Enantioselective degradation of the organophosphorus insecticide isocarbophos in Cupriavidus nantongensis X1T: Characteristics, enantioselective regulation, degradation pathways, and toxicity assessment

Article abstract of DOI:10.1016/j.jhazmat.2021.126024

The chiral pesticide enantiomers often show selective efficacy and non-target toxicity. In this study, the enantioselective degradation characteristics of the chiral organophosphorus insecticide isocarbophos (ICP) by Cupriavidus nantongensis X1^T were investigated systematically. Strain X1^T preferentially degraded the ICP R isomer (R-ICP) over the S isomer (S-ICP). The degradation rate constant of R-ICP was 42-fold greater than S-ICP, while the former is less bioactive against pest insects but more toxic to humans than the latter. The concentration ratio of S-ICP to R-ICP determines whether S-ICP can be degraded by strain X1^T. S-ICP started to degrade only when the ratio (C_S-ICP/C_R-ICP) was greater than 62. Divalent metal cations could improve the degradation ability of strain X1^T. The detected metabolites that were identified suggested a novel hydrolysis pathway, while the hydrolytic metabolites were less toxic to fish and green algae than those from P-O bond breakage. The crude enzyme degraded both R-ICP and S-ICP in a similar rate, indicating that enantioselective degradation was due to the transportation of strain X1^T. The strain X1^T also enantioselectively degraded the chiral organophosphorus insecticides isofenphos-methyl and profenofos. The enantioselective degradation characteristics of strain X1^T make it suitable for remediation of chiral organophosphorus insecticide contaminated soil and water.

Full text of DOI:10.1016/j.jhazmat.2021.126024

Journal of Hazardous Materials 417 (2021) 126024
Contents lists available at ScienceDirect
Journal of Hazardous Materials
journal homepage: www.elsevier.com/locate/jhazmat
Enantioselective degradation of the organophosphorus insecticide
isocarbophos in Cupriavidus nantongensis X1T: Characteristics,
enantioselective regulation, degradation pathways, and toxicity assessment
Liancheng Fanga, Luyuan Xua, Nan Zhanga, Qiongying Shia, Taozhong Shia, Xin Maa,
,
*
Xiangwei Wua, Qing X. Lib, Rimao Huaa
a Anhui Provincial Key Laboratory for Quality and Safety of Agri-Products, School of Resource & Environment, Anhui Agricultural University, Hefei, Anhui 230036,
China
b Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, 1955 East-West Road, Honolulu, HI 96822, United States
A R T I C L E I N F O
Editor: Dr. R. Maria Sonia
A B S T R A C T
The chiral pesticide enantiomers often show selective efficacy and non-target toxicity. In this study, the enan-
tioselective degradation characteristics of the chiral organophosphorus insecticide isocarbophos (ICP) by
Cupriavidus nantongensis X1T were investigated systematically. Strain X1T preferentially degraded the ICP R
isomer (R-ICP) over the S isomer (S-ICP). The degradation rate constant of R-ICP was 42-fold greater than S-ICP,
while the former is less bioactive against pest insects but more toxic to humans than the latter. The concentration
ratio of S-ICP to R-ICP determines whether S-ICP can be degraded by strain X1T. S-ICP started to degrade only
when the ratio (CS-ICP/CR-ICP) was greater than 62. Divalent metal cations could improve the degradation ability
of strain X1T. The detected metabolites that were identified suggested a novel hydrolysis pathway, while the
hydrolytic metabolites were less toxic to fish and green algae than those from P-O bond breakage. The crude
enzyme degraded both R-ICP and S-ICP in a similar rate, indicating that enantioselective degradation was due to
the transportation of strain X1T. The strain X1T also enantioselectively degraded the chiral organophosphorus
insecticides isofenphos-methyl and profenofos. The enantioselective degradation characteristics of strain X1T
make it suitable for remediation of chiral organophosphorus insecticide contaminated soil and water.
Keywords:
Organophosphorus insecticide
Chiral
Enantioselective
Biodegradation
Cupriavidus nantongensis
1. Introduction
(R-ICP) and S-isocarbophos (S-ICP) (Fig. 1) (Zhang et al., 2012). The
insecticidal activities and toxicity of ICP enantiomers for target or
Organophosphorus insecticides (OPs) are among the most commonly
used pesticides worldwide to control agricultural pest insects (Y.N.
