthesis pathway in plants [1]. After being imported to China, fomesafen soon became one of the most frequently used herbicides in soybean and peanut fields because of its high herbicidal activity at low concentrations [2]. However, U.S. Department of Agriculture Soil Conservation Service (USDASCS) [3] reported that fomesafen has a large relative leachability and a medium relative runoff potential. Jumel et al. [4] found that fomesafen has high reproductive toxicity in the freshwater snail Lymnaea stagnalis. Moreover, fomesafen degrades slowly in the soil with a half-life of 100–240 days, and its residue is implicated in phytotoxicity and damage during crop rotation [5]. A significant

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proportion of these pesticides frequently end up in the soil where they undergo a biological and physicochemical transformation [6]. Thus, the intensive use of fomesafen constitutes a potential risk to environmental safety, and may have particularly adverse consequences for soil quality. The diversity of soil organisms is tremendous; one gram of soil contains as many as 1010 –1011 bacteria [7], 6000–50,000 bacterial species [8], up to 200 m fungal hyphae [9] and 3000–11,000 different genomes [10]. Soil microbes are a crucial component of the biosphere because they are responsible for many ecosystem services, such as the promotion of plant growth, the cycling of carbon, nitrogen, and other nutrients and the degradation of pollutants and pesticide [7,11,12]. All of these functions are of great importance to productivity of agricultural soils [13]. Moreover, soil microbes represent the unseen majority in soil and comprise a large portion of the genetic diversity on Earth [14]. Soil microorganisms are very sensitive to any ecosystem perturbation, which rapidly alters their diversity and activity [15]. Properties that reflect the biomass, activity and diversity of microbial community populations in the soil serve as useful indicators of the impact of outside disturbances (including pesticide application) on soil health [16]. However, high inputs of pesticides can cause changes in bacterial diversity [17], and may also influence biogeochemical cycles. While researchers have previously investigated the effect of fomesafen on microbial activity (including microbial biomass C and metabolic quotient (qCO2 ), they have not focused on the structure and function of the microbial community [18]. The aim of this study was to assess the response of soil microbial populations to fomesafen-induced disturbances and to monitor the recovery of soil health after fomesafen treatment. Several soil microbial parameters that have the potential to act as bioindicators of soil quality were determined, including basal respiration, substrate-induced respiration, ecophysiological indices, microbial biomass C, community composition, functional community profiling and the abundance of nitrogen-fixing bacteria. To our knowledge, no previous studies have examined the effect of fomesafen on the composition and functional diversity of microbes in agricultural soils in China.

2. Materials and methods 2.1. Soil characterization and experimental design Soil (top 0–15 cm) was collected from an experimental plot near Lang fang (N39◦ 82 , E116◦ 70 ) in Hebei Province. The field had not been tilled or planted with crops in the last 2 years. The soil was classified as silty loam, and its properties were as follows: 10.0% clay, 78.2% silt, 11.8% sand, 16.9 g organic matter kg−1 soil, 27.8 mg available P kg−1 soil, 308.8 mg available K kg−1 soil, 14.1 mg NH4 + N kg−1 soil, 58.9 mg NO3 − N kg−1 soil and pH 7.36. After collection, the soil samples were mixed, sieved with 2 mm mesh to remove any plant tissue and preincubated under the experimental conditions described below for two weeks in large polyvinyl chloride (PVC) tanks. The effects of fomesafen on soil microbial communities were studied using three different doses: 3.75 mg kg−1 soil dry weight (a.i./dw; T1), 37.5 mg kg−1 soil (a.i./dw; T10) and 375 mg kg−1 soil (a.i./dw; T100). T1 corresponds to the conventional recommended field application dose for spring soybean crops (375 g active ingredient ha−1 ), assuming a soil bulk density of 1 g cm−3 and an effective soil depth of 1 cm. T10 and T100 correspond to10 or 100 times the recommended dosage. For each fomesafen concentration, a set of three replicated mesocosms was prepared by transferring subsamples of 4 kg dry weight soil to separate PVC tanks. Each treatment was artificially contaminated by adding 30 mL of fomesafen (purity, 96.3%, Shandong Binnong

