Subscriber access provided by University of Sunderland
Agricultural and Environmental Chemistry
Lethal toxicity and sub-lethal metabolic interference effects of sulfoxaflor on the earthworm (Eisenia fetida) Song Fang, Yizhi Zhang, Xiangwei You, Peng Sun, Jun Qiu, and Fanyu Kong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04633 • Publication Date (Web): 29 Oct 2018 Downloaded from http://pubs.acs.org on October 30, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 32
Journal of Agricultural and Food Chemistry
1
Lethal toxicity and sub-lethal metabolic interference effects of sulfoxaflor on the
2
earthworm (Eisenia fetida)
3
Song Fang*, Yizhi Zhang, Xiangwei You, Peng Sun, Jun Qiu, Fanyu Kong*
4
Laboratory of Tobacco Quality and Safety Risk Assessment, Ministry of Agriculture and
5
Rural Affairs, Tobacco Research Institute of Chinese Academy of Agricultural Sciences,
6
Qingdao, 266101, China
7
*Corresponding author: Song Fang and Fanyu Kong
8
Tel./fax: +86 53288701916
9
Email:
[email protected];
[email protected] 1
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
10
ABSTRACT: Testing for effects of pesticides on non-target organisms is an integral part
11
of ecological risk assessment. In the present study, the acute toxicity of sulfoxaflor to
12
earthworms was evaluated using an artificial soil toxicity test, and sub-lethal effects were
13
assessed through oxidative stress and metabolomics. Sulfoxaflor is a super toxic pollutant
14
to earthworms that easily bioaccumulates in earthworms, and contains LC2, LC10, and
15
LC50 value of 0.08 (0.04 - 0.13), 0.19 (0.11 - 0.25) and 0.54 (0.45 - 0.65) mg/kg
16
respectively. Sub-lethal doses of sulfoxaflor resulted in oxidative damage to earthworms
17
in which antioxidant enzymatic activities including SOD, CAT, GST were significantly
18
inhibited, and MDA content accumulated. Metabolomics analysis suggested the energy
19
metabolism and the urea cycle in earthworms were significantly activated, while
20
nucleotide metabolism was depressed which could cause DNA damage. Results suggest
21
earthworms have the potential to be a new entry point for sulfoxaflor into the wildlife
22
food chain. Since earthworms significantly contribute to soil function and ecosystems,
23
the high safety risks of sulfoxaflor to the earthworm could extend to the environment. In
24
view of these findings, more attention should be given to the risks sulfoxaflor poses on
25
the environment through its effects on earthworms.
26
KEYWORDS: environmental toxicology; acute toxicity; residue analysis; oxidative
27
stress; metabolomics
2
ACS Paragon Plus Environment
Page 2 of 32
Page 3 of 32
Journal of Agricultural and Food Chemistry
28
INTRODUCTION
29
Neonicotinoid insecticides have played a very important role in agricultural pest
30
management worldwide.1 As a fourth-generation neonicotinoid, sulfoxaflor performs
31
equally well or better against a wide range of sap-feeding insects than other neonicotinoid
32
insecticides, including acetamiprid, imidacloprid, and thiamethoxam.2 Additionally,
33
sulfoxaflor acts on the insect nicotinic acetylcholine receptor (nAChR) in a distinct
34
manner compared to other neonicotinoids, and it is considered an important new tool in
35
insecticide resistance management programs as it lacks insecticidal cross-resistance.3,4
36
Since its commercialization by Dow Agro Sciences in 2013, sulfoxaflor has been
37
registered and approved for use in more than 40 countries worldwide.
38
The impact of pesticides on the environment is a global issue of growing concern, and
39
there is increasing evidence that neonicotinoids have profound effects on non-target
40
organisms, such as honeybees and earthworms.5,6 Although studies have shown that
41
sulfoxaflor is safe for humans, fish and other aquatic species, sulfoxaflor residues can
42
bioaccumulate in such animals’ bodies and have a high long-term risk for the small
43
herbivorous mammals that usually found in field use to grow vegetables or cotton.7
44
Additionally, with the worldwide application of sulfoxaflor, it is bound to affect
45
non-target organisms and the ecological environment.8 Some researchers have shown that
46
sulfoxaflor poses moderate risk to honeybees,9 and sub-lethal concentrations of
47
sulfoxaflor are likely to have a negative impact on ants.10 Up to now, the study of
48
sulfoxaflor mostly focused on biological characterization,11,12 its mechanism of 3
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
49
action,13,14 metabolism and residual detection.15,16 The environmental safety risk of
50
sulfoxaflor on earthworms remains unknown based on the review of past reports.
51
Soil is the most important environmental mediator for the transport and transformation
52
of pesticides into the environment. Earthworms are a vital species in soil ecosystems,
53
playing important roles in pollutant decomposition, soil nutrient mineralization and
54
formation.17 The response of earthworms to soil contaminated with pesticides is an
55
important indicator for assessing soil ecological toxicity.18 Studies on the ecotoxicology
56
of pesticides to earthworms frequently focus on apparent indices, such as mortality,
57
growth and reproduction rates.19,20 These studies investigated the overall toxicity of
58
pesticides, however, they did not reveal the toxicity mechanism in earthworms.
59
Metabolomics, a popular technique for studying metabolic mechanisms, has been used
60
in recent years to study metabolites and their dynamic effects on organisms before and
61
after interference.21,22 The major analytical tools used for metabolomic studies are
62
high-field proton nuclear magnetic resonance (NMR) spectroscopy and chromatography
63
tandem mass spectrometry (MS) based techniques. Compared to NMR techniques, MS
64
methods have higher separation capacity and sensitivity, and can identify small molecule
65
metabolites from complex mixtures in a high-throughput mode.23,24 Although
66
metabolomics have been used in the identification of metabolic pathway perturbations
67
related to environmental heavy metals or organic contaminants exposure,25,26 there is
68
little research investigating the metabolic response of earthworms to sulfoxaflor toxicity.