Zhang et al., 2020). In 2018, about 300,000 tons of pesticides were
applied in China and more than 90% could be finally entered to the soil
and water, causing environmental pollution and endangering human
health (Silver et al., 2017; Gao et al., 2020). OPs are highly toxic and can
inhibit acetylcholinesterase (AChE) in the human nervous system (Jiang
et al., 2019). OPs can also induce blood lymphocytes cells to cause DNA
damage and lead to organ dysfunction (Sandhu et al., 2013;
Karami-Mohajeri et al., 2017).
non-target organisms have been fully studied. The S-ICP was 100-fold
more toxic than R-ICP to Nilaparvata lugens (rice pest, Hemiptera: Del-
phacidae) and Chilo suppressalis (rice pest, Lepidoptera: Crambidae),
while S-ICP was more persistent than R-ICP in rice cultivation (Di et al.,
2019). On the contrary, the R-ICP was 2-fold more toxic than S-ICP to
HepG2 cells (human liver tissue cells) and R-ICP could induce the pro-
duction of reactive oxygen species (Liu et al., 2010). However, most
chiral-OPs are used in racemic-form (Sanganyado et al., 2017). Appli-
cations of the R-ICP that has low insecticidal activity but high
mammalian toxicity can cause environmental pollution and increase the
risk to human health.
Isocarbophos (O-2-isopropoxycarbonylphenyl O-methyl phosphor-
amidothioate, ICP) is one of the most used chiral OPs in rice fields to
control chewing and sucking insects (Yao et al., 2015). ICP has a chiral
center of P atom and contains a pair of enantiomers, R-isocarbophos
Enantioselective degradation of chiral pesticides, especially in soil, is
often attributed to microorganisms and the degradation processes can be
affected by soil type, pH, and microbial populations (Li et al., 2012;
* Corresponding author.
E-mail address: rimaohua@ahau.edu.cn (R. Hua).
https://doi.org/10.1016/j.jhazmat.2021.126024
Received 20 March 2021; Received in revised form 28 April 2021; Accepted 29 April 2021
Available online 7 May 2021
0304-3894/© 2021 Elsevier B.V. All rights reserved.

L. Fang et al.
Journal of Hazardous Materials 417 (2021) 126024
2. Materials and methods
2.1. Chemicals and media
The racemic isocarbophos (Rac-ICP, CAS 24353–61–5) standard and
its metabolites, isopropyl salicylate and salicylic acid were purchased
from Sigma-Aldrich (Shanghai, China). Isocarbophos chiral enantio-
mers, R-ICP and S-ICP, were kindly gifted by Professor Minghua Wang
(Nanjing Agricultural University, China). Rac-isofenphos-methyl and
Rac-profenofos standard were purchased from Dr. Ehrenstorfer (Augs-
burg, Germany). KCl, CaCl2, CrCl3, FeCl3.6H2O, Al2(SO4)3.18H2O,
MnCl2.4H2O, MgCl2.6H2O, ZnCl2, NiSO4.6H2O and CoCl2.6H2O were
purchased from Aladdin Reagent (Shanghai, China). The formic acid,
acetonitrile and acetone in chromatographic grade were purchased from
Tedia Co., Ltd (Ohio, United States).
Fig. 1. Structure of the chiral OPs isocarbophos.
Mineral salt medium (MSM) and Luria-Bertani (LB) medium were
used for the degradation experiments and incubation of strain X1T,
respectively.
Zhang et al., 2018; Kaziem et al., 2020). In paddy soil under natural
conditions, the degradation rate of RS/SR-paichongding was higher than
that of RR/SS-paichongding. After adding the degradation strain
Sphingobacterium sp. P1–3, the degradation rate of RR/SS-paichongding
was significantly increased (Chen et al., 2017). In the paddy soil
collected in Shaoxing (Zhejiang, China), the half-life of R-ICP was
shorter than that of S-ICP, which was caused by soil microorganisms (Di
et al., 2019). A-(ꢀ )-Fosthiazate (an organophosphorus insecticides) was
preferentially enriched in Phaseolus vulgaris Linn soil, while
A-(+)-fosthiazate was preferentially accumulated in Vicia faba Linn soil
(Di et al., 2021). However, very few enantioselective degradation strains
have been isolated from soils. The detailed mechanism of enantiose-
lective microbial degradation of pesticides in the environment is still
unclear.
2.2. Degradation strain
Cupriavidus nantongensis X1T was previously isolated from an acti-
vated sludge of pesticides plant in Nantong (Jiangsu, China). Strain X1T
was deposited in BCCM/LMG with accession number LMG 29818. The
optimal growth conditions for strain X1T were 37 ◦C and pH 7.5 in the
LB medium.
