1 Use of physiologically based kinetic modeling-facilitated reverse dosimetry of combine with in

2 vitro metabolism data to predict the exposure level of imidacloprid in wild wood mice
3
4 Division of Toxicology, Wageningen University and Research, Stippenneg 4, 6708WE
5 Wageningen, The Netherlandss
6 E-Mail:
7 Tel : 031 6 23034881

8 Abstract
9
10
11

12 Keywords:: PBK model, Imidacloprid, Neon-nicotinic Pesticides, wood mice

13 1. Introduction
14 Wildlife is an essential part of the natural environment and a highly valued protection
15 goal. However, agricultural chemicals used, like pesticides, may pose risks to wildlife.
16 As agriculture technology grows, using pesticides becomes more restricted and tech-
17 advanced far more than before, with the usage of pesticides reducing both in amount
18 and variety. However, in some places like China, pesticides still illustrate an
19 increasing trend although local policies have been applied to regulate in, for example,
20 local crop systems, overuse of pesticides to control specific pastes, which can be up to
21 75% (Zhang et al., 2015). The high percentage can potentially result in risks on non-
22 desired effects of the pesticide use.
23 Following these, there has been an increasing recognition on the need to find a new
24 efficient method to reach the risk assessment of large numbers of chemicals in a
25 relatively short time. Among them, the reliance on wild animal liver experimentation
26 has (deleted) ethical, economic, and scientific limitations in assessing human
27 risks (Yoon et al., 2012). Exposure assessment is essential for adequately assessing the
28 risks of chemicals for wildlife. Wildlife is generally exposed to environmental
29 chemicals via oral uptake via food or drinking water. Evaluation of the actual uptake
30 of chemicals by wildlife is therefore influenced by the composition of the animal’s
31 diet, which varies between species and within species. Depending on the availability
32 of food, wildlife’s diet may be very different, for example, in winter and summer or
33 between locations. For the little owl, for instance, the exposure to cadmium in
34 floodplains differed significantly between seasons and between adjacent territories as
35 modeled based on food availability (Schipper et al., 2012) . For small mammals,
36 seasonal variation in diet composition, related to food availability, has also been
37 shown to result in significant differences in exposure (Vermeulen et al., 2010). While
38 differences between species could also be explained based on the differences in
39 diet (van den Brink et al., 2011) . However, it would be very beneficial for risk
40 assessment of chemicals if the exposure of animals could be assessed without the
41 uncertainty of the diet composition.
42 ADME processes may affect the relationships between exposure and internal
43 exposure. Absorption from the body itself, metabolism from blood or another
44 unexpected compartment, and individual difference, (deleted) could have a significant
45 influence on final predictions. The effects of ADME should be considered to link
46 internal concentrations to actual exposure levels. PBK, a physiology-based kinetic
47 modeling, is a mathematical technique (deleted) designed to describe the ADME
48 processes for a target compound in organisms. In this structured mathematical model,
49 organs and tissues are set as standalone compartments, with the process of targeting
50 chemicals inside the body defined as formulations. Previous model studies have
51 revealed that it is possible to quantify the internal concentrations of the chemical in
52 specific target organs from the uptake level for small mammals, for instance,
53 Quercetin concentration in the blood plasma of rats (Rattus norvegicus) to predict
54 their up-take level (Boonpawa et al., 2014) . This approach has been used in human-
55 oriented risk assessment for several years, primarily focused on relating in vitro effect
56 assessment to in vivo risk assessment.
57 Furthermore, this (deleted) platform/parameter tool gives great help in advancing
58 animal-free risk assessment (Bessems et al., 2014) . However, although this approach
59 could be very helpful in linking results from non-invasively collected samples to
60 actual exposure levels, studies for wildlife species still need to be completed.
61 Urea/feces sample PBK modeling-based reverse dosimetry is still an unknown field.