4
ACS Paragon Plus Environment
Page 4 of 32
Page 5 of 32
Journal of Agricultural and Food Chemistry
69
The present study aims to evaluate the lethal toxicity and sub-lethal metabolic
70
interference mechanism of sulfoxaflor in the earthworms. Acute toxicity tests were
71
performed and the lethal concentration (LC) values of sulfoxaflor on earthworms were
72
calculated. The effects of oxidative stress and metabolic disturbance of sulfoxaflor on
73
earthworms were then evaluated under sub-lethal dose exposure. This research
74
contributes to a greater understanding of the toxicity and metabolic interference
75
mechanisms in earthworms, and it provides effective information for risk management of
76
sulfoxaflor.
77
MATERIALS AND METHODS
78
Materials. Sulfoxaflor with 99% purity was obtained from J&K Scientific (China).
79
Mass spectroscopy grade methanol, acetonitrile, ammonium acetate and ammonium
80
hydroxide were obtained from CNW Technologies (Germany). Ultrapure water was
81
prepared using a Milli-Q system of Merck Millipore (Germany). Commercial assay kits
82
for antioxidant enzymatic activities including superoxide dismutase (SOD), catalase
83
(CAT), glutathione peroxidase (GPX) and malonaldehyde (MDA) were obtained from
84
Suzhou Comin Biotech (China). All other chemicals were analytical grade and obtained
85
from Qingdao Quanchang Biotech (China). Earthworms (Eisenia fetida) with visible
86
clitellum and wet weight between 0.3 - 0.5 g were obtained from Shandong Hongda
87
Biotech (China), and acclimated to the experimental conditions for two weeks before the
88
experiment. The artificial soil was consisted of 70% sand, 20% kaolin clay and 10%
89
sphagnum peat moss. 5
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
90
Instruments. The concentration of sulfoxaflor in the soil and earthworms was
91
determined using a UPLC system (Waters, USA) equipped with Orbitrap MS (AB
92
SCIEX, USA). Antioxidant enzymatic activities were detected by ELx 800 TM
93
Microplate Spectrophotometer (Biotek, USA). Metabolomics analysis was performed on
94
a 1290 UHPLC system (Agilent Technology, USA) equipped with a Q Exactive Orbitrap
95
MS (Thermo Fisher Scientific, USA). The System utilized an ACQUITY UPLC HSS T3
96
column (2.1 * 100 mm * 1.8 μm, Waters, USA).
97
Earthworm culture and exposure experiments. Earthworm culture and exposure
98
experiments were performed according to the Organization for Economic Cooperation
99
and Development’s guidelines (OECD).27 Based on previous experimental results, a
100
range of concentrations of test substances was prepared using water as the solvent. For
101
each exposure experiment, the desired amount of sulfoxaflor was mixed into 20 g sand.
102
The mixed sand was set aside for 1 h to evaporate water and then mixed thoroughly with
103
980 g artificial soil (dry weight) in a mixer. The soil was put into a glass beaker, and the
104
moisture of the soil was adjusted to 35%. Earthworms were transferred into the soil and
105
cultured at 20 ± 1 °C for 12 h in light and 12 h in the dark.
106
Acute toxicity tests. The acute toxicity of sulfoxaflor to earthworms was tested by an
107
artificial soil contact toxicity assay. Based on previous experimental results, the
108
determined concentrations of sulfoxaflor in soil sample were 0.05, 0.1, 0.2, 0.4, 0.6, 0.8,
109
and 1 mg/kg. Each soil sample had four replicates at each experimental concentration,
6
ACS Paragon Plus Environment
Page 6 of 32
Page 7 of 32
Journal of Agricultural and Food Chemistry
110
and each treatment contained 18 earthworms. On days 7 and 14, both the mortality rate of
111
earthworms was determined and LC values were calculated.
112
Degradation in soil and bioaccumulation in earthworms. For degradation and
113
bioaccumulation tests, the concentration of sulfoxaflor in soil was 0.2 mg/kg in four
114
replicates experimental samples. Five earthworms and about 10 g soil form each
115
treatment were randomly sampled on days 0, 1, 3, 5, 7, 10, 14, 21 and 28. For soil, 3 mL
116
of distilled water and 5 mL of acetonitrile were added to 5 g soil. For earthworms, five
117
earthworms were weighed and homogenized using 10 mL distilled water. Then, the
118
pretreated sample was vortex treated for 10 min and centrifuged at 4000 g for 5 min.
119
Thereafter, 1.5 mL of supernatant was filtered using a 0.22 μm syringe filter for further
120
UPLC-MS analysis, and the injection volume was 5 μL. The parameters for UPLC and
121
MS are shown in Table S1 and S2 respectively.
122
Oxidative damage test. For oxidative stress testing, the concentrations of sulfoxaflor
123
in soil were 0.01, 0.05 (non-lethal dose), 0.1 (approximate value of LC2) and 0.2
124
(approximate value of LC10) mg/kg. Each of these experimental concentrations were
125
tested in four replicates of soil sample. Five earthworms from each treatment were
126
randomly sampled on days 1, 7, 14 and 28, and the antioxidant enzymatic activities were
127
determined using assay kits according to the kit instructions.
