Determination of Pesticide Minimum Residue Limits in Essential OilsReport No 3A report for the Rural Industries Research andDevelopment CorporationBy Professor R. C. Menary & Ms S. M. GarlandJune 2004RIRDC Publication No 04/023RIRDC Project No UT-23A© 2004 Rural Industries Research and Development Corporation.All rights reserved.ISBN 0642 58733 7ISSN 1440-6845‘Determination of pesticide minimum residue limits in essential oils’, Report No 3Publication No 04/023Project no.UT-23AThe views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report.This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186.Researcher Contact DetailsProfessor R. C. Menary & Ms S. M. GarlandSchool of Agricultural ScienceUniversity of TasmaniaGPO Box 252-54HobartTasmania 7001AustraliaPhone: (03) 6226 2723Fax: (03) 6226 7609Email: r.menary@.auIn submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form.RIRDC Contact DetailsRural Industries Research and Development CorporationLevel 1, AMA House42 Macquarie StreetBARTON ACT 2600PO Box 4776KINGSTON ACT 2604Phone: 02 6272 4819Fax: 02 6272 5877Email: rirdc@.auWebsite: .auPublished in June 2004Printed on environmentally friendly paper by Canprint.FOREWORDInternational regulatory authorities are standardising the levels of pesticide residues present in products on the world market which are considered acceptable. The analytical methods to be used to confirm residue levels are also being standardised. To constructively participate in these processes, Australia must have a research base capable of constructively contributing to the establishment of methodologies and must be in a position to assess the levels of contamination within our own products.Methods for the analysis for pesticide residues rarely deal with their detection in the matrix of essential oils. This project is designed to develop and validate analytical methods and apply that methodology to monitor pesticide levels in oils produced from commercial harvests. This will provide an overview of the levels of pesticide residues we can expect in our produce when normal pesticide management programs are adhered to.The proposal to produce a manual which deals with the specific problems associated with detection of pesticide residues in essential oils is intended to benefit the essential oil industry throughout Australia and may prove useful to other horticultural products.This report is the third in a series of four project reports presented to RIRDC on this subject. It is accompanied by a technical manual detailing methodologies appropriate to the analysis for pesticide residues in essential oils.This project was part funded from RIRDC Core Funds which are provided by the Australian Government. Funding was also provided by Essential Oils of Tasmania and Natural Plant Extracts Cooperative Society Ltd.This report, an addition to RIRDC’s diverse range of over 1000 research publications, forms part of our Essential Oils and Plant Extracts R&D program, which aims for an Australian essential oils and plant extracts industry that has established international leadership in production, value adding and marketing.Most of our publications are available for viewing, downloading or purchasing online through our website:•downloads at .au/fullreports/index.html•purchases at .au/eshopSimon HearnManaging DirectorRural Industries Research and Development CorporationAcknowledgementsOur gratitude and recognition is extended to Dr. Noel Davies (Central Science Laboratories, University of Tasmania) who provided considerable expertise in establishing procedures for chromatography mass spectrometry.The contribution to extraction methodologies and experimental work-up of Mr Garth Oliver, Research Assistant, cannot be underestimated and we gratefully acknowledge his enthusiasm and novel approaches.Financial and ‘in kind’ support was provided by Essential Oils Industry of Tasmania, (EOT).AbbreviationsADI Average Daily IntakeAGAL Australian Government Analytical Laboratoriesingredientai activeAPCI Atmospheric Pressure Chemical IonisationBAP Best Agricultural PracticesenergyCE collisionDETA DiethylenetriamineECD Electron Capture DetectorionisationESI ElectrosprayFPD Flame Photometric DetectionChromatographyGC GasResolutionHR HighChromatographyLC LiquidLC MSMS Liquid Chromatography with detection monitoring the fragments of Mass Selected ionsMRL Maximum Residue LimitSpectrometryMS MassNRA National Registration AuthorityR.S.D. Relative Standard DeviationSFE Supercritical Fluid ExtractionSIM Single Ion MonitoringSPE Solid Phase ExtractionTIC