2.3. Enantioselective degradation of Rac-ICP, R-ICP and S-ICP
Strain X1T was aerobically incubated in 100 mL of LB medium at
37 ◦C to OD600 reached 0.8. The free cells of strain X1T were harvested at
6000 g for 5 min and washed twice with sterilized MSM. Then, the
washed cells were added into 20 mL MSM reaction solution with 50 mg/
L Rac-ICP, 25 mg/L R-ICP or S-ICP, at the final OD600 = 0.1. The reac-
tion solution without strain X1T was used as the negative control. All test
conical flasks were aerobically incubated at 37 ◦C in a rotary shaker at
140 rpm and periodically sampled to measure the residuals of Rac-ICP,
R-ICP and S-ICP via an Acquity™ Ultra Performance Liquid Chromato-
graph (UPLC, E2695, Waters Corp., Milford, MA). All samples were in
triplicate.
Some ICP-degrading strains were isolated from ICP-polluted soil and
identified via cultivation in the past few years. Arthrobacter sp. scl-2,
Bacillus megaterium CM-Z19 and Bacillus pumilus SALL-7 could use ICP
as the only carbon source and energy material for growth, and could
degrade 50–100 mg/L ICP in 18 hꢀ 14 d (Chen et al., 2017, Zhu et al.,
2019). However, these strains could not selectively degrade ICP
enantiomers.
The chiral-selective degradation enzymes are considered to be the
main reason for microbial enantioselective degradation (Huang et al.,
2020). For example, the degradation enzymes RdpA and SdpB from
Sphingobium herbicidovorans MH and Delftia acidovorans MC1 could
preferentially degrade R-dichlorprop and S-dichlorprop, respectively
(Nielsen et al., 2017). Some degrading strains could control the enan-
tiomeric degradation rate through selective absorption, and the degra-
dation enzyme itself did not have enantioselectivity (Lv et al., 2017;
Fang et al., 2020; L. Zhang et al., 2020). However, no selective ab-
sorption transporters or chiral-selective degradation enzymes for OPs
have been reported.
The effects of different cultivation pH (3.0–11.0 at intervals of 1 pH
unit) and metal ions (5 mM K+, Mn2+, Mg2+, Ni2+, Co2+, Ca2+, Fe3+
,
Al3+ and Cr3+) on the biodegradation of Rac-ICP by strain X1T were also
investigated. For the experiments of pH effects, the reaction solution
without strain X1T was used as the negative control. For the experiments
of metal ions effects, the reaction solution without corresponding metals
were used as the negative control. All test conical flasks were aerobically
incubated in a rotary shaker at 140 rpm and periodically sampled to
measure the residuals of Rac-ICP, R-ICP and S-ICP via UPLC. All samples
were in triplicate.
The toxicity, risk assessment and environmental behavior of ICP
enantiomers have been investigated in numerous studies (Liu et al.,
2015). However, the mechanism of enantioselective degradation of ICP
was rarely reported. We previously isolated and identified an OPs
degrading strain, Cupriavidus nantongensis X1T (Sun et al., 2016). The
degradation rate of strain X1T for some chiral OPs was initially fast and
then became slow. Strain X1T showed apparent enantioselectivity for
these chiral enantiomers (Fang et al., 2020). To further research the
characteristics and mechanism of enantioselectivity, strain X1T was used
to (1) characterize the degradation kinetics of Rac-ICP, R-ICP and S-ICP;
(2) measure the effects of pH and metal ions on the enantioselectivity;
(3) determine and identify the degradation metabolites; (4) evaluate the
toxicity of ICP and its metabolites; (5) determine the enantioselective
degradation characteristics of a variety of chiral OPs and (6) explore the
mechanism of preferential degradation of R-ICP by strain X1T.
2.4. Determination and identification of degradation products
The samples were sampled at different degradation times by adding
20 mL acetonitrile to terminate the reaction and extract the residuals of
ICP and its metabolites. The metabolites of Rac-ICP, R-ICP and S-ICP
were analyzed via a Acquity™ UPLC-MS/MS (TQ-S, Waters Corp.). The
enantiomers of ICP and its metabolites were chromatographically
separated on a chiral column (Chiralpak AD-3R, 3 µm, 2.1 × 150 mm) at
40 ◦C. The mobile phase consisted of 0.1% formic acid in water (A) and
acetonitrile (B) (A:B=20:80, v/v). The flow rate and injection volume
were 0.2 mL/min and 5 µL, respectively. The mass spectrometer was
operated at electrospray ionization mode and in a scan range from m/z
50–330.
