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العنوان
RISK ASSESSMENT OF CERTAIN PESTICIDES
USED ON GRAPES UNDER THE EGYPTIAN
ENVIRONMENTAL CONDITIONS /
المؤلف
SAYED, SHAIMAA MOHAMMED.
هيئة الاعداد
باحث / SHAIMAA MOHAMMED SAYED
مشرف / Mohamed Ibrahim Abdel-Megeed
مشرف / Khaled Mohamed Allam
مناقش / Moustafa Abdel-Latif Abbas
تاريخ النشر
2019.
عدد الصفحات
143 P. :
اللغة
الإنجليزية
الدرجة
ماجستير
التخصص
علوم النبات
تاريخ الإجازة
1/1/2019
مكان الإجازة
جامعة عين شمس - كلية الزراعة - قسم وقاية النبات
الفهرس
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Abstract

The aim of this study is estimate of the environmental risks (risk assessment) for imidacloprid insecticide and myclobutanil fungicide on table grape for local use or export, also determination of preharvest intervals (PHI) in order to maintain human health and to avoid damage to the various environmental components biotic and abiotic. In addition to estimating the optional coefficient of the pesticide between the target pests (aphids) and beneficial insects (Apis melifera and Trichogramma).
1. Residues of imidacloprid and myclobutanil in/on grape and soil under field conditions:
Residues and degradation percentages of insecticide imidacloprid in/on grape (leaves and fruits) were investigated. The initial residue deposits, which remained on grape leaves and fruits two hours after treatment were 35.66 and 7.04 mg kg-1, respectively. These amounts decreased to 28.16 and 5.92 mg kg-1 one day after the application indicating degradation percentages of 21.04 and 15.90%, respectively. Residues of imidacloprid in/on grape (leaves and fruits) were gradually decreased to 19.52, 11.84, 6.40, 5.12, 2.88 and ND mg kg-1 corresponding degradation percentages of 45.26, 66.80, 82.05, 85.64, 91.92 and ≈ 100% (leaves) and 4.32, 2.44, 1.64, 1.28, 0.32 and ND mg kg-1corssponding degradation percentages of 38.64, 65.34, 76.71, 81.82, 95.45and ≈ 100% (fruits) after 3, 7, 10, 13, 16 and 21 days of application, respectively. Examination of the considered criteria represented by the established regression lines, i.e. slope, degradation constant (K) and RL50, RL90 proved differences in persistence behavior of the targeted pesticides. The imidacloprid degradation constant (K) values are 0.152 and 0.169 in/on grape (leaves and fruits), respectively. As for RL50, RL90 and PHIs values, imidacloprid showed 4.12, 13.42 and 21.95 days and 5.13, 13.41 and 11.96 days in/on grape leaves and fruits, respectively. These results indicated that the same degradation behavior of imidacloprid insecticide in/on grape leaves and fruits. Data also indicated that despite the low residue half-lives for imidacloprid in grape fruits (5.13 days), it can be consumed safely after 12 days of treatment, concerning health aspects; the maximum residue limit (MRL) of imidacloprid residues in/on grape according to (Codex Aimentarias Commission 2013) was 1 mg kg-1.
Residues and degradation percentages of fungicide myclobutanil in/on grape (leaves and fruits) were investigated. The initial residue deposits, which determined on grape leaves and fruits two hours after treatment were 66.32 and 49.40 mg kg-1, respectively. These amounts decreased to 54.14 and 35.25 mg kg-1 one day recording degradation percentages of 18.36 and 28.64%, respectively. Myclobutanil residues in/on grape (leaves and fruits) were gradually decreased to reach 40.15, 28.65, 12.09, 6.26, 0.85 and 0.11 mg kg-1 corresponding degradation percentages of 39.46, 56.80, 81.77, 90.56, 98.71 and 99.83% (leaves) and 15.86, 11.73, 3.95, 1.73, 0.63 and ND mg kg-1 corresponding degradation percentages of 67.89, 76.25, 92.00, 96.49, 98.72 and ≈ 100% (fruits) after 3, 7, 10, 13, 16 and 21 days of application, respectively. Examination of the considered criteria represented by the established regression lines, i.e. slope, degradation constant (K) and RL50, RL90 proved significant differences in persistence behavior of the targeted pesticides. The myclobutanil degradation constant (K) values are 0.289 and 0.258 in/on grape (leaves and fruits), respectively. As for RL50, RL90 and PHIs values, myclobutanil showed 4.71, 9.38 and 16.31days and 1.97, 9.14 and 14.90 days in/on grape leaves and fruits, respectively. These results indicated that the myclobutanil fungicide was degraded higher in grape fruits than that leaves. Data in the same table indicated that despite the low residue half-lives for myclobutanil in grape fruits (1.97 days) it can be consumed safely after 14.90 days of treatment, concerning health aspects and the maximum residue limit (MRL) of myclobutanil residues in/on grape according to (Codex Aimentarias Commission 2013) was 1 mg kg-1.