128
Metabolic interference tests. For metabolomics experiments, the concentration of
129
sulfoxaflor in soil was 0.2 mg/kg. Eight earthworms from each treatment were randomly
130
sampled on day 14. A method based on previous reports was used to extract and analyze 7
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 8 of 32
131
metabolites in earthworms.28,29 Individual earthworms were placed in a centrifuge tube
132
containing 2 mL of extraction liquid (V methanol: V acetonitrile: V water = 2: 2: 1) and
133
20 μL of internal standard. The mixture was homogenized for 4 min and then underwent
134
ultrasound treatment for 5 min, for a total of three times. After incubating at -20 °C for 1
135
h, the sample was centrifuged at 12000 rpm for 15 min, and the fresh supernatant was
136
dried under a vacuum. Then 200 μL of extraction liquid (V acetonitrile: V water= 1: 1)
137
was added and the extracts were reconstituted. After vortex treatment for 30 s and being
138
sonicated for 10 min, the extracts were centrifuged at 12000 rpm for 15 min. The
139
supernatant (60 μL) was transferred into a glass vial for further UHPLC-MS analysis,
140
with an injection volume of 2 μL. Quality control (QC) samples were obtained by pulling
141
10 μL of supernatant from each sample. The parameters of UHPLC and MS are shown in
142
Table S3 and S4 respectively.
143
Statistical analysis. Probit analysis was used to calculate the LC values of sulfoxaflor
144
for earthworms. ANOVA was performed along with LSD testing for multiple
145
comparisons among treatments (p < 0.05), and results were expressed as mean ± SD. All
146
analyses were performed using SPSS software (V 22.0).
147
For metabolomics analysis, MS raw data files were converted into the mzXML format
148
using ProteoWizard (V 3.0.6428), and XCMS (V 1.46) was employed for peak detection,
149
noise filtering, and peak alignment. The resulting three-dimensional data including the
150
peak number, sample name, and normalized peak area were fed to SIMCA (V 14.1) for
151
principal
component
analysis
(PCA)
and 8
orthogonal
ACS Paragon Plus Environment
projections
to
latent
Page 9 of 32
Journal of Agricultural and Food Chemistry
152
structures-discriminate analysis (OPLS-DA). Differential compounds were screened
153
using variable importance in the projection (VIP > 1) in the first principal component of
154
the OPLS-DA model, and then were analyzed by ANOVA (P < 0.05). The matching
155
information of different metabolites was confirmed by retrieving KEGG and PubChem
156
databases. In addition, commercial databases including KEGG of the corresponding
157
species
158
MetaboAnalyst http://www.metaboanalyst.ca/ were utilized to search for metabolite
159
pathways.
160
RESULTS AND DISCUSSION
Caenorhabditis
elegans
(nematode)
http://www.genome.jp/kegg/
and
161
Acute toxicity in earthworms. In the present study, the mortality of earthworms in the
162
sulfoxaflor exposure treatments showed a significant dose-effect relationship, and linear
163
regression correlation coefficients (R2) were > 0.92 (Figure S1). Table 1 summarizes the
164
results from the specific effect levels (LC2, LC10 and LC50) used in the artificial soil tests.
165
According to the toxicity classes,30 extreme toxicity of sulfoxaflor in earthworms was
166
observed.
167
It has been generally accepted that neonicotinoids pose great safety risks to
168
environmental organisms, such as honeybees and earthworms.5,6 Although sulfoxaflor
169
has little effect on honeybees, it is a super toxic pollutant to earthworms. Additionally,
170
sulfoxaflor was highly toxic to the larvae of a natural predator of sap-feeding pests
171
(Adalia bipunctata),31 and it has not only lethal, but also sub-lethal effects on red
172
imported fire ant (Solenopsis invicta) populations.32 Therefore, more attention should also
173
be given to the ecological risks of sulfoxaflor. 9
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
174
Degradation in soil and bioaccumulation in earthworms. The residual amounts of
175
sulfoxaflor in soil and earthworms during the whole exposure period are shown in Figure
176
1. In the present study, the sulfoxaflor concentration in soil decreased slowly as exposure
177
time increased. On the 21st day, the degradation rate exceeded 90%. The concentration of
178
sulfoxaflor in earthworms first increased and then decreased as exposure time increased.
179
On the 7th day, the concentration of sulfoxaflor in earthworms reached maximum height.
180
Fourteen days later, the concentration of sulfoxaflor in the earthworms was relatively
181
stable, but still at a high level.
182
The degradation of pollutants in soil is influenced by microorganisms, light and soil
183
properties. Studies have shown that microbial degradation is the main mechanism of
184
sulfoxaflor degradation in soil.15 The artificial soil is rich in organic matter, and vigorous
185
microbial activity accelerated the degradation of sulfoxaflor. However, earthworms
186
undergo strong bioaccumulation of sulfoxaflor. The concentration of sulfoxaflor in
187
earthworm remains over 10 times higher than that in soil even after 14 days. Similar
188
results revealed that selective bioaccumulation in adult and juvenile earthworms of other
189
neonicotinoids such as imidacloprid, acetamiorid, and thiachloprid.33 The accumulation
190
of organochlorine pesticides in earthworms was also reported.34 Our study reveals a new
191
potential point of entry of sulfoxaflor into the wildlife food chain.
192
Effects on the oxidative damage to earthworms. The activities of SOD, CAT, GST,
193
and MDA concentration in earthworms are shown in Figure 2. For the non-lethal dose
194
(0.01, 0.05 mg/kg) treatments, sulfoxaflor had few effects on SOD activity during the
10
ACS Paragon Plus Environment
Page 10 of 32
Page 11 of 32
Journal of Agricultural and Food Chemistry
195
exposure period. For sub-lethal (0.1, 0.2 mg/kg) treatments, the SOD activity did not
196
change significantly on the 1st day. With prolonged exposure time, the SOD activity
197
significantly increased and then considerably decreased. A similar biphasic change in
198
CAT and GST activities was also observed. The MDA content did not significantly
199
change in non-lethal dose treatments, however, it increased significantly with the
200
prolongation of exposure time in the sub-lethal treatments.
