Bioremediation by earthworms on soil microbial diversity and partial nitrification processes in oxytetracycline-contaminated soil

Bioremediation by earthworms on soil microbial diversity and partial nitrification processes in oxytetracycline-contaminated soil

Ecotoxicology and Environmental Safety xxx (xxxx) xxxx Contents lists available at ScienceDirect Ecotoxicology and Environmental Safety journal home...

4MB Sizes 0 Downloads 9 Views

Ecotoxicology and Environmental Safety xxx (xxxx) xxxx

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Bioremediation by earthworms on soil microbial diversity and partial nitrification processes in oxytetracycline-contaminated soil Mengli Liua,b,c,1, Jia Caoa,b,c,1, Chong Wanga,b,c,∗ a

College of Resources and Environmental Sciences, China Agricultural University, Beijing, 100193, China Beijing Key Laboratory of Biodiversity and Organic Farming, Beijing, 100193, China c Key Laboratory of Plant-Soil Interactions, MOE, Beijing, 100193, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Oxytetracycline degradation Ammonia oxidizers T-RFLP Bacterial 16S rRNA and fungal 18S rRNA genes Structural equation model

A large proportion (60–90%) of ingested tetracyclines are released to slurry, soils, surface waters and ground water, which has raised extensive concerns and may pose a risk to the soil ecosystem. A 56-day experiment was conducted to study the bioremediation by earthworms on soil microbial diversity and partial nitrification processes in oxytetracycline (OTC)-contaminated soil. The results showed that high OTC concentration significantly decreased the activity of soil bacteria, ammonia-oxidizing bacteria (AOB) and archaea (AOA). Earthworms were found to accelerate the degradation efficiency and rate of OTC, and its main metabolites were 4-epi-oxytetracycline (EOTC) and 2-acetyl-2-decarboxamido-oxytetracycline (ADOTC). Earthworms had an important role in the bioremediation of soil microbial diversity by degrading OTC and its metabolite (EOTC), especially in the high OTC condition. Additionally, the results indicated that the effects of earthworms on the degradation of OTC could remediate the abundances of 16S rRNA and AOB amoA genes and the NO3− content in both low and high OTC-contaminated soils. The structural equation model suggested that earthworms could remediate the microbial diversity, the abundances of 16s rRNA and AOB amoA genes by accelerating the degradation of OTC, which contributed to the bioremediation by earthworms on soil microbial diversity and partial nitrification processes in oxytetracycline-contaminated soil.

1. Introduction Tetracycline is one of the primary antibiotics group used extensively to treat and prevent disease in human and animal medicine, as well as to increase feed efficiency and improve growth rate in livestock and poultry industries (Daghrir and Drogui, 2013). Only a small fraction of ingested antibiotics may be absorbed by animals, and 60–90% of ingested tetracyclines are released through animal excretion to slurry, soils, surface waters and ground water (Li and Zhang, 2016; Zheng et al., 2018), which results in serious environmental problems including ecological risks and damage to human health (Fatta-Kassinos et al., 2011). Soil tetracycline contamination threatens food security and soil ecosystem function worldwide, which has attracted widespread attention (Liu et al., 2018). Therefore, there is growing concern about the potential adverse effects of tetracyclines in soils. Oxytetracycline (OTC) is the earliest and most widely used tetracycline worldwide owing to its high efficiency and low cost (Santaeufemia et al., 2016). Several studies have reported that OTC

negatively affected soil microbial activity, enzyme activity and plant growth (Chitescu et al., 2013; Qin et al., 2019). OTC has also been shown to decrease the rate of nitrification by inhibiting the activity of soil-nitrifying bacteria (Piotrowska-Seget et al., 2008). Our previous results indicated that low OTC concentration decreased the abundance of AOB, decreased nitrogen (N) availability in soil, and reduced the biomass and N content in maize shoots and roots (Cao et al., 2016). Additionally, a variety of OTC metabolites, such as 4-epi-OTC (EOTC) and α-apo-OTC (the antibiotic activity as well as 30% and 7–10% of OTC, respectively) were detected in interstitial water, soil and animal urine and faeces (Halling-Sorensen et al., 2003; Lykkeberg et al., 2004). Therefore, OTC-contaminated soils require remediation to mitigate the hazardous effects. Bioremediation has been used to degrade soil organic pollutants in cost-effective and environmentally friendly manners. Current bioremediation methods mainly focus on adding exogenous degrading strains or compost (Sayara et al., 2009). Earthworms function as ecosystem engineers and show high resistance to organic pollutants (Blouin



Corresponding author. College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193, China. E-mail address: [email protected] (C. Wang). 1 Equal contribution to the research. https://doi.org/10.1016/j.ecoenv.2019.109996 Received 14 July 2019; Received in revised form 16 November 2019; Accepted 20 November 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.

