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Contamination of pyrethroids and atrazine in greenhouse and open-field agricultural soils in China
Journal Pre-proofs Contamination of pyrethroids and atrazine in greenhouse and open-field agricultural soils in China Rongni Dou, Jianteng Sun, Fucai Deng, Pingli Wang, Haijun Zhou, Zi Wei, Meiqin Chen, Zhenxian He, Menglan Lai, Tiancai Ye, Lizhong Zhu PII: DOI: Reference:
S0048-9697(19)34908-3 https://doi.org/10.1016/j.scitotenv.2019.134916 STOTEN 134916
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Science of the Total Environment
Received Date: Revised Date: Accepted Date:
5 August 2019 6 October 2019 8 October 2019
Please cite this article as: R. Dou, J. Sun, F. Deng, P. Wang, H. Zhou, Z. Wei, M. Chen, Z. He, M. Lai, T. Ye, L. Zhu, Contamination of pyrethroids and atrazine in greenhouse and open-field agricultural soils in China, Science of the Total Environment (2019), doi: https://doi.org/10.1016/j.scitotenv.2019.134916
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Contamination of pyrethroids and atrazine in greenhouse and open-field agricultural soils in China
Rongni Doua, Jianteng Suna, b, *, Fucai Denga, Pingli Wanga, Haijun Zhoua, Zi Weia, Meiqin Chena, Zhenxian Hea, Menglan Laia, Tiancai Yea, Lizhong Zhub
a
Guangdong Provincial Key Laboratory of Petrochemical Pollution Processes and
Control, School of Environmental Science and Engineering, Guangdong University of Petrochemical Technology, Maoming, Guangdong, 525000, China b
Department of Environmental Science, Zhejiang University, Hangzhou, Zhejiang,
310058, China
* Corresponding author: Jianteng Sun Tel.: 86 6682923653. Fax: 86 6682923653.
1
E-mail: [email protected]
Abstract A national-scale survey was conducted to assess the levels and distribution of two extensively used pesticides (pyrethroids and atrazine) in greenhouse and open-field soils in 20 provinces across China. Concentrations between 1.30-113 ng/g and 0.5185.4 ng/g for the total pyrethroids (PYs) and of LOD-137 ng/g and LOD-134 ng/g for atrazine were found in greenhouse and open-field soils, respectively. Higher contaminations were found in the greenhouse than in the open fields. The levels of total pyrethroids in 80% of the greenhouses and of atrazine in 60% of the greenhouses were significantly higher than those in the nearby open-field soils (p<0.05), respectively. The contamination of PYs and atrazine was generally more serious in the northern provinces of China, such as Heilongjiang, Jilin, Liaoning, Beijing, and Hebei. Pearson correlation analysis revealed that the contamination of PYs was significantly correlated with the soil total organic carbon (TOC) value in both greenhouse and open-field soils. Canonical correspondence analysis (CCA) showed that PYs might have an impact on the microbial alpha diversity, while cyhalothrin and cypermethrin may be the key factors affecting the microbial community in the greenhouse and open-field soils. The soil samples containing pesticide residues showed distinct taxonomic and functional communities, where an increased diversity and abundance of microorganisms able to degrade pesticides was observed with high-level PYs contamination. These findings provide useful information for evaluating PYs and atrazine pollution and for contamination management in greenhouse agriculture. Keywords: pyrethroids, atrazine, soil properties, microbial diversity, greenhouse soils 2
1. Introduction The use of greenhouses is a widely applied cultivation method for facility agriculture, which is employed to deal with the rapid increase of the population and the shortage of arable land. Greenhouse cultivation was first used in agriculture in the 1950s and introduced to China in the 1970s. By 2011, the greenhouse area in China was 4.67 million hectares, covering 80% of the greenhouse area in the world (Huang et al., 2015; Yang et al., 2013). The development of greenhouse cultivation satisfies the demand for food and achieves a year-round supply of vegetables. Meanwhile, compared with traditional cultivation, the greenhouse cultivation method requires more frequent land use and higher applications of fertilizer and pesticides, which may lead to severe soil contamination (Bojaca et al., 2013). Pesticides have been used in the development of modern agriculture to ensure the quality, safety and yield of agricultural products for several decades. The total amount of pesticides used worldwide per year has reached 3.3×106 tons from 2002 to 2014 (FAO, 2016). The excessive application of pesticides is one of the largest intentional inputs of potentially hazardous compounds into various environmental media and may pose human health and ecological risks (Tsaboula et al., 2016). Agricultural soil is a major reservoir of pesticides and a secondary source of the pollutants to water and air (Hvězdová et al., 2018; Tao et al., 2008). Some pesticides can be stable and persistent in soil, imposing a negative impact on the soil quality and microbial community structure (Hvězdová et al., 2018; Sun et al., 2018). Therefore, it is important to evaluate 3
the levels of persistent and common pesticides in soil to maintain the soil functions and ecosystem. Atrazine (2-chloro-4-ethylamino-6-ethylamino-1,3,5-triazine) is the most commonly used herbicide and is applied worldwide to control pre- and postemergent weeds for agricultural purposes. Atrazine can exist in the environment for a long time, as its persistent and has high water solubility (Barchanska et al., 2017; Moorman et al., 2001; Schwab et al., 2006; Satsuma 2009). In China, the usage volume of atrazine reached 2800 tons in 2000 and then increased by 20% per year (Li et al., 2007). This intensive use has led to frequent occurrences of atrazine in agriculturally dominated areas. Our previous study of 241 farmlands in the Yangtze River Delta (YRD) region found atrazine concentrations ranging from 1.0 to 113 ng/g in soils, with the mean level of atrazine at 5.7 ng/g and a detection rate of 57.7% (Sun et al., 2017). The residue of atrazine in soils could change the biodiversity of the soil bacteria (Chen et al., 2014; Chen et al., 2015), which may pose threat to the soil ecology and in turn have implications for the soil fertility and quality. Human exposure to atrazine could lead to health risks such as eye and skin irritation and endocrine system effects (Fang et al., 2015; Freeman et al., 2011; Lasserre et al., 2009; Zaya et al., 2011). It is necessary to explore the levels of atrazine and its effect on the soil microbial community in agricultural soils. Pyrethroid insecticides (PYs) are a class of widely used synthetic pesticides derived from natural chrysanthemic acid (Radford et al., 2014; Yu and Yang, 2017). As a substitute for many pesticides such as organochlorine pesticides and organophosphorus 4
pesticides, PYs are applied intensely in agricultural fields worldwide. Owing to their widespread use, persistency and environmental mobility, some PYs can accumulate in soil and transfer to multiple environments via irrigation and rainfall, ultimately threatening human health through the food chain (Bayen et al., 2013; Farajzadeh et al., 2014; Bayen et al., 2014; Fernández-Ramos et al., 2014). Previous research indicated that PYs were biotoxic to fish, bees and earthworms in the environment (Brander et
al., 2016; Gill et al., 2012; Song et al., 2015). Female animal studies suggested that early-life PYs exposure might delay the onset of puberty and harm female health (Ye et al., 2017; Li et al., 2018). PYs in contaminated soil can transfer into many kinds of vegetables (Farina., et al., 2018), which may increase the risk of exposure to PYs. Additionally, contamination of PYs in soil can disturb the microbial community structure, which may cause potential ecological risks (Bragança et al., 2019). Knowledge of the levels of PYs and their impacts on agricultural soils is very limited. It is valuable to reveal the contamination of PYs and its influence on microorganisms in agricultural soils. This work aimed to (1) comparison the characterization of the contamination status and spatial distribution of atrazine and PYs in agricultural soils of China between the greenhouse and the open field, (2) reveal the differences of the selected pesticides between greenhouse and traditional cultivation, and (3) evaluate the effect of atrazine and PYs on the structure of soil microflora. As far as we know, this is the first study to survey atrazine and PYs in the greenhouse and open-field soils of China. These results provide a comprehensive understanding of pesticides pollution in greenhouse and open5
field soils at the national scale.