62 The objective of the present study is therefore to develop a way for the exposure level
63 and risk assessment of wildlife exposure to environmental chemicals. This will be
64 focused on a group of pesticides that are most used globally, neonicotinoids,
65 belonging the Imidacloprid. This group of pesticides is heavily debated, due to their
66 potential effects on honeybees, and potential impacts on pollination
(Whitehorn et al., 2012)
67 . However, although they are deemed to be less toxic to mammalian and avian
68 species, some recent studies show effects on mammals and birds at sub-chronic levels.
69 For instance, acetamiprid showed hepatoxicity on male Wistar rats at exposure levels
70 far below LD50 (Chakroun et al., 2016) , while in utero and lactational exposure to
71 low doses of this pesticide resulted in behavioral changes in male offspring in rats
72 (Sano et al., 2016) . It was also shown that effects might be more severe in case of
73 combined exposure to neonicotinoids and stress (Hirano et al., 2015) , a combination
74 often encountered by wildlife under environmental conditions. Studies from
Eng et al., 2019
75 . (2019) demonstrated that even under a single low exposure (1.2mg/kg body
76 mass), it still has adverse effects on songbirds’ behaviors during the migration and
77 decreases food consumption, which could lead to a population decrease.
78 During this study, a PBK model was made to access the exposure level of wild wood
79 mice by using reverse dosimetry. An in-vivo experiment was applied to validate this
80 model by using the exact dosage of imidacloprid in the oral exposure process. The
81 residue concentration of imidacloprid in mice organs was compared with the model-
82 predicted concentration to confirm the function of the model. In the end, it is studied
83 that using the reverse dosimetry method to access the real exposure level of wild mice
84 is possible.
85 2. Materials and methods
86 Chemical and experiment materials
87 Imidacloprid (C9H10CIN5O2, CAS-No.1338261-41-3, purity 99.8%), was purchased
88 from Sigma-Aldrich (Schnelldorf, Germany), and dimethyl sulfoxide (DMSO) from
89 Merck (Darmstadt, Germany). Cages, including living cages and capture cages, also
90 with animal living stocks like peanut butter and hay were purchased from the local
91 store.
92 Animal capture and living
93 Animals were collected from sites (for example, forest areas) where pesticides had not
94 been used in the past, near Sinderhove, Renkum, The Netherlands. Animals were
95 captured using live traps set late at night and checked early in the morning to ensure
96 animals were at minimal stress levels.
97 The traps were filled with hay beds and the specimens were lured with peanut butter
98 and sunflower seeds to feed the mice at night. Sliced apple pieces served as a water
99 source. In the morning, we checked the traps to make sure no shrews or other animals
100 were accidentally captured.After species other than wood mice are released, half are
101 given (uncontaminated) soil and food and water ad libitum for 24 hours of
102 acclimation under outdoor weather conditions to reduce the stress of capture. A wood
103 mouse was placed in the field mesocosm (conform protocol 2017.W-0065.002). The
104 animals are individually housed, which is great because wild-caught animals are
105 territorial and can injure each other when placed together.
106 After capture (placed in a weighing bag), the mice were individually weighed to
107 demonstrate their fitness after the acclimation period. Behavior was monitored hourly
108 by cameras installed above the cages to observe locomotion, maintenance, and use of
109 cage fixtures (shelters or covers provided in the form of a straw). However, mice are
110 mostly nocturnal or active at dawn and dusk, so they are generally invisible. Welfare
111 is then monitored by checking the amount of food eaten and other behaviors (picking
112 hay). For this purpose, a portion of the hay in the cage was cleaned daily, and mouse
113 prints were checked the next day. During the whole period, a life trap filled with hay
114 was placed in the cage and set in safe mode (so the trap would not close). The mice
115 could use this trap as a sleeping quarter. The cage was enriched with vegetation and
116 wheat straw, and the hay was deep enough for the mice to hide.
117
118 Animal chemical exposure and experiment setup

