A monitoring survey and health risk assessment for pesticide residues on Codonopsis Radix in China

In recent years, the safety of Codonopsis Radix (CR) has attracted considerable attention. Pesticide residues is an important index to evaluate the safety of CR. The purpose of this study was to monitor pesticide residues in 164 batches of CR in China and assess dietary risk assessment. Firstly, a combined method of QuEChERS-GC–MS/MS and QuEChERS-LC–MS/MS was established for determination of 155 pesticide residues in CR. Second, 155 Pesticide residues in 3 CR cultivars from Gansu, Shanxi, Hubei, Guizhou and Chongqing were determined by this method. Finally, the risk score of pesticide residues in CR was evaluated, and the dietary health risk was evaluated based on the pesticide residues in CR. The results demonstrated that one or more pesticide residues were detected in 39 batches (23.78%) of 164 batches of CR. Of the 155 pesticide residues, 20 were detected. The most frequently detected pesticide residue was dimethomorph with a detection rate of 5.49%. Risk scores showed that 6 pesticides were at higher risk. Risk assessment based on the hazard quotient/hazard index (HQ/HI) approach revealed that exposure to pesticide residues which detected in CR were far below levels that might pose a health risk.

The usage, toxicity, maximum residue limits (MRLs), acceptable daily intake (ADI) value, acute reference dose (ARfD) value, and detection results of 20 kinds of pesticide residues detected in this experiment are shown in Table 1. Retention time, detected ion pairs, linear equation, correlation coefficient, linearity range, LOQ, mean recovery, intra-day RSD and inter-day RSD for pesticide residue detected in CR. a Represents that the pesticide residues were tested by both LC-MS/MS and GC-MS/MS. *Represents quantitative ion pairs.   Among the 20 kinds of pesticide residues detected, dimethomorph had the highest detection rate, with 9 batches of samples detected, and the detection rate was 5.49%. This was followed by pyridine and diphenylamine with a 3.66% detection rate for 6 batches. metalaxyl and carbendazim were detected in 5 batches of CR with a detection rate of 3.05%. Clothianidin, P,P′-DDT were detected in 4 batches of CR with a detection rate of 2.44%. Thiamethoxam, o-phenylphenol and hexachlorobenzene were detected in 3 batches of CR with a detection rate of 1.83%. Zoxamide was detected in 2 batches of CR with a detection rate of 1.22%. Trifluralin, azoxystrobin, acephate, dipterex, carbofuran, terbufos, phosfolan, tebuconazole, and propargite were detected in only 1 batch of CR with a detection rate was 0.61%, as shown in Fig. 1.
Of the 20 pesticide residues detected, p,p′-DDT, carbofuran, terbufos and phosfolan were required to be detected in the Chinese Pharmacopoeia, with maximum residues of 0.10, 0.05, 0.02 and 0.03 mg/kg, respectively. The content of these 4 pesticide residues detected in this study was lower than the maximum limits, in line with the requirements of the Chinese Pharmacopoeia. Carbendazim, carbofuran and diphenylamine are the pesticide residues with the maximum limits specified in National Food Safety Standard, and the maximum limits are 0.05, 0.05 and 0.01 mg/kg, respectively. The diphenylamine content in 6 batches of samples detected in this project all exceeded the standard. Clothianidin, p,p′-DDT, trifluralin, pyridaben, and propargite are the pesticide residues that are not allowed to be detected in the "Pollution-free standard for ginseng". In this study, clothianidin was detected in 4 batches of samples, p,p′-DDT in 4 batches of samples, and trifluralin in 1 batch of the sample. 6 batches of samples were detected with pyridaben, 1 batch was detected with propargite, and 16 batches of samples detected the above five kinds of undetectable pesticide residues. The maximum limits of pesticide residues for metalaxyl, thiamethoxam, carbendazim, azoxystrobin, hexachlorobenzene, tebuconazole, and dimethomorph are specified in the "Pollution-free standard for ginseng". The maximum limits were 0.05, 0.02, 0.10, 0.50, 0.05, 0.50 and 0.05 mg/kg, respectively. The content of thiamethoxam in 1 batch of the sample exceeded the standard, and the rest met the limit requirements. In conclusion, in 164 batches of CR, pesticide residues are in line with the standard of the Chinese Pharmacopoeia, but part of the samples are not in conformity with the National Food Safety Standard and corporate standards "Pollution-free standard for ginseng". Therefore, we suggest that the control of pesticide residues in CR should be strengthened when it is used as food. According to the catalog and limit of pesticide residues in the three standards, clothianidin, p,p'-DDT, trifluralin, pyridaben, and diphenylamine were not allowed to be detected. 16 batches of 164 samples were considered to be unqualified, and the unqualified rate was 9.76%. Metalaxyl, thiamethoxam, p,p′-DDT, carbendazim, carbofuran, azoxystrobin, diphenylamine, hexachlorobenzene, terbufos, phosfolan, tebuconazole and dimethomorph are the detectable pesticide residues with the specified maximum limits, in this study, thiamethoxam in 1 batch and diphenylamine in 6 batches exceeded the standard, and the unqualified rate was 4.27%, the total unqualified rate was 14.03%.
