Determining Pesticide Minimum Residue Limits in Essential Oils
A report for the Rural Industries Research and Development Corporation by Professor R. C. Menary and Ms S. M. Garland University of Tasmania
November 1999 RIRDC Publication No 99/123 RIRDC Project No UT-13A
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© 1999 Rural Industries Research and Development Corporation All rights reserved. ISBN 0 642 58001 4 ISSN 1440-6845 Determining Pesticide Minumim Residue Limiits in Essential Oils Publication no 99/123 Project no.UT-13A The 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 Details Professor R. C. Menary Ms S. M. Garland School of Agricultural Science University of Tasmania GPO Box 252-54 Hobart TASMANIA 7001 Australia Phone: (03) 6262 6723 Fax: (03) 6262 7609 Email: [email protected] RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6272 4539 Fax: 02 6272 5877 Email: [email protected] Website: http://www.rirdc.gov.au
Published in November 1999 Printed on environmentally friendly paper by Canprint
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FOREWORD
Early in 1990 Essential Oil Industry of Tasmania (EOT) received feedback from one of it’s major purchasers, requesting information about a pesticide residue it had detected in the oil. This was the first incentive to critically assess the implications of the use of pesticides in essential oil crops. Since then, market representatives have repeatedly requested that oils purchased are accompanied by certification detailing analytical screening for pesticide contamination. The National Registration Authority (NRA) oversees the enactment of legislation dealing with the use of pesticides in Australian agriculture. Registration of pesticides for use in crops is an expensive procedure. Economic considerations dictate that that process is only undertaken for crops which have a large demand for chemicals. Smaller, intensive horticultural industries, like the production of essential oils, do not warrant such expenditure. Instead the industry relies on the granting of permits by the NRA for the use of specific pesticides. This temporary arrangement is allowed on the proviso that research continues towards the submission of data supporting the eventual application for pesticide registration. In response to these considerations, the Horticultural Research Group at the University of Tasmania instigated research into the setting of minimum residue limits for pesticides in essential oils. Significant progress in the development of analytical methods, establishment of field trials and the submission of data to the NRA has been achieved in foregoing research funded by RIRDC and industry. With ongoing liaison with industry, research has been able to respond to developments within the industry whilst maintaining the basic objectives, namely: • development and validation of analytical methods • establishment of field trials • monitoring of essential oil harvests • setting of realistic targets for minimum residue levels in essential oil products This is the second in a series of three reports culminating in the production of a technical manual detailing approaches to the analyses for pesticide residues in essential oils and it is hoped the techniques will have applications in other horticultural crops. This report, a new addition to RIRDC’s diverse range of over 400 research publications, forms part of our Essential Oils and Natural Plant extracts R&D Program which aims to support the growth of a profitable and sustainable essential oils and natural plant extracts industry in Australia. Most of our publications are available for viewing, downloading or purchasing online through our website: • downloads at www.rirdc.gov.au/reports/Index.htm • purchases at www.rirdc.gov.au/pub/cat/contents.html Peter Core Managing Director Rural Industries Research and Development Corporation
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Acknowledgements
Our gratitude and recognition is extended to Dr. Noel Davies (Central Science Laboratories, University of Tasmania) who provided considerable expertise in establishing procedures for gas chromatography mass spectrometry. Financial and ‘in kind’ support was provided by Essential Oils Industry of Tasmania, (EOT). Financial support from the Natural Products & Extracts (NPE) group is also gratefully acknowledged. Editorial contributions from Nicola Honeysett were greatly appreciated. Abbreviations
ai active ingredient
AGAL Australian Government Analytical Laboratories
NRA National Registration Authority
GC Gas Chromatography
FPD Flame Photometric Detection
HR High Resolution
MS Mass Spectrometry
MSD Mass Selective Detection
SIM Single Ion Monitoring
BAP Best Agricultural Practices
SFE Supercritical Fluid Extraction
MRL Maximum Residue Limit
ADI Average Daily Intake
ECD Electron Capture Detection
TIC Total Ion Chromatogram
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Contents Foreword......................................................................................................................iii
Acknowledgements......................................................................................................iv
Abbreviations...............................................................................................................iv
Executive Summary....................................................................................................vii
1. Introduction........................................................................................1 1.1 Background to the Project.................... …………................................1 1.2 Objectives...................................................…………….........................3 1.3 Methodology.................................................…………….......................3
2. Experimental Protocols & Results...................………......................5 2.1 Method Development.....................…………........................................5