2. Materials and Methods 2.1 Sample collection In total, 51 pairs of surface soil samples (0-20 cm) were collected from greenhouses and nearby open fields of 20 provinces, municipalities, and autonomous regions across China from November 2014 to February 2015. The 20 provincial administrative units with two or three sampling sites included Heilongjiang (HLJ), Jilin (JL), Liaoning (LN), Beijing (BJ), Shanxi (SX), Hebei (HB), Inner Mongolia (IM), Shandong (SD), Jiangsu (JS), Zhejiang (ZJ), Shanghai (SH), Hunan (HN), Jiangxi (JX), Guangdong (GD), Gansu (GS), Qinghai (QH), Xinjiang (XJ), Yunnan (YN), Sichuan (SC), and Tibet (TB). The sampling map and sites are shown in Figure 1 and the supporting information (Table S1). Detailed information, including the locations of the sampling sites and the protocols of soil sampling and pretreatment, was shown in our previous study (Sun et al., 2018). The greenhouses of the sampling sites have been in use for two to three years, with sizes ranging from 400 m2 to 800 m2. There was a distance of approximately 100200 meters between open fields and corresponding greenhouses. Local vegetables, such as cabbage, beans, tomatoes and watermelon, were planted in the greenhouses. The soil types included black soil, red soil, and brown soil. The main fertilizers used at these sampling sites included organic fertilizer, chemical fertilizer, and mixed fertilizer. During sampling, soil was evenly collected from five cores with a bamboo scoop and then mixed well to form a sample. The samples were packed and immediately transported to the laboratory at low temperature. The soil samples were stored at -20 ℃ 6
for the following study. 2.2 Sample preparation and extraction The extraction of atrazine was conducted according to a previously reported method (Cheng et al. 2015). An aliquot of 10 g soil was spiked with di-2-ethylhexyl phthalate d4 as a surrogate standard. Then, the sample was ultrasonically extracted with dichloromethane/acetone (1:1; v/v) for 60 min. After three extractions, the total extract was concentrated, and the solvent was exchanged with hexane. The sample was further reduced to 2.0 mL in a rotary evaporator (Heidolph 4000, Germany). Then, the final extract was cleaned by a multilayered column (25×1.0 cm), which consisted of 2 cm of Na2SO4, 6 cm of activated florisil, and 2 cm of Na2SO4 from bottom to top and had been precleaned with 30 mL of hexane. The analyte was eluted with hexane/acetone (9:1; v/v) (50 mL) and then evaporated to near-dryness, after which it was redissolved in 0.5 mL of hexane before analysis. The extraction of PYs was adapted following the reported procedure (Trunnelle et al., 2013) with some modification. An aliquot of 5 g soil sample was spiked with 250 ng of 13C6-labeled trans-permethrin and then ultrasonically extracted three times with 15 mL hexane (30 min each step). The extracted solution was separated centrifugally and merged into one sample, which was then concentrated to ~ 2 mL in a rotary evaporator (Heidolph 4000, Germany). The concentrated extracts were further purified through alumina:silica gel (1:1 by weight) column chromatography. The target products were eluted with 50 mL of dichloromethane:hexane (1:1) mixture after the columns were conditioned with hexane. The final extracts were concentrated to 1 mL for the 7
following analysis. 2.3 GC/MS analysis Atrazine and PYs were analyzed independently by a gas chromatograph/mass spectrometer (GC/MS) (Agilent 7890B GC and 5977A MS). Helium was used as the carrier gas at a constant flow of 1.0 mL/min. A HP-5 capillary column (30 m, 0.25 mm) was used for atrazine separation. The GC conditions were set as follows: injection temperature, 250 °C; ion source, 230 °C; and oven program, 80 °C (1 min) to 200 °C at 40 °C /min, followed by heating to 280 °C at 20 °C/min, with a 3-min hold. The postrun was set at 290 °C and held for 1 min. The mass spectrometry was conducted in electron impact (EI) ionization and selected ion monitoring (SIM) mode. The quantification of PYs was conducted in SIM mode. Multiple ions, including one used for quantitation and one or two used for qualification and confirmation, were monitored for each compound. A J&W DB-5 MS fused silica capillary column (30 m × 0.25 mm ID, 0.25 μm film thickness) was used for separation. The injection temperature was 280 °C, with the GC oven temperature set from 80 °C to 100 °C at 20 °C/min and then heated to 300 °C at 10 °C/min, followed by a 10 min hold. The total run time of the quantification analysis of each sample was 31 min. 2.4 Quality assurance and quality control A procedural blank, a spiked blank, and a sample duplicate were processed with each batch of 15 samples to ensure accurate quantification. No selected pesticides were found in the blanks. The recovery rates of the pesticides in the spiked samples ranged from 85.6% to 99.7%. The variations in the concentrations of the pesticides in duplicate 8
samples were lower than 15% (n = 3). Five-point standard calibration curves were employed for quantitative analysis. The recovery rates of the surrogate standards of di2-ethylhexyl phthalate d4 and 13C6-labeled trans-permethrin were 87.4 ± 9.2% and 87.5 ± 11.4%, respectively. The limit of detection (LOD) of the pesticides was defined based on a signal to-noise ratio of 3 using the lowest concentration standard (Table S2). 2.5 Statistical analysis The statistical analysis of the correlation was conducted using SPSS 20.0 (IBM, Armonk, USA). The statistical significance of the differences in the concentrations of the selected pesticides between the greenhouse and open-field soils was tested using an analysis of variance with paired comparisons. Pearson correlation analysis was applied to evaluate the relationships between the physiochemical properties and the concentrations of PYs and atrazine in all soil samples. Then, the relationship between the microbial community structure and the selected pesticides was analyzed through canonical correspondence analysis (CCA). The concentrations of the pesticides were log-transformed to approximate normal distributions prior to the statistical analyses. The concentrations of the selected compounds were expressed on a dry-weight basis.