Finally, the number of pesticide residues in the 3 varieties of CR was statistically analyzed by t-test. The results showed that there was no significant difference in the number of pesticide residues between C. pilosula and C. pilosula var. modesta, but there was significant difference between C. pilosula (C. pilosula var. modesta) and C. tangshen (P < 0.05).
Risk assessment. The risk score of each pesticide residue was calculated according to the criteria in Table 3.
Specific as follows: according to the "Chinese Pharmacopoeia", the daily dosage of CR is 9-30 g. Based on the maximum amount, it can be calculated that the proportion of CR in the diet of Chinese residents is less than 2.5%. Hence, the dietary proportion score (C) of CR is determined to be 0. According to the national standard for the rational use of pesticides, each pesticide can only be used up to 3 times in the CR. CR is a perennial herb, and the root development period is more than 180 days. Therefore, the use frequency of each pesticide calculated using formula (1) is less than 2.5%, and the use frequency score (D) of the pesticide is determined to be 0. Although there are differences in the consumption of CR among different groups in China, there is no relevant data to determine the existence of high exposure groups. Therefore, the score (E) of high exposure groups is determined to be 3. According to the content of each detected pesticide residue, the residue level (F) of 20 kinds  www.nature.com/scientificreports/ tolerance range of humans (Fig. 4a). Furthermore, we calculated the chronic cumulative risk of several kinds of pesticide residues in the samples with the largest number of detected pesticide residues. Three batches of samples, S142, S145 and S163, were found to have the most pesticide residues, and four kinds of pesticide residues were detected respectively. Pyridaben, o-phenylphenol, diphenylamine and hexachlorobenzene were detected in S142, o-phenylphenol, diphenylamine, hexachlorobenzene and p,p′-DDT were detected in S145, and terbufos, phosfolan, tebuconazole and dimethomorph were detected in S163. Among them, pyridaben, diphenylamine, and P,P′-DDT were detected as unqualified samples (S142, S145). Chronic cumulative risk index (HIc) was calculated for the three samples as medicine and food respectively. The results showed that the HIc of S142 as medicine and food were 0.0052 and 0.0008 respectively. The HIc of S145 as medicine and food were 0.0056 and 0.0008 respectively. The HIc of S163 as medicine and food were 0.0310 and 0.0046 respectively. Based on the results, the chronic cumulative risk of CR was within the safe range even if the samples with the most pesticide residues were detected. Finally, we assumed that each batch of CR samples could detect 20 pesticide residues detected in this study, and calculated the HIc values of CR as medicine and food. The results indicated that the HIc was 0.15 when CR was used as medicine and 0.02 when CR was used as food, indicating that the chronic cumulative risk of CR was within the safety range even if the pesticide residues were detected in this study were detected in the same batch of CR. Among the 20 pesticide residues detected, 13 had ARfD values (metalaxyl, clothianidin, thiamethoxam, carbendazim, carbofuran, azoxystrobin, acephate, dipterex, terbufos, phosfolan, tebuconazole, propargite and dimethomorph). HQa values were calculated according to the dosage of CR as medicine and food respectively. The results showed that HQa values of all 13 pesticide residues were < 1, indicating that chronic risks caused by 13 pesticide residues alone were within the tolerance range of humans (Fig. 4b). Further, we calculated the    www.nature.com/scientificreports/ acute cumulative risk of several kinds of pesticide residues in the samples with the largest number of pesticide residues detected (S142, S145 and S163, with 3.2.1 for details). The HIc of S142 as medicine and food is 0.0004 and 0.0000 respectively. The HIc of S145 as medicine and food were 0.0000 and 0.0000 respectively. The HIc of S163 as medicine and food were 0.0148 and 0.0014 respectively. The results indicated that the acute cumulative risk of CR was within the safe range even if the samples with the most pesticide residues were detected. Finally, we assumed that each batch of CR samples could detect 20 pesticide residues detected in this study, and calculated the HIa values of CR as medicine and food. The results showed that when CR was used as medicine or food, the HIa of 20 pesticides were 0.14 and 0.04, which were all less than 1 and indicated that acute cumulative risk should be ignored in the short-term.

