Method optimization and validation
After a series of method optimization proceeding, 1% formic acid acidified acetonitrile was selected as the best extraction solvent owing to its highest recoveries (94.37% − 100.29%) with the relative standard deviation (RSD) being between 1.62% and 4.84% (Fig. 1A). The extraction solvent volume of 10 mL was opted as the best extraction amount since it exhibited a better extraction performance than the other extraction amount (5 mL) with recoveries of 99.28% − 101.01% by purification material npc (Fig. 1B). Npc was chosen as the purification for pesticides FOR, ATP and CAP from strawberries due to its lower absorption capacity and stronger ability to remove impurities than the other three purification materials (Fig. 1C).
Table 3
Linear equation, determination coefficient (R2), matrix effect (ME), LODs and LOQs of fluopyram (FOR), acetamiprid (ATP) and chlorantraniliprole (CAP) in solvent, strawberry samples.
Matrix
|
Linearity range (mg/L)
|
Analyte
|
Linear equation
|
R2
|
ME (%)
|
LODs
(mg/kg)
|
LOQs (mg/kg)
|
acetonitrile
|
0.002 ~ 0.1
|
FOR
|
y = 1944219x + 210
|
0.9993
|
/
|
|
/
|
ATP
|
y = 289656x + 82
|
0.9994
|
/
|
|
/
|
CAP
|
y = 71546x + 26
|
0.9997
|
/
|
|
/
|
strawberry
|
0.002 ~ 0.1
|
FOR
|
y = 1731324x + 300
|
0.9996
|
-10.95
|
0.002
|
0.01
|
ATP
|
y = 257584x-4
|
0.9997
|
-11.07
|
0.002
|
0.01
|
CAP
|
y = 66677x-39
|
0.9990
|
-6.81
|
0.002
|
0.01
|
strawberry x10
|
0.002 ~ 0.1
|
FOR
|
y = 1869814x-581
|
0.9993
|
-3.83
|
0.002
|
0.01
|
ATP
|
y = 298217x + 32
|
0.9999
|
2.96
|
0.002
|
0.01
|
CAP
|
y = 72391x + 12
|
0.9993
|
1.18
|
0.002
|
0.01
|
The linearity examination between the concentrations of tested pesticides and their peak areas revealed that fluopyram, acetamiprid and chlorantraniliprole had good linear relations with determination coefficients (R2) ≧ 0.999 in ACN solvent solution and matrix-matched standard solutions (Table 3). The strawberry matrix weakened the signals of three examined pesticides since the ME values were less than 1(Table 3). As a result, matrix-matched standard solutions were chosen for the quantitative assessment of three examined pesticides in strawberry samples. The LODs and LOQs of FOR, ATP and CAP in strawberry were 3.3 µg kg− 1 and 10 µg kg− 1, respectively. To validate and evaluate the method's accuracy, recoveries of pesticides FOR, ATP and CAP were done at spiked levels of 10, 100 and 1000 mg kg− 1, with five duplicates examined for each level (Table 4). The average recoveries of tested pesticides in strawberries varied from 82.62% (CAP) ~ 107.79% (FOR), with RSDs ranging from 0.58–12.73%. The addition recovery test revealed that the analytical method was linear and accurate, and that it could identify and analyze tested pesticides level in strawberries.
Table 4
Recoveries and relative standard deviations (RSDs) of pesticides fluopyram, acetamiprid and chlorantraniliprole in strawberry samples.
Analyte
|
Spiked level (µg/kg)
|
Mean recoveries (%)
|
RSDs (%) n = 5
|
fluopyram
|
10
|
107.79
|
0.58
|
100
|
101.31
|
2.18
|
1000
|
103.41
|
2.13
|
acetamiprid
|
10
|
99.72
|
4.61
|
100
|
97.91
|
1.00
|
1000
|
93.56
|
2.79
|
chlorantraniliprole
|
10
|
94.60
|
12.73
|
100
|
82.62
|
3.20
|
1000
|
90.84
|
1.28
|
Dissipation and terminal residue levels of pesticides fluopyram, acetamiprid and chlorantraniliprole in strawberries
Dissipation of fluopyram in strawberries
The dissipation curves of fluopyram alone or joint are shown in Fig. 2A, the dissipation equation and correlation coefficient (R2), as well as the T1/2 are listed in Table 5. After carefully examining the R2 values for the fitting curve of fluopyram, the results showed that the connected first plus first order kinetics is the best match model for capturing the kinetics of pesticide dissipation. The R2 value for fluopyram dissipation in a alone application (0.9407) is significantly higher than that in a combination application (0.7775). Fluopyram's dissipation half-life in strawberries was 11.6 (alone) and 12.4 (joint), with no significant differences related to application mode. The initial concentrations of fluopyram were 0.245 ± 0.066 mg kg− 1 (alone) and 0.175 ± 0.028 mg kg− 1 (joint) at 2 h after twice spraying. Within 1 to 3 days of alone application and 3 to 5 days of joint application, the residual concentration of pesticides in strawberries increased slightly, indicating that pesticide absorption by strawberries was greater than their own destruction. The residue in strawberries continued to decrease after that, with ultimate residue amounts of 0.108 ± 0.002 mg kg− 1 (alone) and 0.097 ± 0.030 mg kg− 1 (joint), respectively, which was far lower than the MRLs (minimum residue limits) of China (0.4 mg kg− 1), the European Union (0.1 mg kg− 1) and Australia (1.5 mg kg− 1).