Total Ion ChromatogramContents FOREWORD (III)ACKNOWLEDGEMENTS (IV)ABBREVIATIONS (V)CONTENTS (VI)EXECUTIVE SUMMARY (VII)1. INTRODUCTION (1)1.1B ACKGROUND TO THE P ROJECT (1)1.2O BJECTIVES (2)1.3M ETHODOLOGY (2)2. EXPERIMENTAL PROTOCOLS & DETAILED RESULTS (3)2.1M ETHOD D EVELOPMENT (3)2.2M ONITORING OF H ARVESTS (42)2.3P RODUCTION OF M ANUAL (46)3. CONCLUSIONS (47)IMPLICATIONS & RECOMMENDATIONS (50)BIBLIOGRAPHY (50)Executive SummaryThe main objective of this project was to continue method development for the detection of pesticide residues in essential oils, to apply those methodologies to screen oils produced by major growers in the industry and to produce a manual to consolidate and coordinate the results of the research. Method development focussed on the effectiveness of clean-up techniques, validation of existing techniques, the assessment of the application of gas chromatography (GC) with detection using electron capture detectors (ECD), flame photometric detectors (FPD) and high pressure liquid chromatography (HPLC) with ion trap mass selective (MS) detection.The capacity of disposable C18 cartridges to separate components of boronia oil was found to be limited with the majority of boronia components being eluted on the solvent front, with little to no separation achieved. The cartridges were useful, however, in establishing the likely interaction of reverse phases (RP) C18 columns with components of essential oils, using polar mobile phases . The loading of large amounts of oil onto RP HPLC columns presents the risk of permanently contaminating the bonded phases. The lack of retention of components on disposable SPE C18 cartridges, despite the highly polar mobile phase, presented a good indication that essential oils would not accumulate on HPLC RP columns.The removal of non-polar essential oil components by solvent partitioning of distilled oils was minimal, with the recovery of pesticides equivalent to that recorded for the essential oil components. However application of this technique was of advantage in the analysis of solvent extracted essential oils such as those produced from boronia and blackcurrant.ECD was found to be successful in the detection of terbacil, bromacil, haloxyfop ester, propiconazole, tebuconazole and difenaconzole. However, analysis of pesticide residues in essential oils by application of GC ECD is not sufficiently sensitive to allow for a definitive identification of any contaminant. As a screen, ECD will only be effective in establishing that, in the absence of a peak eluting with the correct retention time, no gross contamination of pesticide residues in an essential oil has occurred . In the situation where a peak is recorded with the correct elution characteristics, and which is enhanced when the sample is fortified with the target analyte, a second means of contaminant identification would be required. ECD, then, can only be used to rule out significant contamination and could not in itself be adequate for a positive identification of pesticide contamination.Benchtop GC daughter, daughter mass spectrometry (MSMS) was assessed and was not considered practical for the detection of pesticide residues within the matrix of essential oils without comprehensive clean-up methodologies. The elution of all components into the mass spectrometer would quickly lead to detector contamination.Method validation for the detection of 6 common pesticides in boronia oil using GC high resolution mass spectrometry was completed. An analytical technique for the detection of monocrotophos in essential oils was developed using LC with detection by MSMS. The methodology included an aqueous extraction step which removed many essential oil components from the sample.Further method development of LC MSMS included the assessment of electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI. For the chemicals trialed, ESI has limited application. No response was recorded for some of the most commonly used pesticides in the essential oil industry, such as linuron, oxyflurofen, and bromacil. Overall, there was very little difference between the sensitivity for ESI and APCI. However, APCI was slightly more sensitive for the commonly used pesticides, tebuconazole and propiconazole, and showed a response, though poor, to linuron and oxyflurofen. In addition, APCI was the preferred ionisation method for the following reasons,♦APCI uses less nitrogen gas compared to ESI, making overnight runs less costly;♦APCI does not have the high back pressure associated with ionisation by ESI such that APCI