2.1.1 Extraction Protocols..............................…..................................5 Supercritical Fluid Extraction of Folicur and Tilt from
Boronia Leaves...............................………........…...............................5
Supercritical Fluid Extraction of Monocrotophos
in Boronia Leaves...........................………......................….................8
Stability of Monocrotophos in Organic Solvents........………...............10
Aspects of Extraction of Tebuconazole from Boronia Leaf..………....12
2.1.2 Extension of Analytical Techniques......................…...........15 Optimisation of Monocrotophos Detection Limit Using GC FPD.…....15 Development of New Analytical Methods of Herbicides in Boronia….17
Method Development for the Analyses of Residues of
Linuron, Stomp, Goal & Sertin......................………............................19
Method Development for the Analyses of Residues of
Allicide, Krovar, Sinbar & Solicam..................………….......................23
Method Development for the Analyses of Residues of
Geasaguard & Verdict.......................…………....................................26
High Resolution Mass Spectra Detection of Herbicides......................28
Analyses of Herbicides with Acidic Moieties..................…..................38
2.1.3 Storage Experiments...............................................................46 Stability of the Active Ingredients of Folicur, Nuvacron and Tilt in
Boronia under Standard Storage Conditions..............………..............46
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Stability of the Active Ingredients of Folicur and Tilt in Peppermint
under Standard Storage Conditions................……….........................49
2.2 Field Trials........................................................……............................51
Degradation of Folicur and Tilt Residues in Peppermint.........………...........51
Field Trials of Folicur and Tilt in Boronia...................................……….........53
Field Trials for Assessment of New Herbicides in Boronia.........………........63
Analysis of Terbacil in Peppermint...................................………..................67
2.3 Monitoring of Harvests.................................………….......................69 Analysis of NZ Boronia Samples for Presence of 9 Pesticides.....……….....69
Pesticides in Tasmanian Boronia...............................................……............71
Monitoring of 1996 Boronia Harvest...................................………................72
Monitoring of 1997 Boronia Harvest...................................………................73
Monitoring of 1997 Boronia Harvest..............................……….....................75
3. Discussion................................................................………..........................77
4. Implications & Recommendations....................……………………..............92
5. Literature Cited..........................................................………….....................93
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Executive Summary To extend the research base of the study of pesticide residues in essential oils, the
development of extraction protocols, analytical methods, harvest monitoring and field trials
were continued.
Extraction Protocols Exhaustive extraction methods to pre concentrate and purify residues of pesticides in the
matrices of essential oils, which range from the light, volatile steam distilled oils to the
complex solvent extracted concretes, is not feasible. However, a simple sample preparation
method was developed whereby solvent extracts, containing a high level of heavy waxes,
were dissolved in a small amount of hexane. Polar solvents were added to effect a partition
such that the non-polar components of the extracts precipitated, leaving the more polar
residues of pesticides in solution.
Solvent-based extraction is the most commonly used sample preparation method in the
analyses of plant material. The consumption of large volumes of expensive solvents, the non
selective extraction conditions and the requirement of pre-concentration steps are a few of the
disadvantages associated with the method.
When gases reach a critical temperature and pressure, they behave as a liquid and are said to
be supercritical fluids. The solvent power of such fluids often exceeds those of standard
liquid solvents. The extract from Supercritical Fluid Extraction (SFE) is separated from the
supercritical fluid by reducing pressure and temperature. The extracting gas is then exhausted
and the extract re dissolved in a minimum solvent volume. The most widely used gas, carbon
dioxide, is non-toxic, non-flammable and inexpensive.
The application of SFE for the extraction of residues of Folicur and Tilt from boronia leaves
was successful, with the matrix having a minimal effect of the recovery of the pesticides. The
selective capabilities of SFE extraction and the effective pre-concentration of elutants were a
few of the advantages of this method.
SFE extraction of the active ingredient of Nuvacron, monocrotophos, had limited application.
Recoveries were low, with a large variation recorded. Monocrotophos is unstable and was
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probably degrading under the extraction conditions. The stability of the analyte in a range of
organic solvents was investigated. No appreciable change in the level of monocrotophos in
any of the solvents was observed over the period of the experiment. It was surmised that
solvents used in the SFE extract protocol could not be the cause for loss of analyte.
Extension of Analytical Techniques Monocrotophos Analyses using GC Flame Photometric Detection (FPD)
The analyses of monocrotophos by GC FPD was optimised. Monocrotophos is thermally
unstable. Results indicate that the gas chromatography of monocrotophos is optimised by
using a slow temperature gradient (~5°C/min). Peak shape and response is improved with the
injector temperature set between 200 and 225°C.