Technology Co., Ltd, China) solution (in methanol). An equal volume of pure methanol (30 mL) was added to fomesafen-free controls (CK). Sub-samples were thoroughly mixed with a rotary mixer (Hana Mixer, AHM-P125B) to assure uniform pesticide distribution and kept for 24 h in a dark room at 22 ± 1 ◦ C to allow the methanol to evaporate. Next, each freshly-treated soil sub-sample was distributed equally (200 g) among brown wide mouth bottles that were 15 cm high and 8 cm in diameter. The water content of the soil was adjusted to 60% capacity and was held constant by the daily addition of deionized water. The pots were covered with porous plastic film and placed in an environmental chamber at 25 ◦ C and 50% humidity. Each experiment was conducted in triplicate. The pots were removed from the environmental chamber after various intervals (7, 15, 30, 60 and 90 d), and the soils were then analyzed for concentration of fomesafen, basal respiration, substrate-induced respiration, microbial biomass carbon, community structure, and functional diversity as described below. 2.2. Fomesafen concentration The fomesafen was extracted from the soil and its levels were determined as previously described by Zhang et al. [19] with some modifications. Briefly, 5.0 g sample of soil was extracted for 2 h in 20 mL acetonitrile (containing 0.5% (v/v) formic acid). After centrifugation, 1.5 mL of the upper layer was transferred into a 2.0 mL dispersive-SPE tubes containing PSA 50 mg and 150 mg MgSO4 . Then the tubes were vortexed for 1 min and centrifuged for 5 min at RCF 2077 g. The resulting supernatants were filtered through 0.22 ␮m nylon syringe filters for UPLC–MS/MS analysis. Analysis of fomesafen was conducted on a triple-quadrupole mass spectrometer (TQD, Waters Crop.) using the multiple reaction monitoring (MRM) mode and negative ESI mode. 437 (m/z) was selected as the precursor ion, and its quantitative and qualitative product ions were 286 (m/z) and 316 (m/z), respectively, when the collision energies were both 23 V. 2.3. Microbial respiration and biomass carbon Soil basal respiration (RB ; an indicator of overall microbial activity) and substrate-induced respiration (SIR; an indicator of potentially active microbial biomass) were measured as previously described by Zeng et al. [20] with some modifications. To measure the basal respiration, 20 g fresh soil was transferred to a flask along with a vial containing 0.1 N NaOH (10 mL) to trap the released CO2 . As a blank, one flask did not contain soil. The soil samples and the blank were all incubated at 25 ◦ C for 24 h. Then NaOH solution was precipitated with 3 N BaCl2 and back-titrated with 0.05 N HCl using Brand Titrette® (Germany). The substrate-induced respiration (SIR) was measured by adding 10,000 mg glucose kg−1 dry weight soil to the soil samples and then measuring CO2 emission 6 h later. Microbial biomass carbon (MBC), an indicator of the overall size of the soil microbial community, was measured by the chloroformfumigation–extraction method [21]. The MBC was calculated using the following equation: MBC = 2.64EC

(1)

where EC = (C extracted from fumigated) − (C extracted from nonfumigated soil) with a conversion factor of 2.64. 2.4. Phospholipid fatty acid (PLFA) profiles The microbial community structure of the soil was determined by analyzing its phospholipid fatty acid (PLFA) composition as previously described by Bossio et al. [22]. The microbial biomass was assessed by analyzing the presence of 17 fatty acids, 14:0, i15:0, a15:0, 15:0, 16:0, i16:0, i17:0, 16:1ω7c, cy17:0, 17:0, 10Me18:0,

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18:2ω6, 18:1ω9c, 18:1ω9t, 18:0, cy19:0 and 20:0. A subset of 13 fatty acids were summed to assess bacterial biomass (14:0, i15:0, a15:0, 15:0, 16:0, i16:0, i17:0, 16:1ω7c, cy17:0, 17:0, 18:0, cy19:0 and 20:0) [23]. The branched phospholipids i15:0, a15:0, i16:0 and i17:0 were used as indicators of GP bacteria [24], while the PLFAs 16:1ω7c, cy17:0, cy19:0 were indicative of GN bacteria [23]. The unsaturated PLFA 18:1ω9c, 18:1ω9t and 18:2ω6 were as used as an indicator of a fungal biomass [25]. The stress level (cyc/precursor) for the microbial community was calculated from the ratio of (cyc17:0 + cyc19:0)/(16:1ω7c + 18:1ω7c) [26]. Lastly, the total PLFA level was calculated by adding together all 17 microbial phospholipids.

2.7. Data analyses The date were considered significant when p < 0.05 (* ), p < 0.01 (** ) or p < 0.001 (*** ). The significance of differences among treatments was tested by Duncan’s multiple-range test. All values reported are the mean ± standard error (SE) of three replicates. A principal component analysis (PCA) was employed to examine the PLFA composition and CLPP among different samples that contained multiple variables. RT-PCR reactions were carried out for all the samples in triplicate and the mean values of these measurements were used for calculations of nifH gene expression. Statistics were calculated using SAS 9.1.

2.5. Community level physiological profiles (CLPPs)

3. Results

Community level physiological profiles (CLPPs) were assessed using Biolog Eco microplates (BIOLOG, Hayward, USA) as previously described by Govaerts et al. [27] with some modifications. Briefly, soil (10 g field-moist weight) samples were shaken with 90 mL of sterilized saline solution (0.85% NaCl, w/v) for 60 min and then brought to 10−3 final dilution. A 150-mL aliquot was inoculated into each microplate well. The plates were incubated at 25 ◦ C in the dark and read every 24 h over a 10-day period. The absorbance values were measured at 72 h and were used to calculate the average well color development (AWCD). Microbial activity in each microplate, expressed as AWCD, was determined as follows:

3.1. Fomesafen degradation Fomesafen degradation appeared to consist of a rapid initial phase and a slower or more stagnant second phase (Fig. 1). The dissipation kinetics were best described using a bi-exponential kinetic model PC (t) = A × exp(−k1 × t) + B × exp(−k2 × t)

(6)

where ODi is the optical density value from each well after subtracting the value of the blank (water). H was calculated as follows:

where PC(t) is the pesticide concentration at t time; A and B are the constants; k1 and k2 are the dissipation kinetic constants for the first and second component of the curve; and t is the time. Fomesafen dissipation was strongly dependent on the application rate with corresponding half-life values of 84.9, 156.3 and 1453.7 days (Table 1). By the end of the experimental period, the concentration of fomesafen remaining in the soil samples after 90 d was 1.67 mg kg−1 , 18.3 mg kg−1 and 256.4 mg kg−1 , respectively.