The above-mentioned results indicate the important role of pesticide type in determining initial deposits as well as residues pattern of the imidacloprid and myclobutanil in/on grape fruits. The fungicide myclobutanil showed the highest initial deposit (66.32 and 49.40 mg kg-1) than insecticide imidacloprid (35.66 and 7.04 mg kg-1) in/on grape (leaves and fruits), respectively. An opposite trend was obtained with residues degradation pattern, as to indicate from RL50 and RL90 values. Myclobutanil showed rapid decline of residues while imidacloprid was the least degradable compound. The RL50 and RL90 values reached 4.71 and 9.38 days (leaves) and 1.97 and 9.14 days (fruits)/myclobutanil and 4.12 and 13.42 days (leaves) and 5.13 and 13.41 days (fruits)/imidacloprid, respectively. It is interesting to notice that, degradation percentages in residues were higher in grape fruits as a comparison with leaves; especially with myclobutanil compared with imidacloprid residues.
Residues and disappearance percentages of insecticide imidacloprid and fungicide myclobutanil on surrounding soil under canopy after successive field applications were investigated. The initial residue deposits, which determined on soil two hours after treatment were 0.98 and 4.60 mg kg-1, respectively. These amounts decreased to 0.72 and 4.32 mg kg-1 one day recording disappearance percentages of 26.51 and 6.09% for the same targeted pesticides, respectively. The longer the time after application the higher the disappearance and vice versa. A steady increase in disappearance percentages occurred by the progression of time until the third week. Pesticide residues were gradually decreased to reach 0.65, 0.56, 0.52, 0.49, 0.41 and 0.30 mg kg-1 corssponding disappearance percentages of 33.47, 42.37, 46.98, 50.87, 58.03 and 69.50% (Imidacloprid) and 3.81, 3.52, 3.31, 3.00, 2.01and 1.78 mg kg-1 corresponding disappearance percentages of 17.17, 23.48, 28.04, 34.78, 56.30 and 61.30% (Myclobutanil) at 3, 7, 10, 13, 16 and 21 days, respectively. Examination of the considered criteria represented by the established regression lines, i.e. slope, disappearance constant (K) and RL50 proved differences in persistence behavior of the imidacloprid and myclobutanil. The disappearance constant (K) values are 0.047 and 0.044, respectively. Results clearly indicate faster disappearance of both targeted pesticides in soil. In this respect, imidacloprid was rapidly dissipated than myclobutanil, showing the RL50 of 11.56 and 15.74 days, respectively. In addition, the relative disappearance of imidacloprid is 1.36 times than myclobutanil.
2. Cytotoxicity of imidacloprid and myclobutanil pesticides on three cancer cell lines:
2.1. Cytotoxicity of the two tested pesticides on cancer cell lines.
Imidacloprid and myclobutanil pesticides were tested for determining the cellular toxicity on three cancer cell lines; HepG-2 (Human Liver Carcinoma), MCF-7 (Human Breast Adenocarcinoma) and PC3 (Prostatic Small Cell Carcinoma) in comparison to doxorubicin as a positive control, by using Neutral Red incorporation assay (NRI Assay). Three cell lines: HepG-2, MCF-7 and PC3were exposed to six concentrations of tested pesticides ranged from 0.5-80 μg ml-1 and incubated for 24 hours.
The results displayed a marked decrease in cell viability of all the three cancer cell lines; HepG-2, MCF-7 and PC3 in a concentration-dependent manner after treatment with imidacloprid pesticide.
The obtained results showed that there was different response with different cell lines where, cell line PC3 found to be more affected and sensitive at low concentrations of IMI (0.5, 5 μg ml-1) in comparison with HepG-2, MCF-7 cell lines. HepG-2 cell line exhibited the highest affected on cell viability at concentrations 15 μg ml-1, 25 μg ml-1 with cell viability 72% and 69.1%, respectively. While, HepG-2, PC3 and MCF-7 exhibited the most affected on cell viability at concentrations 25, 50, 80 μg ml-1, respectively. Maximum reduction in cell viability at 50 and 80 μg ml-1 were found to be 55.2 % and 39.7% for PC3 and MCF-7 cells, respectively.