2

L. Fang et al.
Journal of Hazardous Materials 417 (2021) 126024
2.5. Toxicity assessment of ICP and its metabolites
3. Results and discussion
The toxicity of ICP and its metabolites were predicated with the
Ecological Structure Activity Relationships (ECOSAR, v2.0, Environ-
mental Protection Agency, United States) model. The acute toxicity and
chronic toxicity for fish (Brachydanio rerio) and green algae (Pseudo-
kirchneriella subcapitata) were evaluated as previously reported (Saun-
ders and Fitzpatrick, 2014).
3.1. R-ICP was degraded favorably over S-ICP by strain X1T
The degradation kinetics of 50 mg/L Rac-ICP by strain X1T were
shown in Fig. 2A. Strain X1T completely degraded R-ICP and 30% of S-
ICP in 12 h. The degradation curves were all well fitted with the first-
order kinetics equation (R>0.95). Meanwhile, strain X1T showed high
enantiomer selectivity. R-ICP in the racemic mixture was preferentially
degraded over S-ICP. The degradation rate constant of R-ICP was 42-fold
higher than that of S-ICP. Interestingly, when R-ICP was almost
degraded, the S-ICP started to degrade. The difference in the degrada-
tion rate of the two enantiomers by strain X1T was also performed in the
separate degradation experiments and the degradation kinetic parame-
ters were shown in Table 1.
2.6. Degradation of Rac-ICP by the crude enzyme of strain X1T
The strain X1T were precultured and incubated in 100 mL of LB
medium at 37 ◦C to OD600 reached 0.8. Then, the free cells were har-
vested at 6000 g for 5 min at 4 ◦C and washed twice with sterilized
20 mM PB buffer (pH 7.0). The wet cells were resuspended in 20 mL PB
buffer and sonicated by an ultrasonic homogeniser (Scientz JY92-IIN,
Ningbo Scientz Biotech Company, Ningbo, China) at 300 W for 3 s at
200 times. The crude enzyme in suspension was obtained by centrifu-
gation at 12,000 g for 10 min at 4 ◦C. The crude enzyme assay in vitro
was detected as previously reported (Shi et al., 2019). The 1 mL of
rection solution contained 0.5 mL crude enzyme and 20 mg/L Rac-ICP.
The residuals of R-ICP and S-ICP were measured by UPLC.
About 30–40% of OPs have chiral structure (Gao et al., 2019). The
bioactivity and toxicity of chiral OPs for target organisms are performed
by inhibiting the activity of acetylcholinesterase (AChE) in vivo (Zou
et al., 2018). However, the insecticidal activity of different enantiomers
to target insects is extremely different. Due to the different types of AChE
in insects and mammals, R-ICP has lower bioactivity to target insects and
much higher toxicity to mammals than S-ICP (Liu et al., 2010; Nillos
et al., 2010). Strain X1T could degrade both R-ICP and S-ICP. When
R-ICP and S-ICP coexist in the environment, the former enantiomer,
being more toxic to humans and more harmful to the environment, but
less bioactive to the pests, were preferentially degraded by strain X1T.
2.7. Analytical methods of ICP, isofenphos-methy and profenofos
The residuals of Rac-ICP, R-ICP and S-ICP in degradation experi-
ments’ samples were detected via a UPLC method. The samples were
performed by adding 20 mL acetonitrile to terminate the reaction and
extract the residuals of Rac-ICP, R-ICP and S-ICP. All solutions were
centrifuged at 10,000 g for 2 min to separate the free cells and precipi-
tated crude enzyme, and the supernatants were filtered through 0.22 µm
pore-sized organic phase nylon membranes. The enantiomers of ICP
were separated on a chiral column (Chiralpak AD-3R, 2.1 ×150 mm,
3 µm) at 40 ◦C. The injection volume was 10 µL and the flow rate was
0.3 mL/min. The mobile phase consisted of 0.1% formic acid in water
(A) and acetonitrile (B) (A:B=50:50, v/v). The residuals of isofenphos-
methy in reaction solutions were measured according to a UPLC-MS/
MS method. The enantiomers of isofenphos-methy were separated on
a chiral column (LuxCellulose-3, 4.6 × 250 mm, 5 µm) at 30 ◦C. The
injection volume was 5 µL. The flow rate was 0.8 mL/min. The mobile
phase was 40% aqueous acetonitrile. The residuals of profenofos in re-
action solutions were measured by a UPLC method previously reported
(Fang et al., 2020). The satisfactory recoveries, quality assurance (QA)
and quality control (QC) were described in Supplemental Materials
(Text S1).