H = −

3.2. Soil microbial respiration and biomass

AWCD =

 OD



i

31

Pi × ln (Pi )

(2)

(3)

(ODi ) to where pi is the ratio of microbial activity on each substrate  ODi . the sum of the microbial activities on all substrates 2.6. Real-time PCR DNA was extracted from 0.5 g soil using the FastDNA SPIN Kit for Soil (MP Biomedicals). The final elution volume was 80 ␮L, and the concentration and purity were determined by a spectrophotometer at 260 and 280 nm (Eppendorf AG 22331, Hamburg, Germany). The primer pair PolF (5 -TGCGAYCCSAARGCBGACTC-3 ) and PolR (5 ATSGCCATCATYTCRCCGGA-3 ) was used to amplify the nifH gene [28], and the primer pair 341F (5 -CCTACGGGAGGCAGCAG-3 ) and 518R (5 -ATTACCGCGGCTGCTGG-3 ) was used to amplify the bacterial 16S rRNA gene [29]. Real-time quantitative PCR was performed using the FTC-3000 real-time PCR system (Funglyn Biotech Inc., Toronto, Canada) with fluorescence detection of SYBR green dye. The amplification conditions included one cycle at 95 ◦ C for 30 s for the initial denaturation followed by 45 cycles of 95 ◦ C for 5 s and 60 ◦ C for 20 s. The specificity of the amplified products was confirmed by melting temperatures and dissociation curves after each amplification. The PCR efficiency (E) for each primer pair was determined from the slope of the external calibration curve according to the following equation: E = 10(1/−slope) − 1

The impact of pesticides on various microbial parameters was dependent upon the concentration and incubation time of the pesticide (Table 2). Significant interactions between pesticide concentration and incubation time occurred for all microbial parameters. As shown in Fig. 2, the basal respiration rate in soil treated with 3.75 or 37.5 mg kg−1 fomesafen (T1, T10) was significantly lower (p < 0.05) than the control during the first 15 d, whereas the application of 375 mg kg−1 (T100) of fomesafen resulted in significantly lower basal respiration rates at all incubation times (p < 0.05; Fig. 2A).

(4)

In addition, the real-time PCR detection limit for each target gene was determined using dilutions of sample DNA. The abundance of the nifH gene was expressed as a proportion of total soil bacteria 16S rRNA according to Eq. (5). Relative quantification = 2−(Ct target Ct total bacteria) where Ct represents the threshold cycle.

(5)

Fig. 1. Dynamics of fomesafen degradation in soil. The concentration of fomesafen at a given sampling time is expressed as the % of initial fomesafen concentration. Mean values (n = 3) ± S.E. T1: 3.75 mg fomesafen kg−1 soil dry weight; T10: 37.5 mg fomesafen kg−1 soil dry weight; T100: 375 mg fomesafen kg−1 soil dry weight.

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Fig. 2. (A–E). The effect of fomesafen on basal respiration, substrate-induced respiration, microbial biomass C, qCO2 and QR . (a–c) Means with different letters indicate significant differences (p < 0.05). Mean values (n = 3) ± S.E. qCO2 = RB /MBC: metabolic quotients; QR = RB /SIR: respiratory quotients; CK: without fomesafen addition; T1: 3.75 mg fomesafen kg−1 soil dry weight; T10: 37.5 mg fomesafen kg−1 soil dry weight; T100: 375 mg fomesafen kg−1 soil dry weight.

Table 1 Kinetic parameters of fomesafen degradation in soil at three different initial concentrations: 3.75, 37.5 and 375 mg fomesafen kg−1 soil (a.i./dw) (T1,T10 and T100, respectively). Fomesafen concentration

Aa

k1 b

Bc

k2 d

t1/2 e (d)

r2

T1 T10 T100

1.59 13.74 42.38

−0.00071 0.08017 0.1256

2.03 20.99 265.2

0.09898 0.00147 0.00038

84.9 156.3 1453.7

0.963 0.993 0.999

when compared to the control at days 7, 30 and 60 (Fig. 2D). In addition, treating soil with 375 mg kg−1 (a.i./dw) of fomesafen significantly increased QR values at days 15 and 30 when compared to controls (Fig. 2E). 3.3. PLFA profiles of soil microorganisms No significant difference in PLFAs was observed between control and two levels of fomesafen treatment (T1 and T10; Table 3). However, fomesafen treatment at the highest dosage decreased total PLFAs along with bacterial (both GP and GN) and fungal biomass but increased the microbial stress level (Table 3). A two-dimensional PCA of the PLFA levels (Fig. 3) accounted for 71.6% of the total variance with PC1 (51.8% of total variance) appearing to discriminate by the degree of fomesafen application. T100 samples were concentrated on the left of the origin, while the controls samples were concentrated on the right of the origin (Fig. 3). Higher levels of fomesafen treatment (T100) differed significantly from the controls at a given incubation time with the exception of days 1 and 15. In contrast, control, T1 and T10 treatment did not have a significant effect on PLFA levels within the same incubation time. In total, seven PLFAs were strongly correlated (r ≥ 0.6) to PC1, including i15:0, a15:0, i16:0, i17:0 (an indicator of GP bacteria), 16:1ω7c, cy17:0 (an indicator of GN bacteria) and 14:0.