Table 5 Physicochemical properties, residual kinetics, determination coefficient (R2) and half-life (T1/2) of fluopyram (FOR), acetamiprid (ATP) and chlorantraniliprole (CAP) in strawberries under alone and mixed applications.
Note: Log Kow: logarithm of octanol−water partion coefficient.
Dissipation of acetamiprid (ATP) in strawberries
The dissipation of ATP in alone and joint applications in strawberries followed by first order kinetics were illustrated in Fig. 2B, their half-lives were alike (6.1 days for alone and 6.7 days for joint) (Table 5). The R2 values were in the range of 0.8286 to 0.8676, and showed there was no marked variation whether they were applied alone or joint. The initial concentrations of ATP were 0.089 ± 0.009 mg kg− 1 (alone) and 0.089 ± 0.005 mg kg− 1 (joint) at 2 h after twice spraying. And the dissipation of ATP in strawberries at 1 day was 18.06% by joint, which was remarkable higher than that under alone. The residual concentration of ATP in strawberries increased somewhat after 2 h to 1 day of single application and 3 to 5 days of combined application. The residue in strawberries continued to decrease after that, with ultimate residue amounts of 0.034 ± 0.003 mg kg− 1 (alone) and 0.031 ± 0.009 mg kg− 1 (joint), respectively, which was well below the MRLs of China (1 mg kg− 1), Korea (1 mg kg− 1) and the United States (1 mg kg− 1), and slightly higher than the MRLs of the European Union (0.02 mg kg− 1). The final dissipation of ATP in strawberries were 62.47% (alone) and 64.75% (joint).
Dissipation of chlorantraniliprole (CAP) in strawberries
The concentration curves of CAP were depicted in Fig. 2C, and the dissipation behaviors were estimated using the first-order kinetics equation, with the kinetics parameters provided in Table 5. The dissipation curves of CAP in strawberries alone and in the combination of FOR, CAP and ATP matched well, with R2 values of 0.8666 ~ 0.9041. The residues of CAP were 0.072 ± 0.005 mg kg− 1 (alone) and 0.080 ± 0.007 mg kg− 1 (joint), respectively, with the sampling interval at 2 h after twice spraying. The dissipation of CAP in strawberries were 0.05% (alone) and 17.22% (joint) after 2 h. The residues, on the other hand, can be swiftly eliminated in the first three days, while a portion of the residues are incorporated into cellular components and slowly degraded over time. The final remains of CAP in strawberries were 0.038 ± 0.006 mg kg− 1 (alone) and 0.042 ± 0.003 mg kg− 1 (joint), respectively, which was much lower than the MRLs of CAC (0.5 mg kg− 1), China (2 mg kg− 1) and the United States (0.6 mg kg− 1), and lightly lower than the MRLs of the European Union (0.05 mg kg− 1). After 10 days, CAP dissipation in strawberries were reduced by 47.26% (alone) and 47.70% (joint).