can be run in conjunction with UV-VIS without risk of fracturing the cell, which is pressure sensitive. Analytes that ionised in the negative APCI mode were incorporated into a separate screen which included bromacil, terbacil, and the esters of the fluazifop and haloxyfop acids. Further work using APCI in the positive mode formed the basis for the inclusion of monocrotophos, pirimicarb, propazine and difenaconazole into the standard screen already established. Acephate, carbaryl, dimethoate, ethofumesate and pendimethalin all required further work for enhanced ionisation and / or improved elution profiles. Negative ionisation mode for APCI gave improved characteristics for dicamba, procymidone, MCPA and mecoprop.The thirteen pesticides included in this general screen were monocrotophos, simazine, cyanazine, pirimicarb, propazine, sethoxydim, prometryb, tebuconazole, propiconazole, , difenoconazole and the esters of fluroxypyr, fluazifop and haloxyfop.. Bromacil and terbacil were not included as both require negative ionisation and elute within the same time window as simazine, which requires positive ionisation. Cycling the MS between the two modes was not practical.The method validation was tested against three oils, peppermint, parsley and fennel.Detection limits ranged from 0.1 to 0.5 mgkg-1 within the matrix of the essential oils, with a linear relationship established between pesticide concentration and peak height (r2 greater than 0.997) and repeatabilities, as described by the relative standard deviation (r.s.d), ranging from 3 to 19%. The type of oil analysed had minimal effect on the response function as expressed by slope of the standard curve.The pesticides which have an carboxylic acid moiety such as fluazifop, haloxyfop and fluroxypyr, present several complications in any analytical method development. The commercial preparations usually have the carboxylic acid in the ester form, which is hydrolysed to the active acidic form on contact with soil and vegetation. In addition, the esters may be present in several forms, such as the ethoxy ethyl or butyl esters. Detection using ESI was tested. Preliminary results indicate that ESI is unsuitable for haloxyfop and fluroxypyr ester. Fluazifop possessed good ionisation characteristics using ESI, with responses approximately thirty times that recorded for haxloyfop. Poor chromatography and response necessitated improved mobile phase and the effect of pH on elution characteristics was considered the most critical parameter. The inclusion of acetic acid improved peak resolution.The LC MSMS method for the detection of dicamba, fluroxypyr, MCPA, mecoprop and haloxyfop in peppermint and fennel distilled oils underwent the validation process. Detection limits ranged from 0.01 to 0.1 mgkg-1Extraction protocols and LC MSMS methods for the detection of paraquat and diquat were developed. ESI produced excellent responses for both paraquat and diquat, after some modifications of the mobile phase. Extraction methodology using aqueous phases were developed. Extraction with carbonate buffer proved to be the most effective in terms of recovery and robustness. A total ion chromatogram of the LC run of an aqueous extract of essential oil was recorded and detection using a photodiode array detector confirmed that very little essential oil matrix was co-extracted. The low background noise indicated that samples could be introduced directly into the MS. This presented a most efficient and rapid way for analysis of paraquat and diquat, avoiding the need for specialised columns or modifiers to be included in the mobile phase to instigate ion exchange.The adsorbtion of paraquat and diquat onto glass and other surfaces was reduced by the inclusion of diethylenetriamine (DETA). DETA preferentially accumulates on the surfaces of sample containers, competitively binding to the adsorption sites. All glassware used in the paraquat diquat analysis were washed in a 5% solution of 0.1M DETA, DETA was included in all standard curve preparations, oils were extracted with aqueous DETA and the mobile phase was changed to 50:50 DETA / methanol. The stainless steel tubing on the switching valve was replaced with teflon, further improvingreproducibility. Method validation was undertaken of the analysis of paraquat and diquat using the protocols established. The relationship between analyte concentration and peak area was not linear at low concentrations, with adsorption more pronounced for paraquat, such that the response for this analyte was half that seen for diquat and the 