3. Results and Discussion 3.1 Levels of pesticides in greenhouse and open-field soils The pesticides including four PYs (deltamethrin, cyhalothrin, cypermethrin and fenpropathrin) and atrazine were analyzed, and the results are shown in Table 1. The levels of four selected PYs were in the range of 1.30-113 ng/g and 0.51-85.4 ng/g in the greenhouse and open-field soils, respectively. The mean concentrations of the total 9
PYs in the greenhouse and open-field soils were 31.7 ng/g and 18.2 ng/g, respectively. The detection rates of four PYs ranged from 88.2% to 96.1% and from 66.7% to 80.4% in the greenhouse and open-field soils, respectively. The levels of the four PYs ranged from open-field (10.2 ng/g) for fenpropathrin, greenhouse (5.80 ng/g) > open-field (3.99 ng/g) for cypermethrin, greenhouse (7.10 ng/g) > open-field (3.44 ng/g) for deltamethrin, and greenhouse (0.70 ng/g) > open-field (0.49 ng/g) for cyhalothrin. In 80% of the sampling sites, the levels of total PYs in the greenhouse soils were significantly higher than those in the nearby open field soils (p<0.05). In 70% of the studied provinces, the mean concentrations of total PYs in the soil samples collected from greenhouses were higher than those collected from nearby open fields (p<0.05). The significant difference between the levels of PYs in greenhouse and open-field soils 10
indicated that the cultivation method could influence the contamination. Application to local crops is an important source of PYs in agricultural soils (Campo et al., 2013). On the other hand, wastewater irrigation could also be an important source of PYs (Weston et al., 2013). The high intensity of land use in greenhouses requires higher applications of PYs and wastewater irrigation, which may cause more serious contamination of PYs in the soil. For atrazine, the levels ranged from
(Vonberg et al., 2014). Greenhouse cultivation could enhance the effective use of water irrigation and pesticide application, which also increases the contamination of atrazine in greenhouse soils (Huang et al., 2015). However, relatively high temperatures and humidity will accelerate atrazine degradation (Barrios et al., 2019; Omotayo et al., 2016). At some sampling sites, the concentration of atrazine in open-field soils was higher than that in greenhouse soils. Overall, the contamination of PYs and atrazine in the greenhouse and nearby openfield soils in China is serious. Our previous study revealed that the concentrations of organochlorine pesticides reached 563 ng/g with a mean of 77.2 ng/g in all greenhouse and open-field soils (Sun et al., 2018). As a result, it is necessary to pay more attention to the pollution of pesticides in greenhouse soils. 3.2 Spatial distributions of the pesticides The national spatial distribution of PYs in the greenhouse and open-field soils is shown in Figure 1. The wide application of PYs for the control of insects in agriculture has caused their permanent occurrence in soils, and they can be toxic to soil microorganisms (Hintzenetal et al., 2009; Meyeretal et al., 2013; Oudou and Hansen, 2002). The distinct spatial distribution showed that the total concentrations of PYs in the northern provinces of China (Jilin, Hebei, Inner Mongolia, Liaoning, Beijing, Shanxi and Shandong) were higher than those in the south (Shanghai, Jiangsu, Jiangxi, Zhejiang and Guangdong). The concentrations of PYs at three greenhouse sample sites reached 113, 82.4 and 95.2 ng/g, respectively, in Changchun City, the capital of Jilin Province. Consistent with the high concentrations in Jilin Province, Beijing, the capital 12
of China, also had high detection levels and concentrations (34.7 ng/g and 30.9 ng/g) in the greenhouse and open-field soils. In contrast, the levels of PYs in the greenhouse and open-field soils of Lanzhou (the capital of Gansu Province) and Xining (the capital of Qinghai Province) were generally lower than those in provinces of the north of China. The results of the current study suggest that there are some regional differences in the contamination of PYs in greenhouse and open-field soils of China. The possible reasons behind the differences in the spatial distribution of PYs are the development of agriculture and the amount and frequency of pesticide applications. The regional distribution pattern of atrazine in the greenhouse and open-field soils is shown in Figure 2. The levels of atrazine in the north provinces of China (Heilongjiang, Shandong, Shanxi, Liaoning, Jilin, Beijing and Hebei) were higher than those in the south (Jiangsu, Shanghai, Zhejiang, Jiangxi and Hunan). Like the southern provinces, the western provinces (Gansu, Tibet, Qinghai, Xinjiang and Sichuan) also had low concentrations of atrazine in greenhouse and open-field soils. The highest concentration of atrazine (137 ng/g) was in the greenhouse soil of Harbin, the capital of Heilongjiang Province. Overall, the contamination of atrazine usually occurred in agricultural soils of the north of China, where it is used for weeding control, especially for cornfields (Vonberg et al., 2014). In some heavily polluted provinces such as Shanxi, Hebei and Jilin Provinces, the concentration of atrazine in open-field soils was higher than that in greenhouse soils, which may have been caused by the relatively high temperature and humidity of greenhouses promoting the degradation of atrazine. These results indicated that the distinct distribution of atrazine could be affected by anthropological activity 13
and its chemical properties. 3.3 Effect of soil properties on the levels of pesticides The effect of soil properties (pH value, total nitrogen (TN), total phosphorus (TP), and total organic carbon (TOC)) on the levels of PYs and atrazine can be observed in Figure 3 and Table S3 and S4. The data on the soil properties and organochlorine pesticides (OCPs) were derived from our previous study (Sun et al., 2018). Generally, the levels of organic pollutants are easily affected by soil properties (Barrios et al., 2019; Sun et al., 2018; Zeng et al., 2019). It is necessary to reveal the correlations between the contamination of PYs and atrazine and the soil properties in greenhouse and openfield soils. In this work, cyhalothrin (r = 0.296, p < 0.05), cypermethrin (r = 0.323, p < 0.05), and fenpropathrin (r = 0.282, p < 0.05) were significantly correlated with the TN in the greenhouse soils. At the same time, the cyhalothrin (r = 0.291, p < 0.05) was significantly correlated with the TN in open-field soils. There was no significant correlation between PYs and the TP in any soil samples, except for cypermethrin (r = 0.581, p < 0.01) in greenhouse soils. In addition, cyhalothrin (r = 0.523, p < 0.01), cypermethrin (r = 0.669, p < 0.01), fenpropathrin (r = 0.601, p < 0.01), and the total PYs (r = 0.588, p < 0.01) were significantly correlated with the TOC in greenhouse soils. Cyhalothrin (r = 0.310, p < 0.05), cypermethrin (r = 0.563, p < 0.01) and the total PYs (r = 0.439, p < 0.01) were significantly correlated with the TOC in open-field soils. No significant correlation was observed between PYs and the soil pH in either greenhouse or open-field soils. It has been demonstrated that PYs can strongly bind to soil particles and organic matter with high hydrophobic properties, leading to PYs 14
residues in the soil environment (Jin and Webster., 1998; Xu et al., 2015; Singh and Singh., 2004). These findings suggest that the TOC is the main factor affecting the levels of PYs. For atrazine in greenhouse and open-field soils, there were no significant correlations between atrazine and the soil properties (p > 0.05) (Table S3 and S4). This result indicates that atrazine is less affected by the soil properties, and the residue of atrazine in greenhouse and open-field soils is mainly from external input. On the other hand, the correlation between PYs and atrazine and OCPs was analyzed in soil samples. There were significant correlations between cyhalothrin (r = 0.484, p < 0.01), cypermethrin (r = 0.560, p < 0.01), fenpropathrin (r = 0.501, p < 0.01), PYs (r = 0.535, p < 0.01) and OCPs in the greenhouse soils. In open-field soils, deltamethrin (r = 0.279, p < 0.05), cyhalothrin (r = 0.410, p < 0.01), cypermethrin (r = 0.585, p < 0.01), and fenpropathrin (r = 0.488, p < 0.01), PYs (r = 0.653, p < 0.01) were significantly correlated with OCPs. Meanwhile, atrazine was found to be significantly correlated OCPs in both greenhouse (r = 0.289, p < 0.05) and open-field (r = 0.282, p < 0.05) soil. These pesticides have strong correlations, suggesting that they have similar sources. It is necessary to strengthen the unified control of these pesticides in agriculture to mitigate soil contamination. 3.4 Influences of pesticides on microbial diversity The microbial community composition and diversity in the greenhouse and openfield soils were reported in our previous study (Sun et al., 2018). Bacteria from Bacillus, Kaistobacter, Pseudomonas, Bacteroides, Streptococcus, and Nitrospira were 15