The NRI50 values for imidacloprid were 110.5, 67.7 and 67.6 μg ml-1 for HepG-2, MCF-7 and PC3 cancer cell lines respectively.
The lowest concentration of myclobutanil pesticide tested (0.5 μg ml-1) was toxic to all tested cell lines and toxicity increased as the concentration of myclobutanil was progressively increased. In cell lines treated with myclobutanil pesticide, the PC3 cell was found to be the highest toxicity and has the lowest viability than HepG-2 and MCF-7 cells at lower concentrations (0.5, 5,15 and 25 μg ml-1) while HepG-2 cells showed the lowest cell viability at concentrations 50, 80 μg ml-1.
The NRI50 value, the concentration of myclobutanil resulting in 50% inhibition of cell viability after 24 h exposure to myclobutanil was calculated from concentration-response curves. The NRI50 values were 38.12, 41 and 27.5 μg ml-1 for HepG-2, MCF-7 and PC3 cancer cell lines, respectively.
Depicts the cytotoxic curves from NRI assay showing the survival of HepG-2, MCF-7 and PC3 treated with imidacloprid and myclobutanil pesticides.Comparing the cytotoxic efficacy of imidacloprid and myclobutanil pesticides in different cell lines.
In liver cancer cell line, the higher cytotoxic effect was observed with myclobutanil at concentrations (0.5, 5, 15 μg ml-1), conversely in higher concentrations, imidacloprid at 25, 50 and 80 μg ml-1 showed the highest cytotoxic effect with cell viability percentage 58.8, 26.47 and 0.0% respectively.
Comparing the cytotoxicity of imidacloprid and myclobutanil pesticides in breast cancer cell the result showed that the highest cytotoxicity was observed with myclobutanil pesticide in all concentrations.
The same result was found in Prostatic Small Cell Carcinoma, myclobutanil pesticide was found to be more toxic than imidacloprid.
The results clarify the concentration-dependent inhibitory effect of cell viability and induction of cytotoxicity. Maximum cytotoxic effects were observed at 80 ppm with complete reduction of cell count.
The NRI50 values found for imidacloprid were 110.5, 67.7 and 67.6 μg ml-1 for HepG-2, MCF-7 and PC3 cancer cell lines, respectively. The NRI50 values found for myclobutanil were 38.12, 41 and 27.5 μg ml-1 for HepG-2, MCF-7 and PC3 cancer cell lines, respectively.
As a general trend, the cell line PC3 inhibition was highly observed in myclobutanil with NRI50 value 27.5 μg ml-1 compared to IMI with NRI50 value was 67.6 μg ml-1.
PC3 cell line was the most sensitive cell line, the NRI50 being on average, 27.5 μg ml-1, 38.12 μg ml-1 for PC3 and HepG-2, respectively. The least sensitive with NRI50 value 41 μg ml-1 for MCF-7. Myclobutanil pesticide induced the highest cytotoxicity because it gave the lowest NRI50 value in all tested cell lines than imidacloprid and consider more toxic. Data showed that myclobutanil pesticide was the most toxic in all cell types.
Also, PC3 cell line exhibited higher sensitivity toward the tested pesticides after 48 hrs. In addition, it was observed that the responses of the targeted cell lines (MCF-7 and PC3) were equals toward imidacloprid pesticide treatment with midpoint cytotoxicity value 67.7 and 67.6 μg ml-1 respectively.
The utilized cell lines were suitable as a biological target to evaluate the cytotoxicity of toxic agents. The differences between the determined midpoint cytotoxicity values may be due to the effects of the corresponding metabolites of each compound which exhibit in turn more or less cytotoxic effect compared to the parent substance.
Reviewing the obtained results, it could be concluded that, concentrations of myclobutanil pesticide tested (0.5 – 80 μg ml-1) was toxic to the all tested cell lines and toxicity increased as the concentration of myclobutanil was progressively increased. In cell lines treated with myclobutanil pesticide, the PC3 cell was found to be the highest sensitive and has the lowest viability than HepG-2 and MCF-7 cells at lower concentrations (0.5, 5,15 and 25 μg ml-1) while HepG-2 cells showed the lowest cell viability at concentrations 50, 80 μg ml-1.
The NRI50 value, the concentration of myclobutanil resulting in 50% cellular mortality after 24 h exposure to myclobutanil were 38.12, 41 and 27.5 μg ml-1 for HepG-2, MCF-7 and PC3 cancer cell lines, respectively.