Please cite this article as: Mengli Liu, Jia Cao and Chong Wang, Ecotoxicology and Environmental Safety, https://doi.org/10.1016/j.ecoenv.2019.109996

Ecotoxicology and Environmental Safety xxx (xxxx) xxxx

M. Liu, et al.

Fig. 1. Schematic representation of the effects of earthworms on the soil microbial activity and partial nitrification processes in oxytetracycline-contaminated soil. Ⅰ: Earthworms can accelerate the existing OTC degradation in oxytetracycline-contaminated soil. Ⅱ: Earthworms can improve soil microbial activity by accelerating the existing OTC degradation. Ⅲ: Earthworms can improve partial nitrification processes by accelerating the existing OTC degradation and regulating soil microbial activity.

hypothesized that the addition of earthworms can remediate the microbial activity by accelerating the degradation of OTC, which contributed to the bioremediation by earthworms on soil microbial diversity and partial nitrification processes in oxytetracyclinecontaminated soil (Fig. 1).

et al., 2013; Rodriguez-Campos et al., 2014). Earthworms have been used to promote the bioremediation of contaminants or unrecyclable compounds in soils (Gupta and Garg, 2009; Datta et al., 2016; Vidal et al., 2017). To date, several studies reported that earthworms could enhance the degradation of organic pollutants, such as pesticides (Tejada and Masciandaro, 2011; Lin et al., 2016), veterinary antibiotics (Cao et al., 2015), PAHs and PCBs (Hernandez-Castellanos et al., 2013; Rodriguez-Campos et al., 2014; Deng and Zeng, 2017) under laboratory conditions. Earthworms may directly mineralize of organic contaminants by ingesting and metabolizing the contaminated soils (Martinkosky et al., 2017). The bioturbation of earthworms could regulate the growth, transport and distribution of microorganisms, which in turn would accelerate the degradation of organic contaminants (Rodriguez-Campos et al., 2014; Lin et al., 2016; Liang et al., 2017). Additionally, earthworms can affect the N cycle processes in the contaminated soil (Cao et al., 2016). Soil N cycle processes are predominantly driven by soil microorganisms. Earthworms may remediate soil N cycle by alleviating the toxic effects of pollutants on soil microbial activity and N-cycling microorganisms (Cao et al., 2015, 2018; Deng and Zeng, 2017; Muniz et al., 2017). The effects of earthworms on regulating the soil structure and stability of aggregates, water infiltration and aeration of deep soil layers can indirectly influence the progress of the N cycle (Curry and Schmidt, 2007; Blouin et al., 2013; Zhang et al., 2019). It was reported that inorganic nitrogen (NH4+-N and NO3−-N) in the soil in microcosms inoculated with earthworms was higher than in the earthworm-free controls (Tapia-Coral et al., 2006). However, research on the potential effect of earthworms on microbial activity and nitrogen cycling in soil polluted with antibiotics is still in its infancy. Therefore, a 56-day experiment was conducted using the earthworm Eisenia fetida, which was conducted to investigate the bioremediation by earthworms on soil microbial diversity and partial nitrification processes in oxytetracycline-contaminated soil. We

2. Materials and methods 2.1. Soil, antibiotic and earthworms used Soil was collected from the top 20 cm of long-term non-farmed land at the Shangzhuang experimental station, Beijing (116.25°E, 40.13°N). The soil sample was air-dried and passed through a 2-mm sieve before use. Soil organic matter was determined from K2Cr2O7 colourimetric oxidization (Walkley, 1947). Total N was measured using the Kjeldahl method (Bremner, 1960), and the available phosphorus (P) was extracted with 0.5 mol L−1 NaHCO3 and spectrophotometrically measured at 880 nm according to the procedure described by Olsen et al. (1954). Exchangeable potassium (K) was determined using the procedure described by Metson (1957). The initial soil organic matter content was 12.5 g kg−1, and the total N was 1 g kg−1. The available P and exchangeable K were 13.65 and 143.0 mg kg−1, respectively. The antibiotic used in the study was OTC (Terramycin®, 200 mg mL−1 base as OTC dihydrate). Adult earthworms (approximately one-year-old Eisenia fetida) were used in this study with an average weight of 0.30 g and a mean length of 8.5 cm. Earthworms were provided by Beijing Lvhuan Jingyu Science and Trade Co., Ltd and adapted to the experimental soil for one week before the experiment. The earthworms were placed on moist filter paper for 24 h to allow them to purge their guts and then rinsed with sterile water before use.

2

Ecotoxicology and Environmental Safety xxx (xxxx) xxxx

M. Liu, et al.

Fig. 2. Degradation curve of OTC (a and b), degradation efficiency of OTC (c and d), the kinetic parameters estimated by first-order degradation (e and f), and its degradation product (g and h) inoculated or not inoculated with earthworms.

scenario simulation, and the concentration level which may threaten soil life and health (Gao et al., 2013; Liu et al., 2012). Each group included two treatments: inoculated vs. not inoculated with earthworms. Therefore, we had the following six treatments: (1) CK (soil without OTC and earthworm addition); (2) E (soil with only earthworm addition); (3) OTC1 (soil with only 1 mg kg−1 OTC addition); (4) E + OTC1 (soil with 1 mg kg−1 OTC and earthworm addition); (5) OTC100 (soil with only 100 mg kg−1 OTC addition); and (6) E + OTC100 (soil with

2.2. Experimental design and incubation The experiment was conducted using sterilized 800-mL polypropylene beakers as the culture vessel. There were three groups of beakers that received 0, 1 or 100 mg OTC kg−1 soil DM, respectively. The concentration of 1 mg kg−1 was selected to encompass environmentally relevant concentrations based on a review of the literature (Toth et al., 2011). The concentration of 100 mg kg−1 was the 3

Ecotoxicology and Environmental Safety xxx (xxxx) xxxx

M. Liu, et al.

Fig. 3. The effect of OTC (0, 1 and 100 mg kg−1 OTC) and earthworms (inoculated vs. not inoculated with earthworms) on the bacterial (a and b) and fungal diversity (c and d). Values with different lower case letters were significantly different and represent significant differences between CK, OTC1 and OTC100 treatments. Significance due to the addition of earthworms is analysed in different OTC-polluted soils. NS not significant; ***p < 0.001; **p < 0.01; *p < 0.05.

