Abstract |
Aim of this study was to develop and validate new analytical methods for the
detection and quantification of parent and metabolite compounds of cypermethrin
and imidacloprid pesticides in various biological samples. Also their toxicity was
investigated in-vivo. Pyrethroids and neonicotinoids are two classes of pesticides
widely and extensively used the last decades. The interest concerning these
substances is growing, since they are used as replacement for the organophosphorus
and carbamates pesticides. Of a great concern is also the biomonitoring of
population to these chemicals, especially for occupationally exposed individuals who
are exposed longer and in higher levels. Cretan population consists of a mixture of
rural and urban residents, while many individuals dealing with agricultural activities
as amateurs. Furthermore, the residence of cretan population is offen near
agricultural facilities such as open fields and green houses. This results multiple risk
and burden to the aforementioned pesticides. In literature even increasing reports of
acute or chronic toxicity cases of these substances can be found. The escalating
concerns of the international scientific community on the effects of these substances
on populations, gave rise to this study.
Cypermethrin is a synthetic pyrethroid pesticide used for both domestic and
agricultural applications. Its chemical structure is relative to the natural pyrethrin. It
is supposed to be relatively low toxic for human and other mammals, while acts as
neurotoxin when contact with insects. The specific metabolites for cypermethrin are
cis- and irons-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acids (cisand
trans-Cl2CA) and the non-specific metabolite 3-phenoxybenzoic acid (3-PBA),
which is common for at least ten different pyrethroids. Neonicotinoids are also a
group of synthetic pesticides with insecticidal act that found a lot of application
mainly in agriculture, the last three decades. Imidacloprid is one of the most
common neonicotinoid, which is metabolized to the non-specific 6-chloronicotinic
acid (6-ClNA). Although limited references can be found concerning neonicotinoids,
it is proven that the toxicity of these pesticides must be examined.
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In-vivo experiments were executed on rabbits. Laboratory animals of same
gender and age were used and were exposed orally with sub-acute dosages of the
aforementioned pesticides. The administrated levels were calculated in order to
represent the 1/15 and 1/30 of the LD50 (lethal dose) when it was possible. Duration
of the experiment was about 6 months. During experiment, samplings of biological
samples (urine, hair, blood) were executed (before beginning the administration and
after completing two months).
Analytical methods for the detection of the parent compound and the
metabolites were developed, using techniques of chromatography and mass
spectrometry. The developed methods were validated by calculating several
analytical parameters (linearity, recovery, precision, accuracy, limits of detection-
LOD, limits of quantification-LOQ). Furthermore, the results were compared with
contamination parameters (administrative dosage, duration of exposure).
Briefly, for the detection of imidacloprid and its metabolite 6-ClNA, an
analytical method using liquid chromatography-mass spectrometry was developed.
Limit of detection for imidacloprid was 0.02 ng/mg and 0.01 ng/mg for 6-ClNA in hair
samples, while in urine samples the corresponding values were 0.002 μg/ml for
imidacloprid and 0.008 μg/ml for 6-ClNA. Satisfactory chromatographic separation
was achieved with retention time for imidacloprid 11.13 min and 12.46 min for 6-
ClNA. Linearity of both standard solutions and spiked samples was more than
satisfactory for both hair and urine developed methods (&γτ0.99). Mean recovery for
imidacloprid was calculated to be 97.13% (n=9) and 91.42% (n=9) for 6-ClNA in hair
samples, while in urine samples the corresponding values were 96.11% (n=9) and
103.44% (n=9). The method accuracy was ΄&γτ98.71% for imidacloprid and ΄&γτ100.25%
for 6-ClNA in hair samples method and ΄&γτ98.93% for imidacloprid and ΄&γτ97.82% for 6-
ClNA in urine samples method. The corresponding precision values were fluctuated
΄&λτ12.43%.
Results of the analysis of in-vivo hair samples indicated statistically important
differences in the detected levels between the different treatment groups (control,
low dose, high dose) for imidacloprid (P΄&λτ0.001) but not for 6-ClNA (p=0.571). Found
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levels of imidacloprid in hair samples of control group were ranged from 0.62 to 1.61
ng/mg, for low dose group from 0.99 to 41.91 ng/mg and for high dose from 0.26 to
58.87 ng/mg. The corresponding concentrations of 6-ClNA in urine samples of
control group were ΄&λτLOD during the experiment, for low dose group found levels
were ranged from ΄&λτLOD to 0.21 ng/mg and for high dose group from ΄&λτLOD to 2.88
ng/mg.
Results regarding urine samples of exposed rabbits indicated statistical
important differences between the different treatment groups for both imidacloprid
(p=0.008) and 6-ClNA (p=0.032). Levels of imidacloprid and 6-ClNA in urine samples
of control group were ΄&λτLOD during the administration period. For low dose group
found concentration of imidacloprid were ranged from ΄&λτLOD to 125.02 μg/ml and for
high dose group from ΄&λτLOD to 343.17 μg/ml. Found values for 6-ClNA were from
΄&λτLOD to 1.29 μg/ml and from ΄&λτLOD to 4.25 μg/ml for low and high dose groups,
respectively. Compared to 6-ClNA, the parent compound was indicated as more
suitable biomarker of exposure to the pesticide imidacloprid for both urine and hair
samples.