3.2. Enantioselective degradation of ICP was not caused by inter-
conversion
Interconversions can often occur among enantiomers of chiral pes-
ticides in the environment. These chiral enantiomeric conversions are
closely related to the structure of pesticides and environmental condi-
tions. At high temperature and protic solvents (H2O and methanol), the
chiral center of α-C atom in pyrethroid insecticides could be epimerized,
which causes conversion between the enantiomers (Liu et al., 2005).
Some chiral OPs, like malathion and phenthoate, can be racemized in
protic solvents. This conversion efficiency is related to the acidity of the
proton attached to the chiral center (phosphorus atom) (Li et al., 2010).
However, for those the chiral center was not attached with protons, like
profenofos and ICP, the enantiomeric conversion was not reported
before.
To identify whether the enantiomers of ICP could be mutually con-
verted during the degradation process by strain X1T. The degradation
kinetics of 25 mg/L R-ICP and S-ICP were measured separately (Fig. 2 B
and C). No enantiomeric conversion was detected, which indicated that
the degradation of S-ICP by strain X1T was not caused by enantiomeric
conversion.
2.8. Data calculation
The degradation curve of Rac-ICP and its enantiomers were fitted
with first order kinetics equation, Ct = C0 × e-kt, where C0 and Ct are the
concentration of ICP at the initial time and end time, k is the degradation
rate constant and t is the reaction time. Enantiomer fraction equation,
3.3. The degradation of S-ICP by strain X1T could be controlled by
adjusting the ratio of R-ICP and S-ICP
CR
To further explore the biodegradation characteristics of the S-ICP by
strain X1T, after complete degradation of R-ICP, additional R-ICP was
added to the reaction solution to monitor the degradation of R-ICP and
S-ICP. The result showed that the S-ICP starts to degrade only upon
complete degradation of the R-ICP (3, 7 and 13 h). After adding R-ICP
again (6 and 12 h), the degradation of S-ICP stopped immediately
(Fig. 3A). The experiment of adding R-ICP when R-ICP was not
completely degraded showed the same result (Fig. S1). When most R-ICP
was degraded, the S-ICP started to degrade. It showed that the degra-
dation of the S-ICP by strain X1T was related to the ratio of the two chiral
enantiomers. To clarify the threshold ratio for S-ICP degradation, the
initial ratios of the two enantiomers (EFR-ICP) were adjusted to 0.1 and
0.25 under the condition of keeping the concentration of S-ICP constant,
and the degradation kinetics of the two enantiomers by strain X1T were
EF(R) = C +C , was used to evaluate the selective ratio of R isomer and S
R
S
isomer, where CR and CS are the concentration of R isomer and S isomer,
respectively. An EF(R) > 0.5 indicates the preferential degradation of S
isomer, and EF(R) < 0.5 indicates the preferential degradation of R
isomer. The relative activities of strain X1T in different metal ions so-
lution were calculated by the following equation,
Degradation rate containing the corresponding metalions
Relative activities =
Degradation rate in deionized water
3

L. Fang et al.
Journal of Hazardous Materials 417 (2021) 126024
Fig. 2. Enantioselective degradation of 50 mg/L Rac-ICP (A), 25 mg/L R-ICP (B) and S-ICP (C) by strain X1T. (A) Degradation kinetics of R-ICP (organ line) and S-ICP
(red-line) in Rac-ICP by strain X1T. The dotted line represents the negative control without strain X1T. (B) Degradation kinetics of 25 mg/L R-ICP (orange line) and
negative control (dotted line). (C) Degradation kinetics of 25 mg/L S-ICP (red line) and negative control (dotted line).
Table 1
Degradation kinetic parameters of Rac-ICP, R-ICP, and S-ICP by strain X1T.
Concentration
Enantiomer
First-order kinetic equation
Degradation rate constant (k) (h¡1
)
DT50 (h)
R2
50 mg/L (Rac-ICP)
R-ICP
S-ICP
R-ICP
S-ICP
Ct = 24.80eꢀ 1.58 t
Ct = 24.41eꢀ 0.038 t
Ct = 25.05eꢀ 0.55 t
Ct = 24.56eꢀ 0.015 t
1.58 ± 0.11
0.037 ± 0.002
0.55 ± 0.08
0.44
18
0.99
0.99
0.98
0.92
25 mg/L
25 mg/L
1.3
0.015 ± 0.002
46
DT50 stands for degradation half-lives.
measured. The results showed that with the degradation of R-ICP, EFR
was also decreased (Fig. 3B and C). When the EFR-ICP values were
approximately lower than 0.016 and 0.014 (corresponding ratios of CS-
ICP / CR-ICP: 62 and 70), the S-ICP started to degrade (the concentration
points of S-ICP before and after had a significant difference, p < 0.01).