Fomesafen degradation in soil was described by the bi-exponential model PC(t) = A × exp(−k1 × t) + B × exp(−k2 × t), where PC(t) = pesticide concentration at t time; a A = Constants. b k1 = Dissipation kinetic constants for the first component of the curve and t = time. c B = Constants. d k2 = Dissipation kinetic constants for the second component of the curve and t = time. e t1/2 = Half-life or time required for a 50% dissipation of the initial fomesafen concentration.

No clear differences in substrate-induced respiration were observed between control and pesticide-treated soil samples at 3.75 and 37.5 mg kg−1 (a.i./dw). In contrast, applying 375 mg kg−1 (a.i./dw) of fomesafen resulted in the reduction of substrateinduced respiration at all incubation times (p < 0.05; Fig. 2B). Similarly, treating the soil with 100 times the recommended dosage of fomesafen had a negative effect on the microbial biomass carbon at all incubation times (p < 0.05; Fig. 2C). When measuring ecophysiological indices, treating the soil with 375 mg kg−1 (a.i./dw) of fomesafen resulted in a higher qCO2 value

3.4. Substrate utilization and functional diversity of soil microorganisms As shown in Fig. 4, fomesafen treatment had a significant positive effect on AWCD especially at the higher application rates.

Table 2 Two-way ANOVA for soil microbial properties as affected by fomesafen concentration (treat), incubation time (time) and their interaction (treat × time). Factor Treat Time Treat × time

RB

SIR ***

11.3 193.2*** 2.76***

MBC ***

13.8 5.8** 2.0*

***

23.6 72.1*** 3.5**

qCO2

QR ***

127.2 533.3*** 54.9***

Total PLFA ***

112.1 375.6*** 49.9***

***

32.9 11.7*** 2.9**

Bacterial ***

33.1 17.1*** 3.1**

Fungal ***

17.3 23.0*** 2.2*

GP

GN ***

42.8 47.6*** 4.4***

Stress ***

47.6 28.5*** 3.4**

***

41.6 5.2** 2.1*

H (BIOLOG) 24.1*** 24.7*** 4.2**

The categorical factors are treatment (CK, T1, T10, T100), incubation time (7, 15, 30, 60, 90 days). Presented are the F-values with the level of significance. Significant differences were accepted at * p < 0.05,** p < 0.01 or *** p < 0.001.

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Table 3 The total biomass, bacterial biomass, fungal biomass and stress level expressed as the amount of marker phospholipid fatty acids (nmol PLFA g−1 of soil) in fomesafen-treated soil. Incubation time (d)

7

15

30

60

90

Dosages

CK T1 T10 T100 CK T1 T10 T100 CK T1 T10 T100 CK T1 T10 T100 CK T1 T10 T100

Total PLFAs

17.82 ± 0.63ab 18.14 ± 0.85ab 19.86 ± 0.97a 16.46 ± 0.56b 19.12 ± 0.43a 18.91 ± 0.99a 19.23 ± 0.86a 15.93 ± 0.73b 20.67 ± 0.42a 20.64 ± 0.56a 20.04 ± 1.04a 17.37 ± 0.15b 23.01 ± 1.42a 22.89 ± 1.36a 20.41 ± 0.83ab 16.94 ± 0.78b 22.18 ± 0.14a 24.61 ± 0.41a 22.47 ± 0.91a 15.52 ± 1.11b

Bacteria Total bacteria

Gram-negative bacteria

Gram-positive bacteria

14.02 ± 0.55ab 14.18 ± 0.61ab 15.67 ± 0.82a 12.77 ± 0.43b 14.30 ± 0.29a 14.02 ± 0.79a 14.08 ± 0.74a 12.10 ± 0.71b 15.17 ± 0.33a 15.29 ± 0.42a 14.80 ± 0.80a 12.54 ± 0.11b 17.48 ± 1.23a 17.69 ± 0.98a 15.95 ± 0.51a 12.88 ± 0.65b 17.70 ± 0.22b 19.79 ± 0.41a 18.03 ± 0.63ab 12.12 ± 0.95c

3.21 ± 0.18ab 3.10 ± 0.16ab 3.66 ± 0.43a 2.54 ± 0.13b 3.20 ± 0.19a 2.89 ± 0.11a 3.20 ± 0.15a 2.23 ± 0.12b 3.19 ± 0.06a 3.33 ± 0.25a 3.14 ± 0.17a 2.31 ± 0.07b 3.62 ± 0.31a 3.56 ± 0.24a 3.22 ± 0.11a 2.31 ± 0.04b 4.47 ± 0.16b 5.07 ± 0.08a 4.37 ± 0.13b 2.60 ± 0.18c

4.48 ± 0.21ab 4.74 ± 0.24ab 4.96 ± 0.26a 4.07 ± 0.17b 4.48 ± 0.13a 4.54 ± 0.25a 4.71 ± 0.27a 3.53 ± 0.19b 5.07 ± 0.09a 5.15 ± 0.19a 4.96 ± 0.26a 3.63 ± 0.06b 5.73 ± 0.40a 5.70 ± 0.33a 5.15 ± 0.24a 3.50 ± 0.12b 6.71 ± 0.10b 7.51 ± 0.10a 6.65 ± 0.16b 4.25 ± 0.35c