119
120 Figure 1 Wild wood mice catch and treatment procedure

121 A food mixture of peanut butter and the compound imidacloprid has been used for
122 oral exposure. The feed itself is 100% peanut butter, mixed with an imidacloprid
123 solution containing a certain amount of imidacloprid, thoroughly mixed to ensure that
124 the imidacloprid is uniform in the feed tray.
125 The initial setup was designed at 20mg/kg BW per Wood mice. The body weight of
126 the mice was assumed to be an average of 20 g, and the oral intake was set at 1 g, the
127 food itself was set at 0.18 mg/g imidacloprid/peanut butter. All food is balanced in the
128 bowl before being added. In addition, to avoid disturbance from other food sources,
129 the mice were starved overnight by being fed an apple slice. The next day, all
130 leftovers were removed and moved to display shelves. A live camera was installed on
131 the cage’s roof to observe the mice eating at T-0. After the rat took the food once, the
132 food bowl was reweighed to assess the amount of food taken. Actual exposure levels
133 are lower than known to show no adverse effects on animals in single acute
134 exposures. (Farag et al., 2001; Kapoor et al., 2014; Vohra et al., 2014).
135 Experiment exposure periods were set primely as 0/3/6/9/24/48 hours, though exact
136 hours and minutes may differ cause of the uncertainty from wild mice diet hobbies. In
137 vitro results indicate that most clearance of imidacloprid occurs within 24 hours,
138 especially in the first 12 hours. For the sacrifice of the animals, each wood mice was
139 anesthetized with isoflurane gas by inhalation, Blood was collected as a terminal
140 procedure and under anesthesia by heart puncture in Eppendorf tubes with EDTA
141 anticoagulant. Tissues such as Liver, Kidney were collected, then frozen under -80 for
142 further analysis.
143 Animal organ sample treatment and analysis
144 Mice organ samples stored at -80 °C were removed from the freezer, then left at room
145 temperature to normal, cut a tissue piece of 0.1-0.3 g, and mixed with 2 ml of
146 phosphate buffer pH 7.4 in a dedicated glass tube for sampling. Drawing for 30
147 seconds until no viscera are visible. Organs were weighed before homogenization.
148 After fully homogeneous, the whole solution was removed from the glass tube, into a
149 10ml tube that contained 6ml ACN to help solve imidacloprid into solution. Then
150 2.4g of CaSO4 was added in to help settle down organ mixes. The full tube was then
151 moved to a rotter machine at rpm 50/minute for 60 minutes. in the end, centrifuge the
152 tube at 3500 rpm for 30 minutes. A 100ul suspensions were collected for further
153 LCMS analysis to detect the imidacloprid and 5-Hydroxy-imidacloprid residue
154 amount.
155 A pilot trial experiment, with chicken organs as target tissue instead of mice organ,
156 was performed before the exposure experiment to ensure the analytic way of the
157 target compound’s concentration test method was capable of the expected results.
158 PBK modeling and reverse dosimetry approach
159 In general, to form a model that could predict the risk assessment of certain
160 compounds through concentration-response data by in-vitro methods, the following
161 steps will be taken:
162 (1): Establish in vitro hepatic metabolite to generate a rate of selected compound in
163 the target organ’s microsomes, in this case, liver microsomes and S9 fractions of rats.
164 (2): Development of a PBK model that could simulate how the selected compound
165 "absorption, distribution, metabolism, and excretion” in the target body. Each target
166 organ will be formalized as a unique mass balance equation compartment.
167 Intake/excretion of the compound was carried out by blood, which was also set as a
168 mass balance equation. Physiology constants of the PBK model were gathered from
169 previous study and (1)’s experiments. In this study, the output of the model will be the
170 concentration of the selected compound in blood, and the input will be the uptake
171 dose of the selected compound and body weight.
172 (3): Model validation, compared the output data from the PBK model and existing in-
173 vivo experiment data. First, adjust model constants to an existing in-vivo
174 experiment’s data. Then secondly, validate the output of the model by comparing its
175 calculation results with the in-vivo experiment data. This study acquired physiology
176 data from the previous chapter with the same compound and physiology parameters.
177 During this study, physiology data of Mice Imidacloprid metabolism were required in
178 earlier chapters. In vivo physiology data will be used for model validation.

179
180 Figure 2 Wood mice imidacloprid PBK model schematic

181 Mice Imidacloprid physiology data and PBK model

182 Mice physiology data, e.g. metabolic rate of Imidacloprid, including its selected phase
183 II metabolic compound 5-Hydroxy-IMI, were obtained from previous chapter 1, for
184 detail, see Appendix.
185 Two kinds of wood mice imidacloprid PBK model were created to describe the
186 metabolism pathway, the concentration of imidacloprid, and its main metabolic 5-
187 Hydroxy-imidacloprid in different compartments. One general model, which contains
188 literature gained physiology data combined with lab in-vitro metabolism data, and
189 another individual model, which contained some of the physiology data like
190 liver/kidney compartment weight ratio were specified to each cached mice to
191 subdivide every single model. These two models were made to help determine and
192 calibrate the differences between standardization lab physiology data and wild animal
193 physiology data in the model.
194 Model validation
195 Model validation by comparing the compartment concentration for Imidacloprid and
196 5-Hydroxy-Imidacloprid between model calculated prediction and in-vivo wood mice
197 exposure experiment results. Also, the general model and individual model prediction
198 results were compared.
 Quantify the compound Parent compound and selected metabolites in
concentration in rats' Blood sample concentration
blood
Based on the ratio of concentration between the
Uptake time parent compound and the selected metabolites in
locating blood, speculate the last uptake time

Uptake dose
Reverse dosemtry calcualte the exposure level by inputing
calculation body weight, time, and blood compound concentation in the
model.