Assessment of New Herbicides in Boronia Harvests
The introduction of herbicides to reduce production costs for such harvests as boronia were to
be assessed for efficacy by industry. Before these pesticides could be used in a commercial
crop, information on the possible contamination of oil products with chemical residues was
sought. Preliminary field trials were established and analytical methods to detect possible
residues in the treated crops were developed. The large number of herbicides to be
investigated limited the extent of method validation undertaken for each of the analyses
developed. The field trials were not designed to present as a comprehensive study. However
results served as an indicator as to which pesticides warrant further investigation in terms of
weed control and risk for contamination of oils with residues.
Method development for analyses in boronia leaf was successful for Sinbar (terbacil), Goal
(oxyflurofen), Krovar (bromacil and diuron) Allicide (chloropham), Solicam (norflurazon),
Stomp (pendimethalin) Lorsban (chlorpyriphos), Friontier (dimethenamid), Sertin
(sethoxydim) and Simazine. Detection limits for these chemicals ranged from 2 ppb to 100
ppb.
The detection residues of the ai of Orthene, acephate, was achieved, however, the high signal
to noise ratio, and the poor peak resolution, meant that the detection limit was relatively high.
Some of the of herbicides investigated had an ai containing an acidic functional groups. In
the commercial formulations the parent chemicals are usually present as an ester. When these
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esters come into contact with the soil, they are cleaved so that the corresponding carboxylic
acids are formed and the herbicide becomes bioactive. Both the parent molecule and the acid
can present residue problems in essential oils. Herbicides of this type investigated were
Garlon (trichlopyr) Lontrel (clopyralid), Dicamba, MCPA, and Verdict (haloxyfop). Semi
quantitative analytical methods were developed for each of these pesticides. The parent
esters were able to be analysed directly by GC HR MS. The carboxylic acids required
derivatisation with diazomethane before GC analyses. Again method validation was not
undertaken as the experiment was designed only to give an indication of the risk of residue
contamination of crops treated with the herbicides.
Stability of the Active Ingredients of Folicur, Nuvacron and Tilt in Boronia under Standard
Storage Conditions
Validation of the methods used in the field trials of the fungicides, Tilt and Folicur, in
peppermint and boronia and the insecticide, Nuvacron, in boronia require that the effect of
leaf and oil storage on residue concentration be assessed. This data is also required to meet
the specification set by NRA for the registration of pesticides in essential oil crops.
Comprehensive experiments were conducted for all three chemicals and results indicate that
the residual pesticides in field samples do not degrade under the storage conditions trialed.
Field Trials Degradation of Folicur and Tilt Residues in Peppermint and Boronia
Field trials of Folicur and Tilt were established in commercial boronia crops. Leaf samples
were collected before and after each application of fungicide, then at 1, and 4 days after the
final application. Further samples were collected at 1, 2 and 4 weeks, then at monthly
intervals until harvest.
Results indicated that the ai of Folicur, tebuconazole, degrades more quickly in peppermint
than in boronia. The rate of dissipation in the first 2 weeks is more rapid that in the
remaining weeks. The flowers of the boronia from the field trials were harvested in spring,
around 14 weeks after the pesticides had been applied to the vegetative material in the
previous late summer. Tebuconazole is a systemic fungicides and was translocated into the
flowers through the growing season. The amount of pesticide detected in the oils was related
to that originally present in the flowers from which they were produced. The residue level
was then compared to the concentration of tebuconazole detected in the leaves collected on
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the same day. Between 0.3 to 1.7% of tebuconazole had been translocated from the leaves to
the flowers.
Tilt (ai porpiconazole) is registered for use in boronia crops and as such extensive field trials,
as required by the NRA for registration, were not conducted. However, samples were
collected over the growing season of a commercial boronia crop. In addition, results from
previous trials of Tilt in peppermint were reprocessed to allow for comparison with results
obtained in this study.
The dissipation of propiconazole in peppermint was well defined within two time periods.
Two weeks after application only 20% of the propiconazole originally detected remained. The
remaining 20% dissipate over the remaining season in a linear relationship relative to time.
In boronia, however, propiconazole residues do not so clearly follow this two tiered pattern.
Results indicate that propiconazole dissipates more rapidly in peppermint crops than it does in
boronia.
It is proposed that two mechanisms of residue dissipation predominate in the two distinct time
periods. After pesticide application the ai of the sprays dry on the surface of the vegetative
material. The interactive effects of temperature, humidity, wind and sunlight significantly
contribute to the dissipation of propiconazole. After external factors have removed residual
pesticide from the leaf surfaces, a second combination of effects including metabolism and
catabolism within the vegetative tissue determine the rate of dissipation after two weeks.