frequently observed to show differences between greenhouse and open-field soils. For most sample sites, the OTU number in greenhouse soil was relatively higher than that in open-field soil. However, there was no significant difference between the overall microbial diversity in greenhouse soils relative to that in open field soils (p > 0.05). In this study, we further evaluated the relative influences of the pesticides on the overall microbial diversity. There was no significant correlation between the pesticides and the number of microbial phylotypes (p > 0.05) in the greenhouse soils. In the openfield soils, deltamethrin (r = 0.387, p < 0.01), cypermethrin (r = 0.393, p < 0.01) and the total PYs (r = 0.400, p < 0.01) were significantly correlated with the number of microbial phylotypes. The results suggested that PYs and atrazine have little influence on the microbial alpha diversity in the greenhouse soils, while PYs might have impact on microbial alpha diversity in open-field soils. CCA was conducted to quantify the influence of the selected pesticides (PYs and atrazine) on the microbial community composition in the greenhouse and open-field soils (Figure 4). The first two axes of the CCA explained 4.24% and 2.07% of the variation in the greenhouse soils and 8.12% and 5.25% of that in the open-field soils. The PYs in all the soil samples had effects on the composition of the microbial phylotypes. Cyhalothrin and cypermethrin were the key factors influencing the composition of the microbial community in the greenhouse and open-field soils. However, atrazine in all soil samples had the least impact on the microbial phylotypes. Smith et al. (2005) showed that atrazine affects the soil community structure at concentrations over 40 ppm. The effects of pesticides on microbial communities are also controlled by other 16
factors such as the soil properties and the persistence, concentration and toxicity of the applied pesticide (Hao et al., 2009; Kim et al., 2016). In addition, the coexistence of different pesticides and other contaminants might have different effects on the microbial activity and diversity. To better understand the effect of pesticides on the soil bacterial community in greenhouse and open field soil, three localities (HLJ, JL and SH) with relatively high or low concentrations of PYs and atrazine were selected to further analyze the soil microbial community. The composition of the most abundant phyla (>1%) present in the soils is shown in Figure 5. A similar pattern could be found between samples collected in greenhouses and open fields. A total of 7 representative phylogenetic groups including Proteobacteria, Firmicutes, Acidobacteria, Chloroflexi, Actinobacteria, Bacteroidetes, and Gemmatimonadetes were identified in the six soil samples. The distribution of sequences among the known bacterial phyla revealed that Proteobacteria predominated in all samples. The soil samples from SH with low levels of PYs and atrazine were dominated by the phylum Firmicutes (>26%), followed by Proteobacteria
(>22%),
Bacteroidetes
(>21%),
Actinobacteria
(>10%)
and
Acidobacteria (>4%). For the soil samples from HLJ with low levels of PYs and high levels of atrazine, an increase was observed of Bacteroidetes (>31%), along with a decrease of Actinobacteria (>5%), while Proteobacteria (23.4%) remained at a similar percentage. Striking differences in the proportions of the microbial composition could be observed in the soil samples from JL, which contained high concentrations of PYs and atrazine. Acidobacteria (>26%), Chloroflexi (>13%), Gemmatimonadetes (>11%) and Actinobacteria (>8%), all of which are usually found to be the dominant category 17
in organic and inorganic contaminants bioremediation and produce bioactive molecules (Alvarez et al., 2017; Deng et al., 2016), were more dominant than Firmicutes (<1%) and Bacteroidetes (<4%), in addition to Proteobacteria (>23%). This suggested that the effect of atrazine on the composition of the soil microbial community is limited, while PYs at high concentrations can induce changes of the soil microbial community. A more detailed composition of the most abundant microbial genus (>0.5%) can be observed in Figure 6. In general, the effects of different concentrations PYs or atrazine on the soil microbial taxon composition (both at phyla and genus) in open field samples are consistent with those in greenhouse samples. The dominant genera of the bacterial community in the open field soil samples with low concentrations of PYs and atrazine (SH-OF) were Prevotella (19%), followed by Bacillus (11.7%), Pseudomonas (10.4%), Megamonas (10.3%), Bacteroides (10.2%), Phascolarctobacterium (8.5%) and Bifidobacterium (5.3%). By contrast, the soil samples with high PYs and low atrazine concentrations (JL-OF) were characterized by a low abundance of Prevotella (0.85%) and high abundances of Streptococcus (12%), Kaistobacter (10%), Nitrospira (22%), Candidatus (16%), Rhodoplanes (15%), Bacillus (8%) and Pseudomonas (6%), along with the disappearance of Megamonas, Bacteroides, Phascolarctobacterium and Bifidobacterium. In the soil samples with high concentrations of PYs and atrazine (HLJOF),
Prevotella
(22%),
Megamonas
(12.9%),
Bacteroides
(15%),
Phascolarctobacterium (8%), Bifidobacterium (14%) and Bacillus (5%) were dominant. The natural microbial community can hold the first responsibility for pesticide degradation in soil (Raj et al., 2019), while the correlation between pollutant 18
concentrations and the microbial community could reveal the microorganism potential to biodegrade such compounds. Many different microorganisms have been demonstrated to degrade PYs and their metabolites (Bragança et al., 2019). Streptomyces sp. was reported to secrete monooxygenase that could utilize βcypermethrin as the growth substrate (Chen et al., 2013). Rhodococcus was the most representative genera of Actinobacterial and has been proposed to be involved in the biodegradation of soluble pesticides and atrazine (Gajendiran et al., 2018; Singh and Singh, 2014). Bacillus (belongs to phylum Firmicutes) was recognized for its potential in bioremediation for soils contaminated with PYs, namely, through catabolizing the pyrethroid pesticide fenpropathrin, as described by Liu et al. (2015). Pseudomonas with soil microflora or alone was able to efficiently degrade pyrethroid residues and atrazine, as well as their metabolites (Gajendiran et al., 2018; Kolekar et al., 2019). Overall, PYs residues had effects on the composition of the microbial community of the soil, while the effect of atrazine was not significant. The effects of different concentrations PYs or atrazine on the soil microbial taxon composition (at both the phyla and genus levels) in greenhouses were consistent with those in open fields. High levels of PYs contamination in soil could result in an increase of the diversity and abundance of microorganisms which have the ability to degrade pesticides. Similarly, Raj et al. (2019) found that the long-term exposure of pesticide led to selectively enriching specific taxa, influencing microbial community by curtailing microbial diversity and limiting community richness. The microbial populations existing in contaminated sites can obtain degradative and other metabolic properties by the 19
constant evolution. Changes in soil microbial community after the exposure to pesticides can also been found in other reports (Chen et al., 2015; Bragança et al., 2019). The slight differences between two cultivation methods may be caused by the differences in farming methods (e.g., cover crop incorporation). These influencing factors could play a role in the variance of the composition of the microbial community in soil environments.
Conclusions Four pyrethroids (deltamethrin, cyhalothrin, cypermethrin and fenpropathrin) and atrazine were found to be almost ubiquitous in agricultural soils from two cultivation modes (greenhouse vs. open field) at the national scale. The concentration varied from 1.30 to 113 ng/g and 0.51 to 85.4 ng/g for PYs and from
Acknowledgments 20
This work was jointly supported by the National Key Research and Development Program of China (2018YFC1800704, 2017YFA0207003), National Natural Science Foundations of China (21836003, 41701357), and Guangdong Key Research and Development Program (2019B110207002).
Appendix A. Supplementary data Supplementary data to this article can be found online.
21
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Figure Legend: Figure 1: Spatial distributions of pyrethroids in the greenhouse (left column) and openfield (right column) soils Figure 2: Spatial distributions of atrazine in the greenhouse (left column) and openfield (right column) soils Figure 3: The effect of soil properties on selected pesticides in the greenhouse (left) and open-field (right) soils through redundancy analysis. The length of arrow for soil properties demonstrated the strength of relationship with selected pesticides profiles and the angles indicated the correlations between soil properties and selected pesticides. Figure 4: Canonical correspondence analysis of microbial dominant lineages and the variables of selected pesticides. Figure 5: Relative abundance (%, mean) of microbial phyla in the open field (OF) and the greenhouse (GH) soils with different concentrations of pesticides (concentration of PY/atrazine in the soil samples of SH, HLJ and JL were low/low, low/high, high/high, respectively) Figure 6: Relative abundance (%, mean) of microbial genera in the open field (OF) and the greenhouse (GH) soils with different concentrations of pesticides (concentration of PY/atrazine in the soil samples of SH, HLJ and JL were low/low, low/high, high/high, respectively)
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Highlights Higher concentrations of pesticides were found in the greenhouse than in the openfields. pyrethroids contamination was found associated with total organic carbon. Cyhalothrin and cypermethrin are important factors affecting microbial community in soil. High-level pyrethroid increase the diversity and the abundance of pesticidedegrading microorganisms.
