Ecotoxicology and Environmental Safety 189 (2020) 109996

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Bioremediation by earthworms on soil microbial diversity and partial T

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
b
Beijing Key Laboratory of Biodiversity and Organic Farming, Beijing, 100193, China
c
Key Laboratory of Plant-Soil Interactions, MOE, Beijing, 100193, China

A R T I C LE I N FO A B S T R A C T

Keywords: A large proportion (60–90%) of ingested tetracyclines are released to slurry, soils, surface waters and ground
Oxytetracycline degradation water, which has raised extensive concerns and may pose a risk to the soil ecosystem. A 56-day experiment was
Ammonia oxidizers conducted to study the bioremediation by earthworms on soil microbial diversity and partial nitrification pro-
T-RFLP cesses in oxytetracycline (OTC)-contaminated soil. The results showed that high OTC concentration significantly
Bacterial 16S rRNA and fungal 18S rRNA genes
decreased the activity of soil bacteria, ammonia-oxidizing bacteria (AOB) and archaea (AOA). Earthworms were
Structural equation model
found to accelerate the degradation efficiency and rate of OTC, and its main metabolites were 4-epi-oxyte-
tracycline (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 mi-
crobial 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 in-
gested 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 atten-
tion (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 tetra-
cycline 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 bior-
emediation methods mainly focus on adding exogenous degrading
strains or compost (Sayara et al., 2009). Earthworms function as eco-
system 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: wangchong@cau.edu.cn (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
Available online 28 November 2019
0147-6513/ © 2019 Elsevier Inc. All rights reserved.
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.
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 con-
taminants by ingesting and metabolizing the contaminated soils
(Martinkosky et al., 2017). The bioturbation of earthworms could reg-
ulate 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 pre-
dominantly driven by soil microorganisms. Earthworms may remediate
soil N cycle by alleviating the toxic effects of pollutants on soil micro-
bial 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 infiltra-
tion and aeration of deep soil layers can indirectly influence the pro-
gress 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
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Ecotoxicology and Environmental Safety 189 (2020) 109996
hypothesized that the addition of earthworms can remediate the mi-
crobial activity by accelerating the degradation of OTC, which con-
tributed to the bioremediation by earthworms on soil microbial di-
versity and partial nitrification processes in oxytetracycline-
contaminated soil (Fig. 1).
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 ex-
tracted with 0.5 mol L−1 NaHCO3 and spectrophotometrically mea-
sured at 880 nm according to the procedure described by Olsen et al.
(1954). Exchangeable potassium (K) was determined using the proce-
dure described by Metson (1957). The initial soil organic matter con-
tent 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 (approxi-
mately 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.
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.
2.2. Experimental design and incubation
The experiment was conducted using sterilized 800-mL poly-
propylene 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 en-
vironmentally relevant concentrations based on a review of the litera-
ture (Toth et al., 2011). The concentration of 100 mg kg−1 was the
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Ecotoxicology and Environmental Safety 189 (2020) 109996
scenario simulation, and the concentration level which may threaten
soil life and health (Gao et al., 2013; Liu et al., 2012). Each group in-
cluded 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 addi-
tion); (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
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 cal-
culated to approximate an agronomic nitrogen-based manure applica-
tion 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 li-
quid. 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. 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 determi-
nations. The soils were stored at −70 °C for soil microbial diversity (T-
RFLP) and the abundance of bacteria, fungi, AOA and AOB (Q-PCR)
analyses, and at −20 °C for NH4+-N and NO3−-N analyses.
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Ecotoxicology and Environmental Safety 189 (2020) 109996
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 de-
termined 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 com-
munity 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
M. Liu, et al. Ecotoxicology and Environmental Safety 189 (2020) 109996

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
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 treat-
ments. 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.
DNA Sequencer (Applied Biosystems, Inc., USA). The relative abun-
dance of individual terminal restriction fragments (T-RFs) was calcu-
lated 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 bac-
teria generating a 491-bp fragment and Arch-amoA F (5′-STAATGGT-
CTGGCTTAGACG-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).
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
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Ecotoxicology and Environmental Safety 189 (2020) 109996
between treatment means at the 5% level. OTC degradation by-pro-
ducts were determined using the ratio of Ct/C0, where Ct is the con-
centration of substrate at any given point in time and C0 is the initial
substrate concentration. Half-life values (t1/2 = ln2/k) of the con-
taminants 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-re-
lationships or causal relationships (Eisenhauer 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 earth-
worm-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 de-
gradation rate constant values were 0.0231 and 0.034 day−1, respec-
tively. The half-life values were 31.5 d and 33 d in low and high OTC
M. Liu, et al.
Fig. 6. Correlation between EOTC ratio and soil bacterial diversity (a) and fungal diversity (b) at high OTC concentration.
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 mor-
tality 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 or-
ganic 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 degrada-
tion of OTC by increasing soil aeration and moisture retention, mod-
ifying soil chemistry and stimulating degradation-associated bacteria
(Cao et al., 2015; Lin et al., 2019). Burrowing by the earthworms im-
proved soil porosity, mixed the soil, increased the soil surface area and
the contact between contaminant and the autochthonous microorgan-
isms (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 de-
grading microorganisms (Schaefer and Juliane, 2007). Therefore,
7
Ecotoxicology and Environmental Safety 189 (2020) 109996
earthworms may accelerate the degradation of OTC by the above-
mentioned 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-oxy-
tetracycline (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 (Halling-
Sorensen 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).
3.2. The bioremediation by earthworms on microbial activity in OTC-
contaminated 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.,
M. Liu, et al.
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 sig-
nificant 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 earth-
worms could restore soil microbial diversity and activity by the de-
gradation 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 re-
mediated the diversity and activity of soil microbes in oxytetracycline-
contaminated 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 bur-
rowing, which are attributed to the improvement of soil physical
structure, decomposition of organic matter and the releasing of secre-
tions (Dey et al., 2017). The improvement of soil physical structure
8
Ecotoxicology and Environmental Safety 189 (2020) 109996
Fig. 7. A structural equation model (SEM)
(a) showing the causal relationship between
OTC residual, soil microbial diversity (bac-
terial and fungal diversity), the abundances
of 16S rRNA and AOB amoA genes, and
NO3−-N content in oxytetracycline-con-
taminated 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 ar-
rows. The R2 values above every variable
explain the variance proportion.
could provide spatially heterogeneous microhabitats for microorgan-
isms 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' in-
testinal microorganisms may produce co-metabolism with soil in-
digenous 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.
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 oxytetracy-
cline system (Zhang et al., 2015; Wang et al., 2018). Further, the ad-
dition 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 ac-
celerating the degradation of OTC (Fig. 7), which may be due to re-
duced toxicity of OTC to ammonia-oxidizers (Cao et al., 2015). Fur-
thermore, 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
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 earth-
worms increased the NO3− content during the incubation and de-
creased the NH4+ content in the later stage of incubation (Fig. 4b and
d), which indicated that earthworms could accelerate the transforma-
tion 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 nitrifica-
tion, 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 ni-
trification processes by accelerating the degradation of OTC in oxyte-
tracycline-contaminated soil (Fig. 7).
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 bior-
emediation 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 effec-
tively 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, Super-
vision, Project administration, Funding acquisition.
Acknowledgments
This work was funded by the National key research and develop-
ment 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 as-
sistance.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ecoenv.2019.109996.
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