Discussion
In this study, GC-MS/MS and LC-MS/MS methods were used to quantitatively analyze 155 pesticide residues in 164 batches of CR from 3 varieties and 5 major producing areas in China, and a total of 20 pesticide residues were detected. According to the three criteria referenced in this experiment, the unqualified rate of 164 batches of samples was 14.03%. The detection rate of pesticide residues in 3 varieties of CR was compared and analyzed. According to the results, the detection rate of pesticide residues in C. pilosula var. modesta was lower, but that in C. tangshen was higher. The producing environment of C. pilosula var. modesta and C. tangshen were investigated respectively. The main producing area of C. pilosula var. modesta, Wenxian, belongs to the transition zone from subtropical to warm temperate. C. pilosula var. modesta is most likely to grow in the middle of high mountains, where the climate is mild and cool, and the invasion of pests and grasses is less, the frequency of use of pesticides may be relatively less. C tangshen is mainly distributed in subtropical areas, where the air is humid and the precipitation is abundant, so traditional Chinese medicine is easy to be attacked by bacteria, fungi and algae, so the use of fungicides is relatively frequent.
In this study, 20 kinds of pesticide residues detected in CR were risk-scored by referring to the veterinary drug residue risk ranking standard of the British Veterinary Drug Residues Committee. The results showed that there were 6 pesticides with higher risk, namely: diphenylamine, terbution, phosfolan, propargite, carbofuran, and trichlorphon. Among them, trichlorphon is a carcinogen announced by the World Health Organization's International Agency for Research on Cancer. Studies have shown that trichlorfon causes oxidative stress, neurotoxicity, and immune responses in carp 24. Therefore, we believe that the pesticide residues in CR should not be ignored.
In addition, the study used the Health Risk Assessment Model (2000) developed by the U.S. Environmental Protection Agency to evaluate the chronic and acute health risks of CR as a drug and food. The results demonstrated that when CR was used as medicine or food, the HQc and HQa of 20 pesticide residues were less than 1, indicating that the risks of 20 pesticide residues were acceptable. At the same time, we calculated the cumulative risk index HI of CR as medicine and food. The results exhibited that HIc and HIa values of CR were less than 1 when they were used as medicine or food, indicating that the cumulative risk of CR was within the safety range even if all pesticide residues detected in this study could be detected by the same batch of CR. This experiment proves that when CR is used as a medicine, it will not cause chronic or acute health hazards to the human body if it is within the dose range (9-30 g) prescribed by the Chinese Pharmacopoeia, and it will not cause chronic or acute health hazards to the human body if it is taken for 15 years for adults and 60 days a year. When CR is utilized as food, according to the questionnaire, daily consumption is 20 g 25 . It is employed for 50 years in a person's life and takes 260 days a year. It will not lead to chronic or acute health hazards to humans.