100 mg kg−1 OTC and earthworm addition). Each treatment had four replicates. Fifteen earthworms with similar fresh weights (0.30 ± 0.03 g) were added to the relevant treatment pots. The antibiotic was spiked to cattle manure. After thoroughly mixing with a sterile wooden spatula, the spiked manures were mixed the prepared soil. Soil and manure were not sterilized. The final soil-manure mixture consisted of 35 g cattle manure per kilogram of DM soil, and the manure/soil ratios were calculated to approximate an agronomic nitrogen-based manure application of 180 kg N ha−1. The mixtures were then brought to 80% of field capacity (soil field capacity was 31.3% gravimetric water content), having accounted for the water contained in manure and antibiotic liquid. After thorough mixing, portions of 500 g were weighted into sterilized 800-mL polypropylene beakers and the beakers were covered in parafilm to retard moisture loss. All of the containers were randomly arranged in an artificial climate incubator with a temperature of 25 °C, and humidity of approximately 60%. Throughout the incubation, moisture content was monitored weekly and maintained by adding sterile deionized water.

2.3.1. Determination of OTC concentration and its products The concentration of OTC was determined by an HPLC method. Detailed measurement information can be found in our previous study (Cao et al., 2015). The degradation by-products of OTC were determined using an Agilent 1200 series HPLC coupled to an Agilent 6420 triple quadrupole mass spectrometer with electrospray ionization mode as described by Cao et al. (2018). 2.3.2. Soil NH4+-N and NO3−-N content Soil NH4+-N and NO3−-N contents were extracted with 1 mol L−1 KCl (solution: soil = 4:1) for 1 h in an orbital shaker (175 r min−1; 20 °C) and then analysed using a Bran + Luebbe Auto Analyser 3. 2.3.3. Terminal restriction fragment length polymorphism (T-RFLP) analysis Soil DNA was extracted from 0.5 g fresh soil with the PowerSoil® DNA Isolation Kit (MOBIO Laboratories Inc., Carlsbad, CA, USA). Polymerase chain reaction (PCR) amplification of the bacterial community was performed using the bacterial universal primers 27F (5′AGA GTT TGA TCC TGG CTC AG-3′) and 907R (5′-CCG TCA ATT CCT TTG AGT TT-3′). Fungal community amplification was performed with the primer pair EF3RCNL (5′-CAA ACT TGG TCA TTT AGA GGA-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′). The PCR and product purification for bacteria and fungi were performed following the methods reported by Cao (Cao et al., 2015). All PCR products were purified with an OMEGA kit (Beijing, China), and bacterial and fungal products were digested with the restriction enzymes MspI and HinfI (Takara), respectively. The 5′ ends of primers (27F and EF3RCNL) were labelled with fluorescent dye 6-FAM (6-carboxyfluorescein; Sangon Biotech, Shanghai, China) to detect terminal fragments on an ABI 373

2.3. Sampling and analysis After incubations of 1, 3, 7, 14, 28 and 56 d, the whole soils in the beakers in each treatment were collected. The soil samples were sieved (< 2 mm) and air-dried for residual OTC and OTC products determinations. The soils were stored at −70 °C for soil microbial diversity (TRFLP) and the abundance of bacteria, fungi, AOA and AOB (Q-PCR) analyses, and at −20 °C for NH4+-N and NO3−-N analyses.

4

Ecotoxicology and Environmental Safety xxx (xxxx) xxxx

M. Liu, et al.

Fig. 4. The effect of OTC (0, 1 and 100 mg kg−1 OTC) and earthworms (inoculated vs. not inoculated with earthworms) on the abundance of bacteria (a and b), fungi (c and d), AOA (e and f) and AOB (g and h). Values with different lower case letters were significantly different and represent significant differences between CK, OTC1 and OTC100 treatments. Significance due to the addition of earthworms is analysed in different OTC-polluted soils. NS not significant; ***p < 0.001; **p < 0.01; *p < 0.05.

5

Ecotoxicology and Environmental Safety xxx (xxxx) xxxx

M. Liu, et al.

Fig. 5. The effect of OTC (0, 1 and 100 mg kg−1 OTC) and earthworms (inoculated vs. not inoculated with earthworms) on soil NH4+-N (a and b) and NO3−-N content (c and d). Values with different lower case letters were significantly different and represent significant differences between CK, OTC1 and OTC100 treatments. Significance due to the addition of earthworms is analysed in different OTC-polluted soils. NS not significant; ***p < 0.001; **p < 0.01; *p < 0.05.

between treatment means at the 5% level. OTC degradation by-products were determined using the ratio of Ct/C0, where Ct is the concentration of substrate at any given point in time and C0 is the initial substrate concentration. Half-life values (t1/2 = ln2/k) of the contaminants were estimated based on the kinetics equation (Ct = C0e-kt, k is degradation rate constant values). Multivariate analysis of variance (MANOVA) was performed to determine the effects of earthworm and OTC concentrations on soil microbial activity, soil NH4+-N and NO3−N content in oxytetracycline-contaminated soil. A structural equation model (SEM) was applied using AMOS 21.0 to investigate the causal relationships between OTC residual, soil microbial diversity, the abundances of 16S rRNA and AOB amoA genes, and the soil NO3−-N content in oxytetracycline-contaminated soil. The SEM (structural equation model) is a multivariate statistical method that can provide causal understanding by testing hypothesized networks of path-relationships or causal relationships (Eisenhauer et al., 2015).