Fungi

Stress level

3.40 ± 0.07a 3.57 ± 0.23a 3.74 ± 0.19a 3.32 ± 0.13a 4.32 ± 0.14a 4.43 ± 0.20a 4.65 ± 0.11a 3.42 ± 0.04b 5.01 ± 0.14a 4.84 ± 0.17ab 4.78 ± 0.24ab 4.41 ± 0.12b 4.95 ± 0.17a 4.65 ± 0.36ab 3.98 ± 0.33bc 3.71 ± 0.19c 4.09 ± 0.12a 4.36 ± 0.04a 4.07 ± 0.29a 3.17 ± 0.15b

0.59 ± 0.014b 0.61 ± 0.023b 0.60 ± 0.032b 0.79 ± 0.033a 0.77 ± 0.033ab 0.71 ± 0.044b 0.81 ± 0.059ab 0.88 ± 0.022a 0.61 ± 0.027b 0.63 ± 0.037b 0.66 ± 0.039b 0.93 ± 0.010a 0.61 ± 0.012b 0.67 ± 0.029b 0.59 ± 0.013b 0.92 ± 0.049a 0.64 ± 0.025b 0.63 ± 0.068b 0.58 ± 0.041b 1.03 ± 0.132a

CK: without fomesafen addition; T1: 3.75 mg fomesafen kg−1 soil dry weight; T10: 37.5 mg fomesafen kg−1 soil dry weight; T100: 375 mg fomesafen kg−1 soil dry weight. (a–c) Means within a same column in each sampling day with different letters indicate significant differences (p < 0.05). Mean values (n = 3) ± S.E.

Fig. 3. A principal component plot was generated from the PLFA profiles of the CK, T1, T10 and T100 on days 7, 15, 30, 60 and 90. CK: without fomesafen addition; T1: 3.75 mg fomesafen kg−1 soil dry weight; T10: 37.5 mg fomesafen kg−1 soil dry weight; T100: 375 mg fomesafen kg−1 soil dry weight.

AWCD in soil treated with 375 mg kg−1 fomesafen (T100) was higher than that in the control samples at days 7, 15, 30, 60 and 90. AWCD in T100 samples increased rapidly after 24 h; however, AWCD in the control samples increased rapidly after 96 h in all the sampling days. Moreover, six substrate categories (amino acids, carbohydrates, carboxylic acids, polymers, amines and

miscellaneous) were strongly utilized in the T100 treatment condition at a statistically greater rate compared to the controls (Table 4, 72 h data). Microbial community diversity in the soil (H ) significantly increased upon fomesafen treatment during the initial 30 d incubation period (Fig. 5). The PCA plot of the CLPP explained 39.8% of the total variance, and PC1 accounted for 29.7%. The first

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Fig. 4. AWCD of soil samples after the addition of fomesafen at different sampling times. Mean values (n = 3) ± S.E. AWCD: average well color development; CK: without fomesafen addition; T1: 3.75 mg fomesafen kg−1 soil dry weight; T10: 37.5 mg fomesafen kg−1 soil dry weight; T100: 375 mg fomesafen kg−1 soil dry weight.

principal component accounts for more variability than the second. Two distinct groups (control and T100) were tested. At days 15, 30, 60 and 90, the T100-treated sampleswere located to the right of the origin, whereas the controls samples were located to the left of the origin (Fig. 6). 3.5. Quantification of nifH gene expression Administering fomesafen at the highest dosage (T100) decreased the relative abundance of the nifH gene over the entire incubation period. Treatment with fomesafen at the middle dosage (T10) also decreased the relative abundance of the nifH gene at days 15 and 30, but these differences were not observed at days 7, 60 and 90. In contrast, the relative abundance of the nifH gene was higher in the T1-treated samples at day 7, although that difference did not persist at later time points (Fig. 7). 4. Discussion Fomesafen degradation appeared to consist of a rapid initial phase and a slower ormore stagnant second phase. At the beginning, microorganism was able to use the pesticides as carbon source for growth because a fraction of the added pesticides was still in the dissolved phase and/or weakly adsorbed [30], resulting in a rapid degradation. Then a slower degradation phase was observed which may be attributed to different adsorption sites or increased adsorption over time after the pesticide was addition [31]. A similar biphasic pattern of degradation has also been observed with other pesticides [32,33]. No significant differences among the basal respiration, substrate-induced respiration or microbial biomass carbon were observed in either the control or T1-treated samples, which suggest that fomesafen is innocuous to soil microorganisms at the recommended dose. Our results were consistent with previous studies that examined the effects of other herbicides, including glyphosate [34,35], mesotrione [36], and imazethapyr [25]. In contrast, Santos et al. [18] observed a decrease in microbial biomass carbon after applying 250 g a.i. ha−1 fomesafen. These conflicting results may