Moreover, for the detection of the cypermethrin metabolites, cis-Cl2CA,
trans-Cl2CA και 3-PBA, an analytical method using gas chromatography-mass
spectrometry (GC-MS) was validated and used. The limits of detection achieved for
hair samples were 4.0 pg/mg for cis-Cl2CA, 3.9 pg/mg for trans-Cl2CA and 1.0 pg/mg
for 3-PBA, while for urine samples the corresponding limits were 1.4 ng/ml for cis-
Cl2CA, 0.8 ng/ml for trans-Cl2CA and 0.3 ng/ml for 3-PBA. Linearity was good for
standard solutions and spiked samples in the investigated ranges (΄&γτ0.99). Mean
recovery for cis-Cl2CA was 84.8% (n=8), for trans-Cl2CA 87.2% (n=8) and for 3-PBA
was 96.4% (n=8) for hair samples, while for urine samples the corresponding
recoveries were 102.1% (n=3), 111.2% (n=3) and 87.1% (n=3). Accuracy was
estimated to be ΄&γτ91.8% for cis-Cl2CA, ΄&γτ96.7% for trans-Cl2CA and ΄&γτ90.5% for 3-PBA
for hair samples method and ΄&γτ89.2% for cis-Cl2CA, ΄&γτ71.6% for trans-Cl2CA and
΄&γτ86.0% for 3-PBA for urine samples method. The corresponding precision values
were fluctuated ΄&λτ13.6%.
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Results concerning hair samples of laboratory animals indicated statistically
significant differences between the different treatment groups (control, low dose,
high dose) for cis-Cl2CA (p=0.009), trans-Cl2CA (p=0.003) and 3-PBA (p=0.012). Found
concentrations of cis-Cl2CA in hair samples of control group were ranged from ΄&λτLOD
to 112.6 pg/mg, for low dose group from ΄&λτLOD to 1022.0 pg/mg and for high dose
group from ΄&λτLOD to 5361.4 pg/mg. The corresponding values for trans-Cl2CA in hair
samples of control group were from ΄&λτLOD to 112.0 pg/mg, for low dose group from
΄&λτLOD to 1367.0 pg/mg and for high dose group from ΄&λλτLOD to 5448.2 pg/mg. Finally
for 3-PBA found levels were ranged from 16.5 to 42.8 pg/mg for control group, from
΄&λτLOD to 146.7 pg/mg for low dose group and from ΄&λτLOD to 659.9 pg/mg for high
dose group.
Levels of all cypermethrin metabolites in urine samples of control group were
΄&λτLOD during the experiment. For low dose group levels of cis-Cl2CA were ranged
from ΄&λτLOD to 1365.6 ng/ml and for high dose group from ΄&λτLOD to 5396.9 ng/ml. The
corresponding values for trans-Cl2CA were from ΄&λτLOD to 2311.4 ng/ml and from
΄&λτLOD to 7453.9 ng/ml. Finally for 3-PBA found levels in urine samples of low dose
group were ranged from ΄&λτLOD to 813.1 ng/ml and for high dose group from ΄&λτLOD to
2072.8 ng/ml.
Additionally, the influence of cypermethrin and imidacloprid in oxidative
stress was investigated in-vivo by measuring TBARS, protein carbonyls, TAC levels in
blood samples from exposed rabbits. Moreover the genotoxicity effects of
imidacloprid in the administrated dosages were examined in-vivo. Briefly, results
indicated that cypermethrin does not affect any oxidative stress biomarker in the
administrated dosages. On the other hand, imidacloprid influences oxidative stress,
since a significantly difference (p΄&λτ0.05) in TBARS levels between exposed and control
groups was observed. For the estimation of the genotoxic effects of the pesticide
imidacloprid a Cytokinesis Block Micronuclei (CBMN) method was applied. Results
indicated statistically significant differences between exposed and control group.
The population study focused on different population groups in the wider
region of Cretem, which is a mixture of exposed (rural population, sprayers-
31
applicators) and general population (without occupational exposure to pesticides).
Hair samples were collected from each participant and filled alongside appropriate
questionnaire, which recorded basic socio-economic and clinical evidence, potential
exposure to pesticides, dietary habits, as well as chronic health problems. Head hair
samples were collected from the scalp, as close to the skin. In total, 58 hair samples
were collected, of which 32 from residents of rural areas of Crete and 26 samples
from urban areas of Crete. Average age of participants of urban areas was 35 years,
while 50% of the residents of the rural area were older than 50 years. 38.5% of
residents of the urban area were males, while the rate for residents of the rural area
was 21.9%. The analysis was performed on all the available length of the hair in
order to study the burden of the studied substances over time. From the analysis of
the samples observed detectable levels of imidacloprid only for residents of the rural
area. However, no statistically significant difference was observed by examining the
population group by gender or age. The detection rate of imidacloprid for residents
of the rural area was quite high (65.6%), which is based on the questionnaires
answered can be attributed to the different lifestyle but also engage them with
agricultural activities or the proximity of his residence in rural activities.
The protocols developed in this thesis can be applied successfully to estimate
risk or biomonitoring in specific plant protection substances and their possible
association with health problems or other adverse side effects. Also these protocols
can be used to investigate incidents of acute poisoning in order to assess the clinical
picture of the patient. In both cases, the importance of measurability of the above
substances in hair samples can be useful and provide important information for the
past and/or chronic exposure to these pesticides. Such an evaluation was not yet
possible due to the non-existence of those protocols sofar.
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