DNA Sequencer (Applied Biosystems, Inc., USA). The relative abundance of individual terminal restriction fragments (T-RFs) was calculated as the percentage of total peak height in a given T-RFLP profile. Only those T-RFs with a relative abundance > 1% and fragment lengths in the range of 50–500 bp were used in analyses. The matrix of the relative height of peaks detected from T-RFs was used to calculate richness and diversity indices as described by Lang et al. (2012). 2.3.4. Real-time PCR quantification of bacteria, fungi, and amoA genes for AOB and AOA Real-time quantitative PCR of bacterial 16S rRNA and fungal 18S rRNA genes was performed to estimate the abundance of the total bacterial and fungal communities. Primer pairs used for bacterial 16S rRNA genes were 341F (5′-CCTACGGGAGGCAGCAG-3′) and 534R ( 5′-ATTACCGCGGCTGCTGGCA-3′). For fungal 18S rRNA genes, primer pairs were FR1 (5′-AICCATTCAATCGGTAIT-3′) and FF390 (5′-CGATA ACGAACGAGACCT-3′). Real-time quantitative PCR of amoA genes was performed to estimate the abundance of the archaeal and bacterial amoA genes. The primers amoA-1 F (5′-GGGGTTTCTACTGGTGGT-3′) and amoA-2 R (5′-CCCCTCKGSAAAGCCTTCTTC-3′) were used for bacteria generating a 491-bp fragment and Arch-amoA F (5′-STAATGGTCTGGCTTAGACG-3′) and Arch-amoA R (5′-GCGGCCATCCATCTGTA TGT-3′) were used for archaea generating a 628-bp fragment. The qPCR and standard curve determination were conducted as described in our previous study (Cao et al., 2015).

3. Results and discussion 3.1. Impact of earthworm on OTC degradation The concentration of OTC in soils was determined to test the residue of OTC in soil (Fig. 2a and b). As expected, earthworms enhanced OTC degradation. The degradation efficiency of OTC significantly increased after inoculation with earthworms, especially in the high OTC condition (Fig. 2c and d). A faster degradation rate was observed in the earthworm-inoculated soil than in the non-inoculated soil; the degradation rate constant values were 0.0225 and 0.021 day−1 in low and high OTC treatments, respectively, and in earthworm inoculated soil, the degradation rate constant values were 0.0231 and 0.034 day−1, respectively. The half-life values were 31.5 d and 33 d in low and high OTC

2.4. Data analysis Analysis of variance was conducted using SPSS software, version 17.0 (SPSS Institute, Inc., Cary, NC, USA). Fisher's LSD (Least Significant Difference) test was used to test significant differences 6

Ecotoxicology and Environmental Safety xxx (xxxx) xxxx

M. Liu, et al.

Fig. 6. Correlation between EOTC ratio and soil bacterial diversity (a) and fungal diversity (b) at high OTC concentration.

earthworms may accelerate the degradation of OTC by the abovementioned mechanisms, which contributed to the bioremediation goals in OTC-contaminated soils. Two degradation products were detected during the incubation time for the high OTC treatment, which could be interpreted as 4-epi-oxytetracycline (EOTC) and 2-acetyl-2-decarboxamido-oxytetracycline (ADOTC) (Fig. S1). The earthworm-inoculated treatments had a lower EOTC ratio at day 56 compared to the control, while earthworms had no effect on the ratio of ADOTC (Fig. 2g and h). EOTC possesses higher polarity and water solubility than its parent compound, likely resulting in easier leaching to and contamination of groundwater (HallingSorensen et al., 2003; Lykkeberg et al., 2004). Thus, the degradation of EOTC suggested that earthworm may remediate the OTC-contaminated soils by accelerating the degradation of the OTC metabolite (EOTC).

treatments, respectively, and in earthworm inoculated soil, the half-life values were 30.13 d and 20.38 d, respectively (Fig. 2e and f). These results suggested that earthworms could accelerate the degradation of OTC, which is agreement with another study conducted by Ravindran and Mnkeni (2017). Earthworms (Eisenia fetida) are highly adaptable and have a large reproductive capacity and are capable of tolerating and resisting high concentrations of numerous organic pollutants (Rodriguez-Campos et al., 2014; Lin et al., 2016). No earthworm mortality was observed in the 1 or 100 mg kg−1 OTC-contaminated soil in our current and previous studies (Cao et al., 2015, 2018). Previous studies have documented that earthworms could directly degrade organic contaminants, i.e., hydrocarbons and widespread antibiotics, such as OTC, by metabolizing the ingested contaminated soils (Martinkosky et al., 2017). Additionally, earthworms could indirectly stimulate the degradation of OTC by increasing soil aeration and moisture retention, modifying soil chemistry and stimulating degradation-associated bacteria (Cao et al., 2015; Lin et al., 2019). Burrowing by the earthworms improved soil porosity, mixed the soil, increased the soil surface area and the contact between contaminant and the autochthonous microorganisms (Rodriguez-Campos et al., 2014; Lin et al., 2016; Liang et al., 2017). Beneficial microbes, such as bacteria that fix nitrogen, might accumulate in the worm cast and improve the nutrient supply to degrading microorganisms (Schaefer and Juliane, 2007). Therefore,

3.2. The bioremediation by earthworms on microbial activity in OTCcontaminated soil T-RFLP analysis was used to analyze microbial diversity in the present study. Although T-RFLP analysis was of lower resolution than high-throughput sequence and that results obtained did not represent actual taxonomic information (Karczewski et al., 2017). It was reported that T-RFLP analysis was an effective, rapid and inexpensive tool for characterizing microbial diversity (Prakash et al., 2014; Seshan et al., 7