The results indicated that the degradation of S-ICP by strain X1T was
affected by the ratio of the two enantiomers. When CS-ICP was 62 greater
than CR-ICP, S-ICP started to degrade by strain X1T.
Mn2+, Mg2+, Ni2+, Co2+, Ca2+, Fe3+, Al3+ or Cr3+) were measured
(Fig. 4 A). The results showed that divalent metal cations such as Mn2+
,
Mg2+, Ni2+, Co2+ and Ca2+ could significantly increase the degradation
rate (p < 0.01) relative to that in ddH2O. The relative activities of Ca2+
was 10.7-fold greater than that in ddH2O. The EFR-ICP were also calcu-
lated and EFR-ICP values were negatively correlated with relative activ-
ities. The results indicate that the divalent metal cations can improve the
degradation of R-ICP in strain X1T.
In biodegradation, the metal ions mainly affect the degradation ef-
ficiency of strains by affecting the activity of corresponding enzymes
(Haque et al., 2018). In previous studies, an organophosphorus
degrading gene (opdB), encoding organophosphate hydrolase (OpdB),
was found in the original plasmid of strain X1T by complete genome
sequencing (Fang et al., 2016). According to the different substrates,
3.4. Divalent metal cations improved the degradation ability of strain X1T
To determine the effect of the main metal cations in the environment
on the enantiomer selective degradation of ICP, the relative degradation
activities by strain X1T in different metal cation solution (5 mM K+,
4

L. Fang et al.
Journal of Hazardous Materials 417 (2021) 126024
Fig. 3. (A) Degradation kinetics of R-ICP (red line) and S-ICP (black line) in Rac-ICP with continuous addition of R-ICP. (B) Degradation kinetics of R-ICP (red line)
and S-ICP (black line) under the initial EF(R-ICP) was 0.10. (C) Degradation kinetics of R-ICP (red line) and S-ICP (black line) under the initial EF(R-ICP) was 0.25. The
blue line showed the change of the EF(R-ICP)
.
Fig. 4. (A) Effects of different metal ions and (B) pH on the biodegradation of Rac-ICP in strain X1T. A: The degradation activity in H2O was defined as 1 unit (red
dotted line). The blue line showed the EF(R-ICP) value under different metal ion conditions. B: The organ curve fitted by Gaussian equation represented the degra-
dation rate of Rac-ICP by strain X1T and the blue curve fitted by Gaussian equation represented the Log (CFU).
organophosphorus degradation enzymes (phosphoric triester hydro-
lases) could be divided into two categories, aryldialkylphosphatase (EC
hydrolases reported so far are metalloenzymes and the catalytically
active center contains two divalent metal cations (Schenk et al., 2016).
Different divalent metal cation composition could significantly affect the
activity and catalytic properties of enzymes. The degradation effect of
OPAA with Mn2+-Mn2+ as the catalytic center on P-F bond was
3.1.8.1,
like
organophosphate
hydrolase,
OPD)
and
diisopropyl-fluorophosphatase (EC 3.1.8.2, like paraoxonase, PON)
(Chen et al., 2010; Namini et al., 2015). All the organophosphorus
5

L. Fang et al.
Journal of Hazardous Materials 417 (2021) 126024
significantly higher than that of PON with Ca2+-Ca2+ as the catalytic
center (Vyas et al., 2010). In vitro experiments, replacing the Fe2+-Zn2+
combination of OPD to Co2+-Co2+ could increase the pH tolerance of the
enzyme (Ely et al., 2011). Similar to these enzymes, divalent metal
cations could promote the degradation efficiency of OpdB by strains
X1T. However, unlike these enzymes, Ca2+-Ca2+ were the optimal
combination of active center for OpdB in strain X1T. Unfortunately,
phylogenetic tree analysis showed that opdB was the newly subclass of
organophosphorus degrading (OPD class) enzyme, and there was no
corresponding crystal structure of OpdB reported before. The effects of
divalent metal cations on OpdB in strain X1T remain an interest in
further elucidation.
affected the degradation rate by affecting the number of alive cells of
strain X1T in the degradation process.
