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Determination of Pesticide Minimum Residue Limits in Essential Oils
— Report No 4by Sandra M. Garland, Prof. Robert C. Menary and Garth S. Oliver
June 2004
RIRDC Publication No 04/104 RIRDC Project No UT-36A
Method Development
Clean-up Technology
The limitations for developing
sensitive and specific screens for pesticides in essential oils stem from
the complexity of the matrices. As essential oils are extracts themselves,
it follows that any contaminant must have similar properties such as solubility,
polarity and volatility for it to be coextracted.
Hence isolation and pre-concentration of such contaminants can be very difficult, requiring complex chromatographic steps which are usually specific for each type of pesticide. Nonpolar components of essential oils were removed from extracts by partitioning the more polar pesticide contaminants between a two phase, non-polar / polar solvent system. This resulted in a general purpose clean-up method particularly effective for solvent extracted oils such as boronia and blackcurrant extracts. A solvent / solvent partition method using methanol, water and hexane was successfully optimised, with good recoveries and repeatabilities. Methanol is relatively non-toxic and the methodology enables the introduction of complex extracts into the liquid chromatography (LC) and gas chromatography (GC) systems with reduced risk of column damage and fouling of the detectors.
The solvent partition method, however, still equated to the dilution of oils into larger volumes of solvents and did not include a pre-concentration step. A solid phase extraction (SPE) method was developed where analytes were isolated and pre-concentrated. With fewer essential oil components co-extracted, larger sample volumes were introduced into analytical equipment.
Hexane and diethyl ether were used in the silica based SPE method to remove non-polar essential oil components. The target pesticides were eluted with acetone. In peppermint oil and boronia oil, 42% and 81% of volatiles, respectively, were removed. This in effect allowed the pre-concentration of a 0.5 g sample of essential oil into a final sample volume of 200 µL whilst the sample volume introduced into the LC MS/MS was increased from 10 µL to 25 µL, owing to the lower amount of contaminants. The recoveries of pesticides in peppermint oil by SPE were low but the standard errors recorded at each concentration level were acceptable.
Ion exchange chromatography was investigated as a possible clean-up technique for acidic pesticides in essential oils. Methylation of the acids dicamba, clopyralid, fluroxypyr and haloyxfop on ion exchange media were successful, enabling analyses by GC with an electron capture detector (ECD).
Preliminary experiments indicated the potential to detect residues of these herbicides to levels below 0.1 mgkg-1. Ion exchange discs were also applied to the isolation and methylation of mancozeb derivatives with further development of this methodology having the potential to provide a sensitive and specific analysis for dithiocarbamates.
Detection of mancozeb derivatives was also successful using phase transfer reagents to move the salt derivative of partly neutralised EDTA extracts of mancozeb into an organic phase containing a methylating agent. Recoveries, however, were poor when mancozeb was extracted from essential oils. Experimentation indicated that co-extracted components of essential oil were interfering with the derivatisation process, possibly competitively binding with methyl iodide.
Due to the multitude of complications arising from the LC MS/MS method using phase transfer reagents and methylation, attention was diverted to the monitoring of manganese levels in essential oils to indicate mancozeb contamination. A partly neutralised ethylenediaminetetraacetic acid (EDTA) wash of essential oils fortified with mancozeb was found to liberate manganese ions by chelation, allowing them to solvate into the aqueous phase. The samples were then submitted for analysis using an inductively coupled plasma optical emission spectrophotometer (ICP-OES) for manganese analyses. The method was successfully applied to the monitoring of the dissipation of mancozeb in peppermint crops and used to establish whether mancozeb co-distilled with peppermint oil.
The application of NPD to the detection of pesticides in essential oil.
The selective response of a nitrogen phosphorus detector (NPD) to nitrogen and phosphorus containing pesticides was shown to be sufficient to detect pesticides in distilled oils without the inclusion of clean-up methods. The presence of oil has limited effect on the retention time but suppressed the response of the NPD by over 80 % for all the pesticides. However, despite the quenching of the response, the selectivity of the NPD was encouraging and the analyte peaks stood well above the background signal. In conjunction with SPE methodology, GC NPD may prove to be a cost effective screen for pesticides residues containing atoms of nitrogen or phosphorus.
Pesticides in tea tree oil The behaviour of pesticides in tea tree oil did not differ from that recorded in other essential oils tested. The retention characteristics, signal suppression and fragmentation patterns observed were identical to those of peppermint, parsley, fennel and dill oils in all the analytical methodologies trialed.
Field trials
Sinbar in peppermint
Field trails established
to determine the dissipation rates of terbacil (the active ingredient (ai)
of Sinbar) in peppermint showed that after 56 days residues of the ai were
below detection levels. The most rapid loss occurred within one day with
71% of the terbacil originally detected, degraded or removed by abrasion.
The dissipation rate thereafter was less dramatic. Oils were distilled
from leaves collected at 4 days and at the time of peppermint harvest.
Terbacil levels in oil of 1.7 ± 0.3 mgkg-1 were obtained from the
distillation of leaves contaminated with terbacil to the order of 120 ±
24 mgkg-1. This amounts to only 0.03 ± 0.01 % of terbacil co-distilling
with the oil, however it is evident that peppermint vegetative material
that is significant contaminated will produce contaminated distilled oils.
Dithane in peppermint The dissipation of mancozeb (ai of Dithane) in peppermint crops was established by the coordination of a field trial. Mancozeb was still present on the leaf surface almost one month after application but had dissipated to below detection levels after 56 days. Distillation of leaf contaminated at 740 ± 76 mgkg-1 mancozeb produced oils, which did not have elevated levels of manganese. The poor solubility of mancozeb in all solvents, it would appear, precludes the co-distillation of residues in controlled laboratory distillations, even when extreme contamination is present. Field based stills however, have a higher propensity for carryover of particulate matter as water droplets are often blasted through to the condenser at the point of breakthrough.
Annual monitoring of commercially
produced oils
Over the harvest years 2000,
2001, 2002 and 2003 the problems with residues of propiconazole continued.
Contamination levels as high as 43 mgkg-1 were found. With the industry
again alerted to the high levels of residues, the 2001/2002 harvest provided
boronia concretes mostly below the 1 mgkg-1 level with the highest not
exceeding 12 mgkg-1. However this year also saw the emergence of the positive
detection of difenaconazole in six boronia samples, though levels did not
exceed 2 mgkg- 1. Relatively good results continued into the 2002/2003
year for residue levels of propiconazole in boronia with most oils containing
between 0 and 2 mgkg-1. The highest level did not exceed 10 mgkg-1.
Pesticide residues in distilled oils presented few problems. Although positive results were recorded for pirimicarb in parsley and fennel oil in 2002/2003, levels were well below 0.1 mgkg-1. All tea tree oils tested were also found to be free of pesticide contamination for the analytes included in the screens undertaken.
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