Ecotoxicology and Environmental Safety xxx (xxxx) xxxx

M. Liu, et al.

Fig. 7. A structural equation model (SEM) (a) showing the causal relationship between OTC residual, soil microbial diversity (bacterial and fungal diversity), the abundances of 16S rRNA and AOB amoA genes, and NO3−-N content in oxytetracycline-contaminated soil. χ2 = 0.782, df = 4, p = 0.941; CFI = 1, RMSEA = 0.00. Direct, indirect and total effect coefficients of each variable in relation to soil NO3−-N content (B). The path coefficients are above the arrows. The R2 values above every variable explain the variance proportion.

could provide spatially heterogeneous microhabitats for microorganisms that vary in organic matter stabilization, water potential and oxygen flux (Jiang et al., 2013; Rillig et al., 2017). The excretion and mucus of earthworms are nutrient resources for microbial activity, which may stimulate the growth of some microorganisms, changing the diversity and the abundance of soil microbes (Cao et al., 2015, 2018; Deng and Zeng, 2017; Muniz et al., 2017). Probably, earthworms' intestinal microorganisms may produce co-metabolism with soil indigenous microorganisms, which can regulate soil microbial diversity and partial nitrification processes (Hong et al., 2011; Medina-Sauza et al., 2019). Thus, earthworms could remediate soil microbial diversity and activity in oxytetracycline-contaminated soil by both direct and indirect pathways.

2014). In the present study, it was found that OTC had no effect on bacterial diversity but significantly decreased the diversity of fungi in the early stages of incubation (days 1, 3, 7, 14) (p < 0.05, Fig. 3a and c). Earthworms increased bacterial and fungal diversity in high OTC treatments (Fig. 3b and d). Both earthworms and OTC concentrations had significant effects on soil bacterial diversity (Table S1). High OTC significantly decreased the abundance of bacteria (p < 0.05, Fig. 4a). Earthworms significantly increased the abundance of 16S rRNA gene in low and high OTC-contaminated soil, while the abundance of 18S rRNA gene at low OTC-contaminated soil significantly increased (p < 0.05; Fig. 4b and d). Both earthworms and OTC concentrations had significant effects on the abundance of 16S rRNA gene, while only earthworms significantly affected the abundance of 18S rRNA gene (Table S1). It was reported that OTC was proposed as a bacterial inhibitor (Bailey et al., 2003), which may change the soil microbial diversity and activity by selecting for resistant populations and decreasing sensitive bacterial groups (Xiong et al., 2018). Earthworms function as ecosystem engineers and can modify ecosystem functions and services, such as soil microbial diversity and activity. Previous studies indicated that earthworms could restore soil microbial diversity and activity by the degradation of OTC, which was attributed to reducing the toxicity of OTC to microorganisms in oxytetracycline-contaminated soil (Chen et al., 2013; Rodriguez-Campos et al., 2014). Our results indicated that the degradation of OTC and its metabolite (EOTC) by earthworms remediated the diversity and activity of soil microbes in oxytetracyclinecontaminated soil, especially in the high OTC condition (Figs. 6 and 7). Additionally, earthworms could stimulate the growth of some microbes and improve the microbial activity by feeding, digestion and burrowing, which are attributed to the improvement of soil physical structure, decomposition of organic matter and the releasing of secretions (Dey et al., 2017). The improvement of soil physical structure

3.3. The bioremediation by earthworms of partial nitrification processes in OTC-contaminated soil In the present study, both OTC concentrations and earthworms had significant effects on the abundance of AOB (Table S1). The abundance of AOB and AOA amoA genes were significantly inhibited in high OTC treatments (Fig. 4e and g), which is consistent with previous studies showing low AOA and Arch-amoA gene abundances in the oxytetracycline system (Zhang et al., 2015; Wang et al., 2018). Further, the addition of earthworms significantly increased the abundance of AOB in low and high OTC treatments and increased the abundance of AOA in the high OTC treatment (Fig. 4f and h). The results of the SEM indicated that earthworms improved the activity of ammonia-oxidizers by accelerating the degradation of OTC (Fig. 7), which may be due to reduced toxicity of OTC to ammonia-oxidizers (Cao et al., 2015). Furthermore, ammonia oxidation is typically an obligatorily aerobic process (Francis et al., 2007). Earthworms could increase soil aeration and porosity by regulating soil structure, which would contribute to the 8

Ecotoxicology and Environmental Safety xxx (xxxx) xxxx

M. Liu, et al.

growth of the aerobic ammonia-oxidizers (Curry and Schmidt, 2007). Previous research showed that the antibiotic sulfadiazine changes nitrogen turnover (Hammesfahr et al., 2011a, 2011b). In the present study, the higher concentrations of NH4+ and lower concentrations of NO3− in the early stages of incubation with low OTC could imply that OTC may decrease rates of nitrification (Fig. 5a and c). While earthworms increased the NO3− content during the incubation and decreased the NH4+ content in the later stage of incubation (Fig. 4b and d), which indicated that earthworms could accelerate the transformation from NH4+ to NO3− in OTC-contaminated soil. The conversion of ammonia to nitrate is performed primarily by bacteria that live in the soil and by other nitrifying bacteria. In the primary stage of nitrification, both AOB and AOA are the drivers for the first and rate-limiting step in nitrification processes (Zhang et al., 2015). The high NO3− content is consistent with elevated nitrifying bacterial (AOB, AOA) populations indicating autotrophic nitrification in the presence of earthworms (Araujo et al., 2004; Blouin et al., 2013). There was a significantly positive relationship between the abundance of AOB and soil NO3− content (Fig. 7), which suggested that earthworms could accelerate the transformation from NH4+ to NO3- by increasing the abundance of AOB, resulting in higher NO3− content. Additionally, earthworms can enhance organic N mineralization by feeding, digestion and casting activities, which often lead to increased soil inorganic N (Araujo et al., 2004). Overall, the results of the SEM indicated that earthworms could remediate soil microbial diversity and partial nitrification processes by accelerating the degradation of OTC in oxytetracycline-contaminated soil (Fig. 7).