Field Trials for Assessment of New Herbicides in Boronia
During the course of the experiment visual assessments of the efficacy of the pesticides were
conducted by field staff. It is emphasised that the method establishment was preliminary and
validation of all analyses are required. The results are estimates only, an indicator as to
whether any of the pesticides trialed present potential residue problems.
Two methods of pesticide application were trialed. Directed sprays refer to the application
directed on the area at the base of boronia trees over the trial plot. Cover sprays were applied
over the entire trial area, including the existing foliage of the boronia crops. For all
herbicides for which significant residues were detected, it was evident that cover sprays
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resulted in contamination levels ten times that resulting from directed spraying in some
instances. Allicide, Sinbar 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 Frontier presented only low residue levels in
extracted flowers with adequate control for specific weeds reported by weedicide efficacy
assessments conducted by field staff.
Goal and Krovar presented only low levels of residues when used as a directed spray and was
effective as both a post and pre emergent herbicide.
Only very low levels of residues of both Sertin and Solicam were detected in boronia oil
produced in crops treated with directed spray applications. Sertin was effective as a cover
spray for grasses whilst Solicam showed potential as herbicide to be used in combination with
other chemicals such as Diuron and Gramoxone.
All of the herbicides with acidic moieties showed few traces of residue contamination in
boronia oil. This advantage, however appears to be offset by the relatively poor weed
control.
Both Stomp and Verdict showed good weed control. Both, however, present problems with
chemical residues in boronia oil and should only be used as a directed spray.
Terbacil in Peppermint
Sinbar (terbacil) is applied as pre emergence herbicide, or at early signs of regeneration on
peppermint annually. A simple experiment was conducted to determine whether terbacil is
accumulating in peppermint over the seasons, retarding plant vigour. Leaves were analysed
which had been collected from the standard commercial harvest and from plants propagated
from root stock, one and two generations removed from the original peppermint which had
been treated with Sinbar. The level recorded in the peppermint leaves were comparatively
low. It is unlikely that terbacil carry over is the cause for the lack of vigour in young
peppermint fields.
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Monitoring of Harvests Pesticides in Boronia Concrete Produced in New Zealand
Samples from boronia concretes produced in New Zealand were tested for the presence of
residues of Tilt, Nuvacron, Goal, Tramat, Linuron, Stomp, Gesaguard, Fusilade and Folicur.
Residues of the herbicides Stomp and Linuron were detected. No other pesticides were
detected in the New Zealand extracts. The detection limit for the majority of the pesticides
investigated were below 50ppb with the exception of Nuvacron (monocrotophos). Nuvacron
was not present above the 1ppm level.
Pesticides in Tasmanian Boronia
Concretes and absolutes of boronia flowers were provided by the essential oils industry in
Tasmania. The oils were selected so that there were representative samples from each of
major growers. Strict guidelines are circulated to farmers by the processors of the oils, yet
previous annual screenings indicated high levels of residues of the fungicide Tilt
(propiconazole).
Boronia oils produced in 1996, 1997 and 1998 were screened for pesticides using the
analytical methods developed. High levels of residues of Tilt 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. Outcomes
for the seasonal monitoring were communicated to industry and steps have been taken to
ensure best agricultural practices (BAP) are adopted.
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1. Introduction 1.1 Background to the Project Research undertaken by the Horticultural Research Group at the University of Tasmania
into pesticide residues in essential oils has been ongoing for several years. In December
1994, a review of the literature determined that many analytical techniques had been
developed to detect pesticide residues in aqueous media such as vegetables and water. Few
references dealt with the problems specific to the analysis of residues within the matrix of
essential oils.
In consultation with industry, the importance of a range of pesticides used in the essential
oils industry were assessed. Mancozeb (Dithane), prometryn (Gesaguard), Mecoprop,
glyphosate (Roundup) fluroxypyr (Starane), clopyralid (Lontrel), haloxyfop (Verdict)
monocrotophos (Nuvacron) and fluazifop-p-butyl (Fusilade) were identified. Of these, a
method for the simultaneous analysis of nine of the pesticides was developed.
High resolution gas chromatography mass spectrometry (GCMS) allowed for the
simultaneous analysis of boronia samples for residues of Tilt, Folicur, Goal, Tramat
Gesaguard, Verdict, Fusilade, Nuvacron and Stomp without the need for pre-concentration.