An analysis of the uncertainty in exposure assessment is necessary for this experiment to properly interpret the assessment results. First of all, the consumption of CR as a drug comes from the dosage recommended by the Chinese Pharmacopoeia, not based on the actual dosage, which will lead to higher or lower risk estimates. Therefore, when accurate consumption data is available, a more precise risk assessment should be carried out. Second, The HI values in this study are additive and hypothetical, so the results of the cumulative health risks will be revised as further work on the mechanisms of interaction of these pesticide residues is clarified. Third, the HI value in this study only considered the risk accumulation of multiple pesticide residues in a single sample. However, when CR is used as a medicine, it is often mixed and decocted with other traditional Chinese medicines, which means that the cumulative HI value of pesticide residues in various traditional Chinese medicines may exceed 1.

Conclusions
China is an important CR producer and commercial region. Therefore, it is necessary to know the actual situation of pesticide residues for CR at the national or regional level and its impact on health of the consumers. In this study, two methods (GC-MS/MS and LC-MS/MS) were established for the determination of 155 pesticide residues in CR. Based on the results, the new methods are suitable for the determination of pesticide residues in the CR. In this study, 40 pesticide residues were determined by two methods, and the results showed that LC-MS/MS had a lower detection limit. Among the pesticide residues detected, 6 repeated pesticide residues were detected by LC-MS/MS method. The monitoring results indicated that 164 CR samples were collected from the three varieties containing one or multiple pesticide residues in 23.78% of samples. Of the monitored samples, 14.03% were still substandard. The results of dietary risk assessment showed that when CR was used as a medicine or food, the HQc, HQa, HIc, and HIa values of the 20 pesticide residues detected were less than 1, indicating that the health risks caused by the detected 20 pesticide residues were acceptable. The 20 pesticides detected were ranked for their risk of ingestion according to a pre-set ranking matrix. The results reflected that 6 pesticides, diphenylamine, terbufos, thiocyclophosphine, propargite, carbofuran, and trichlorfon, had higher risks. Hence, we recommend: (1) The government should strengthen the management of banned and restricted pesticides, and speed up the process of delisting highly toxic pesticides. (2) (3) It is recommended that local farmers learn more about physical and biological control methods and avoid the extensive use of pesticides, especially highly toxic pesticides.
There are 5 main planting areas of CR, including Gansu province, Shanxi province, Hubei province, Guizhou province and Chongqing municipality. Gansu province mainly contains two varieties, C. pilosula and C. pilosula var. modesta. The Source of CR planted in Shanxi province was C. pilosula. The source of CR cultivated in Hubei province, Guizhou province, and Chongqing municipality was C. tangshen. After nearly 3 years of field research, 164 batches of samples from core producing areas of the above five provinces or municipalities were collected in this experiment. The CR samples collected in this study can represent the quality status of CR in various producing areas. The area of CR in Gansu province accounts for more than 80% of the whole of China, therefore, there are many CR samples collected in Gansu. Information of 164 batches of samples is shown in Table 4, and the details are given in Table S3. Samples of CR were gathered in the field between 2018 and 2020, and at least 2 kg were collected in each batch. After the fresh samples were washed with water, they were processed following the processing methods in the Chinese Pharmacopoeia, then the medicinal materials of CR (moisture ≤ 16.0%) were obtained. CR was stored in a sealed bag in a refrigerator at -20℃ until analysis, and samples were stored in the CR Research Institute of Lanzhou University. Preparation of matrix-matched mixed standard working solution: Firstly, a blank matrix solution was prepared by taking a sample of CR without any pesticide residues. Then, measure 1.0 mL of pesticide mixed standard solution, dilute it to 10 mL with CR blank matrix solution, and prepare a series of matrix-matched mixed standard working solutions.
Sample preparation. Pre-treatment methods are extremely important for pesticide residue monitoring.