Blouin, M., Hodson, M.E., Delgado, E.A., Baker, G., Brussaard, L., Butt, K.R., Dai, J., Dendooven, L., Peres, G., Tondoh, J.E., Cluzeau, D., Brun, J.J., 2013. A review of earthworm impact on soil function and ecosystem services. Eur. J. Soil Sci. 64, 161–182. Bremner, J., 1960. Determination of nitrogen in soil by the Kjeldahl method. J. Agric. Sci. 55, 11–33. Cao, J., Ji, D.G., Wang, C., 2015. Interaction between earthworms and arbuscular mycorrhizal fungi on the degradation of oxytetracycline in soils. Soil Biol. Biochem. 90, 283–292. Cao, J., Wang, C., Dou, Z.X., Ji, D.G., 2016. Independent and combined effects of oxytetracycline and antibiotic-resistant Escherichia coli O157:H7 on soil microbial activity and partial nitrification processes. Soil Biol. Biochem. 98, 138–147. Cao, J., Wang, C., Dou, Z.X., Liu, M.L., Ji, D.G., 2018. Hyphospheric impacts of earthworms and arbuscular mycorrhizal fungus on soil bacterial community to promote oxytetracycline degradation. J. Hazard Mater. 341, 346–354. Chen, W., Liu, W., Pan, N., Jiao, W., Wang, M., 2013. Oxytetracycline on functions and structure of soil microbial community. J. Soil Sci. Plant Nutr. 13, 967–975. Chitescu, C.L., Nicolau, A.I., Stolker, A.A.M., 2013. Uptake of oxytetracycline, sulfamethoxazole and ketoconazole from fertilised soils by plants. Food Addit. Contam. A 30, 1138–1146. Curry, J.P., Schmidt, O., 2007. The feeding ecology of earthworms - a review. Pedobiologia 50, 463–477. Daghrir, R., Drogui, P., 2013. Tetracycline antibiotics in the environment: a review. Environ. Chem. Lett. 11, 209–227. Datta, S., Singh, J., Singh, S., Singh, J., 2016. Earthworms, pesticides and sustainable agriculture: a review. Environ. Sci. Pollut. Res. 23, 8227–8243. Deng, S.G., Zeng, D.F., 2017. Removal of phenanthrene in contaminated soil by combination of alfalfa, white-rot fungus, and earthworms. Environ. Sci. Pollut. Res. 24, 7565–7571. Dey, M.D., Das, S., Kumar, R., Doley, R., Bhattacharya, S.S., Mukhopadhyay, R., 2017. Vermiremoval of methylene blue using Eisenia fetida: a potential strategy for bioremediation of synthetic dye-containing effluents. Ecol. Eng. 106, 200–208. Eisenhauer, N., Bowker, M.A., Grace, J.B., Powell, J.R., 2015. From patterns to causal understanding: structural equation modeling (SEM) in soil ecology. Pedobiologia 58, 65–72. Fatta-Kassinos, D., Meric, S., Nikolaou, A., 2011. Pharmaceutical residues in environmental waters and wastewater: current state of knowledge and future research. Anal. Bioanal. Chem. 399, 251–275. Francis, C.A., Beman, J.M., Kuypers, M.M.M., 2007. New processes and players in the nitrogen cycle: the microbial ecology of anaerobic and archaeal ammonia oxidation. ISME J. 1, 19–27. Gao, M.L., Song, W.H., Zhou, Q., Ma, X.J., Chen, X.J., 2013. Interactive effect of oxytetracycline and lead on soil enzymatic activity and microbial biomass. Environ. Toxicol. Pharmacol. 36, 667–674. Gupta, R., Garg, V.K., 2009. Vermiremediation and nutrient recovery of non-recyclable paper waste employing Eisenia fetida. J. Hazard Mater. 162, 430–439. Halling-Sorensen, B., Lykkeberg, A., Ingerslev, F., Blackwell, P., Tjornelund, J., 2003. Characterisation of the abiotic degradation pathways of oxytetracyclines in soil interstitial water using LC-MS-MS. Chemosphere 50, 1331–1342. Hammesfahr, U., Bierl, R., Thiele-Bruhn, S., 2011a. Combined effects of the antibiotic sulfadiazine and liquid manure on the soil microbial-community structure and functions. J. Plant Nutr. Soil Sci. 174, 614–623. Hammesfahr, U., Kotzerke, A., Lamshoft, M., Wilke, B.M., Kandeler, E., Thiele-Bruhn, S., 2011b. Effects of sulfadiazine-contaminated fresh and stored manure on a soil microbial community. Eur. J. Soil Biol. 47, 61–68. Hernandez-Castellanos, B., Ortiz-Ceballos, A., Martinez-Hernandez, S., Noa-Carrazana, J.C., Luna-Guido, M., Dendooven, L., Contreras-Ramos, S.M., 2013. Removal of benzo (a) pyrene from soil using an endogeic earthworm Pontoscolex corethrurus (Muller, 1857). Appl. Soil Ecol. 70, 62–69. Hong, S.W., Lee, J.S., Chung, K.S., 2011. Effect of enzyme producing microorganisms on the biomass of epigeic earthworms (Eisenia fetida) in vermicompost. Bioresour. Technol. 102, 6344–6347. Jiang, Y.J., Sun, B., Ji, C., Wang, F., 2013. Soil aggregate stratification of nematodes and microbial communities affects the metabolic quotient in an acid soil. Soil Biol. Biochem. 60, 1–9. Karczewski, K., Riss, H.W., Meyer, E.I., 2017. Comparison of DNA-fingerprinting (T-RFLP) and high-throughput sequencing (HTS) to assess the diversity and composition of microbial communities in groundwater ecosystems. Limnologica 67, 45–53. Lang, J.J., Hu, J., Ran, W., Xu, Y.C., Shen, Q.R., 2012. Control of cotton Verticillium wilt and fungal diversity of rhizosphere soils by bio-organic fertilizer. Biol. Fertil. Soils 48, 191–203. Li, J., Zhang, H., 2016. Adsorption-desorption of oxytetracycline on marine sediments: kinetics and influencing factors. Chemosphere 164, 156–163. Liang, J., Xia, X.Q., Zaman, W.Q., Zhang, W., Lin, K.F., Hu, S.Q., Lin, Z.F., 2017. Bioaccumulation and toxic effects of decabromodiphenyl ether in the presence of nanoscale zero-valent iron in an earthworm soil system. Chemosphere 169, 78–88. Lin, Z., Bai, J., Zhen, Z., Lao, S.Q., Li, W.Y., Wu, Z.H., Li, Y.T., Spiro, B., Zhang, D.Y., 2016. Enhancing pentachlorophenol degradation by vermicomposting associated bioremediation. Ecol. Eng. 87, 288–294. Lin, Z., Zhen, Z., Liang, Y.Q., Li, J., Yang, J.W., Zhong, L.Y., Zhao, L.R., Li, Y.T., Luo, C.L., Ren, L., Zhang, D.Y., 2019. Changes in atrazine speciation and the degradation pathway in red soil during the vermiremediation process. J. Hazard Mater. 364, 710–719. Liu, W., Pan, N., Chen, W., Jiao, W., Wang, M., 2012. Effect of veterinary oxytetracycline on functional diversity of soil microbial community. Plant Soil Environ. 58, 295–301. Liu, X.H., Lv, Y., Xu, K., Xiao, X.X., Xi, B.D., Lu, S.Y., 2018. Response of ginger growth to a