3. Genotoxic effects of imidacloprid and myclobutanil against Drosophila melanogaster insect:
In this part of study, insecticide imidacloprid (IMI) and fungicide Myclobutanil (MYC) were tested for pesticidal effects in D. melanogaster adults. The potential cytotoxic effects of tested pesticides were estimated on Drosophila Adult after acute treatment (18h) exposure via adult feeding treatment at various concentrations, ranging from 5 ppm to 50 ppm to find out the major concentration-dependent effects.
The LC50 was determined at a broad range of sublethal concentrations specifically 5, 10, 15, 25 and 50 ppm against the Drosophila. For this data, the toxicity regression line was plotted in form of log/probit relation using Sigma Plot Program software 2.0 (Jandel 1996).
from the data, it was revealed that the two pesticides have a different range of toxic effects varying from 5 to 50 ppm due to structural differences. Their bioactivity data is presented in the graphic representation and the LC50 was determined by using Sigma Plot Software as shown. A significantly less number of flies were enough to successfully delimit upon exposure to higher concentrations of tested pesticides as compared to control flies. Gradual mortality in larvae was observed with increasing pesticidal concentration. Treatment of pesticides also resulted in a delay in the developmental process of Drosophila.
For the higher concentrations, results revealed typical sigmoid curves for which mortality increased sharply with concentrations in a concentration-dependent manner. Reliable concentration-response relationships could be determined for all concentrations.
In the control experiments (the same solvent used to dissolve the pesticides), the average survival rate of adult flies was 87% at the acute treatment. The LC50 values estimated by using probit analysis, concentrations of imidacloprid which cause mortality to 50% (LC50) a of the treated D. melanogaster adults, after acute treatments, imidacloprid was about three times as effective in killing the adults than myc fungicide at the same concentration.
The results showed reduced larval and pupal survival. Over 50% pupal lethality up to 37 ppm concentration for myc at which 50% of the tested flies were unable to emerge from pupae and to finish the developmental cycle. Results obtained from Sigma plot software, probit analysis indicated the value of LC50 which was 13 ppm, 37 ppm concentration for IMI and MYC, respectively and this concentration was used for carcinogenicity analysis. That LC50 concentration for IMI and myc induced a powerful toxic effect resulting in a significant decrement in the number of larvae and pupae.
The carcinogenic activity of IMI and MYC were tested consulting tumor body spots assay using wts-based SMART in D. melanogaster. Induction of tumors using wts assay allows one to draw a closer analogy between the activity of a substance in a SMART test in Drosophila and its potential carcinogenic hazard to humans.
Wild type males were mated to wts/TM3 females resulting in F1 females and males with two phenotypes (♀♂ wts/+, ♂♀ +/TM3, Sb). F1 heterozygous larvae of wts cross were exposed to tested pesticides by feeding treatment. The wts gene is located near the tip of the right arm of chromosome III and only genotype wts/+ males and females were scored for tumor induction.
Four groups of crosses were applied as follows: a control group and two pesticides and positive control groups for different treatments. Mitomycin C (MMC) was used as a powerful genotoxic agent for tumor induction using larval feeding method at concentration of 20 ppm media.
In the negative control experiment (using standard media containing the previous solvent without tested pesticides), the frequency of spontaneous tumors was low, where 27 flies which carry small tumors were scored after screening of 1070 flies of wts/+ genotype. Most of the scored tumors were found on wings and thorax. The frequency of spontaneous tumors (total number of scored tumors/total number of screened flies) was 0.0252. No induction of warts tumors were found in the control siblings TM3/+, while the untreated flies yielded a very low frequency of spontaneous tumors.
On the other hand, Warts tumors were induced in wts/+ males and females in the frequency of 1.024 and 1.274 per fly by 20 ppm MMC (highly significant above the control according to χ2 test at P≤0.05. MMC treatment recorded the highest frequency of tumor induction compared with control which proofs high sensitivity of tumor assay. These tumors arose in every part of the fly analyzed. In some cases, tumors in the head may be scored as tumors in the eye and vice versa. The size of the tumors varied and therefore the number of detected tumors may be slightly affected if scored by different investigators (this has no effect on relative increase/ decrease after treatment). MMC induced large tumors.
After treatment with MMC (20 ppm), the females showed a higher frequency of wts clones (1.2739 spot/fly) than their brothers (1.0237 spot/fly). Most of the extra spots in these females were probably due to mitotic recombination. The fre¬quency of mitotic recombination is related to the distance of the wts gene to the centromere on the third chromosome.