0.1 mgkg-1 level.The development of a method for the detection of the dithiocarbamate, mancozeb was commenced. Disodium N, N'-ethylenebis(dithiocarbamate) was synthesised as a standard for the derivatised final analytical product. An LC method, with detection using MSMS, was successfully completed. The inclusion of a phase transfer reagent, tetrabutylammonium hyrdrogen sulfate, required in the derivatisation step, contaminated the LC MSMS system, such that any signal from the target analyte was masked. Alternatives to the phase transfer reagent are now being investigated.Monitoring of harvests were undertaken for the years spanning 1998 to 2001. Screens were conducted covering a range of solvent extracted and distilled oils. Residues tested for included tebuconazole, simazine, terbacil, bromacil, sethoxydim, prometryn, oxyflurofen, pirimicarb, difenaconazole, the herbicides with acidic moieties and paraquat and diquat. Problems continued for residues of propiconazole in boronia in the 1998 / 1999 year with levels to 1 mgkg-1 still being detected. Prometryn residues were detected in a large number of samples of parsley oil.Finally the information gleaned over years of research was collated into a manual designed to allow intending analysts to determine methodologies and equipment most suited to the type of the pesticide of interest and the applicability of analytical equipment generally available.1. Introduction1.1 Background to the ProjectResearch undertaken by the Horticultural Research Group at the University of Tasmania, into pesticide residues in essential oils has been ongoing for several years and has dealt with the problems specific to the analysis of residues within the matrix of essential oils. Analytical methods for pesticides have been developed exploiting the high degree of specificity and selectivity afforded by high resolution gas chromatography mass spectrometry. Standard curves, reproducibility and detection limits were established for each. Chemicals, otherwise not amenable to gas chromatography, were derivatised and incorporated into a separate screen to cover pesticides with acidic moieties.Research has been conducted into low resolution GC mass selective detectors (MSD and GC ECD. Low resolution GC MSD achieved detection to levels of 1 mgkg-1 in boronia oil, whilst analysis using GC ECD require a clean-up step to effectively detect halogenated chemicals below 1mgkg-1.Dithane (mancozeb) residues were digested using acidified stannous chloride and the carbon disulphide generated from this reaction analysed by GC coupled to FPD in the sulphur mode.Field trials in peppermint crops were established in accordance with the guidelines published by the National Registration Authority (NRA), monitoring the dissipation of Tilt and Folicur residues in peppermint leaves and the co-distillation of these residues with hydro-distilled peppermint oils were assessed.Development of extraction protocols, analytical methods, harvest monitoring and field trials were continued and were detailed in a subsequent report. Solvent-based extractions and supercritical fluid extraction (SFE) was found to have limited application in the clean-up of essential oilsIn conjunction with Essential Oils of Tasmania (EOT), the contamination risk, associated with the introduction of a range of herbicides, was assessed through a series of field trials. This required analytical method development to detect residues in boronia flowers, leaf and oil. The methodology for a further nine pesticides was successful applied. Detection limits for these chemicals ranged from 0.002 mgkg-1 to 0.1 mgkg-1. In addition, methods were developed to analyse for herbicides with active ingredients (ai) whose structure contained acidic functional groups. Two methods of pesticide application were trialed. Directed sprays refer to those directed on the stems and leaves of weeds at the base of boronia trees throughout the trial plot. Cover sprays were applied over the entire canopy. For all herbicides for which significant residues were detected, it was evident that cover sprays resulted in contamination levels ten times those occurring as a result of directed spraying in some instances. Chloropropham, terbacil and simazine presented potentially serious residue problems, with translocation of the chemical from vegetative material to the flower clearly evident.Directed spray applications of diuron and dimethenamid presented only low residue levels in extracted flowers with adequate control of weeds. Oxyflurofen and the mixture of bromacil and