4. Conclusion OTC could affect soil microbial activity and inhibit soil nitrification processes by suppressing ammonia oxidizers, especially with high OTC concentration. Our study provides clear and strong evidence that earthworms could enhance the degradation of OTC and the metabolite (EOTC) and thus fulfil the aim of the bioremediation approach. Furthermore, earthworms showed an important role in the bioremediation of soil microbial activity and partial nitrification processes in oxytetracycline-contaminated soil by accelerating the existing OTC degradation. All these factors indicate that earthworms could effectively improve bioremediation efficiency and reduce the risks posed by OTC to some extent. Mengli Liu: Formal analysis, Writing - Original Draft, Visualization. Jia Cao: Conceptualization, Investigation, Writing - Original Draft. Chong Wang: Conceptualization, Writing - Review & Editing, Supervision, Project administration, Funding acquisition. Acknowledgments This work was funded by the National key research and development program(2016YFE0101100)and the Da Bei Nong's Youth Researchers Program (2413002). We also acknowledge Dr. Zhenjun Sun and Dr. Xiaolin Li at China Agricultural University for laboratory assistance. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2019.109996. References Araujo, Y., Luizao, F.J., Barros, E., 2004. Effect of earthworm addition on soil nitrogen availability, microbial biomass and litter decomposition in mesocosms. Biol. Fertil. Soils 39, 146–152. Bailey, V.L., Smith, J.L., Bolton, H., 2003. Novel antibiotics as inhibitors for the selective respiratory inhibition method of measuring fungal : bacterial ratios in soil. Biol. Fertil. Soils 38, 154–160.