201
Antioxidant system played an important role in maintaining the function of system,
202
and the induction of the antioxidant enzymatic activities has been considered a biomarker
203
of environmental pollution in earthworm toxicology.35 SOD and CAT are the first line of
204
antioxidative defense against reactive oxygen species (ROS) in earthworms, which may
205
cause oxidative damage to biological macromolecules, like DNA, protein and lipid.
206
Normally, ROS levels and the antioxidant enzyme contents coexist in a dynamic balance.
207
However, excessive ROS production exceeding the antioxidant capacity causes oxidative
208
stress in the organism.36 In the present study, the antioxidant enzymatic activities in
209
earthworms increased, and then decreased after longer sub-lethal exposure. The activities
210
of antioxidant enzymes were activated by sulfoxaflor, which could resist antioxidant
211
damage. However, longer sub-lethal dose exposure caused irreversible antioxidant
212
damage, and the activities of antioxidant enzymes were subsequently inhibited.
213
Antioxidant damage further leads to lipid peroxidation, which damages the cell
214
membrane and leads to destruction of membrane lipids. In addition, as the product of
215
lipid peroxidation, MDA can react with deoxyadenosine and deoxyguanosine in DNA, 11
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
216
forming DNA adducts which can cause mutagenesis and carcinogenesis.37 Our results
217
indicated that exposure sulfoxaflor resulted in serious oxidative damage to earthworms.
218
Effect on metabolic interference to earthworms. Good data quality and repeatability
219
of analytical methods are the basis for metabolomics. Base peak chromatograms (BPC)
220
showed that the retention time and peak area of QC samples overlapped well, and no
221
significant peaks were detected in the blank samples (Figure S2). The results of PCA
222
analysis showed the distribution of QC samples was intensive and the results of PCA-X
223
are distributed within 2 STD (Figure S3). The OPLS-DA model showed clear separation
224
and discrimination, revealing a visible perturbation of the earthworm metabolic profiles
225
between the two groups (Figure 3). All samples fell within the 95% confidence interval
226
and the results of a permutation test (n = 200) confirmed the good quality of the
227
OPLS-DA model, which has good robustness and no over-fitting. All results indicated
228
that the stability and repeatability of the analytical methods were acceptable.
229
Differential metabolites were screened through comprehensive analysis by VIP of the
230
OPLS-DA model and P values of ANOVA. This screening was confirmed by retrieving
231
data from the KEGG and PubChem databases. A total of 26 metabolites were identified,
232
and their information, including VIP values, P values and fold changes, are given in
233
Table 2. As is considered common knowledge, complex metabolic reactions are affected
234
by complex metabolic pathways and networks. Through a comprehensive pathway
235
analysis, some key metabolic interference pathways were found and summarized, as
236
shown in the Figure 4. The sub-lethal dose exposure of sulfoxaflor mainly affected 12
ACS Paragon Plus Environment
Page 12 of 32
Page 13 of 32
Journal of Agricultural and Food Chemistry
237
energy metabolism, urea cycle, homeostasis of amino acid metabolism and nucleotide
238
metabolism of the earthworms.
239
Specifically, metabolite alterations in the carbohydrate metabolism, TCA cycle, and
240
changes in some amino acids indicated that sulfoxaflor affects earthworm energy
241
metabolism. A carbohydrate metabolism related metabolite, glucose-6-phosphate, was
242
up-regulated. Glucose-6-phosphate is a common intermediate product of carbohydrate
243
metabolism, and it is the crossing point of various metabolic pathways38. Up-regulation
244
of malic acid and citric acid showed inhibition of the TCA cycle. The TCA cycle is the
245
hinge of sugar, fat and protein metabolism, and it is also the main metabolic process for
246
supplying energy39. The metabolism of some amino acids and derivatives have obviously
247
changed. Glutamate, glutamine, and histidine were also up-regulated. The α-ketoglutaric
248
acid generated by the decomposition of amino acids can be converted into sugar or lipids,
249
and it can resynthesize some nonessential amino acids. Additionally, α-ketoglutaric acid
250
and can also participate in the energy metabolism of the TCA cycle.40 Leucine and valine
251
were up-regulated as well. The branched chain amino acid can be an important source of
252
calories, and it is superior as a fuel to the ubiquitous intravenous glucose.41 All these
253
results showed that energy metabolism of earthworms was affected after sulfoxaflor
254
exposure. Similar to our results, interference in energy metabolism were also observed in
255
earthworms42, land snail43 and zebrafish larvae44 after exposure to other pesticides.
256
Some metabolites related to the urea cycle had obvious disturbances, which is the main
257
way to remove ammonia toxicity in animals. In the earthworms, aspartic acid and 13
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 14 of 32
258
asparagine were down-regulated, while arginine and ornithine were up-regulated.
259
Aspartic acid is the donor of nitrogen during the urea synthesis process, and the urea
260
cycle is linked to the TCA cycle by aspartic acid and fumaric acid. Arginine and ornithine
261
are important intermediate products in the urea cycle and play an important role in urea
262
synthesis.45 The disturbances in all these metabolites reflect the up-regulation of the urea
263
cycle, which possibly causes the abnormality of the excretory function in earthworms.
264
The purine and pyrimidine metabolisms were affected based on relevant metabolites
265
indicating the perturbation of nucleotide metabolism. Deoxyguanosine, deoxyinosine,
266
deoxyuridine and guanine, which are all nucleoside components of DNA, were
267
down-regulated. Deoxyguanosine can be converted into 8-hydroxydeoxyguanosine,
268
which is regarded as a critical biomarker of oxidative stress and oxidative DNA
269
damage.46 The formation of 8-hydroxydeoxyguanosine induces the reduction of
270
deoxyguanosine, indicating that oxidative stress and DNA damage occurred after
271
sulfoxaflor exposure.47 Guanine is transformed to guanosine monophosphate by
272
hypoxanthine
273
down-regulation of HGPRT activity imply that the DNA base repair might be blocked.48
274
The changes in these metabolites indicate that sulfoxaflor interfered with nucleotide
275
metabolism and could cause DNA damage in earthworms. DNA damage is also
276
significantly increased through exposure of earthworms to thiacloprid.49 Therefore, one
277
of the reasons for long-term toxicity of neonicotinoids to earthworms may be the DNA
278
damage.
guanine
phosphoribosyl
transferase
14
ACS Paragon Plus Environment
(HGPRT)
catalysis.