3.6. A novel hydrolysis degradation pathway of ICP by strain X1T was
detected
The analytical results of ICP metabolites showed that ICP could be
degraded to isopropyl salicylate by strain X1T under acidic or neutral
conditions (pH 6, Fig. 5A-e, f). In addition to isopropyl salicylate, two
novel metabolites (compound I and II) were detected under alkaline
conditions (pH 11, Fig. 5A-b, c). The extracted mass spectra verified that
the two metabolites had chiral structure (Fig. 5B, Table S2). Compounds
I and II had the same characteristic fragment ion peaks at m/z = 230 [M-
17], 198 [M-49], 142 [M-105] and 60 [M-187], and were identified as 2-
((amino(methoxy)phosphorothioyl)oxy)benzoic acid (AMPOBA). The
structure of AMPOBA has a phosphorus chiral center. To further confirm
the absolute configuration of the two enantiomers of AMPOBA (com-
pound I and II), R-ICP and S-ICP were individually degraded by strain
X1T under alkaline conditions. The results showed that the metabolite R-
AMPOBA of R-ICP corresponded to compound II, while the metabolite S-
AMPOBA of S-ICP corresponded to compound I (Fig. S2). The degra-
dation experiment showed that isopropyl salicylate and AMPOBA could
be further degraded by strain X1T (Fig. S3). However, unlike ICP, the
degradation rates of the two chiral enantiomers of AMPOBA were the
same. The results indicated that the degradation of AMPOBA by strain
X1T was not enantioselective and the catabolic pathways of ICP in
3.5. Medium pH controlled the degradation rate through affecting the
growth of strain X1T
The degradation rate of 100 mg/L Rac-ICP in 3 h under different pH
(3ꢀ 11) by strain X1T were detected (Fig. 4B). Strain X1T had good
degradation activity at pH 6–10 (degradation rate > 70%). The curve
could be well fitted with the Gaussian equation and the optimal pH for
Rac-ICP degradation was 7.5, which was the same with the optimal
growth pH of strain X1T previously reported (Fang et al., 2019). The
number of alive cells of strain X1T (colony-forming unit, CFU) in the
reaction solutions after 3 h incubation were also detected. The CFU
curve of strain X1T was performed well in correlation with the degra-
dation rate curve at different pH. The results indicated that pH mainly
Fig. 5. (A) UPLC chromatograms of Rac-ICP after varying periods under pH 6 and 11, (B) mass spectra of compound I and II, and (C) proposed degradation pathways
of ICP in strain X1T. (++) main degradation pathways under alkaline conditions, (+) minor degradation pathways under alkaline conditions.
6

L. Fang et al.
Journal of Hazardous Materials 417 (2021) 126024
different pH by strain X1T were shown in Fig. 5 C.
were 0.0407 ± 0.004 and 0.0396 ± 0.004 hꢀ 1, which did not have a
significant difference (p > 0.5). Unlike the strain X1T whole cells pref-
erentially to degrade R-ICP, crude enzyme degraded ICP enantiomers
unselectively.
The hydrolysis of P-O or P-S bond was a main degradation reaction of
OPs (Shi et al., 2019). An alkaline condition promoted the hydrolysis of
P-O or P-S bond (Ji et al., 2016). For example, profenofos, which was
also a chiral organophosphorus insecticide, was degraded by hydro-
lysing the P-O bond (Fang et al., 2020). The half-life of phosmet under an
acidic condition (pH 4.0, 15 d) (i.e., P-S breakage) was 1636-fold longer
than that under an alkaline condition (pH 9.0, 0.22 h). However, ICP
was preferentially degraded to break the ester (C-O) bond rather than
the P-O bond under alkaline condition.
The chiral degrading enzymes are considered to be the main reason
for the enantioselective degradation of microorganisms (Huang et al.,
2020). However, the enantioselective degradation of ICP by strain X1T
was not caused by enzyme’s substrate selectivity. The difference be-
tween free cells and the crude enzyme was only the destruction of cells’
structure, which indicated that the enantioselectivity of strain X1T to
R-ICP and S-ICP occurred in the absorption process. The OPs were pre-
sumably degraded in the periplasm of the cells. The transport of the OPs
into the cells was an active process and the active transport relies on the
corresponding transport protein (Stasi et al., 2019). The difference in the
binding between the transporter and the chiral enantiomer may cause
this difference in absorption. The phosphonate transporter phnC iden-
tified in Escherichia coli K-12 could transport phosphate and phosphite
esters (Wanner and Metcalf, 1992). The phnC was also an ATP-binding
cassette transporter (type II). The transmembrane transport of phos-
phate requires ATP. The extracapsular portion of the phnC contains a
substrate binding protein phnD, which was responsible for binding the
substrate phosphate (Alicea et al., 2011). However, it is not reported
that phnC and phnD could transport organophosphorus insecticides.