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Graphical abstract
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Table 1 Concentrations of pyrethroids and atrazine in the greenhouse and open-field soil samples across China (dry weight). Greenhouse soils (n=51) Compounds
Detection Mean Median
Open-field soils (n=51) Min
(%)
(ng/g)
(ng/g)
Deltamethrin
96.1
7.10
4.63
ND
Cyhalothrin
88.2
0.70
0.50
Cypermethrin
90.2
5.80
Fenpropathrin
90.2
ΣPyrethroids Atrazine
Max
(ng/g) (ng/g)
Detection Mean Median
Min
Max
(%)
(ng/g)
(ng/g)
31.2
72.5
3.44
2.07
ND
17.5
ND
2.78
72.5
0.49
0.36
ND
2.09
4.75
ND
17.9
80.4
3.99
3.02
ND
14.7
18.1
13.8
ND
66.6
66.7
10.2
4.22
ND
62.0
100
31.7
24.3
1.30
113
100
18.2
11.1
0.51
85.4
82.4
15.7
7.3
ND
137
54.9
10.8
3.18
ND
134
ND: not detected
35
(ng/g) (ng/g)
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
36
S0048-9697(19)34908-3 https://doi.org/10.1016/j.scitotenv.2019.134916 STOTEN 134916
To appear in:
Science of the Total Environment
Received Date: Revised Date: Accepted Date:
5 August 2019 6 October 2019 8 October 2019
Please cite this article as: R. Dou, J. Sun, F. Deng, P. Wang, H. Zhou, Z. Wei, M. Chen, Z. He, M. Lai, T. Ye, L. Zhu, Contamination of pyrethroids and atrazine in greenhouse and open-field agricultural soils in China, Science of the Total Environment (2019), doi: https://doi.org/10.1016/j.scitotenv.2019.134916
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Contamination of pyrethroids and atrazine in greenhouse and open-field agricultural soils in China
Rongni Doua, Jianteng Suna, b, *, Fucai Denga, Pingli Wanga, Haijun Zhoua, Zi Weia, Meiqin Chena, Zhenxian Hea, Menglan Laia, Tiancai Yea, Lizhong Zhub
a
Guangdong Provincial Key Laboratory of Petrochemical Pollution Processes and
Control, School of Environmental Science and Engineering, Guangdong University of Petrochemical Technology, Maoming, Guangdong, 525000, China b
Department of Environmental Science, Zhejiang University, Hangzhou, Zhejiang,
310058, China
* Corresponding author: Jianteng Sun Tel.: 86 6682923653. Fax: 86 6682923653.
1
E-mail: [email protected]
Abstract A national-scale survey was conducted to assess the levels and distribution of two extensively used pesticides (pyrethroids and atrazine) in greenhouse and open-field soils in 20 provinces across China. Concentrations between 1.30-113 ng/g and 0.5185.4 ng/g for the total pyrethroids (PYs) and of LOD-137 ng/g and LOD-134 ng/g for atrazine were found in greenhouse and open-field soils, respectively. Higher contaminations were found in the greenhouse than in the open fields. The levels of total pyrethroids in 80% of the greenhouses and of atrazine in 60% of the greenhouses were significantly higher than those in the nearby open-field soils (p<0.05), respectively. The contamination of PYs and atrazine was generally more serious in the northern provinces of China, such as Heilongjiang, Jilin, Liaoning, Beijing, and Hebei. Pearson correlation analysis revealed that the contamination of PYs was significantly correlated with the soil total organic carbon (TOC) value in both greenhouse and open-field soils. Canonical correspondence analysis (CCA) showed that PYs might have an impact on the microbial alpha diversity, while cyhalothrin and cypermethrin may be the key factors affecting the microbial community in the greenhouse and open-field soils. The soil samples containing pesticide residues showed distinct taxonomic and functional communities, where an increased diversity and abundance of microorganisms able to degrade pesticides was observed with high-level PYs contamination. These findings provide useful information for evaluating PYs and atrazine pollution and for contamination management in greenhouse agriculture. Keywords: pyrethroids, atrazine, soil properties, microbial diversity, greenhouse soils 2
1. Introduction The use of greenhouses is a widely applied cultivation method for facility agriculture, which is employed to deal with the rapid increase of the population and the shortage of arable land. Greenhouse cultivation was first used in agriculture in the 1950s and introduced to China in the 1970s. By 2011, the greenhouse area in China was 4.67 million hectares, covering 80% of the greenhouse area in the world (Huang et al., 2015; Yang et al., 2013). The development of greenhouse cultivation satisfies the demand for food and achieves a year-round supply of vegetables. Meanwhile, compared with traditional cultivation, the greenhouse cultivation method requires more frequent land use and higher applications of fertilizer and pesticides, which may lead to severe soil contamination (Bojaca et al., 2013). Pesticides have been used in the development of modern agriculture to ensure the quality, safety and yield of agricultural products for several decades. The total amount of pesticides used worldwide per year has reached 3.3×106 tons from 2002 to 2014 (FAO, 2016). The excessive application of pesticides is one of the largest intentional inputs of potentially hazardous compounds into various environmental media and may pose human health and ecological risks (Tsaboula et al., 2016). Agricultural soil is a major reservoir of pesticides and a secondary source of the pollutants to water and air (Hvězdová et al., 2018; Tao et al., 2008). Some pesticides can be stable and persistent in soil, imposing a negative impact on the soil quality and microbial community structure (Hvězdová et al., 2018; Sun et al., 2018). Therefore, it is important to evaluate 3
the levels of persistent and common pesticides in soil to maintain the soil functions and ecosystem. Atrazine (2-chloro-4-ethylamino-6-ethylamino-1,3,5-triazine) is the most commonly used herbicide and is applied worldwide to control pre- and postemergent weeds for agricultural purposes. Atrazine can exist in the environment for a long time, as its persistent and has high water solubility (Barchanska et al., 2017; Moorman et al., 2001; Schwab et al., 2006; Satsuma 2009). In China, the usage volume of atrazine reached 2800 tons in 2000 and then increased by 20% per year (Li et al., 2007). This intensive use has led to frequent occurrences of atrazine in agriculturally dominated areas. Our previous study of 241 farmlands in the Yangtze River Delta (YRD) region found atrazine concentrations ranging from 1.0 to 113 ng/g in soils, with the mean level of atrazine at 5.7 ng/g and a detection rate of 57.7% (Sun et al., 2017). The residue of atrazine in soils could change the biodiversity of the soil bacteria (Chen et al., 2014; Chen et al., 2015), which may pose threat to the soil ecology and in turn have implications for the soil fertility and quality. Human exposure to atrazine could lead to health risks such as eye and skin irritation and endocrine system effects (Fang et al., 2015; Freeman et al., 2011; Lasserre et al., 2009; Zaya et al., 2011). It is necessary to explore the levels of atrazine and its effect on the soil microbial community in agricultural soils. Pyrethroid insecticides (PYs) are a class of widely used synthetic pesticides derived from natural chrysanthemic acid (Radford et al., 2014; Yu and Yang, 2017). As a substitute for many pesticides such as organochlorine pesticides and organophosphorus 4
pesticides, PYs are applied intensely in agricultural fields worldwide. Owing to their widespread use, persistency and environmental mobility, some PYs can accumulate in soil and transfer to multiple environments via irrigation and rainfall, ultimately threatening human health through the food chain (Bayen et al., 2013; Farajzadeh et al., 2014; Bayen et al., 2014; Fernández-Ramos et al., 2014). Previous research indicated that PYs were biotoxic to fish, bees and earthworms in the environment (Brander et
al., 2016; Gill et al., 2012; Song et al., 2015). Female animal studies suggested that early-life PYs exposure might delay the onset of puberty and harm female health (Ye et al., 2017; Li et al., 2018). PYs in contaminated soil can transfer into many kinds of vegetables (Farina., et al., 2018), which may increase the risk of exposure to PYs. Additionally, contamination of PYs in soil can disturb the microbial community structure, which may cause potential ecological risks (Bragança et al., 2019). Knowledge of the levels of PYs and their impacts on agricultural soils is very limited. It is valuable to reveal the contamination of PYs and its influence on microorganisms in agricultural soils. This work aimed to (1) comparison the characterization of the contamination status and spatial distribution of atrazine and PYs in agricultural soils of China between the greenhouse and the open field, (2) reveal the differences of the selected pesticides between greenhouse and traditional cultivation, and (3) evaluate the effect of atrazine and PYs on the structure of soil microflora. As far as we know, this is the first study to survey atrazine and PYs in the greenhouse and open-field soils of China. These results provide a comprehensive understanding of pesticides pollution in greenhouse and open5
field soils at the national scale.