The
Page 15 of 32
Journal of Agricultural and Food Chemistry
279
In summary, sulfoxaflor is a super toxic pollutant to earthworms. Earthworms
280
bioaccumulate sulfoxaflor in high concentrations and may be a new potential point of
281
entry of sulfoxaflor into the wildlife food chain. Sub-lethal doses of sulfoxaflor resulted
282
in oxidative damage, which mainly affected energy metabolism, the urea cycle,
283
homeostasis of amino acid metabolism and nucleotide metabolism within earthworms.
284
The lethal toxicity results and sub-lethal metabolic interference effects on earthworms
285
may have implications for requiring a better assessment of the soil environmental risks of
286
sulfoxaflor. Due to the earthworm’s significant contribution to soil function and the
287
ecosystem, more attention should be given to the high safety risks sulfoxaflor may pose
288
to the environment at large.
289
ACKNOWLEDGMENTS
290
This work was financially supported by the Agricultural Science and Technology
291
Innovation Program (ASTIP-TRIC06).
292
Supporting Information
293
The parameters of UPLC and MS for detecting the sulfoxaflor concentration in soil
294
and earthworms (Table S1, S2), the parameters of UHPLC and MS for detecting
295
metabolites in earthworms (Table S3, S4), linear regression curve of sulfoxaflor toxicity
296
to earthworm (Figure S1), Base peak chromatograms (BPC) of QC and blank samples
297
(Figure S2), PCA and PCA-X distribution diagram of QC samples (Figure S3).
298
Notes
299
The authors declare no competing financial interest. 15
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
300
REFERENCES
301
(1) Jeschke, P.; Nauen, R.; Schindler, M.; Elbert, A. Overview of the status and global
302
strategy for neonicotinoids. J. Agric. Food Chem. 2011, 59, 2897-2908.
303
(2) Zhu, Y. M.; Loso, M. R.; Watson, G. B.; Sparks, T. C.; Rogers, R. B.; Huang, J. X.;
304
Gerwick, B. C.; Babcock, J. M.; Kelley, D.; Hegde, V. B.; Nugent, B. M.; Renga, J. M.;
305
Denholm, I.; Gorman, K.; DeBoer, G. J.; Hasler, J.; Meade, T.; Thomas, J. D. Discovery
306
and characterization of sulfoxaflor, a novel insecticide targeting sap-feeding pests. J.
307
Agric. Food Chem. 2011, 59, 2950-2957.
308
(3) Watson, G. B.; Loso, M. R.; Babcock, J. M.; Hasler, J. M.; Letherer, T. J.; Young,
309
C. D.; Zhu, Y.; Casida, J. E.; Sparks, T. C. Novel nicotinic action of the sulfoximine
310
insecticide sulfoxaflor. Insect biochem. Mol. Biol. 2011, 41, 432-439.
311
(4) Sparks, T. C.; Watson, G. B.; Loso, M. R.; Geng, C. X.; Babcock, J. M.; Thomas, J.
312
D. Sulfoxaflor and the sulfoximine insecticides: chemistry, mode of action and basis for
313
efficacy on resistant insects. Pestic. Biochem. Physiol. 2013, 107, 1-7.
314
(5) Wang, X.; Anadón, A.; Wu, Q. H.; Qiao, F.; Ares, I.; Martínez-Larrañaga, M. R.;
315
Yuan, Z. H.; Martínez, M. A. Mechanism of neonicotinoid toxicity: impact on oxidative
316
stress and metabolism. Annu. Rev. Pharmacol. Toxicol. 2018, 58, 471-507.
317
(6) Morrissey, C. A.; Mineau, P.; Devries, J. H.; Sanchez-Bayo, F.; Liess, M.;
318
Cavallaro, M. C.; Liber, K. Neonicotinoid contamination of global surface waters and
319
associated risk to aquatic invertebrates: A review. Environ. Int. 2015, 74, 291-303.
16
ACS Paragon Plus Environment
Page 16 of 32
Page 17 of 32
320 321 322 323
Journal of Agricultural and Food Chemistry
(7) European Food Safety Authority. Conclusion on the peer review of the pesticide risk assessment of the active substance sulfoxaflor. EFSA Journal. 2014, 12, 3692. (8) Fairbrother, A.; Purdy, J.; Anderson, T.; Fell, R. Risks of neonicotinoid insecticides to honeybees. Environ Toxicol. Chem. 2014, 33, 719-723.
324
(9) Wu, S. G.; Xu, J. Y.; Rao, H. X.; Liu, X. J.; An, X. H.; Lv, L.; Guan, W. B.; Zhao,
325
X. P. Acute toxicity and risk assessment of pesticides used in strawberry for controlling
326
aphid to honeybees. Asian. J. Ecotoxicol. 2017, 12, 222-227.
327
(10) Pan, F. X.; Lu, Y. Y.; Wang, L. Toxicity and sublethal effects of sulfoxaflor on
328
the red imported fire ant, Solenopsis invicta. Ecotoxicol. Environ Saf. 2017, 139,
329
377-383.
330
(11) Babcock, J. M.; Gerwick, C. B.; Huang, J. X.; Loso, M. R.; Nakamura, G.;
331
Nolting, S. P.; Rogers, R. B.; Sparks, T. C.; Thomas, J.; Watson, G. B.; Zhu, Y.