be caused by adjuvant-induced toxicity in which the commercial formulation of the herbicide results in the depression of overall microbial activity. Crouzet et al. [36] also observed that applying the herbicide mesotrione at 45 mg kg−1 dry weight soil markedly stimulated overall microbial activity. The commercial formulation had a lesser effect on overall microbial activity and slower rates of dissipation than did pure mesotrione. In addition, some adjuvants such as alcohol ethoxylates and alkylamine ethoxylates can be toxic to microeukaryotes at low concentrations and to bacteria at high concentrations [37,38]. Applying high levels of fomesafen (especially T100), in contrast, strongly inhibit the microbial biomass carbon, which might be explained by the fact that high levels of fomesafen could directly harm some bacteria or fungi. This adverse impact was also reflected by the observed reduction in microbial basal respiration and substrate-induced respiration. Ecophysiological indices such as qCO2 and QR have been used to quantify environmental effects on the microbial community in soils [39,40] and indicate a negative impact on soil quality. An increase in QR or qCO2 was observed in fomesafen-treated soil samples at 375 mg kg−1 (a.i./dw), suggesting that the pesticide has a harmful effect on microorganisms and forces them to use a higher amount of their energetic resources to survive, which in turn results in less organic C incorporation into the microbial biomass [40]. Overall levels of soil microbial activity cannot elucidate any potential heterogeneity of the microbial community. Moreover, changes in the composition of the microbial community structure resulting from fomesafen contamination can also affect soil functionality. PLFA profiles can provide some insight into the microbial community structure because the relative abundance of certain PLFAs differs considerably among specific groups of microorganisms [41]. The results from our PLFA analysis revealed that applying 100 times the recommended dose of fomesafen has an inhibitory effect on bacterial and fungal as well as total PLFA levels. These findings suggest that using a higher concentration of an herbicide can inhibit the growth of some bacteria or fungi, which could also explain the reduction in basal respiration, substrate-induced respiration and microbial biomass carbon observed in the present study. Moreover, we found that GP bacteria

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Table 4 Biolog substrate utilization potential (OD) of microbial communities following the addition of fomesafen. Incubation time (d)

Carboxylic acids 7 15 30 60 90 Carbohydrates 7 15 30 60 90 Amines 7 15 30 60 90 Amino acids 7 15 30 60 90 Polymers 7 15 30 60 90 Miscellaneous 7 15 30 60 90