2. Materials and Methods 2.1 Sample collection In total, 51 pairs of surface soil samples (0-20 cm) were collected from greenhouses and nearby open fields of 20 provinces, municipalities, and autonomous regions across China from November 2014 to February 2015. The 20 provincial administrative units with two or three sampling sites included Heilongjiang (HLJ), Jilin (JL), Liaoning (LN), Beijing (BJ), Shanxi (SX), Hebei (HB), Inner Mongolia (IM), Shandong (SD), Jiangsu (JS), Zhejiang (ZJ), Shanghai (SH), Hunan (HN), Jiangxi (JX), Guangdong (GD), Gansu (GS), Qinghai (QH), Xinjiang (XJ), Yunnan (YN), Sichuan (SC), and Tibet (TB). The sampling map and sites are shown in Figure 1 and the supporting information (Table S1). Detailed information, including the locations of the sampling sites and the protocols of soil sampling and pretreatment, was shown in our previous study (Sun et al., 2018). The greenhouses of the sampling sites have been in use for two to three years, with sizes ranging from 400 m2 to 800 m2. There was a distance of approximately 100200 meters between open fields and corresponding greenhouses. Local vegetables, such as cabbage, beans, tomatoes and watermelon, were planted in the greenhouses. The soil types included black soil, red soil, and brown soil. The main fertilizers used at these sampling sites included organic fertilizer, chemical fertilizer, and mixed fertilizer. During sampling, soil was evenly collected from five cores with a bamboo scoop and then mixed well to form a sample. The samples were packed and immediately transported to the laboratory at low temperature. The soil samples were stored at -20 ℃ 6
for the following study. 2.2 Sample preparation and extraction The extraction of atrazine was conducted according to a previously reported method (Cheng et al. 2015). An aliquot of 10 g soil was spiked with di-2-ethylhexyl phthalate d4 as a surrogate standard. Then, the sample was ultrasonically extracted with dichloromethane/acetone (1:1; v/v) for 60 min. After three extractions, the total extract was concentrated, and the solvent was exchanged with hexane. The sample was further reduced to 2.0 mL in a rotary evaporator (Heidolph 4000, Germany). Then, the final extract was cleaned by a multilayered column (25×1.0 cm), which consisted of 2 cm of Na2SO4, 6 cm of activated florisil, and 2 cm of Na2SO4 from bottom to top and had been precleaned with 30 mL of hexane. The analyte was eluted with hexane/acetone (9:1; v/v) (50 mL) and then evaporated to near-dryness, after which it was redissolved in 0.5 mL of hexane before analysis. The extraction of PYs was adapted following the reported procedure (Trunnelle et al., 2013) with some modification. An aliquot of 5 g soil sample was spiked with 250 ng of 13C6-labeled trans-permethrin and then ultrasonically extracted three times with 15 mL hexane (30 min each step). The extracted solution was separated centrifugally and merged into one sample, which was then concentrated to ~ 2 mL in a rotary evaporator (Heidolph 4000, Germany). The concentrated extracts were further purified through alumina:silica gel (1:1 by weight) column chromatography. The target products were eluted with 50 mL of dichloromethane:hexane (1:1) mixture after the columns were conditioned with hexane. The final extracts were concentrated to 1 mL for the 7
following analysis. 2.3 GC/MS analysis Atrazine and PYs were analyzed independently by a gas chromatograph/mass spectrometer (GC/MS) (Agilent 7890B GC and 5977A MS). Helium was used as the carrier gas at a constant flow of 1.0 mL/min. A HP-5 capillary column (30 m, 0.25 mm) was used for atrazine separation. The GC conditions were set as follows: injection temperature, 250 °C; ion source, 230 °C; and oven program, 80 °C (1 min) to 200 °C at 40 °C /min, followed by heating to 280 °C at 20 °C/min, with a 3-min hold. The postrun was set at 290 °C and held for 1 min. The mass spectrometry was conducted in electron impact (EI) ionization and selected ion monitoring (SIM) mode. The quantification of PYs was conducted in SIM mode. Multiple ions, including one used for quantitation and one or two used for qualification and confirmation, were monitored for each compound. A J&W DB-5 MS fused silica capillary column (30 m × 0.25 mm ID, 0.25 μm film thickness) was used for separation. The injection temperature was 280 °C, with the GC oven temperature set from 80 °C to 100 °C at 20 °C/min and then heated to 300 °C at 10 °C/min, followed by a 10 min hold. The total run time of the quantification analysis of each sample was 31 min. 2.4 Quality assurance and quality control A procedural blank, a spiked blank, and a sample duplicate were processed with each batch of 15 samples to ensure accurate quantification. No selected pesticides were found in the blanks. The recovery rates of the pesticides in the spiked samples ranged from 85.6% to 99.7%. The variations in the concentrations of the pesticides in duplicate 8
samples were lower than 15% (n = 3). Five-point standard calibration curves were employed for quantitative analysis. The recovery rates of the surrogate standards of di2-ethylhexyl phthalate d4 and 13C6-labeled trans-permethrin were 87.4 ± 9.2% and 87.5 ± 11.4%, respectively. The limit of detection (LOD) of the pesticides was defined based on a signal to-noise ratio of 3 using the lowest concentration standard (Table S2). 2.5 Statistical analysis The statistical analysis of the correlation was conducted using SPSS 20.0 (IBM, Armonk, USA). The statistical significance of the differences in the concentrations of the selected pesticides between the greenhouse and open-field soils was tested using an analysis of variance with paired comparisons. Pearson correlation analysis was applied to evaluate the relationships between the physiochemical properties and the concentrations of PYs and atrazine in all soil samples. Then, the relationship between the microbial community structure and the selected pesticides was analyzed through canonical correspondence analysis (CCA). The concentrations of the pesticides were log-transformed to approximate normal distributions prior to the statistical analyses. The concentrations of the selected compounds were expressed on a dry-weight basis.