199
200 Mice uptake dose reverse dosimetry

201
202 Figure 3 Wild wood mice imidacloprid exposure reverse dosimetry method

203 To accomplish the objective of reverse dosimetry and predict the target compound's
204 absorbed dose in wild-type mice, the plasma concentration ratios of imidacloprid and
205 5-hydroxy-imidacloprid were measured compared to help estimate contact time.
206 Then, by matching the compound's predicted compound blood concentrations with the
207 blood sample's actual concentrations, it is possible to reveal a reasonably up-to-date
208 dose range, which can be reduced to a bad level.
209 3. Results

210 The PBK model codes for both the General model and the Individual model are
211 presented in the supplementary material. Model physiology parameters were obtained
212 from previous chapters. In addition, body parameters measured in mice are included
213 in the appendix.
214 Mice organ sample treatment

215 The chicken liver pilot trial experiment was performed to determine the recovery ratio
216 of Imidacloprid and 5-Hydroxy-IMI from the chicken liver organ residue after a
217 single inject of imidacloprid and 5-Hydrxoy-IMI. This trial experiment showed that
218 the recovery of Imidacloprid and 5-Hydroxy-IMI from chicken organs was stable at
219 55%.
220 Wild wood mice single Imidacloprid exposure results

221 21 wild wood mice were cached, and pre-setup exposure times from 0 to 2,4,6,9,24,48
222 hours were applied to mice. However, detailed exposure hours may differ from pre-
223 setup, e.g., needed to be increased. This is because uncertainty about the
224 behavior/physiology of animals in the wild is limited.

Model calculated Imidacloprid concen-

1000.00 1000.00

Model calculated 5-Hydroxy-IMI

concentration in log scale (μmol)
tration in log scale (μmol) 100.00
100.00
10.00
10.00 1.00
0.10
1.00
0.01
0.10
0.00
00 01 10 00 .0
0 00 .0
0
0.01 0. 0. 0. 1. 10 0. 00
0.01 0.10 1.00 10.00 100.00 1000.00 10 10
In-vivo Imidacloprid concentration in log scale In-vivo 5-Hydroxy-IMI concentration in log scale
(μmol) (μmol)

225
226 Figure 4 The concentration of imidacloprid(left), 5-Hydroxy-Imidacloprid(right) in Liver, in-vivo exposure results vs plotted
227 model calculation results(●), after oral administration on wild wood mice. The upper hardline ( 一) stands for 1 to 5, and the
228 middle dot(….) line stands for 1 to 1, lower hardline(一) stands for 1 to 0.2.

229 Imidacloprid and 5-Hydroxy-imidacloprid concentrations in the liver, kidney, and

230 blood are shown in Figures 4,5 and 6. The model calculated concentration points
231 using the general model were plotted with actual in-vivo sample residue
232 concentrations in each graph. The hardline on the top is the 1 to 5 fold, and the bottom
233 is 5 to 1 fold, the dotted line in the middle stands for the 1 to 1 ratio. After a single
234 oral exposure, imidacloprid and its main metabolite, 5-hydroxy-imidacloprid’s
235 concentration, appeared in all compartments.
236 In the liver compartment, the imidacloprid concentration stayed in a stable 1 to 1 ratio
237 between the model-calculated and sample-measured results. The 5-Hydroxy-IMI on
238 the other hand were highly underestimated from model calculated results to sample
239 measured results, illustrating a 5 fold in all samples, though the distribution was very
240 even.
Model calculated Imidacloprid concen-

1000.00 1000.00
Model calculated 5-Hydroxy-IMI
concentration in log scale (μmol)

100.00
tration in log scale (μmol)

100.00
10.00
10.00 1.00
0.10
1.00
0.01
0.10
0.00
00 01 10 00 .0
0 00 00
0.01 0. 0. 0. 1. 10 0. 0.
0.01 0.10 1.00 10.00 100.00 1000.00 10 10
0

In-vivo Imidacloprid concentration in log scale In-vivo 5-Hydroxy-IMI concentration in log scale
(μmol) (μmol)

241
242 Figure 5 The concentration of imidacloprid(left), 5-Hydroxy-Imidacloprid(right) in Kidney, in-vivo exposure results vs plotted
243 model calculation results(●), after oral administration on wild wood mice. The upper hardline ( 一) stands for 1 to 5, the middle
244 dot(….) line stands for 1 to 1, lower hardline(一) stands for 1 to 0.2.