Comparison of pesticide dissipation in strawberries
The initial deposits of FOR, ATP and CAP in strawberries were alike when used alone and joint. The concentrations of ATP and CAP under alone application were slightly higher than those of joint application. The FOR, ATP and CAP residues in strawberries decreased to 0.149 ± 0.0001 mg kg− 1, 0.048 ± 0.002 mg kg− 1, 0.056 ± 0.004 mg kg− 1 for alone application after 5th day, but increased to 0.135 ± 0.009 mg kg− 1, 0.054 ± 0.010 mg kg− 1, 0.060 ± 0.0001 mg kg− 1 for joint application. The dissipation of FOR, ATP and CAP in strawberry residues in the beginning were slower than those in the end. The terminal concentrations (after 10th day) of FOR, ATP and CAP in strawberries were no significant difference whether used in combination or alone. Besides, the dissipation half-life values in strawberries were 11.6 and 12.4 days for FOR, 6.1 and 6.7 days for ATP, and 11.7 and 10.9 days for CAP at alone and joint application, respectively. The dissipation half-life of combination application increased by 0.07 (FOR) and 0.10 (ATP) times and decreased by 0.07 (CAP) times when compared to alone application. The results showed that differences caused by alone and joint applications in half-life were not statistically significant. The results of this investigation on FOR in pomegranate (Matadha et al., 2021) were consistent with its half-life of 7.3 ~ 15.0 days. Xiao et al. reported the t1/2 of 5.37 days in ATP in honeysuckle (Xiao et al., 2022), Moreover, the t1/2 of 5.78 days in pigeonpea (Kansara et al., 2021), 6.50 days in celery (Zhang et al., 2022), and 10.0 days in cabbage(Lee et al., 2019) was stated for CAP.
Residues on greenhouse plants peaked in the hours following spraying, then gradually decreased over the next few days, resulting from pesticide degradation (Matadha et al., 2021). Pesticide degradation in plants is primarily influenced by the pesticide's nature, plant metabolic transformation, and microbial degradation (Li et al., 2020). After application, the three pesticides' residues are in the following order: FOR (0.108 ± 0.002 ~ 0.097 ± 0.030 mg kg− 1) > CAP (0.038 ± 0.006 ~ 0.042 ± 0.003 mg kg− 1) > ATP (0.034 ± 0.003 ~ 0.031 ± 0.009 mg kg− 1), respectively. Pesticide dissipation can also be affected by differences in enzymes and microorganisms (Diez et al., 2017; Muñoz-Leoz et al., 2013). The T1/2 of the tested pesticides also followed the sequence: FOR (11.6 ~ 12.4) > CAP (10.9 ~ 11.7) > ATP (6.1 ~ 6.7), respectively. The T1/2 orders of the three pesticides in this greenhouse trial were consistent with Kow (Table 5). In contrast, FOR has the highest residual and persistent properties that can lead to potential groundwater contamination, and ATP has a lower risk of dissipation than FOR and CAP. For joint application, there are no statistically pronounced variations in the T1/2 and final residue levels of the three pesticides, which suggests that there is no substantial interaction between the three pesticides, and that the aggregate effect of the three pesticide mixes is solely dependent on individual effects (Xiaoqiang et al., 2008).
Dietary risk assessment
Chronic dietary exposure assessment
In addition to comparing ultimate residue concentrations and MRL values, dietary intake risk assessment could be used to evaluate the safety of FOR, CAP and ATP in strawberries. The ADI values of FOR, ATP and CAP were 0.01, 0.07 and 2 mg (kg b.w.)−1, respectively. For distinct groups of male and female consumers in China (this chapter selects 10 typical groups), body weights and daily intakes were 17.9–65.0 kg and 75.5–229.1 g/d, respectively (Wang et al., 2021). Tables S1-S3 (Supplementary information) illustrate the RQc caused by three pesticide residues found on strawberries in distinct age and genders groups. The values of RQc ranged from 2.06–7.63% after spraying FOR at 1, 3, 5, 7 and 10 days (Table S1). And the RQchronic values of ATP were between 0.096% and 0.63% (Table S2), the RQchronic of CAP ranged between 0.0041% and 0.016% (Table S3). For three pesticides, the risk of chronic dietary exposure to strawberry residues was highest in the aged 8 to 12-year-old group. The chronic risks of three pesticides intake were ranked in the order of FOR > ATP > CAP. The chronic risks (low to high) of the different groups were in an order of over 65 year old males < 20–50 year old males < over 65 year old females < 51–65 year old females < 51–65 year old males < 20–50 year old females < 13–19 year old females < 13–19 year old males < 4.5% < 2–7 years old group < 8–12 years old group (FOR used alone after 3 d).