Fomesafen concentration CK

T1

T10

T100

0.148 ± 0.120b 0.454 ± 0.262b 0.169 ± 0.031b 0.141 ± 0.034c 0.110 ± 0.045b

0.157 ± 0.092b 0.336 ± 0.143b 0.156 ± 0.052b 0.077 ± 0.005c 0.129 ± 0.007b

1.646 ± 0.079a 0.212 ± 0.101b 0.295 ± 0.134b 0.545 ± 0.195b 0.156 ± 0.033b

1.523 ± 0.202a 1.291 ± 0.174a 1.881 ± 0.229a 1.554 ± 0.048a 1.90 ± 0.175a

0.282 ± 0.245a 0.043 ± 0.012c 0.094 ± 0.027c 0.356 ± 0.043b 0.233 ± 0.059b

0.408 ± 0.080a 0.214 ± 0.088b 0.153 ± 0.024bc 0.362 ± 0.053b 0.186 ± 0.048b

0.502 ± 0.155a 0.304 ± 0.089b 0.353 ± 0.068b 0.483 ± 0.116b 0.205 ± 0.060b

0.678 ± 0.099a 0.475 ± 0.044a 0.686 ± 0.121a 0.875 ± 0.301a 0.921 ± 0.036a

0.069 ± 0.069a 0.009 ± 0.01b 0.019 ± 0.004b 0.014 ± 0.003b 0.017 ± 0.013b

0.081 ± 0.050a 0.014 ± 0.043b 0.042 ± 0.026b 0.015 ± 0.003b 0.020 ± 0.005b

0.051 ± 0.032a 0.019 ± 0.010b 0.017 ± 0.009b 0.031 ± 0.004b 0.075 ± 0.051ab

0.182 ± 0.014a 0.137 ± 0.029a 0.269 ± 0.168a 0.319 ± 0.072a 0.277 ± 0.141a

0.057 ± 0.057b 0.077 ± 0.034c 0.064 ± 0.015b 0.063 ± 0.026b 0.163 ± 0.135b

0.709 ± 0.111a 0.072 ± 0.065bc 0.046 ± 0.003b 0.123 ± 0.028b 0.076 ± 0.039b

0.181 ± 0.051b 0.383 ± 0.113ab 0.256 ± 0.22b 0.225 ± 0.153b 0.356 ± 0.130b

0.641 ± 0.056a 0.511 ± 0.030a 0.893 ± 0.137a 1.001 ± 0.160a 1.085 ± 0.039a

0.236 ± 0.212b 0.321 ± 0.120b 0.106 ± 0.030b 0.088 ± 0.03b 0.472 ± 0.047b

0.960 ± 0.065a 0.560 ± 0.054ab 0.132 ± 0.035b 0.108 ± 0.006b 0.157 ± 0.034c

0.231 ± 0.106b 0.394 ± 0.056ab 0.195 ± 0.047b 0.611 ± 0.213a 0.190 ± 0.046c

1.056 ± 0.032a 0.885 ± 0.153a 1.107 ± 0.082a 0.868 ± 0.027a 1.349 ± 0.077a

0.004 ± 0.004c 0.025 ± 0.017b 0.081 ± 0.063b 0.008 ± 0.005b 0.016 ± 0.009b

0.039 ± 0.039bc 0.102 ± 0.063b 0.089 ± 0.042b 0.040 ± 0.038b 0.028 ± 0.008b

0.200 ± 0.103ab 0.435 ± 0.044a 0.362 ± 0.056a 0.303 ± 0.130ab 0.104 ± 0.064b

0.381 ± 0.031a 0.356 ± 0.01a 0.476 ± 0.004a 0.548 ± 0.182a 0.598 ± 0.075a

CK: without fomesafen addition; T1: 3.75 mg fomesafen kg−1 soil dry weight; T10: 37.5 mg fomesafen kg−1 soil dry weight; T100: 375 mg fomesafen kg−1 soil dry weight. (a–c) Means within a same row with different letters indicate significant differences (p < 0.05). Mean values (n = 3) ± S.E.

were more sensitive to high fomesafen concentrations (T100) than GN bacteria. It is possible that GN bacteria might have more tolerance to environmental stresses [42]. Cycon´ et al. [43] also observed a decrease in the variety of PLFAs that typically characterize GP bacteria, while the PLFAs that characterize GN bacteria were

Fig. 5. Shannon diversity (H ) of microbial communities following the addition of fomesafen with an incubation time of 72 h. (a–c) Means with different letters indicate significant differences (p < 0.05). Mean values (n = 3) ± S.E. CK: without fomesafen addition; T1: 3.75 mg fomesafen kg−1 soil dry weight; T10: 37.5 mg fomesafen kg−1 soil dry weight; T100: 375 mg fomesafen kg−1 soil dry weight.

unaffected after the application of 1.5 mg teflubenzuron kg−1 (10-fold of the recommended field rate, 10 × FR) on 14 day of incubation. And another reason for the more sensitivity of GP bacteria than GN bacteria could be that the GN bacteria can also multiply rapidly in the presence of additional carbon sources [44]. Wang et al. [24] observed an increase in GN bacteria after 4 years of repeated applications of the methamidophos at 23.8 mg kg−1 , however, GP bacterial numbers remain unchanged. In addition, fungal growth was lower in the presence of higher fomesafen concentrations (T100). Similar results have also been observed when other pesticides are used, including insecticides such as azadirachtin and methamidophos [24,45], fungicides such as tebuconazole and cypermethrin [32,42] and herbicides such as imazethapyr and napropamide [25,46]. Blakely et al. [47] also found that polycyclic aromatic hydrocarbon contamination decreased fungal biomass in soil. The reduction in fungal biomass resulting from these various compounds suggests that fungi are more sensitive to chemical stresses. In contrast, higher levels of pesticides have been observed to increase microbial stress level, which causes the bacteria to alter the composition of their cell membrane in response [48]. This conversion of monoenoic precursors (prec) into cyclopropyl fatty acids (cyc) helps to maintain a functional living membrane by minimizing the loss of membrane lipids and resulting changes in membrane fluidity [49,50]. The PCA of the PLFA levels revealed significant differences in the PLFA pattern between the control and herbicide treatment (T100) at days 30, 60 and 90. Several studies have reported a larger influence of incubation time versus concentration [51,52]. Although similar results were observed in this

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Fig. 6. Principal component analysis of the CLPP following the addition of fomesafen at different sampling times. CK: without fomesafen addition; T1: 3.75 mg fomesafen kg−1 soil dry weight; T10: 37.5 mg fomesafen kg−1 soil dry weight; T100: 375 mg fomesafen kg−1 soil dry weight.

study, shifts in treated samples also occurred due to fomesafen addition. At the end of our experiment, the microbial communities in both T100-treated and control soil samples had significantly different PLFA profiles, which indicates that fomesafen treatment caused lasting changes (>90 days) in the structure of the microbial communities. Applying high levels of fomesafen reduces soil respiration and microbial biomass, and this effect may suppress decomposition rates, resulting in an increase in soil carbon storage. Moreover, we found evidence for a decline in fungal and bacterial abundance as indicated by PLFA, suggesting a reduced potential for the microbial community to metabolize C. However, microbial adaptation or a change in microbial communities could lead to an upward adjustment of the efficiency of carbon use, counteracting the decline in microbial biomass and accelerating soil–carbon loss [53]. Results from several field studies demonstrate that although soil

Fig. 7. The N2 -fixing microbial population in the soil relative to total bacterial 16S rRNA after the addition of fomesafen at different sampling times. (a–c) Means with different letters indicate significant differences (p < 0.05). Mean values (n = 3) ± S.E. CK: without fomesafen addition; T1: 3.75 mg fomesafen kg−1 soil dry weight; T10: 37.5 mg fomesafen kg−1 soil dry weight; T100: 375 mg fomesafen kg−1 soil dry weight.