Alignment with the complete genome date of strain X1T (Fang et al.,
2016) with the gnomic database indicated that the phnC and phnD genes
are located in chromosome 2 of strain X1T, which suggested that the
enantioselective degradation of ICP in strain X1T occur in the transport
process. The responding transport mechanism in strain X1T warrants
further studies.
3.7. Toxicity assessment of ICP and its metabolites
The different degradation pathway (break ester bond) of ICP in an
alkaline environment would cause different environmental behaviors.
The acute and chronic toxicity of ICP and its metabolites for fish and
green algae were estimated by the ECOSAR model (Fig. 6). The results
showed that ICP would have low toxicity to fish and green algae with the
LC50 and EC50 of 14.9 and 11.0 mg/L, respectively. Isopropyl salicylate,
a major metabolite under acidic conditions, was more toxic for fish and
green algae than ICP with the LC50 and EC50 of 3.15 and 1.87 mg/L,
respectively. On the contrary, AMPOBA, a main metabolite under
alkaline conditions, was much less toxic to fish and green algae than
isopropyl salicylate. Because the degradation rate of isopropyl salicylate
and AMPOBA to salicylic acid were much lower in strain X1T, which
would cause the isopropyl salicylate and AMPOBA to be accumulated. At
the same level of exposure, an alkaline environment was safer for non-
target organisms.
Isocarbophos-oxon (ICPO) is a proximate toxicant and an important
metabolite of ICP in mammals (Gao et al., 2020). S-ICPO possesses
higher hepatotoxic effects compared with R-ICPO. However, ICPO was
not be detected in strain X1T, which indicates a suitability of using strain
X1T to remediate ICP contaminated sites.
3.9. Strain X1T enantioselectively degraded other OPs
Strain X1T could also preferentially degrade R-isofenphos-methyl
and S-profenofos (Table 2). However, the degradation rates of
isofenphos-methyl and profenofos were slower than ICP. Meanwhile, the
enantioselectivity of profenofos was not as obvious as ICP and
isofenphos-methyl. Interestingly, as with ICP, the degradation of
isofenphos-methyl and profenofos by the crude enzyme did not have
enantioselectivity, which further verified that the enantioselective
degradation of chiral OPs by strain X1T occurred in the absorption
3.8. Crude enzyme degraded R-ICP and S-ICP in a similar rate
The degradation of 20 mg/L R-ICP and S-ICP by crude enzyme from
strain X1T were measured separately (Fig. 7). The crude enzyme could
hydrolyse both R-ICP and S-ICP to isopropyl salicylate. Interestingly, the
degradation rate constant (k) of the crude enzyme for R-ICP and S-ICP
Fig. 6. Toxicity of isocarbophos and its metabolites isocarbophos (A), isopropyl salicylate (B1), AMPOBA (B2), and salicylic acid(C) to fish and green algae. The
numbers represent the LC50 or EC50 values (mg/L). Toxicity level: High-toxicity (x < 1 mg/L, red), moderate-toxicity (1 <x < 10 mg/L, orange), low-toxicity
(10 <x < 100 mg/L, brown green) and no-harmful (x > 100 mg/L, green).
7

L. Fang et al.
Journal of Hazardous Materials 417 (2021) 126024
Fig. 7. Degradation of 20 mg/L R-ICP (A) and S-ICP (B) by crude enzyme.
the work reported in this paper.
Table 2
Changes of ratios of R- and S-enantiomers of ICP, isofenphos-methyl and pro-
Acknowledgment
fenofos after 24 h of incubation.
Enantiomers
Concentrations (mg/L)
EFR-enatiomer
This work was supported in part by grants from the National Natural
Science Foundation of China (31972314), Collaborative Innovation
Projects of Anhui Province (GXXT-2019–034), High-level Talent Intro-
duction Project of Anhui Agricultural University (rc522101) and the
USDA (Hatch project HAW5032-R).
0 (h)
24 (h)
R-ICP
25.14 ± 0.67
25.53 ± 0.54
23.51 ± 0.64
0.55 ± 0.21
13.88 ± 0.87
1.85 ± 0.49
From the initial 0.50–0.04
after 24 h of incubation
From the initial 0.51–0.15
after 24 h of incubation
S-ICP
R-isofenphos-
methyl
S-isofenphos-
methyl
22.76 ± 1.29
10.67 ± 0.20
Appendix A. Supporting information
R-profenofos
S-profenofos
24.92 ± 1.39
25.17 ± 1.59
19.62 ± 1.04
10.01 ± 1.13
From the initial 0.50–0.66
after 24 h of incubation
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.jhazmat.2021.126024.
EF stands for the ratios of the two enantiomers.
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