FOR had the highest chronic intake risk of the three pesticides, which was primarily attributed to the initial application dose. In the case of ATP and CAP, the lower chronic intake risk for CAP (2 mg (kg bw)−1) was mainly due to its much higher ADI than ATP (0.07 mg (kg bw)−1). The residual data proved that the greenehouse-cultured strawberries were safe for consumption after being sprayed with three pesticides (FOR, ATP and CAP). Furthermore, no difference was observed between the RQchronic of pesticides alone and joint application in strawberries gathered from a greenhouse trial. The results revealed that the dietary intake risk of three pesticides in strawberries grown in the greenhouse could be ignored, and the strawberries were safe for Chinese customers even though the tested pesticides were applied in combination and sprayed in the light of the suggested method.
Whole diet risk assessment
Table 6
The Chinese dietary model and risk probability of fluopyram (alone application) in strawberry and the corresponding MRLs registered by various countries.
Food classification
|
Fi
(kg)
|
Reference limits (mg/kg)
(mg/kg
0
|
Sources
|
NEDI
(mg)
|
ADI (mg)
|
Risk probability (%)
|
Rice and its products
|
0.2399
|
|
|
|
ADI×63
|
|
Flour and its products
|
0.1385
|
|
|
|
Other cereals
|
0.0233
|
|
|
|
Tubers
|
0.0495
|
0.03
|
China
|
0.001485
|
Dried beans and their products
|
0.016
|
0.07
|
China
|
0.00112
|
Dark vegetables
|
0.0915
|
2
|
China
|
0.183
|
Light vegetables
|
0.1837
|
1
|
China
|
0.1837
|
Pickles
|
0.0103
|
|
|
|
Fruits
|
0.0457
|
0.145
|
STMR5
|
0.008683
|
Nuts
|
0.0039
|
0.04
|
China
|
0.000156
|
Livestock and poultry
|
0.0795
|
|
|
|
Milk and its products
|
0.0263
|
|
|
|
Egg and its products
|
0.0236
|
|
|
|
Fish and shrimp
|
0.0301
|
|
|
|
Vegetable oil
|
0.0327
|
1
|
China
|
0.0327
|
Animal oil
|
0.0087
|
|
|
|
Sugar, starch
|
0.0044
|
0.04
|
China
|
0.000176
|
Salt
|
0.012
|
|
|
|
Soy sauce
|
0.009
|
0.07
|
China
|
0.00063
|
Total
|
1.0286
|
|
|
0.40961
|
0.63
|
65.02%
|
Note: Fi is the dietary reference intake for a certain kind of food used to plan and assess the nutrient intakes of healthy Chinese people. NEDI is the national estimated daily intake. The average body weight of an adult is 63 kg. STMR5 is the supervised trial median residue of fluopyram in strawberries with a pre-harvest interval. |
According to the existing results of FOR with the highest chronic risk, the whole diet risk of FOR was further assessed. Table S4 illustrates the MRLs of three pesticides for strawberries approved by various countries. The reference MRLs for this paper were chosen based on the following sequence: China comes first, followed by the CAC, the EU, Japan, Korea and America. Table 6 shows the Fi and the Chinese dietary model (Li et al., 2021). The food classifications of FOR registered in China contain tubers, dried beans and their products, dark vegetables, light vegetables, fruits, nuts, vegetable oil, sugar, starch and soy sauce. Strawberries are categorized as fruits. The assessment results showed that the risk of dietary FOR intake (alone and mix application) was less than 100% (Table 6 and Table S5). Furthermore, the population meal statistics shown in this dietary risk evaluation came from 2002. However, the public's daily dietary intake may have altered significantly over the years, and the consequences might be undervalued as a matter of fact.
Acute diet exposure assessment
The WHO (World Health Organization) has ascertained that ARfDs (acute reference doses) for some pesticides, which include CAP, are unnecessary. The ARfDs values of the remaining pesticides, FOR and ATP, were 0.5 and 0.1 mg kg− 1, respectively. Table S6 exhibits the RQa of the pesticides FOR and ATP in six consumption groups with body weights of 13.4–61.3 kg and daily intakes of 339.4-510.2 g/d, respectively (Chu et al., 2020). The values of RQacute ranged from 0.32–1.48% after spraying FOR in different groups. And the RQacute of ATP ranged from 0.77–3.14% in 60–70 year old males and 2–4 year old females, respectively. The acute risks (low to high) of the six groups were in the sequence of 60–70 year old males < 18–30 year old males < 60–70 year old females < 18–30 year old females < 2–4 year old males < 3.0% < 2–4 year old females (ATP used alone). Females were at a slightly higher risk of pesticide exposure from consumed strawberries than males. The RQacute of FOR and ATP were far less than 100%, which is within the scope of safe consumption.