Standard curves, reproducibility and detection limits were established for each.
The remaining eight chemicals were not found to be amenable to direct analysis by GC MS.
Monitoring of the degradation product was an acceptable method for preliminary screening
for residues of Linuron and Carbaryl.
Roundup (glyphosate), Lontrel (clopyralid) and Mecoprop were simultaneously derivatised
by BSTFA (N,O-bis(trimethylsilyl)trifluroacetamide) to products amenable to gas
chromatography. The active ingredient of Starane, fluroxypyr acid, was derivatised with
diazomethane for GC MS analysis. The parent molecule, fluroxypyr ester, was amenable to
GC and did not require derivatisation.
Dithane (mancozeb) residues were digested using acidified stannous chloride and the carbon
disulphide analysed by GC coupled to FPD in the sulphur mode.
2
A series of detection methods was trialed using low resolution GC MS and GC ECD
(electron capture detection). Low resolution GC MS achieved detection to levels of 1
mg/kg in boronia oil, whilst analyses using GC ECD would require a clean-up step to
effectively detect halogenated chemicals below 1ppm.
In November 1995, field trials in peppermint crops were established in accordance with the
guidelines published by the National Registration Authority (NRA). Results for Tilt and
Folicur residues in peppermint leaves showed a marked degradation of propiconazole and
tebuconazole respectively, with levels falling from as high as 30 mg/kg to below 2 mg/kg
(wet weight) within two weeks after application. Both propiconazole and tebuconazole
were found to co-distill during the production of hydro-distilled peppermint oil.
Peppermint oils distilled from crops treated with Starane were analysed for the presence of
the parent molecule of the active ingredient, a fluroxypyr ester, and the active ingredient
itself, fluroxypyr acid. Results showed that although the acid residues were below detection
limit, the parent ester could be detected to levels as high as 3 mg/kg.
3
1.2 Objectives • Continue the development of analytical methods for the detection of pesticide
residues in essential oils.
• Validate the methods developed.
• Conduct field trials in accordance with the NRA.
• Develop comprehensive research base with facilities and methods adaptable for the
assessment of new pesticides trialed in essential oil crops.
• Provide industry with data supporting assurances of quality for all exported products.
1.3 Methodology Three 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.
Sample Preparation Techniques
Aspects of the protocols for extraction of pesticide residues from essential oil crops and
products were examined. Solvents and standards used in sample preparation were
modified and assessed.
Solvent-based extraction is the most commonly used sample preparation method in the
analyses of plant material. However large volumes of solvents are usually consumed in
this process and the extraction is non-selective, requiring further pre-concentration.
Carbon dioxide is an inexpensive, non-toxic gas which, when used under controlled
conditions of pressure and temperature, has excellent solvating powers which can be
easily adjusted to meet specific extraction requirements. This process of supercritical
fluid extraction (SFE) was investigated for application in the extraction of pesticides from
boronia leaves.
4
The effect of storage on the concentration of pesticide residues in field samples were
investigated as part of the validation of analytical methods as required by the NRA,
Analytical Methods
Communications with the essential oil industry identified a range of herbicides which
may reduce production costs for such harvests as boronia. Before these pesticides could
be used in a commercial crop, information on the possible hazards introduced by way of
contamination of oil products, was sought. Little to no data was available on many of the
chemicals of interest. In response, preliminary analytical methods were developed using
gas chromatography high resolution mass spectrometry (GC HR MS) for the detection of
an extensive range of herbicides within the matrices of boronia leaves and flowers.
Derivatisation techniques were employed where required.
• Conduct field trials to determine the degradation profile of pesticides in essential oil
crops.
Trials were established for the study of Folicur and Tilt in boronia. Experiments were
designed to meet the requirements of the NRA for the registration of Folicur in boronia.
Boronia leaf and flower samples were collected and analysed from basic field trials
established by Essential Oils of Tasmania (EOT), to assess the efficacy of the herbicides
and establish which of the chemicals present as residues in the final extract of boronia
plants.
• Provide industry with data supporting assurances of quality for all exported products.
One 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).
5
2. Experimental Protocols & Detailed Results The experimental conditions and results are presented under the following headings:
• Method Development
• Field Trials
• Monitoring of Commercial Harvests
2.1 Method Development Research in method development was undertaken to improve aspects of existing
techniques, such as extraction and sample preparation protocols and the extension of
analytical capabilities of the research project to include a range of new pesticides.