245 Imidacloprid inner organ concentrations went steady in a linear ratio range in the in-
246 vivo sample results compared to model prediction results in the liver, kidney, and
247 blood compartments. These trends illustrate the accuracy of the model in-vitro to in-
248 vivo prediction in Imidacloprid. However, there is one point in the kidney
249 compartment over 5 fold, which is from teenage mice that have the lowest body
250 weight, 9g. The model calculated that 5-Hydroxy-IMI concentrations were
251 underestimated in all three compartments. Most of the models predict 5-Hydroxy-IMI
252 concentrations were all larger than 5 fold. This also has an influence in the later
253 reverse dosimetry prediction part, where the ratio of 5-Hydroxy-IMI/Imidacloprid
254 played an important role.
Model calculated Imidacloprid concen-

1000.00 1000.00

Model calculated 5-Hydroxy-IMI

concentration in log scale (μmol)
100.00
tration in log scale (μmol)

100.00
10.00
10.00 1.00
0.10
1.00
0.01
0.10
0.00
00 01 10 00 .0
0 00 .0
0
0.01 0. 0. 0. 1. 10 0. 00
0.01 0.10 1.00 10.00 100.00 1000.00 10 10
In-vivo Imidacloprid concentration in log scale In-vivo 5-Hydroxy-IMI concentration in log scale
(μmol) (μmol)

255
256 Figure 6 The concentration of imidacloprid(left), 5-Hydroxy-Imidacloprid(right) in Kidney, in-vivo exposure results vs plotted
257 model calculation results (●), after oral administration on wild wood mice. The upper hardline ( 一) stands for 1 to 5, the middle
258 dot (….) line stands for 1 to 1, lower hardline(一) stands for 1 to 0.2.

259 Wood mice general and individual PBK model for Imidacloprid and 5-Hydroxy-IMI

260 A general model and 21 individual models were coded. The difference in each model
261 was the liver/kidney’s weight versus body weight. Each mice’s individual model has a
262 unique liver/body and kidney/body weight ratio, these ratios will slightly alternate the
263 mass balance calculation in model prediction.
100.00 100.00
In-vivo mouse Liver IMI concentration
In-vivo mouse Liver IMI concentration

10.00
10.00
umol/(l or g) in log scale
umol/(l or g) in log scale

1.00

1.00
0.10

0.01 0.10
0.10 1.00 10.00 100.00 0.10 1.00 10.00 100.00

Model calculated Liver IMI concentration Model calculated Liver IMI concentration
264 umol/(l or g) in Log scale umol/(l or g) in Log scale

265 Figure 7 Prediction of Imidacloprid concentration in the Liver by General(left) and Individual(right) model versus
266 In-vivo data.

267 Cross-comparison of the inner organ Imidacloprid concentration prediction results vs

268 organ sample results from General and individual models were shown in figure 7 and
269 table 1. The numbers above the plots are exposure hours. Generally, the prediction
270 results are similar between general and individual model accuracy, though the general
271 model provides slightly better results. Other organs’ results are shown in Table 1.
272 Imidacloprid is shown a higher co-respond accuracy in all three main sample organs,
273 while 5-Hydroxy-Imidalcoprid was not on the same level. The general model reveals
274 a slightly higher prediction accuracy through all the organs compared to in-vivo
275 results.
276 Table 1 differentials calculation results between wild wood mice individual model and general model

Imidacloprid
Compartment General vs In-vivo Individual vs In-vivo R2 differential
Liver 0.9483 0.8760 0.0723
Kidney 0.9315 0.8414 0.0901
Blood 0.9606 0.9478 0.0128
5-Hydroxy-Imidacloprid
Compartment General vs In-vivo Individual vs In-vivo R2 differential
Liver 0.8353 0.8462 0.0109
Kidney 0.1416 0.1717 0.0301
Blood 0.4554 0.6283 0.1729
277