3. Results and Discussion 3.1 Levels of pesticides in greenhouse and open-field soils The pesticides including four PYs (deltamethrin, cyhalothrin, cypermethrin and fenpropathrin) and atrazine were analyzed, and the results are shown in Table 1. The levels of four selected PYs were in the range of 1.30-113 ng/g and 0.51-85.4 ng/g in the greenhouse and open-field soils, respectively. The mean concentrations of the total 9
PYs in the greenhouse and open-field soils were 31.7 ng/g and 18.2 ng/g, respectively. The detection rates of four PYs ranged from 88.2% to 96.1% and from 66.7% to 80.4% in the greenhouse and open-field soils, respectively. The levels of the four PYs ranged from
indicated that the cultivation method could influence the contamination. Application to local crops is an important source of PYs in agricultural soils (Campo et al., 2013). On the other hand, wastewater irrigation could also be an important source of PYs (Weston et al., 2013). The high intensity of land use in greenhouses requires higher applications of PYs and wastewater irrigation, which may cause more serious contamination of PYs in the soil. For atrazine, the levels ranged from
(Vonberg et al., 2014). Greenhouse cultivation could enhance the effective use of water irrigation and pesticide application, which also increases the contamination of atrazine in greenhouse soils (Huang et al., 2015). However, relatively high temperatures and humidity will accelerate atrazine degradation (Barrios et al., 2019; Omotayo et al., 2016). At some sampling sites, the concentration of atrazine in open-field soils was higher than that in greenhouse soils. Overall, the contamination of PYs and atrazine in the greenhouse and nearby openfield soils in China is serious. Our previous study revealed that the concentrations of organochlorine pesticides reached 563 ng/g with a mean of 77.2 ng/g in all greenhouse and open-field soils (Sun et al., 2018). As a result, it is necessary to pay more attention to the pollution of pesticides in greenhouse soils. 3.2 Spatial distributions of the pesticides The national spatial distribution of PYs in the greenhouse and open-field soils is shown in Figure 1. The wide application of PYs for the control of insects in agriculture has caused their permanent occurrence in soils, and they can be toxic to soil microorganisms (Hintzenetal et al., 2009; Meyeretal et al., 2013; Oudou and Hansen, 2002). The distinct spatial distribution showed that the total concentrations of PYs in the northern provinces of China (Jilin, Hebei, Inner Mongolia, Liaoning, Beijing, Shanxi and Shandong) were higher than those in the south (Shanghai, Jiangsu, Jiangxi, Zhejiang and Guangdong). The concentrations of PYs at three greenhouse sample sites reached 113, 82.4 and 95.2 ng/g, respectively, in Changchun City, the capital of Jilin Province. Consistent with the high concentrations in Jilin Province, Beijing, the capital 12
of China, also had high detection levels and concentrations (34.7 ng/g and 30.9 ng/g) in the greenhouse and open-field soils. In contrast, the levels of PYs in the greenhouse and open-field soils of Lanzhou (the capital of Gansu Province) and Xining (the capital of Qinghai Province) were generally lower than those in provinces of the north of China. The results of the current study suggest that there are some regional differences in the contamination of PYs in greenhouse and open-field soils of China. The possible reasons behind the differences in the spatial distribution of PYs are the development of agriculture and the amount and frequency of pesticide applications. The regional distribution pattern of atrazine in the greenhouse and open-field soils is shown in Figure 2. The levels of atrazine in the north provinces of China (Heilongjiang, Shandong, Shanxi, Liaoning, Jilin, Beijing and Hebei) were higher than those in the south (Jiangsu, Shanghai, Zhejiang, Jiangxi and Hunan). Like the southern provinces, the western provinces (Gansu, Tibet, Qinghai, Xinjiang and Sichuan) also had low concentrations of atrazine in greenhouse and open-field soils. The highest concentration of atrazine (137 ng/g) was in the greenhouse soil of Harbin, the capital of Heilongjiang Province. Overall, the contamination of atrazine usually occurred in agricultural soils of the north of China, where it is used for weeding control, especially for cornfields (Vonberg et al., 2014). In some heavily polluted provinces such as Shanxi, Hebei and Jilin Provinces, the concentration of atrazine in open-field soils was higher than that in greenhouse soils, which may have been caused by the relatively high temperature and humidity of greenhouses promoting the degradation of atrazine. These results indicated that the distinct distribution of atrazine could be affected by anthropological activity 13
and its chemical properties. 3.3 Effect of soil properties on the levels of pesticides The effect of soil properties (pH value, total nitrogen (TN), total phosphorus (TP), and total organic carbon (TOC)) on the levels of PYs and atrazine can be observed in Figure 3 and Table S3 and S4. The data on the soil properties and organochlorine pesticides (OCPs) were derived from our previous study (Sun et al., 2018). Generally, the levels of organic pollutants are easily affected by soil properties (Barrios et al., 2019; Sun et al., 2018; Zeng et al., 2019). It is necessary to reveal the correlations between the contamination of PYs and atrazine and the soil properties in greenhouse and openfield soils. In this work, cyhalothrin (r = 0.296, p < 0.05), cypermethrin (r = 0.323, p < 0.05), and fenpropathrin (r = 0.282, p < 0.05) were significantly correlated with the TN in the greenhouse soils. At the same time, the cyhalothrin (r = 0.291, p < 0.05) was significantly correlated with the TN in open-field soils. There was no significant correlation between PYs and the TP in any soil samples, except for cypermethrin (r = 0.581, p < 0.01) in greenhouse soils. In addition, cyhalothrin (r = 0.523, p < 0.01), cypermethrin (r = 0.669, p < 0.01), fenpropathrin (r = 0.601, p < 0.01), and the total PYs (r = 0.588, p < 0.01) were significantly correlated with the TOC in greenhouse soils. Cyhalothrin (r = 0.310, p < 0.05), cypermethrin (r = 0.563, p < 0.01) and the total PYs (r = 0.439, p < 0.01) were significantly correlated with the TOC in open-field soils. No significant correlation was observed between PYs and the soil pH in either greenhouse or open-field soils. It has been demonstrated that PYs can strongly bind to soil particles and organic matter with high hydrophobic properties, leading to PYs 14
residues in the soil environment (Jin and Webster., 1998; Xu et al., 2015; Singh and Singh., 2004). These findings suggest that the TOC is the main factor affecting the levels of PYs. For atrazine in greenhouse and open-field soils, there were no significant correlations between atrazine and the soil properties (p > 0.05) (Table S3 and S4). This result indicates that atrazine is less affected by the soil properties, and the residue of atrazine in greenhouse and open-field soils is mainly from external input. On the other hand, the correlation between PYs and atrazine and OCPs was analyzed in soil samples. There were significant correlations between cyhalothrin (r = 0.484, p < 0.01), cypermethrin (r = 0.560, p < 0.01), fenpropathrin (r = 0.501, p < 0.01), PYs (r = 0.535, p < 0.01) and OCPs in the greenhouse soils. In open-field soils, deltamethrin (r = 0.279, p < 0.05), cyhalothrin (r = 0.410, p < 0.01), cypermethrin (r = 0.585, p < 0.01), and fenpropathrin (r = 0.488, p < 0.01), PYs (r = 0.653, p < 0.01) were significantly correlated with OCPs. Meanwhile, atrazine was found to be significantly correlated OCPs in both greenhouse (r = 0.289, p < 0.05) and open-field (r = 0.282, p < 0.05) soil. These pesticides have strong correlations, suggesting that they have similar sources. It is necessary to strengthen the unified control of these pesticides in agriculture to mitigate soil contamination. 3.4 Influences of pesticides on microbial diversity The microbial community composition and diversity in the greenhouse and openfield soils were reported in our previous study (Sun et al., 2018). Bacteria from Bacillus, Kaistobacter, Pseudomonas, Bacteroides, Streptococcus, and Nitrospira were 15
frequently observed to show differences between greenhouse and open-field soils. For most sample sites, the OTU number in greenhouse soil was relatively higher than that in open-field soil. However, there was no significant difference between the overall microbial diversity in greenhouse soils relative to that in open field soils (p > 0.05). In this study, we further evaluated the relative influences of the pesticides on the overall microbial diversity. There was no significant correlation between the pesticides and the number of microbial phylotypes (p > 0.05) in the greenhouse soils. In the openfield soils, deltamethrin (r = 0.387, p < 0.01), cypermethrin (r = 0.393, p < 0.01) and the total PYs (r = 0.400, p < 0.01) were significantly correlated with the number of microbial phylotypes. The results suggested that PYs and atrazine have little influence on the microbial alpha diversity in the greenhouse soils, while PYs might have impact on microbial alpha diversity in open-field soils. CCA was conducted to quantify the influence of the selected pesticides (PYs and atrazine) on the microbial community composition in the greenhouse and open-field soils (Figure 4). The first two axes of the CCA explained 4.24% and 2.07% of the variation in the greenhouse soils and 8.12% and 5.25% of that in the open-field soils. The PYs in all the soil samples had effects on the composition of the microbial phylotypes. Cyhalothrin and cypermethrin were the key factors influencing the composition of the microbial community in the greenhouse and open-field soils. However, atrazine in all soil samples had the least impact on the microbial phylotypes. Smith et al. (2005) showed that atrazine affects the soil community structure at concentrations over 40 ppm. The effects of pesticides on microbial communities are also controlled by other 16
