| Since its application in agricultural production,chemical pesticides have brought great benefits to humans while also bringing negative effects.Long-term low-dose exposure and intake of pesticides in the human population can accumulate in the body and cause chronic damage.Epidemiological studies have shown that the occurrence of infertility in women is associated with pesticide exposure.However,there are few reports of systematic multi-pesticide testing for infertile populations in China,and the types of pesticides studied have mostly focused on organochlorine pesticides that have been banned.Therefore,in this paper,we analyzed the burden levels of pesticides in follicular fluid and serum samples from infertile women,and studied the ovarian toxicity and tissue distribution of rats in which the pesticide difenoconazole was detected,to serve as a guide for evaluating the effects of pesticides exposure on human health.The key research findings are listed below:1.Analytical methods for 123 pesticides in human follicular fluid and serum samples were established.In this study,123 pesticides were identified,and the samples were determined by GC-MS/MS with the matrix solution calibrated for matrix effects.The pretreatment methods of follicular fluid and serum samples were investigated,and eventually the pretreatment technique of "direct extraction+GCB/NH2 solid phase extraction column" was decided.The recoveries of most of pesticides for follicular fluid samples at three spiked concentrations of 2,5 and 10 ng/mL and for serum samples at three spiked concentrations of 10,20 and 50 ng/mL ranged from 60%to 120%with RSD values less than 20%.The calibration curves’correlation coefficients(r)were all more than 0.99.Pesticides in follicular fluid samples had limits of quantitation(LOQs)of 0.1-2 ng/mL and limits of detection(LODs)of 0.05-0.5 ng/mL.Pesticides in serum samples had limits of quantitation(LOQs)of 0.510 ng/mL and limits of detection(LODs)of 0.1-2 ng/mL.The technique worked well and was dependable.It can be used to analyze 123 pesticides in serum and follicular fluid.2.The detection of 123 pesticides in 63 pairs of human follicular fluid and serum samples was completed.The results showed that 24 pesticides were detected in serum and follicular fluid samples,and 10 prohibited pesticides and their metabolites were detected in serum and follicular fluid samples.The pesticides with more than 50%detection rate in the serum samples were p,p’-DDE(ND-6.14 ng/mL),acetochlor(ND1.81 ng/mL),fipronil sulfone(ND-2.40 ng/mL),chlorpyrifos(ND-1.65 ng/mL),βHCH(ND-0.89 ng/mL)and quintozene(ND-3.13 ng/mL);the pesticides with more than 50%detection rate in follicular fluid samples were fipronil sulfone(ND-1.00 ng/mL),p,p’-DDE(ND-2.64 ng/mL)and quintozene(ND-0.53 ng/mL),respectively.The concentration of pesticides in serum was roughly three times higher than that in follicular fluid,with median amounts of 2.84 and 0.74 ng/mL in serum and in follicular fluid samples,respectively,which may suggest that serum samples are a more suitable assay matrix for understanding pesticide burden levels in the population.DDTs and HCHs were significantly lower compared to previous data in China.The results of this study suggest that it is important to pay attention not only to the human health risks of already banned persistent organochlorine pesticides,but also to the human health risks of in-use pesticides such as chlorpyrifos,chlorpyrifos,acetochlor and difenoconazole.3.A tissue distribution study of subacute oral exposure to difenoconazole in female SD rats was completed.In this study,the LC-MS/MS method for the determination of difenoconazole in rat organs was investigated.The homogenization method of organ tissues such as liver was examined,and the purification method and extraction solvent were examined with liver tissues as a representative methodological examination.After verifying the sensitivity,accuracy,precision,range and linearity of the method,it was determined that "acetonitrile direct extraction+GCB/NH2 solid phase extraction column cleanup" as the pretreatment method could meet the requirements of the analytical determination.The distribution of difenoconazole in rat organ tissues under subacute oral exposure was investigated.The results showed that the distribution of difenoconazole was mainly in the ovaries and uterus of female reproductive organs and in the liver and kidney,suggesting that these organs are the main target organs for short-term repeated exposure to difenoconazole.The results provide toxicokinetic support in the elucidation of the toxicity mechanism of difenoconazole.4.The ovarian toxicity of subacute oral exposure to difenoconazole in rats was examined,while a preliminary assessment of the ovarian safety of oral exposure to difenoconazole in humans was conducted.The ovarian toxicity of the priority pesticide,difenoconazole,was investigated by considering the detection of pesticides in human follicular fluid and serum samples and taking into account the exceedance of pesticide standards during practical application.The results showed that the exposure dose of 200 mg/(kg bw·d)increased the progesterone level and interfered with the development of primordial follicles to growing follicles in female rats;at 100 mg/(kg bw·d),there was no significant ovarian toxicity in rats.Based on the results of the present study,the toxicological safety dose of difenoconazole to the ovaries was estimated to be 0.33 mg/(kg bw·d)for long-term repeated oral exposure in humans,which was much higher than the ADI value(0.01 mg/(kg bw·d)).Therefore,it was tentatively determined that repeated oral exposure to difenoconazole is not expected to affect human ovaries under the premise of safe use of difenoconazole.In this thesis,we firstly screened 123 pesticide residues in follicular fluid and serum of infertile women and established the GC-MS/MS analytical method.24 pesticides and their metabolites were detected,among which 10 prohibited pesticides and metabolites were detected.The results showed that the pesticides can be distributed in the ovaries,uterus,liver and kidney,with higher levels in the ovaries and uterus.A preliminary risk assessment based on the results of toxicological experiments concluded that oral dietary exposure to difenoconazole in humans would not significantly affect the ovaries. |
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