332
Biological characterization of sulfoxaflor, a novel insecticide. Pest Manage. Sci. 2011,
333
67, 328-334.
334
(12) Buysse, A. M.; Nugent, B. M.; Wang, N. X.; Benko, Z.; Breaux, N.; Rogers, R.;
335
Zhu, Y. M. Studies toward understanding the SAR around the sulfoximine moiety of the
336
sap-feeding insecticide sulfoxaflor. Pest Manag. Sci. 2017, 73, 731-742.
337
(13) Nugent, B. M.; Buysse, A. M.; Loso, M. R.; Babcock, J. M.; Johnson, T. C.;
338
Oliver, M. P.; Martin, T. P.; Ober, M. S.; Breaux, N.; Robinson, A.; Adelfinskaya, Y.
339
Expanding the structure-activity relationship of sulfoxaflor: the synthesis and biological
340
activity of N - heterocyclic sulfoximines. Pest Manage. Sci. 2015, 71, 928-936. 17
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
341
(14) Wang, N. X.; Watson, G. B.; Loso, M. R.; Sparks, T. C. Molecular modeling of
342
sulfoxaflor and neonicotinoid binding in insect nicotinic acetylcholine receptors: impact
343
of the Myzus β1R81T mutation. Pest Manag. Sci. 2016, 72, 1467-1474.
344
(15) Chen, Z. L.; Dong, F. S.; Pan, X. L.; Xu, J.; Liu, X. G.; Wu, X. H.; Zheng, Y. Q.
345
Influence of uptake pathways on the stereoselective dissipation of chiral neonicotinoid
346
sulfoxaflor in greenhouse vegetables. J. Agric. Food Chem. 2016, 64, 2655-2660.
347
(16) Tian, C. Y.; Xu, J.; Dong, F. S.; Liu, X. G.; Wu, X. H.; Zhao, H. H.; Ju, C.; Wei,
348
D. M.; Zheng, Y. Q. Determination of sulfoxaflor in animal origin foods using dispersive
349
solid-phase extraction and multiplug filtration cleanup method based on multiwalled
350
carbon nanotubes by ultraperformance liquid chromatography/tandem mass spectrometry.
351
J. Agric. Food Chem. 2016, 64, 2641-2646.
352 353 354 355
(17) Datta, S.; Singh, J.; Singh, S.; Singh, J. Earthworms, pesticides and sustainable agriculture: a review. Environ. Sci. Pollut. R. 2016, 23, 8227-8243. (18) Paoletti, M. G. The role of earthworms for assessment of sustainability and as bioindicators. Agr. Ecosyst. Environ. 1999, 74, 137-155.
356
(19) Li, J.; Zhang, W.; Chen, L.; Liang, J.; Lin, K. F. Biological effects of
357
decabromodiphenyl ether (BDE209) and Pb on earthworm (Eisenia fetida) in a soil
358
system. Environ. Pollut. 2015, 207, 220-225.
359
(20) Li, L. L.; Yang, D.; Song, Y. F.; Shi, Y.; Huang, B.; Yan, J.; Dong, X. X. Effects
360
of bifenthrin exposure in soil on whole-organism endpoints and biomarkers of earthworm
361
Eisenia fetida. Chemosphere. 2017, 168, 41-48. 18
ACS Paragon Plus Environment
Page 18 of 32
Page 19 of 32
362 363
Journal of Agricultural and Food Chemistry
(21) Bundy, J. G.; Davey, M. P.; Viant, M. R. Environmental metabolomics: a critical review and future perspectives. Metabolomics. 2009, 5, 3-21.
364
(22) Shi, Y. J.; Xu, X. B.; Chen, J.; Liang, R. Y.; Zheng, X. Q.;Shi, Y. J.; Wang, Y. R.
365
Antioxidant gene expression and metabolic responses of earthworms (Eisenia fetida)
366
after exposure to various concentrations of hexabromocyclododecane. Environ. Pollut.
367
2018, 232, 245-251.
368
(23) Ma, Y.; Tanaka, N.; Vaniya, A.; Kind, T.; Fiehn, O. Ultrafast polyphenol
369
metabolomics of red wines using microLC-MS/MS. J. Agric. Food Chem. 2016, 64,
370
505-512.
371
(24) Hu, X. Q.; Thakur, K.; Chen G. H.; Hu, F.; Zhang J. G.; Zhang, H. B.; Wei, Z. J.
372
Metabolic effect of 1-deoxynojirimycin from mulberry leaves on db/db diabetic mice
373
using liquid chromatography-mass spectrometry based metabolomics. J. Agric. Food
374
Chem. 2017, 65, 4658-4667.
375
(25) Huang, S. S. Y.; Benskin, J. P.; Chandramouli, B.; Butler, H.; Helbing, C. C.;
376
Cosgrove, J. R. Xenobiotics produce distinct metabolomic responses in zebrafish larvae
377
(Danio rerio). Environ. Sci. Technol. 2016, 12, 6526-6535.
378
(26) Gillis, J. D.; Price, G. W.; Prasher, S. Lethal and sub-lethal effects of triclosan
379
toxicity to the earthworm Eisenia fetida assessed through GC-MS metabolomics. J.
380
Hazard. Mater. 2016, 323, 203-211.
381 382
(27) Organization for Economic Co-operation and Development (OECD). Test 207: earthworm, acute toxicity tests. OECD guidelines for testing of chemicals. 1984, 1-9. 19
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
383
(28) Dunn, W. B.; Broadhurst, D.; Begley, P.; Zelena, E.; Francis-McIntyre, S.;
384
Anderson, N.; Brown, M.; Knowles, J. D.; Halsall, A.; Haselden, J. N.; Nicholls, A. W.;
385
Wilson, I. D.; Kell, D. B.; Goodacre, R. Procedures for large-scale metabolic profiling of
386
serum and plasma using gas chromatography and liquid chromatography coupled to mass
387
spectrometry. Nat. Protoc. 2011, 6, 1060-1083.