Experimental parameters and detailed results are presented in three sections:
• Extraction & Sample Preparation Protocols
• Stability of Residues in Samples from Field Trial Experiments
• Extension of Analytical Techniques
2.1.1 Extraction Protocols Supercritical Fluid Extraction of Folicur and Tilt from Boronia Leaves Solvent-based extraction is the most commonly used sample preparation method in the
analyses of plant material. The consumption of large volumes of expensive solvents, the
non-selective extraction conditions and the requirement of pre-concentration steps are a
few of the disadvantages.
When gases reach a critical temperature and pressure they behave as a liquid and are said
to be supercritical fluids. The solvent power of such fluids often exceed those of standard
liquid solvents. There are several advantages to using SFE. The most widely used gas,
carbon dioxide has a critical temperature of 304.20K, a critical pressure of 73.86 bar and
a critical density of 0.468 g/mL (IUPAC, 1976). Thus the critical point is accessible and
carbon dioxide is non-toxic, non-flammable and inexpensive. The extract from SFE can
be separated from the supercritical fluid by reducing pressure, removing the evaporation
6
step required in solvent extraction. In addition, the solvating properties of SFE can be
adjusted by controlling the temperature, pressure and solvent composition. Additions of
small amounts of polar modifiers, such as methanol, allow for the variation of extracting
power.
In this experiment we investigate the extraction of residues of the pesticides Tilt and
Folicur from the matrix of boronia leaves.
Experimental
Firstly the analytes were extracted from filter paper. Whatman # 3 filter paper (3cm)
were rolled into 2 x 10cm stainless steel extraction chambers and spiked with 100µl of a
solution containing 0.2mg/mL of each of propiconazole (Tilt) and tebuconazole (Folicur).
Leaves of boronia, which had never been treated with Tilt or Folicur, were frozen in
liquid nitrogen and ground in a stainless steel mortar and pestle. Hydromatrix, 25%, was
added to the ground plant material and the approximately 2g was packed into each of 2 x
10cm extraction chambers. These were also spiked with 100µL of 0.2mg/mL of each of
propiconazole and tebuconazole.
The receiving vessels for the SFE extraction were standard GC vials. Six GC vials were
spiked with 1mg of octadecane, to be used as an internal standard. Two of the GC vials
were spiked with 20µg of propiconazole and tebuconazole and made up to 1mL with
methanol, to be used as reference standards for the recovery calculations.
Methanol was selected as the modifier solvent. The conditions of extraction were as
follows:-
7
SFE Conditions
Extraction: static mode: 40°C for 1min., 8% modifier, 150atms dynamic mode: 40°C 1 mL /min., 8% modifier, 150 atms Collection trap temperature -20°C desorb at 10°C volume of methanol desorb: 1.0mL at 0.5mL/min flush with 3.0mL methanol at 1,0mL/min.
Samples were analysed by GC MSD.
Analytical Conditions
Equipment: HP5890 GC coupled via an open split interface to a HP 5970B mass selective detector (MSD) Injection: 1 µL, split automatic injections Column: 25 m HP1, 0.22 mm id, 0.25 µm film thickness Head pressure: 15 psi. Temp. program 50°C (held for 1 min), ramped to 220°C at 30°C/min and then at
10°C/min to 290°C (held for 5 minutes) Injection Temp: 250°C Detector: 290°C Ions monitored: 254 (C18), 259 (propiconazole) and 250 (tebuconazole)
Results
Table 1 lists the percentage recoveries recorded for propiconazole and tebuconazole
extracted by SFE.
Table 1. % Recoveries for SFE Extraction of Propiconazole and Tebuconazole in
Boronia Sample prop./C18 teb./C18 propiconazole tebuconazole
ratio ratio % recovery % recovery filter 1. 0.40 0.28 74.2 88.7 filter 2. 0.46 0.30 85.8 98.1 boronia 1. 0.39 0.29 72.7 92.7 boronia 2. 0.45 0.33 84.6 107.3
mean 79 97 std. dev. 7 8
8
Supercritical Fluid Extraction of Monocrotophos in Boronia Leaves The detection limit of monocrotophos using GC FPD or HR MS is comparatively high, as
the analyte has a poor peak shape when analysed by GC. The potential of SFE to extract
and pre-concentrate the analyte was investigated. Monocrotophos was spiked into
boronia leaves and extracted using SFE.