F1 heterozygous larvae of wts cross were exposed to LC50 (13 & 37 ppm) of IMI and Myc pesticides respectively by feeding treatment.
In the imidacloprid experiment, larvae feeding treatment 1078 flies were screened of wts/+ and 180 flies which carry tumors were observed. Most of scored tumors were found on wings (55%), thorax (11%), head (9.5%), eye (9%), leg (6%) and abdomen (3%). The frequency of induced tumors (total numbers of scored tumors / total number of screened flies was 0.167.
In the LC50 of MYC (37 ppm), 146 flies which carry tumors were detected after screening of 1118 flies of wts/+. Most of the scored tumors were found on the thorax, especially on wings. Most of scored tumors were found on wings (51%), leg (15%), eye (14%), thorax (9.5%), head (8%) and abdomen (2%). The frequency of induced tumors (total number of scored tumors/total number of screened flies) was 0.123. According to χ2 test, this was insignificant differences compared with the control.
There were significant differences in the frequency of induced tumors between flies treated with pesticides and negative control. The frequencies of tumor induction exhibited highly significant increased after feeding of Drosophila larvae with IMI and MYC and MMC, where the frequencies of (0.167) and (0.123) and (1.1516) tumor/fly, respectively were observed in comparison with the corresponding negative control.
Imidacloprid and myclobutanil treatments resulted in a highly significant increase in the frequencies of induced wts clone spots in both females and males (0.183, 0.154, 0.140 and 0.10 respectively). Screening for the carcinogenicity of imidacloprid and myclobutanil indicated that two pesticides have evidence of genotoxic potential using SMART assays and showed a clearly positive result.
Imidacloprid shows highly genotoxic and carcinogenic activity by acute treatment to Drosophila than myclobutanil.
In most experiments, the frequency of induced warts tumors was slightly lower in males compared to females and it was considered statistically insignificant. This difference might be explained by the smaller size of the males and possibly a smaller number of target cells at the time of exposure.
Therefore, the current awareness of the real or potential hazards of pesticides considering their cytotoxic/ genotoxic actions cannot be neglected. Thus, further investigations are needed to gain a comprehensive and complete knowledge of the possible mechanisms through which imidacloprid and myclobutanil exert their cytotoxic/genotoxic effects.
4. Impact of imidacloprid insecticide and myclobutanil fungicide on non-target organisms (Honey bees):
The most dangerous pesticide is imidacloprid insecticide which caused the highest percentages of mortality 23.4 and 36.2 % at the tested concentrations of 1 and 10 ppm after four hours of treatment, respectively. In contrast, the myclobutanil fungicide showed zero percentage of mortality at the same mentioned tested time and concentrations (1 and 10 ppm).
In addition, imidacloprid through the experimental time (24 - 96 hours) had mortality percentages between (21.73 - 72.72), (52.17 - 88.63) and (58.69 - 93.16) at three tested concentrations (0.1, 1 and 10 ppm), respectively. Whereas the lowest mortality percentages (%) were obtained in myclobutanil ranged from (17.39 - 38.64), (23.91 - 45.46) and (28.26 - 54.55) at the same mentioned time intervals and tested concentrations, respectively.
On the other hand, resulted that lethal time (LT50) after exposure of honeybee workers to the tested pesticides after 96 hours at different three concentrations (0.1, 1 and 10 ppm) were 59.50, 10.90 and 13.50 hours (imidacloprid) and 181.26, 125.16 and 76.98 hours (myclobutanil) for three tested concentrations, respectively.
from illustrated results it concluded that the imidacloprid insecticide was the most effecting compared with myclobutanil fungicide and had the exhibited a high mortality percentages of honey bees.
5. Determination of the selectivity factor of insecticide imidacloprid between target and non-target organism:
The effects of imidacloprid insecticide at different concentrations on grape aphids and the emergence percentage of Trichogramma egg parasitoid adults from eggs 3 and 5 days old. When the insecticide was used with concentration 266 ppm, the mortality percentage of grape aphids was 87% and the average rate of adult’s parasitoid emergence was 94%. When the minimal concentration of 4.156 ppm was used, the mortality percentage of grape aphids was 13% and the average rate of adult’s parasitoid emergency was 98.3%. The results showed that increasing the concentration of the insecticide was associated with an increase in the mortality percentage of grape aphids and didn’t impact the emergence percentage of parasitoid rate. The recommended concentration (25 g active substance/ha) of imidacloprid insecticide caused 90.67% emergence rate of Trichogramma chilonis adults