Abstract |
The main aim of the present study was the investigation of the possibility to use hair
analysis as a tool for the assessment of chronic and low level exposure to pesticides,
employing in vitro and in vivo experiments. The studied substances belong to the families of
organophosphates, carbamates and organochlorines. A literature review revealed that no such
studies have been conducted in the past.
The disposition of organochlorine pollutants and pesticides in hair was also studied.
The organochlorine pesticides under investigation are no longer used and the PCBs are
byproducts of industrial activities. Exposure of the organisms to the aforementioned
substances occurs either through the diet or from the environment.
The first and crucial step was the development and validation of the analytical
methods for the detection and quantification of the analytes of interest in a difficult matrix like
hair.
The pesticide diazinon was the model compound for the study of organophosphates
incorporation in hair. Sample preparation included methanolic extraction of the pesticide from
the hair matrix, at 37 0C overnight, followed by liquid-liquid extraction with ethyl acetate,
evaporation of the solvent under nitrogen, resuspension of the residue in 50 μl of methanol
and analysis by GC-MS or GC-NPD. GC-MS analysis was performed in full scan and SIM
mode. Analysis in SIM mode offered much better selectivity and sensitivity. Two ionisation
modes were also utilised; electronic ionisation and negative chemical ionisation with methane.
Chemical ionisation mode offered much lower detection limits than the electronic ionisation
mode.
The disposition of diazinon in hair from the environment was studied using fortified
hair samples. Three parameters that would probably affect the detected concentration of
diazinon in hair were studied; the concentration of diazinon in the exposure media, the time of
exposure and the colour of the hair. Initially hair samples coming from the same person were
immersed in aqueous solutions diazinon at different concentrations, for varying time periods.
The samples were analysed by EI GC-MS following a decontamination rinse with water,
drying, pulverization, methanolic extraction in an ultrasonic bath, liquid-liquid extraction with
ethyl acetate, evaporation of the solvent and resuspension in 50 μl of methanol. It was found
that the concentration of the pesticide detected in hair was proportional to the concentration of
the exposure media and to the time of exposure.
The same experiment was repeated using hair of different shades brown, coming from
different people. The hair samples were immersed in aqueous solution of the pesticide at the
same concentration and for the same time period. Our results indicated that in the case of
external disposition of the pesticide in hair, the concentration of the pesticide is not related to
the melanin content and hence the colour of the hair. On the other hand, other factors like the
lipid content of the hair or the diameter of the hair fiber may play a more important role in the
amount of pesticide detected.
The next step was the study of incorporation of diazinon in hair in vivo. Rats and
rabbits were used as our experimental animals. It is obvious by the difference in the literature
reported LD50 values that rabbits are more susceptible to diazinon toxicity than rats. Only a
very small fraction of the administered doses was detected in the hair of the animals. The
concentration of the pesticide detected in the hair of the rats was much higher than that
detected in the hair of the rabbits even though the rabbits received higher doses than the rats.
This may be explained by the different metabolic profile of the two animal species.
Two methods were used for the detection of methomyl in the hair of experimental
animals. The screening of the samples was performed by ELISA while the results were
confirmed by HPLC. Even though ELISA gave acceptable results with methomyl there was
significant background noise. Analysis with GC-MS was not possible due to the instability of
the molecule
The exposure of the population to certain organochlorine compounds through the diet
was also studied. The compounds studied were two organochlorine pesticides, DDT and
lindane, and the following PCB congeners: PCB 28, 52, 99, 101, 118, 138, 149, 153, 156,
170, 180 and 187. A comparative study of the population exposure of three countries, Greece,
Romania and Belgium was also conducted. The hair matrix was dissolved by incubation in
HCl, and the samples were initially liquid-liquid extracted in a mixture of hexane:
dichloromethane (4:1) and subsequently cleaned by SPE. Finally they were analysed by GCECD
and GC-MS for confirmation.
The Greek samples were found to be the most contaminated ones by organochlorine
pesticides while they carried the smallest PCBs burden. Belgian samples were more
contaminated by PCBs but carried a smaller pesticide burden while the samples from
Romania contained less pesticide than the Greek ones but more than the Belgian ones.
Three sensitive and selective analytical methods were developed and validated during
the study of incorporation of diazinon in hair. They were based on GC-MS and GC-NPD
detection and they were successful in detecting and quantitating diazinon in hair in the pg/mg
level. It was confirmed by the in vivo studies in white rats and rabbits that diazinon may be
detected in hair once it enters blood circulation, at concentrations proportional to the
administered dose. When the detected pesticide concentrations in hair were compared, it
became evident that metabolic species differences influence the quantity of non metabolized
pesticide circulating in the blood and hence the concentration detected in hair.
The in vitro studies indicated that diazinon is deposited in hair not only in the case of
exposure through the blood stream, but also in the case of external exposure. The
concentration of the exposure media as well as the time of exposure does play a role in the
detected pesticide concentration. On other hand the colour of the hair is not such a crucial
parameter as it happens when substances are incorporated in the hair shaft from the sweat and
sebum that bathe the shaft during the hair growth.
The incorporation of methomyl in hair was studied using rabbits as experimental
animals. It was confirmed that once it enters the blood stream it is detected in hair. Segmental
hair analysis that was performed indicated that methomyl binds to specific sites and does not
migrate along the hair shaft. This can be used in human exposure studies to obtain an accurate
record of exposure, bearing in mind that human head hair grows approximately 1 cm/month..
The main conclusion reached by the study of the organochlorine compounds
was that contamination of the samples with the aforementioned substances was universal and
that organochlorine concentration detected in the samples was related to the origin of the
examined population. The Greek and occupationally exposed samples carried more DDTs,
and less PCBs. The HCHs burden was similar for all the samples.
In summary, during the present study sensitive and selective methods for the detection
of minute quantities of pesticides in complex matrices were developed and validated, it was
confirmed that the metabolic profile of an organism influences the concentration of pesticides
detected in hair, and when external contamination of hair was studied, it was deduced that the
concentration of pesticide in the exposure media as well as the time of exposure influences the
concentration of the detected pesticides in hair. Segmental hair analysis that was performed in
the case of methomyl and may be performed in human hair may give an accurate record of
past exposure to pesticides.
As a future development, hair disposition of certain characteristic metabolites of
pesticides should be studied. Also it would be interesting to examine hair disposition of other
commonly used pesticides. This is a potentially valuable additional tool for the assessment of
chronic exposure to pesticides.
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