Experimental
5cm discs of clean filter paper were rolled and inserted into 3 stainless steel, 10mL SFE
tubes and spiked with 100µL of 0.16mg/mL solution of monocrotophos. Boronia leaf,
known to be free of contamination with monocrotophos, was ground in a stainless steel
mortar and pestle under liquid nitrogen. 2 x 10mL SFE tubes were packed with
approximately 4g of the blank boronia leaves and 0.1g of hydro matrix. The leaf samples
were spiked with 100µL of 0.16mg/mL of monocrotophos.
Boronia leaves were treated with the equivalent of 80mL/ha of Nuvacron. The
insecticide was applied with a Buchmeister backpack attached to a Matabi 1.4m boon
with a 4 nozzle cone spray. Samples were collected immediately after the spray had
dried on the leaf. Samples were ground under liquid nitrogen and 4g transferred into SFE
tubes along with 0.1g of hydromatrix.
Samples were extracted on a Varian SFE using the following conditions:-
Modifier 8% acetone Static mode 0.0g/min for 1minute at 150 atms and 40˚C Dynamic mode 1g/min – 20g CO2at 150 atms and 40˚C Collection trap 20 ˚C Desorption 1mL of acetone at 0.5mL/min at 10˚C Trap flush 3mL acetone at 1.0mL/min
9
A standard curve of monocrotophos was established by diluting 10, 0.5 and 1 µg into
1mLof acetone. All GC vials of samples and standards were spiked with 10µg
octadecane. All samples were analysed by GC MSD under the conditions listed below.
Analytical Conditions
Equipment: HP5890 GC coupled via an open split interface to a HP 5970B mass selective detector (MSD) Injection: 1 µL, split automatic injections Column: 25 m HP1, 0.22 mm id, 0.25 µm film thickness Head pressure: 15 psi. Temp. program: 50°C (held for 1 min), ramped to 220°C at 30°C/min and then at
10°C/min to 290°C (held for 5 minutes) Injection Temp: 250°C Detector: 290°C Ions monitored: 254.2973 (C18), 97.0527, 127.0160 (Nuvacron).
Results
The results for the extraction of monocrotophos using super critical fluid extraction are
listed in Table 2.
Table 2. Recoveries of Monocrotophos by SFE Sample matrix mono. int std monocrot. ratio recovery
µg peak area peak area % Standard 1 acetone 10.36 41315 522545 12.65 Standard 2 acetone 5.18 41171 302975 7.36 Standard 3 acetone 1.04 35844 103211 2.88 SFE1 filter 10.36 52420 218020 4.16 32.88 SFE2 filter 10.36 51779 663624 12.82 101.33 SFE3 bor leaves 10.36 54385 230458 4.24 33.50 SFE4 bor leaves 10.36 52746 185241 3.51 27.77 SFE5 field sple peak moved SFE6 field sple peak moved
The response factors are much lower than expected with a large variation recorded for the
recovery of monocrotophos from filter paper. Monocrotophos is unstable and is probably
degrading under the SFE conditions. A study into the stability of monocrotophos in
different solvents would provide some insight into this problem.
10
Initially no peak was recorded for the analyte extracted from the boronia leaves but it
appears that the retention time is altered by the matrix, eluting up to 0.2 minutes slower in
the boronia matrix compared to that of monocrotophos in acetone. The retention time
changed yet again when extracted from field trial samples.
A total ion trace was produced from one of the SFE extracted boronia samples to give an
indication of the components co-extracting with the monocrotophos. It was observed that
many of the chemicals seen in the normal solvent extraction of boronia are also extracted.
Stability of Monocrotophos in Organic Solvents The previous SFE experiment indicated monocrotophos may be labile in organic
solvents. This simple experiment is to determine the stability the insecticide.
Experimental
Three x 30mL scintillation vials were spiked with approximately 2 to 4 mg of pure
monocrotophos. Analytical grade acetone, 25mLs, was added to the first scintillation
vial, then 1mL was quantitatively transferred to each of 2 GC vials, labelled 1 and 4.
Chloroform, 25mLs, was added to scintillation vial 2 and 25mLs of benzene was added to
scintillation vial 3. Again 1mL of each solution was transferred to GC vials labelled 2
and 3 respectively. Each of the 4 GC vials were spiked with 25µL of a 0.222mg/mL
solution of octadecane. 10mg of boronia concrete was added to GC vial 4. 1µL of each
vial was analysed by GC MSD under the conditions listed below. Each of the 4 samples
was analysed over a period of 24 hours at intervals of approximately 1 hour.