diuron (Krovar) presented only low levels of residues when used as a directed spray and were effective as both post and pre-emergent herbicides. Only very low levels of residues of both sethoxydim and norflurazon were detected in boronia oil produced in crops treated with directed spray applications. Sethoxydim was effective as a cover spray for grasses whilst norflurazon showed potential as herbicide to be used in combination with other chemicals such as diuron, paraquat and diquat. Little contamination of boronia oils by herbicides with acidic moieties was found. This advantage, however, appears to be offset by the relatively poor weed control. Both pendimethalin and haloxyfop showed good weed control. Both, however, present problems with chemical residues in boronia oil and should only be used as a directed sprayThe stability of tebuconazole, monocrotophos and propiconazole in boronia under standard storage conditions was investigated. Field trials of tebuconazole and propiconazole were established in commercial boronia crops and the dissipation of both were monitored over time. The amount of pesticide detected in the oils was related to that originally present in the flowers from which the oils were produced.Experiments were conducted to determine whether the accumulation of terbacil residues in peppermint was retarding plant vigour. The level recorded in the peppermint leaves were comparatively low. Itis unlikely that terbacil carry over is the cause for the lack of vigour in young peppermint plants.Boronia oils produced in 1996, 1997 and 1998 were screened for pesticides using the analytical methods developed. High levels of residues of propiconazole were shown to persist in crops harvested up until 1998. Field trials have shown that propiconazole residues should not present problems if the fungicide is used as recommended by the manufacturers.1.2 Objectives♦Provide the industry, including the Standards Association of Australia Committee CH21, with a concise practical reference, immediately relevant to the Australian essential oil industry♦Facilitate the transfer of technology from a research base to practical application in routine monitoring programs♦Continue the development of analytical methods for the detection of metabolites of the active ingredients of pesticide in essential oils.♦Validate the methods developed.♦Provide industry with data supporting assurances of quality for all exported products.♦Provide a benchmark from which Australia may negotiate the setting of a realistic maximum residue limit (MRL)♦Determine whether the rate of uptake is relative to the concentration of active ingredient on the leaf surface may establish the minimum application rates for effective pest control.1.3 MethodologyThree approaches were used to achieve the objectives set out above.♦Continue the development and validation of analytical methods for the detection of pesticide residues in essential oils. Analytical methods were developed using gas chromatography high resolution mass spectrometry (GC HR MS), GC ECD, GC FPD and high pressure liquid chromatography with detection using MSMS.♦Provide industry with data supporting assurances of quality for all exported products.♦Coordinate research results into a comprehensive manual outlining practical approaches to the development of analytical proceduresOne aspect of the commissioning of this project was to provide a cost effective analytical resource to assess the degree of the pesticide contamination already occurring in the essential oils industry using standard pesticide regimens. Oil samples from annual harvests were analysed for the presence of pesticide residues. Data from preceding years were collated to determine the progress or otherwise, in the application of best agricultural practice (BAP).2. Experimental Protocols & Detailed ResultsThe experimental conditions and results are presented under the following headings:♦Method Development♦Monitoring of Commercial Harvests♦Production of a Manual2.1 Method DevelopmentMethod development focussed on the effectiveness of clean-up techniques, validation of existing techniques, the assessment of the application of GC ECD and FPD and high pressure liquid chromatography with ion trap MS, MS detection.2.1.1 Clean-up Methodologies2.1.1.i. Application of Disposable SPE cartridges in the clean-up of pesticide residues in essentialoilsLiterature reviews provided limited information with regards to the separation of contaminants within essential oils. The retention characteristics of disposable C18 cartridges were trialed.Experiment 1;Aim : To assess the capacity of disposable C18 cartridges to the separation of boronia oil components. Experimental : Boronia concrete (49.8 mg) was dissolved in 0.5 mL of acetone and 0.4 mL of chloroform was added. 