The QuEChERS method was introduced in 2003 as a pre-treatment for pesticide monitoring and has been widely used by several governments and scientific standards organizations 26 . At present, QuEChERS method has been broadly applied in analysis of pesticide multi-residues in fruits and vegetables owing to its simplicity, low cost, speed and broad applicability to a wide range of analytes 27,28 . In this study, QuECHERS method was selected for the pretreatment of CR samples. The sample pretreatment of the GC-MS/MS and LC-MS/MS analysis procedures includes the following steps: (1) a portion of 2.0 g of pulverized CR sample was added into a 50 mL centrifuge tube. (2) 10 mL of ultrapure water was added into the tube and the tube was mixed evenly and soaked for 30 min. (3) 10 mL of acetonitrile and two ceramic homogenizers were added, and then the tube www.nature.com/scientificreports/ was shaken vigorously for 6 min, followed by adding QuEChERS extraction package and kept shaking for the same minutes. (4) the tube was centrifuged at 3900 rpm for 10 min and the supernatant was transferred into a QuEChERS purification tube containing decontaminant. (5) the tube was vortexes for 3 min and centrifuged at 3900 rpm for 10 min, and take the supernatant for use. (6) 2 mL of the supernatant was dried with nitrogen (40 °C) and then dissolved in 1 mL of n-hexane for GC-MS/MS analysis. (7) 2 mL of the supernatant was dried with nitrogen (40 °C) and then dissolved in 1 mL of 60% acetonitrile for LC-MS/MS analysis. Before the analysis, all of them were to be filtered through a nylon filter (0.22 μm).
GC-MS/MS conditions. Gas chromatography separation was performed on an HP-5MS UI gas chromatography column (30 m × 0.25 mm × 0.25 μm). The gradient heating program was performed as follows: the initial temperature kept at 60 °C for 1 min; raising to 170 °C at 40 °C/min; raising to 310 °C at 10 °C/min and for 3 min. The inlet temperature was set at 280 °C and the carrier gas was high purity nitrogen at a flow rate of 1.2 mL/min. An aliquot of 1 mL of sample extract or standards was injected into the column without shunting.
The following general MS parameters were employed: EI source. The source temperature and gas chromatography-tandem mass spectrometry transmission line temperatures were 250 °C and 280 °C, respectively. The electron energy was 70 eV and the multiple reaction monitoring (MRM) scanning mode was adopted. Agilent Mass Hunter is a working software that was used for data processing and more details were shown in Tables S1 and S2. Mass analysis was performed using an ESI source. The nozzle voltage, dry temperature, dry gas flow rate and capillary voltage were 30 Psi, 500 °C, 900 L/h and 3800 V respectively.

LC-MS
Risk scoring of pesticide residues. The matrix ranking scheme was developed by the Veterinary Residues Committee of the UK 29,30 . Use toxicity index instead of drug property index. Five other indicators, pesticide toxicity effect (ADI value), dietary ratio (percentage of CR in the total residents' diet, unit: %), frequency of use, high exposure population, and pesticide residue level, adopt the original assignment standards 31 . The assignment criteria for each indicator are shown in Table 3. Toxicity adopts acute oral toxicity and is divided into four categories: Extremely high, high, moderate, and low toxicity according to the oral median lethal dose (LD 50 ). The LD 50 of each pesticide is obtained from the China Pesticide Information Network 32 . The ADI value is obtained from the National Standard Network. The frequency of pesticide use (FOD) was calculated using formula (1). The residue risk score (S) for each pesticide in the sample is calculated using formula (2). The residue risk score of each pesticide is calculated as the average of the pesticide residue risk score in all samples, the higher the value, the greater the residue risk.
In formula (1, 2), P represents the growth days of CR (the time from transplanting to maturity, unit: d), T represents the number of times the pesticide was used during the growth of CR, A is the toxicity score, B is the score of toxic potency, C is the score of the CR diet proportion in total, D is the score of the frequency of dosing with a particular pesticide, E is the score of the evidence of high exposure groups, and F is the score of the detectable pesticide residue level.

Health risk assessment. According to the health risk assessment model established by US Environmental
Protection Agency (2000), the chronic and acute risks caused by pesticide residues in CR were evaluated. The Entropy of chronic hazard (HQc) and the Entropy of acute Hazard (HQa) assess chronic and acute health risks, respectively. When HQ < 1 is considered an acceptable risk, it does not pose a health threat in the long or short term. The higher the HQc or HQa value, the greater the health risk 33 . Further, the cumulative risk of detected pesticides was assessed by HI method. HI is the sum of the HQ of each pesticide 34 . Where HI < 1 is considered an acceptable risk and does not pose a health threat, and HI > 1 is considered an unacceptable risk.