388
(29) Wang, J. L.; Zhang, T.; Shen, X. T.; Liu, J.; Zhao, D. L.; Sun, Y. W.; Wang, L.;
389
Liu, Y. J.; Gong, X. Y.; Liu, Y. X.; Zhu, Z. J.; Xue, F. Z. Serum metabolomics for early
390
diagnosis of esophageal squamous cell carcinoma by UHPLC-QTOF/MS. Metabolomics.
391
2016, 12, 116-124.
392
(30) Standardization Administration Committee of China (SACC). Test guidelines on
393
environmental safety assessment for chemical pesticides-part 15: earthworm acute
394
toxicity test (GB/T 31270.15). 2014, 6.
395
(31) Garzón, A.; Medina,P.; Amor, F.; Viñuela, E.; Budia, F. Toxicity and sublethal
396
effects of six insecticides to last instar larvae and adults of the biocontrol agents
397
Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) and Adalia bipunctata (L.)
398
(Coleoptera: Coccinellidae). Chemosphere. 2015, 132, 87-93.
399 400
(32) Pan, F. X.; Lu, Y. Y.; Wang, L. Toxicity and sublethal effects of sulfoxaflor on the red imported fire ant, Solenopsis invicta. Ecotox. Environ Safe. 2017, 139, 377-383.
401
(33) Chevillot, F.; Convert, Y.; Desrosiers, M.; Cadoret, N.; Veilleux, E.; Cabana, H.;
402
Bellenger, J. Selective bioaccumulation of neonicotinoids and sub-lethal effects in the
20
ACS Paragon Plus Environment
Page 20 of 32
Page 21 of 32
Journal of Agricultural and Food Chemistry
403
earthworm Eisenia andrei exposed to environmental concentrations in an artificial soil.
404
Chemosphere. 2017, 186, 839-847.
405
(34) Miglioranza, K. S. B.; Aizpún de Moreno, J. E.; Moreno, V. J.; Osterrieth, M. L.;
406
Escalante, A. H.; Fate of organochlorine pesticides in soils and terrestrial biota of “Los
407
Padres” pond watershed, Argentina. Environ. Pollut. 1999, 105, 91-99.
408
(35) Capolupo, M.; Valbonesi, P.; Kiwan, A.; Buratti, S.; Franzellitti, S. Use of an
409
integrated biomarker-based strategy to evaluate physiological stress responses induced by
410
environmental concentrations of caffeine in the Mediterranean mussel Mytilus
411
galloprovincialis. Sci. Total Environ. 2016, 563, 538-548.
412
(36) Ziech, D.; Franco, R.; Georgakilas, A. G.; Georgakila, S.; Malamou-Mitsi, V.;
413
Schoneveld, O.; Pappa, A.; Panayiotidis, M. I. The role of reactive oxygen species and
414
oxidative stress in environmental carcinogenesis and biomarker development. Chem.
415
Biol. Interact. 2010, 188, 334-339.
416
(37) Bartsch, H.; Nair, J. Ultrasensitive and specific detection methods for exocyclic
417
DNA adducts: markers for lipid peroxidation and oxidative stress. Toxicology. 2000, 153,
418
105-114.
419
(38) Bundy, J. G.; Sidhu, J. K.; Rana, F.; Spurgeon, D. J.; Svendsen, C.; Wren, J. F.;
420
Stürzenbaum, S. R.; Morgan, A. J.; Kille, P. 'Systems toxicology' approach identifies
421
coordinated metabolic responses to copper in a terrestrial non-model invertebrate, the
422
earthworm Lumbricus rubellus. BMC. Biology. 2008, 6, 1-25.
21
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
423
(39) Ratnasekhar, C.; Sonane, M.; Satish, A.; Mudiam, M. K. Metabolomics reveals
424
the perturbations in the metabolome of Caenorhabditis elegans exposed to titanium
425
dioxide nanoparticles. Nanotoxicology. 2015, 9, 994-1004.
426
(40) Wang, Y.; Teng, M. M.; Wang, D. Z.; Yan, J.; Miao, J. Y.; Zhou, Z. Q.; Zhu, W.
427
T. Enantioselective bioaccumulation following exposure of adult zebrafish (Danio rerio)
428
to epoxiconazole and its effects on metabolomic profile as well as genes expression.
429
Environ. Pollut. 2017, 229, 264-271.
430
(41) Song, Y.; Chai, T. T.; Yin, Z. Q.; Zhang, X. N., Zhang, W.; Qian, Y. Z.; Qiu, J.
431
Stereoselective effects of ibuprofen in adult zebrafish (Danio rerio) using
432
UPLC-TOF/MS-based metabolomics. Environ. Pollut. 2018, 241, 730-739.
433
(42) Wu, S. J.; Xu, X.; Zhao, S. L.; Shen, F. C.; Chen, J. M. Evaluation of
434
phenanthrene toxicity on earthworm (Eisenia fetida): An ecotoxicoproteomics approach.
435
Chemosphere. 2013, 93, 963-971.
436
(43) Radwan, M. A.; Mohamed, M. S. Imidacloprid induced alterations in enzyme
437
activities and energy reserves of the land snail, Helix aspersa. Ecotox. Environ. Saf. 2013,
438
95, 91-97.
439
(44) Wang, C.; Qian, Y.; Zhang X. F.; Chen, F.; Zhang, Q.; Li, Z. Y.; Zhao, M. R. A
440
metabolomic study of fipronil for the anxiety-like behavior in zebrafish larvae at
441
environmentally relevant levels. Environ. Pollut. 2016, 241, 252-258.