278 The model calculated Blood 5-Hydroxy-imidacloprid/Imidacloprid concentration

279 ratio, and the measured blood sample ratio curve was plotted in figure 8, compared
280 with real in-vivo sample exposure concentration. The dotted line is the model
281 calculated 5-Hydroxy-IMI/Imidacloprid ratio, and the hardline is the in-vivo sample
282 measured 5-Hydroxy-IMI/Imidacloprid ratio, which formed from in-vivo blood
283 sample results. The model-calculated ratio curve was much flatter than an in-vivo
284 curve, this means the metabolite rates of 5-Hydroxy-IMI are overestimated in the
285 model. In this case, the in-vivo curve was used because the in-vitro model calculated
286 curve was not reliable and calculable for the estimated exposure T, though the in-vitro
287 calculated results will also be presented as a compare.
0.5

0.45

0.4
5HY/IMI blood concentration ratio

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0
1 2 3 4 5 6 7 8 9 10

Hours

288
289 Figure 8 Blood 5-Hydroxy-Imidacloprid/Imidacloprid concentration ratio curve after single exposure by model calculation(…)
290 and in-vivo(一) sample results.

291 The exposure time could be estimated using the ratio curve trend line formula reverse
292 calculated. In this case, the in-vivo sample measured ratio was used to calculate the
293 exposure T and the reverse dosimetry dose as the method. As shown in figure 9, the
294 in-vitro reverse calculated exposure time T has a much larger error margin, over 5
295 fold, than the exact exposure time T. Also, the in-vitro curve calculated exposure T
296 has already surplus the limited of 48 hours, which is the maximum single exposure
297 time in this experiment, whereas in the model, the absolute amount of Imidacloprid
298 and 5-Hydrxoy-IMI is nearly non. The in-vivo measured ratio calculated T has much
299 higher coordinates, which stayed in a 1 to 1 ratio.
100.0 10000
Ratio predicted exposure time (hours)

Ratio predicted exposure time (hours)

1000

10.0 100

10

1.0 1

0.1 0
0.1 1.0 10.0 100.0 0.0 0.1 1.0 10.0 100.0 1000.0
In-vivo exposure time (hours) In-vivo exposure time (hours)

300
301 Figure 9 In-vivo measured ratio calculated exposure time(●)(left), and in-vitro ratio calculated exposure time(●)(right)
302 compared to the real exposure time. The upper hardline (一) stands for 1 to 5. The middle dot(….) line stands for 1 to 1, lower
303 hardline(一) stands for 1 to 0.2.

304 Adding in the exposure time, combined with the measured wild wood mice body
305 weight and Imidacloprid blood residue concentration measured from the blood
306 sample. Figure 10 presents the reverse calculated exposure dose from the setup
307 calculation method, plotted with the exact exposure dose. Most of the calculated
308 exposure dose results are within the range of 2 fold. This is the consequence of the
309 overestimated imidacloprid concentration in the blood compartment from the model
310 calculation.
1000.0
Model predicted exposure dose mg/kg BW in log scale

100.0

10.0

1.0

0.1
0.1 1.0 10.0 100.0 1000.0
In-vivo exposure dose mg/kg BW in log scale

311
312 Figure 10 Reverse dosimetry calculated exposure level(●) compared to real exposure level. The upper hardline ( 一) stands for 1
313 to 5, the middle dot(….) line stands for 1 to 1, lower hardline(一) stands for 1 to 0.2.