1mg of octadecane was added as an internal standard. A C18 Sep-Pak Classic cartridge (short body) was pre- conditioned with 1.25 mL of methanol, which was passed through the column at 7.5 mLmin-1, followed by 1.25 mL of acetone, at the same flow rate. The boronia samplewas then applied to the column at 2 mLmin-1 flow and eluted with 1.25 mL of acetone / chloroform (5/ 4) and then eluted with a further 2.5 mL of chloroform. 5 fractions of 25 drops each were collected. The fractions were analysed by GC FID using the following parametersAnalytical parameters6890PackardHewlettGCcolumn: Hewlett Packard 5MS 30m, i.d 0.32µmcarrier gas instrument grade nitrogeninjection volume: 1µL (split)injector temp: 250°Cdetector temp: 280°Cinital temp: 50°C (3 min), 10°Cmin-1 to 270°C (7 mins)head pressure : 10psi.Results : Table 1 record the percentage volatiles detected in the fractions collectedFraction 1 2 3 4 5 % components eluting 18 67 13 2636%monoterpenes 15%sesquiquiterpenes 33 65 2%high M.W components 1 43 47 9Table 1. Percentage volatiles eluting from SPE C18 cartridgesDiscussion : The majority of boronia components eluted on the solvent front, effecting minimal separation. This area of SPE clean-up of essential oils requires a wide ranging investigation, varying parameters such as cartridge type and polarity of mobile phase.Experiment 2.Aim : For the development of methods using LC MSMS without clean-up steps, the potential for oil components to accumulate on the reverse phase (RP) column must be assessed. The retention of essential oil components on SPE C18 cartridges, using the same mobile phase as that to be used in theLC system, would provide a good indication as to the risk of contamination of the LC columns withoil components.Experimental: Parsley oil (20-30 mg) was weighed into a GC vial. 200 µL of a 10 µgmL-1 solution (equivalent to 100mgkg-1 in oil) of each of sethoxydim, simazine, terbacil, prometryn, tebuconazoleand propiconazole were used to spike the oil, which was then dissolved in 1.0 mL of acetonitrile. The solution was then slowly introduced to the C18 cartridge (Waters Sep Pac 'classic' C18 #51910) using a disposable luer lock, 10 mL syringe, under constant manual pressure, and eluted with 9 mLs of acetonitrile. Ten, 1 mL fractions were collected and transferred to GC vials. 1mg of octadecane was added to each vial and the samples were analysed by GC FID under the conditions described in experiment 1.The experiment was repeated using C18 cartridges which had been pre-conditioned with distilled waterfor 15 mins. Again, parsley oil, spiked with pesticides was eluted with acetonitrile and 5 x 1 mL fractions collected.Results: The majority of oil components and pesticides were eluted from the C18 cartridge in the firsttwo fractions. Little to no separation of the target pesticides from the oil matrix was achieved. Table2 lists the distribution of essential oil components in the fractions collected.Fraction 1 2 3 4 5 % components eluting 18 67 13 2663%monoterpenes 15%sesquiquiterpenes 33 65 2%high M.W components 1 43 47 9water conditioned% components eluting 35 56 8 12%monoterpenes 3068%sesquiquiterpenes 60 39 1 0%high M.W components 0 50 42 7Table 2. Percentage volatiles eluting for SPE C18 cartridgesFigure 1 shows a histogram of the percentage distribution of components from the oil in each of the four fractions.Figure 1. Histogram of the percentage of volatiles of distilled oils in each of four fraction elutedon SPE C18 cartridges (non-preconditioned)Figure 2. Histogram of the percentage of volatiles of distilled oils in each of four fraction elutedon SPE C18 cartridges (preconditioned)Discussion : The chemical properties of many of the target pesticides, including polarity, solubility in organic solvents and chromatographic behaviour, are similar to the majority of essential oil components. This precludes the effective separation of analytes from such matrices through the use of standard techniques, where the major focus is pre-concentration of pesticide residues from water or water based vegetative material. However, this experiment served to provide a good indication that under HPLC conditions, where a reverse phase C18 column is used in conjunction with acetonitrile / water based mobile phases, essential oil components do not remain on the column.。