factors such as the soil properties and the persistence, concentration and toxicity of the applied pesticide (Hao et al., 2009; Kim et al., 2016). In addition, the coexistence of different pesticides and other contaminants might have different effects on the microbial activity and diversity. To better understand the effect of pesticides on the soil bacterial community in greenhouse and open field soil, three localities (HLJ, JL and SH) with relatively high or low concentrations of PYs and atrazine were selected to further analyze the soil microbial community. The composition of the most abundant phyla (>1%) present in the soils is shown in Figure 5. A similar pattern could be found between samples collected in greenhouses and open fields. A total of 7 representative phylogenetic groups including Proteobacteria, Firmicutes, Acidobacteria, Chloroflexi, Actinobacteria, Bacteroidetes, and Gemmatimonadetes were identified in the six soil samples. The distribution of sequences among the known bacterial phyla revealed that Proteobacteria predominated in all samples. The soil samples from SH with low levels of PYs and atrazine were dominated by the phylum Firmicutes (>26%), followed by Proteobacteria
(>22%),
Bacteroidetes
(>21%),
Actinobacteria
(>10%)
and
Acidobacteria (>4%). For the soil samples from HLJ with low levels of PYs and high levels of atrazine, an increase was observed of Bacteroidetes (>31%), along with a decrease of Actinobacteria (>5%), while Proteobacteria (23.4%) remained at a similar percentage. Striking differences in the proportions of the microbial composition could be observed in the soil samples from JL, which contained high concentrations of PYs and atrazine. Acidobacteria (>26%), Chloroflexi (>13%), Gemmatimonadetes (>11%) and Actinobacteria (>8%), all of which are usually found to be the dominant category 17
in organic and inorganic contaminants bioremediation and produce bioactive molecules (Alvarez et al., 2017; Deng et al., 2016), were more dominant than Firmicutes (<1%) and Bacteroidetes (<4%), in addition to Proteobacteria (>23%). This suggested that the effect of atrazine on the composition of the soil microbial community is limited, while PYs at high concentrations can induce changes of the soil microbial community. A more detailed composition of the most abundant microbial genus (>0.5%) can be observed in Figure 6. In general, the effects of different concentrations PYs or atrazine on the soil microbial taxon composition (both at phyla and genus) in open field samples are consistent with those in greenhouse samples. The dominant genera of the bacterial community in the open field soil samples with low concentrations of PYs and atrazine (SH-OF) were Prevotella (19%), followed by Bacillus (11.7%), Pseudomonas (10.4%), Megamonas (10.3%), Bacteroides (10.2%), Phascolarctobacterium (8.5%) and Bifidobacterium (5.3%). By contrast, the soil samples with high PYs and low atrazine concentrations (JL-OF) were characterized by a low abundance of Prevotella (0.85%) and high abundances of Streptococcus (12%), Kaistobacter (10%), Nitrospira (22%), Candidatus (16%), Rhodoplanes (15%), Bacillus (8%) and Pseudomonas (6%), along with the disappearance of Megamonas, Bacteroides, Phascolarctobacterium and Bifidobacterium. In the soil samples with high concentrations of PYs and atrazine (HLJOF),
Prevotella
(22%),
Megamonas
(12.9%),
Bacteroides
(15%),
Phascolarctobacterium (8%), Bifidobacterium (14%) and Bacillus (5%) were dominant. The natural microbial community can hold the first responsibility for pesticide degradation in soil (Raj et al., 2019), while the correlation between pollutant 18
concentrations and the microbial community could reveal the microorganism potential to biodegrade such compounds. Many different microorganisms have been demonstrated to degrade PYs and their metabolites (Bragança et al., 2019). Streptomyces sp. was reported to secrete monooxygenase that could utilize βcypermethrin as the growth substrate (Chen et al., 2013). Rhodococcus was the most representative genera of Actinobacterial and has been proposed to be involved in the biodegradation of soluble pesticides and atrazine (Gajendiran et al., 2018; Singh and Singh, 2014). Bacillus (belongs to phylum Firmicutes) was recognized for its potential in bioremediation for soils contaminated with PYs, namely, through catabolizing the pyrethroid pesticide fenpropathrin, as described by Liu et al. (2015). Pseudomonas with soil microflora or alone was able to efficiently degrade pyrethroid residues and atrazine, as well as their metabolites (Gajendiran et al., 2018; Kolekar et al., 2019). Overall, PYs residues had effects on the composition of the microbial community of the soil, while the effect of atrazine was not significant. The effects of different concentrations PYs or atrazine on the soil microbial taxon composition (at both the phyla and genus levels) in greenhouses were consistent with those in open fields. High levels of PYs contamination in soil could result in an increase of the diversity and abundance of microorganisms which have the ability to degrade pesticides. Similarly, Raj et al. (2019) found that the long-term exposure of pesticide led to selectively enriching specific taxa, influencing microbial community by curtailing microbial diversity and limiting community richness. The microbial populations existing in contaminated sites can obtain degradative and other metabolic properties by the 19
constant evolution. Changes in soil microbial community after the exposure to pesticides can also been found in other reports (Chen et al., 2015; Bragança et al., 2019). The slight differences between two cultivation methods may be caused by the differences in farming methods (e.g., cover crop incorporation). These influencing factors could play a role in the variance of the composition of the microbial community in soil environments.
Conclusions Four pyrethroids (deltamethrin, cyhalothrin, cypermethrin and fenpropathrin) and atrazine were found to be almost ubiquitous in agricultural soils from two cultivation modes (greenhouse vs. open field) at the national scale. The concentration varied from 1.30 to 113 ng/g and 0.51 to 85.4 ng/g for PYs and from
Acknowledgments 20
This work was jointly supported by the National Key Research and Development Program of China (2018YFC1800704, 2017YFA0207003), National Natural Science Foundations of China (21836003, 41701357), and Guangdong Key Research and Development Program (2019B110207002).
Appendix A. Supplementary data Supplementary data to this article can be found online.
21
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Figure Legend: Figure 1: Spatial distributions of pyrethroids in the greenhouse (left column) and openfield (right column) soils Figure 2: Spatial distributions of atrazine in the greenhouse (left column) and openfield (right column) soils Figure 3: The effect of soil properties on selected pesticides in the greenhouse (left) and open-field (right) soils through redundancy analysis. The length of arrow for soil properties demonstrated the strength of relationship with selected pesticides profiles and the angles indicated the correlations between soil properties and selected pesticides. Figure 4: Canonical correspondence analysis of microbial dominant lineages and the variables of selected pesticides. Figure 5: Relative abundance (%, mean) of microbial phyla in the open field (OF) and the greenhouse (GH) soils with different concentrations of pesticides (concentration of PY/atrazine in the soil samples of SH, HLJ and JL were low/low, low/high, high/high, respectively) Figure 6: Relative abundance (%, mean) of microbial genera in the open field (OF) and the greenhouse (GH) soils with different concentrations of pesticides (concentration of PY/atrazine in the soil samples of SH, HLJ and JL were low/low, low/high, high/high, respectively)
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Highlights Higher concentrations of pesticides were found in the greenhouse than in the openfields. pyrethroids contamination was found associated with total organic carbon. Cyhalothrin and cypermethrin are important factors affecting microbial community in soil. High-level pyrethroid increase the diversity and the abundance of pesticidedegrading microorganisms.
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Graphical abstract
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Table 1 Concentrations of pyrethroids and atrazine in the greenhouse and open-field soil samples across China (dry weight). Greenhouse soils (n=51) Compounds
Detection Mean Median
Open-field soils (n=51) Min
(%)
(ng/g)
(ng/g)
Deltamethrin
96.1
7.10
4.63
ND
Cyhalothrin
88.2
0.70
0.50
Cypermethrin
90.2
5.80
Fenpropathrin
90.2
ΣPyrethroids Atrazine
Max
(ng/g) (ng/g)
Detection Mean Median
Min
Max
(%)
(ng/g)
(ng/g)
31.2
72.5
3.44
2.07
ND
17.5
ND
2.78
72.5
0.49
0.36
ND
2.09
4.75
ND
17.9
80.4
3.99
3.02
ND
14.7
18.1
13.8
ND
66.6
66.7
10.2
4.22
ND
62.0
100
31.7
24.3
1.30
113
100
18.2
11.1
0.51
85.4
82.4
15.7
7.3
ND
137
54.9
10.8
3.18
ND
134
ND: not detected
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(ng/g) (ng/g)
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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