314 4. Discussion

315 Neonicotinoids are broad-spectrum insecticides that bind almost irreversibly to the
316 organism's nicotinic acetylcholinergic receptors (nAChRs) (Simon-Delso et al., 2015).
317 Neonicotinoids stimulate nAChRs by blocking CNS receptors, leading to paralysis
318 and eventual death (Asita et al., 2022). Application routes include soil treatment, foliar
319 spray, soil drenching, watering, stem spraying, seed mulching, and seedling
320 drenching (Ma et al., 2021) . Seed coating and seedling immersion minimize the drift
321 of pesticides into the surrounding environment and limit exposure to non-target
322 invertebrates that do not directly ingest the treated crop. The cellular properties of
323 neonicotinoids facilitate plants’ uptake and systematic activity, supporting these
324 applications. These Plant protection products (PPPs) are currently one of the most
325 stringent chemical products in terms of regulation, marketing, and licensing of
326 molecules based on an intensive environmental risk assessment (ERA), post-
327 processing monitoring, and registration (Fritsch et al., 2022; Ma et al., 2021) . Their
328 breakdown products are still present in the environment due to legacy contamination
329 and long persistence, and they can be remobilized due to current practices on arable
330 soils (Sabatier et al., n.d.).Small mammals occupy a central place in the ERA process
331 for PPP registration as the standard toxicity test. The toxicity data for humans and
332 mammals are determined based on animal testing, mainly performed on rats and
333 mice
(“Guidance on the Risk Assessment of Plant Protection Products on Bees (Apis Mellifera, Bombus S
334 . Small
335 mammals are largely represented in the list of “mammalian indicator species”, and
336 “generic focal species” in the first tiers of ERA. A critical step is to characterize the
337 exposure of non-target species to these compounds under realistic conditions. Still,
338 information about the contamination of wildlife by these pesticides needs to be
339 included. measurements of small mammal exposure to PPP under actual field
340 conditions are rare. Small mammals have a major functional role in terrestrial
341 ecosystems, and some species are considered beneficial organisms in agroecosystems
342 by feeding on weed seeds and invertebrates
(Delibes-Mateos et al., 2011; Tschumi et al., 2018)
343 .
344 The present study aimed to use the physiology-based kinetic model-based reverse
345 dosimetry method to translate the inner compound concentration into wood mice
346 exposure intake level. Physiology data of Imidacloprid and its main metabolite 5-
347 Hydroxy-Imidalcoprid were acquired from previous studies, then input into the PBK
348 model. Reverse dosimetry calculations were applied to predict the oral exposure level
349 of wild wood mice by matching the sample blood imidacloprid concentration with the
350 model-calculated concentration. Input values for the model were required in-vitro
351 metabolism incubations, combined with a blood imidacloprid/5-hydroxy-imidacloprid
352 concentration ratio. The results show that in the examined exposure hours, which are
353 between 1 to 9, the in-vitro calculated curve was not able to quantify the exposure
354 hour, but the in-vivo curve showed very high accuracy. This means for this
355 compound, within the application domain of 1 hour and then up to 9 hours, it is
356 possible to use this method to reverse calculate the exposure level from wild wood
357 mice blood samples. However, the 5-Hydroxy-IMI prediction varies from in-vivo
358 measured samples for a fold of 5.
359 Inherit from the previous chapters from Carbendazim, the exposure time estimation
360 method was based on the blood concentration ratio of the Parent compound and its
361 metabolite, in this case, imidacloprid and 5-Hydroxy-Imidacloprid. The difference
362 from the previous one was that the ratio curve used to reverse calculate the exposure
363 time was based on in-vivo samples, not the model calculation itself. The reason for
364 this could be the unstable condition of Phase II metabolites in organs in rodents’ liver
365 by amino acid conjugation, acetylation, and methylation
(Zamek-Gliszczynski et al., 2006)
366 . Time points between phase II take reaction and sample analysis period could
367 also lead to the metabolism of Phase II compounds. Besides the model itself, the
368 Neonicotinoids in rats Phase II reaction and its metabolism need more relative studies
369 and detective methods to fulfill the blank knowledge field (Khidkhan et al., 2021) , as
370 the phase II reaction plays the same important role in neonicotinoids clearance in
371 rodent’s body (Shi et al., 2009) . This kind of inconformity has not been shown in the
372 previous study on carbendazim, which means the model calculated ratio between
373 parent compound and metabolites did not apply for every compound.
374 In conclusion, the results of this study suggest that a generic PBK model with a basic
375 predefined structure is a useful tool for reverse calculation prediction of the
376 environmental exposure to local small mammals like wood mice, with minimum
377 parameterization, stemming from in vitro and silico sources, whereas the development
378 of bespoke probabilistic PBK models for use in a thorough in vitro/in silico-based risk
379 assessment. Nevertheless, the number of chemicals applied was limited, more
380 substances shall be evaluated according to the format laid down in this manuscript.
381 Future research should focus on defining better the applicability domain of such
382 models and developing guidance criteria for their performance capacity. Global
383 differential research should also be performed to determine the most influential input
384 parameters and the impacts of variabilities on the model output. Furthermore, more
385 empirical data on Phase II biotransformation and partitioning in mice and rats are
386 needed to refine the current PBK models and improve estimations. It is encouraged to
387 refine further and apply our generalized wood mice PBK model in chemical risk
388 assessment for wild mice and rats, given the goal of protecting the whole ecosystem
389 in the context of animal health, ecosystem integrity, and food safety.
390
391
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