9

Ecotoxicology and Environmental Safety xxx (xxxx) xxxx

M. Liu, et al.

Sayara, T., Sarra, M., Sanchez, A., 2009. Preliminary screening of co-substrates for bioremediation of pyrene-contaminated soil through composting. J. Hazard Mater. 172, 1695–1698. Schaefer, M., Juliane, F., 2007. The influence of earthworms and organic additives on the biodegradation of oil contaminated soil. Appl. Soil Ecol. 36, 53–62. Seshan, H., Goyal, M.K., Falk, M.W., Wuertz, S., 2014. Support vector regression model of wastewater bioreactor performance using microbial community diversity indices: effect of stress and bioaugmentation. Water Res. 53, 282–296. Tapia-Coral, S.C., Luizao, F.J., Barros, E., Pashanasi, B., del Castillo, D., 2006. Effect of Pontoscolex corethrurus Muller, 1857 (Oligochaeta : glossoscolecidae) inoculation on litter weight loss and soil nitrogen in mesocosms in the Peruvian Amazon. Caribb. J. Sci. 42, 410–418. Tejada, M., Masciandaro, G., 2011. Application of organic wastes on a benzo(a)pyrene polluted soil. Response of soil biochemical properties and role of Eisenia fetida. Ecotoxicol. Environ. Saf. 74, 668–674. Toth, J.D., Feng, Y., Dou, Z., 2011. Veterinary antibiotics at environmentally relevant concentrations inhibit soil iron reduction and nitrification. Soil Biol. Biochem. 43, 2470–2472. Vidal, A., Quenea, K., Alexis, M., Tu, T.T.N., Mathieu, J., Vaury, V., Derenne, S., 2017. Fate of C-13 labelled root and shoot residues in soil and anecic earthworm casts: a mesocosm experiment. Geoderma 285, 9–18. Walkley, A., 1947. A critical examination of a rapid method for determining organic carbon in soils—effect of variations in digestion conditions and of inorganic soil constituents. Soil Sci. 63, 251–264. Wang, L.J., Wang, J.H., Zhu, L.S., Wang, J., 2018. Toxic effects of oxytetracycline and copper, separately or combined, on soil microbial biomasses. Environ. Geochem. Hlth. 40, 763–776. Xiong, W.G., Wang, M., Dai, J.J., Sun, Y.X., Zeng, Z.L., 2018. Application of manure containing tetracyclines slowed down the dissipation of tet resistance genes and caused changes in the composition of soil bacteria. Ecotoxicol. Environ. Saf. 147, 455–460. Zhang, W.W., Wang, C., Liu, M.L., Yu, Y.C., 2019. Integrated reclamation of saline soil nitrogen transformation in the hyphosphere by earthworms and arbuscular mycorrhizal fungus. Appl. Soil Ecol. 135, 137–146. Zhang, Y., Tian, Z., Liu, M.M., Shi, Z.J., Hale, L., Zhou, J.Z., Yang, M., 2015. High concentrations of the antibiotic spiramycin in wastewater lead to high abundance of ammonia-oxidizing archaea in nitrifying populations. Environ. Sci. Technol. 49, 9124–9132. Zheng, W., Zhang, Z.Y., Liu, R., Lei, Z.F., 2018. Removal of veterinary antibiotics from anaerobically digested swine wastewater using an intermittently aerated sequencing batch reactor. J. Environ. Sci. 65, 8–17.

tetracycline-contaminated environment and residues of antibiotic and antibiotic resistance genes. Chemosphere 201, 137–143. Lykkeberg, A.K., Sengelov, G., Cornett, C., Tjornelund, J., Hansen, S.H., Halling-Sorensen, B., 2004. Isolation, structural elucidation and in vitro activity of 2-acetyl-2-decarboxamido-oxytetracycline against environmental relevant bacteria, including tetracycline-resistant bacteria. J. Pharm. Biomed. Anal. 34, 559–567. Martinkosky, L., Barkley, J., Sabadell, G., Gough, H., Davidson, S., 2017. Earthworms (Eisenia fetida) demonstrate potential for use in soil bioremediation by increasing the degradation rates of heavy crude oil hydrocarbons. Sci. Total Environ. 580, 734–743. Medina-Sauza, R.M., Álvarez-Jiménez, M., Delhal, A., Reverchon, F., Blouin, M., Guerrero-Analco, J.A., Cerdan, C.R., Guevara, R., Villain, L., Barois, I., 2019. Earthworms building up soil microbiota, a review. Front. Environ. Sci. 7, 81. Metson, A.J., 1957. Methods of chemical analysis for soil survey samples. Soil Sci. 83, 245. Muniz, S., Gonzalvo, P., Valdehita, A., Molina-Molina, J.M., Navas, J.M., Olea, N., Fernandez-Cascan, J., Navarro, E., 2017. Ecotoxicological assessment of soils polluted with chemical waste from lindane production: use of bacterial communities and earthworms as bioremediation tools. Ecotoxicol. Environ. Saf. 145, 539–548. Olsen, S.R., Cole, C.V., Watanabe, S., Dean, L.A., 1954. Estimation of available phosphorous in soils by extraction with sodium bicarbonate. In: USDA Circular, vol. 939. pp. 1–8. Piotrowska-Seget, Z., Engel, R., Nowak, E., Kozdroj, J., 2008. Successive soil treatment with captan or oxytetracycline affects non-target microorganisms. World J. Microbiol. Biotechnol. 24, 2843–2848. Prakash, O., Pandey, P.K., Kulkarni, G.J., Mahale, K.N., Shouche, Y.S., 2014. Technicalities and glitches of terminal restriction fragment length polymorphism (TRFLP). Indian J. Microbiol. 54, 255–261. Qin, J.M., Xiong, H.Y., Ma, H.T., Li, Z.J., 2019. Effects of different fertilizers on residues of oxytetracycline and microbial activity in soil. Environ. Sci. Pollut. Res. 26, 161–170. Ravindran, B., Mnkeni, P.N.S., 2017. Identification and fate of antibiotic residue degradation during composting and vermicomposting of chicken manure. Int. J. Environ. Sci. Technol. 14, 263–270. Rillig, M.C., Muller, L.A.H., Lehmann, A., 2017. Soil aggregates as massively concurrent evolutionary incubators. ISME J. 11, 1943–1948. Rodriguez-Campos, J., Dendooven, L., Alvarez-Bernal, D., Contreras-Ramos, S.M., 2014. Potential of earthworms to accelerate removal of organic contaminants from soil: a review. Appl. Soil Ecol. 79, 10–25. Santaeufemia, S., Torres, E., Mera, R., Abalde, J., 2016. Bioremediation of oxytetracycline in seawater by living and dead biomass of the microalga Phaeodactylum tricornutum. J. Hazard Mater. 320, 315–325.

10