Chronic risk assessment. HQc was calculated using Eqs. (3) and (4). In the equation, EDI represents the daily intake of pesticide residues (μg/kg bw) in CR, HQc represents the entropy of chronic hazard.
EF is equal to the number of exposure to toxic substances per year (d). Ed is lifetime exposure time, i.e. lifetime exposure years (y). IR is the daily intake of CR (g). C 1 is the concentration of single pesticide residue detected in CR (mg/kg), and the average concentration of each pesticide residue is used in this equation. AT refers (1) FOD = T/P × 100  35 . Thinking that CR is a medicinal material of the same origin as both medicine and food, it can be used as both medicine and food. Therefore, the risk assessment is based on the evaluation of the two situations. Based on the results of the questionnaire, EF = 60 d, Ed = 15 y, and IR 19.5 g were calculated according to the average daily dosage of CR in the Chinese Pharmacopoeia (2020 edition) when CR is used as medicine. When CR is utilized as food, EF = 260 d, Ed = 50 y, IR is 20 g according to the questionnaire 25 . SF stands for the safety factor. When CR was used as a medicine, according to the safety factors stipulated in Sect. 4 of the Chinese Pharmacopoeia (2020 edition) "Guidelines for establishing limits of harmful residues of traditional Chinese medicine (9302)", SF means that the daily pesticide residues ingested from traditional Chinese medicine and its products should not exceed 1% of the total daily exposure (including food and drinking water), that is, SF = 100. When CR is used as food, SF = 1. The ADI represents the oral reference dose for pesticide residues and is the dose at which an individual can be continuously exposed to this level for a long period without harm 36 . Chronic exposure risk assessment could not be performed for hexachlorobenzene because there was no available data on AID value. The ADI values of the other 19 pesticide residues detected in this experiment are shown in Table 2.
Acute risk assessment. HQa was calculated using formulas (5) and (6). In the equation, EDI represents the daily intake of pesticide residues (μg/kg bw) in CR, HQa represents the entropy of acute hazard. C 2 is the detected concentration of a single pesticide residue in CR (mg/kg), where the maximum concentration of each pesticide residue is taken. When CR was used as a medicine, IR is 30 g according to the maximum daily dosage of CR in the Chinese Pharmacopoeia (2020 edition). When CR was used as food, IR took 20 g according to the questionnaire results. The meanings and values of other symbols are the same as 5.8.1.
ARfD represents the acute reference dose of pesticide residues in medicinal materials 35 . Acute exposure risk assessment could not be performed for p,p'-DDT, trifluralin, pyridaben, o-phenylphenol, diphenylamine, hexachlorobenzene, and zoxamide because the ARfD values had been deemed unnecessary for these compounds or because there were no available data on ARfD. The ARfD values of the other 13 pesticide residues detected in this study are given in Table 2.
Cumulative risk assessment. Exposure to two or more chemicals may lead to additives or other interactions, and additive risk usually requires that all components act according to the same mechanism. However, for the quantitative risk assessment of various chemicals, a risk additional hypothesis must be adopted. Therefore, the cumulative health risk caused by pesticide residues in CR was considered to evaluate the total hazard entropy of risks to health caused by multiple pesticide residues. The cumulative health risk represented by HI is calculated using Formula (7). HI < 1 is considered acceptable cumulative risk and does not pose a health threat, while HI > 1 is considered to pose an unacceptable risk 34 .
Sample collection. The authors declare that they have a license to collect three varieties of Codonopsis Radix (Codonopsis pilosula (Franch.) Nannf, Codonopsis pilosula Nannf. var. modesta (Nannf.) L. T. Shen, Codonopsis tangshen Oliv). The authors declare that they comply with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.

Data availability
All data generated or analysed during this study are included in this published article and its supplementary information files.