442 443
(45) Tang, F. C.; Chan, C. C. Contribution of branched-chain amino acids to purine nucleotide cycle: a pilot study. Eur. J. Clin. Nutr. 2017, 71, 587-593. 22
ACS Paragon Plus Environment
Page 22 of 32
Page 23 of 32
Journal of Agricultural and Food Chemistry
444
(46) Guo, Y.; Weck, J.; Sundaram, R.; Goldstone, A. E.; Buck L. G.; Kannan, K.
445
Urinary concentrations of phthalates in couples planning pregnancy and its association
446
with 8-hydroxy-2-deoxyguanosine, a biomarker of oxidative stress: longitudinal
447
investigation of fertility and the environment study. Environ. Sci. Technol. 2014, 48,
448
9804-9811.
449
(47) Aguirre-Martinez, G. V.; Del Valls, T. A.; Martin-Diaz, M. L. Identification of
450
biomarkers responsive to chronic exposure to pharmaceuticals in target tissues of
451
Carcinus maenas. Mar. Environ. Res. 2013, 7, 1-11.
452
(48) Song, Q. Q.; Zheng, P. F.; Qiu, L. G.; Jiang, X.; Zhao, H. W.; Zhou, H. L.; Han,
453
Q.; Diao, X. P. Toxic effects of male perna viridis gonad exposed to BaP, DDT and their
454
mixture: a metabolomic and proteomic study of the underlying mechanism. Toxicol. Lett.
455
2016, 240, 185-195.
456
(49) Feng, L.; Zhang, L.; Zhang, Y. N.; Zhang, P.; Jiang, H. Y. Inhibition and recovery
457
of biomarkers of earthworm Eisenia fetida after exposure to thiacloprid. Environ. Sci.
458
Pollut. Res. Int. 2015, 22, 9475-9482.
23
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
459
Table legends
460
Table 1. LC values of sulfoxaflor for earthworms.
461
Table 2. Information of the characteristic metabolites in earthworms.
24
ACS Paragon Plus Environment
Page 24 of 32
Page 25 of 32
Journal of Agricultural and Food Chemistry
462
List of Tables
463
Table 1. LC values of sulfoxaflor for earthworms Pesticide days sulfoxaflor
LC Values(mg/kg dry soil)
Toxicity grade
LC2
LC10
LC50
7
0.12 (0.04 - 0.19)
0.24 (0.13 - 0.33)
0.61 (0.49 - 0.76)
Extremely toxic
14
0.08 (0.04 - 0.13)
0.19 (0.11 - 0.25)
0.54 (0.45 - 0.65)
Extremely toxic
25
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
465
Page 26 of 32
Table 2. Information of the characteristic metabolites in earthworms. Compound
KEGG ID
VIP Values
P-Values
Fold change
L-Arginine
C00062
1.79
0.018
2.07
L-Histidine
C00135
1.55
0.042
1.72
L-Leucine
C00123
1.99
0.028
1.30
L-Methionine
C00073
1.51
0.049
1.21
D-Glutamine
C00819
1.45
0.048
1.19
L-Glutamate
C00025
1.95
0.020
1.17
L-Asparagine
C00152
1.80
0.019
0.87
L-Valine
C01799
1.67
0.025
1.32
L-Phenylalanine
C00079
1.27
0.050
0.74
L-Aspartic acid
C00049
1.54
0.037
0.81
L-Ornithine
C00077
1.80
0.032
1.37
Spermidine
C00315
1.65
0.042
1.39
N-Acetylglutamic acid
C00624
2.09
0.004
1.25
D-Glucose-6-phosphate
C00092
1.83
0.017
1.68
Malic acid
C00711
1.60
0.033
1.24
Maleic acid
C01384
1.66
0.036
1.16
Hydroxypropionic acid
C01013
1.26
0.050
0.83
Citric acid
C00158
1.90
0.019
1.38
Propionic acid
C00163
1.57
0.040
0.73
4-Pyridoxic acid
C00847
1.56
0.040
1.27
Deoxyguanosine
C00330
1.28
0.045
0.71
Deoxyinosine
C05512
1.91
0.010
0.69
Deoxyuridine
C00526
1.47
0.033
0.84
Guanine
C00242
1.46
0.049
0.82
Serotonin
C00780
1.25
0.032
0.74
Xanthurenic acid
C02470
2.08
0.026
1.42
26
ACS Paragon Plus Environment
Page 27 of 32
Journal of Agricultural and Food Chemistry
467
Figure captions
468
Figure 1. Sulfoxaflor residues in artificial soil and earthworms.
469
Figure 2. Effects of sulfoxaflor on the oxidative damage to earthworms. Data with
470
different letters means significant difference at P < 0.05.
471
Figure 3. Score scatter plot and permutation test of OPLS-DA model for earthworms.
472
Figure 4. Perturbed metabolic pathways and metabolites in earthworms.
27
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
473
List of Figures
474 475
Figure 1. Sulfoxaflor residues in artificial soil and earthworms.
28
ACS Paragon Plus Environment
Page 28 of 32
Page 29 of 32
Journal of Agricultural and Food Chemistry
476 477
Figure 2. Effects of sulfoxaflor on the oxidative damage to earthworms. Data with
478
different letters means significant difference at P < 0.05.
29
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
479 480
Figure 3. Score scatter plot and permutation test of OPLS-DA model for earthworms.
30
ACS Paragon Plus Environment
Page 30 of 32
Page 31 of 32
Journal of Agricultural and Food Chemistry
481 482
Figure 4. Perturbed metabolic pathways and metabolites in earthworms.
31
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
483
TOC Graphic
484
32
ACS Paragon Plus Environment
Page 32 of 32