respiration is initially stimulated by warming, this effect often diminishes over time, with elevated soil respiration in chronically warmed soils returning to ambient levels within a few years [54–56]. Carney et al. [57] observed that the decline in carbon storage was driven by changes in soil microbial composition and activity. In general, fomesafen application can affect belowground ecosystem processes such as carbon storage and carbon cycling by altering soil microbial community structure. The functional diversity of soil microbes that can be inferred from the CLPP data is useful in monitoring changes in microbial diversity that are caused by environmental fluctuations and pollution [58]. In the present study, the BIOLOG results indicated that some fast-growing heterotrophic bacteria were enriched by the high fomesafen concentration. More substrates from a variety of classes (carbohydrate, carboxylic acid, amine, amino acid, polymers and miscellaneous) were utilized at the higher concentration when compared to untreated soil. However, these BIOLOG results did not agree with our other results. One explanation is that each method analyzes a different aspect of the soil microbial community. CLPP measures the metabolic diversity of culturable, primarily fast-growing bacteria [59]. These bacteria likely compose only a small fraction of the microbial community in the soil; therefore, fungi and slow-growing bacteria may have a minimal influence on the CLPP. In contrast, the PLFA pattern was used as an integrated measure of all living microorganisms present in the soil regardless of their growth rate or metabolic capacity [60]. Total PLFA levels are closely correlated with microbial biomass carbon. All microbes in the soil contribute to the total PLFA level and microbial biomass carbon; however, this is not the case for the CLPP. These opposing results between the BIOLOG data and the PLFA levels (or the microbial biomass carbon) have been observed ˜ previously. For example, Munoz-Leoz et al. [32] found that basal respiration, substrate-induced respiration and microbial biomass C levels were all inhibited at concentrations of 50 and 500 mg tebuconazole kg−1 soil at all incubation times. In contrast, BIOLOG profiles from the same soil samples show the opposite trend. A similar finding was reported by Wang et al. [61] who observed that applying methamidophos at a concentration of 15.5 mg kg−1 reduced microbial biomass but enhanced functional diversity. Similarly, Yao et al. [62] also observed that the utilization of C as the

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sole energy sources as measured by the BIOLOG assay was not correlated with PLFA levels. Therefore, we hypothesize that higher AWCD and H values following fomesafen application may be the result of some fungal or bacteria populations dying and the resultant increase in fast-growing bacteria. This switch could result from (i) a decrease in competition for nutrients among the fast-growing bacterial populations or (ii) the release of available nutrients and energy sources from dead fungal or bacteria populations [63,64]. Moreover, the PCA plot of the CLPP revealed a distinct difference in both the control and herbicide treatments (T100). This shift in functional diversity of the soil bacteria is a long-term response (>90 days) and was not reversed as the herbicide dissipated. A clearer understanding of changes in expression levels of some key functional genes can help us to better understand how specific functional microbial populations change in response to fomesafen. In this study, using a higher dosage of fomesafen resulted in a long-term (>90 days) adverse effect on the abundance of N2 -fixing bacteria. This result suggests that large amounts of fomesafen (375 mg kg−1 ) might have direct toxic effects on the microbial population, which resulted in the mass elimination of N2 -fixing bacteria. Moreover, the decrease in the abundance of N2 -fixing bacteria leaded to the disturbance of the interaction between atmosphere nitrogen and N2 -fixing bacteria by fomesafen, and consequently reduced the ability of bacteria to utilize N2 as a nitrogen source. Fox et al. [65] reported that organochlorine pesticides reduced nitrogenase activity and inhibit nodule formation. Nitrogen fixation is considered to be a very important source of nitrogen in the soil [66]. Therefore, these small fractions of bacteria are sensitive to pesticide application and could therefore be used as a potential indicator of soil quality. At higher concentrations, non-target effects were more pronounced. The health of the soil (as reflected by the values of the soil microbial properties measured here) did not recover after higher doses of fomesafen were applied to the soil during the 90-day incubation period. This observation highlights the importance of considering increasing pesticide concentrations when assessing the environmental impact and potential non-target effects of pesticides on soil microbial communities and therefore soil quality in a shortterm mesocosm experiment. The low degradation rates ultimately lead to a progressive accumulation of pesticide in the soil upon repeated application. At the same time, fomesafen also was found to be toxic to soil microorganisms and caused changes in microbial communities after 5 years of repeated applications of the herbicide fomesafen at the recommended rate in field experiments and fomesafen residue in soil was 3.5 mg kg−1 (unpublished data). Repeated or prolonged use of fomesafen even at a recommended dose, can lead to adverse effects on the soil ecosystem. In general, excessive use of fomesafen not only causes decline in soil quality but also affects carbon cycling. Therefore, soil microbial properties may be a powerful tool to monitor and assess the impact of fomesafen treatment.

5. Conclusions From this study, it was clear that a higher dose of fomesafen (T100) results in significantly reduced overall microbial activity, lower bacterial (both GP and GN) and fungal biomass and an abundance of the nifH (N2 -fixing bacteria) gene. Moreover, fomesafen application at high levels resulted in severe changes in microbial community structure and functional diversity that did not recover even after a> 90-day period of incubation. In addition, high inputs of fomesafen affect carbon cycling and biological nitrogen fixation. Therefore, soil microbial properties can be a useful monitoring tool to assess the ecological risk of fomesafen application on soil health.

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Acknowledgments We would like to thank Ying Zhang and Shenpeng Wang for their assistance in collecting and processing soil samples. This study was supported by grants from the Nature Science Foundation of China (NSFC, 31171879) and Special Fund for Agro-scientific Research in the Public Interest (201203098).

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