11
Analytical conditions
Equipment: HP5890 GC coupled via an open split interface to a HP 5970B mass selective detector (MSD)
Injection: 1 µL, split automatic injections Column: 25 m HP1, 0.22 mm id, 0.25 µm film thickness Head pressure: 15 psi. Temp program: 50°C (held for 1 min), ramped to 220°C at 30°C/min and then at
10°C/min to 290°C (held for 5 minutes) Injection temp: 250°C Detector: 290°C Ions monitored: 254.2973 (C18), 97.0527, 127.0160 (Nuvacron).
Results
Initially no appreciable change in the level of monocrotophos was observed over that
period. Instead, a slight priming effect was evident with subsequent injection of
monocrotophos consistently recording a higher peak area than the one immediately
preceding. Towards the end of the experiment a reduction in concentration of
monocrotophos was observed, however, 90% remained in solution after 24 hours. From
this experiment it can be assumed that the loss of pesticide observed in the SFE process
cannot be due to the effect of solvents alone.
Aspects of Extraction of Tebuconazole from Boronia Leaf Previous experiments detailed in RIRDC Final Report 96, had dealt with the analysis of
oils dissolved in hexane and using octadecane as the internal standard. Field trial
samples of tebuconazole in boronia, however, involve the extraction of wet leaf samples.
As such a relatively polar solvent was required to ensure partitioning does not occur
between the residual water and the extracting solvent. Previously octadecane had been
used as the internal standard in analyses using GC HR MS. This was successful because,
the amount of octadecane used to spike the peppermint samples was low.
The suitability of using polar extraction solvents in conjunction with a non-polar internal
standard was investigated.
Approximately 2-3g of ground boronia samples were extracted in 5 mLs of methanol.
12
1mL was subsampled and spiked with 1.2167 mg of octadecane. Chloroform, 0.4mL,
was added to aid the solvation of the internal standard.
A standard curve was constructed using 4.79g of boronia leaves which had never been
treated with fungicides. These were was extracted in 10mL of methanol. Five GC vials
were spiked with 100µl, 10µL, 1µL and 0µL of 0.212mg/mL of tebuconazole and 1.2167
mg of octadecane. 1mL of the blank leaf sample extract and 0.4 mL of chloroform were
added to each vial and analysed using the same conditions as those to be used for the
field trial samples of Folicur in boronia.
Analytical Conditions
Equipment: HP5890 GC coupled via an open split interface to a HP 5970B mass selective detector (MSD) Injection: 1 µL, split automatic injections Column: 25 m HP1, 0.22 mm id, 0.25 µm film thickness Head pressure: 15 psi. Temp. program 50°C (held for 1 min), ramped to 220°C at 30°C/min and then at
10°C/min to 290°C (held for 5 minutes) Injection Temp: 250°C Detector: 290°C Ions monitored: 254 (C18), and 250 (tebuconazole) Analysis proceeded and the results for the standard curves are presented below in Table
3.
Table3. Standard Curve for Folicur Sample Folicur C18 Area Folicur Folic ratio
wt. (µg ) Area FT std 1 21.2 17658820 11042935 0.62535 FT std 2 2.12 17789825 908385 0.05106 FT std 3 0.212 16637787 110180 0.00662 FT std 4 0 17189967 0 0.00000
Regression Statistics
R Square 1.000 x1 33.951
From the results shown above it appeared that the use of polar extracting solvents and
non-polar internal standard would give consistent results. However, when analyses were
13
undertaken, the response for the internal standard was very erratic. This variability in the
response for octadecane was most likely due to precipitation of the chemical when the
water content of the leaf sample increased the polarity of the extracting solvent.
Endosulfan has similar volatility to tebuconazole. A solution of endosulfan was prepared
by dissolving 4.8mg of the purified pesticide in 25mLs of acetone. The solution was
used to spike 1mL of chloroform containing 2.12µg of tebuconazole and an acetone
extract of boronia containing a similar quantity of tebuconazole. The solutions were
analysed by GC MSD using the same conditions listed above, with the exception that ion
194 was monitored for an ion fragment of endosulfan, instead of ion 254 (octadecane).
Midway through the experiment the MSD was retuned The change in response after this
event emphasises the need to re-establish the standard curve with each experiment .
Despite this the mean tebuconazole area to endosulfan area was consistent within the
time frames of the two different instrument tunings as shown in Table 4.
Table 4. Peak Areas and Ratios for Folicur and Endosulfan Time Endosulfan Folicur Ratio
area area Thurs 11:57am
1625876 3487242 2.14
Thurs 12:14pm
1637882 3716514 2.27
Thurs 12:32pm
1638461 3425711 2.09
Thurs 12:50pm
1578592 3313062 2.10
mean 2.151 st.dev. 0.08 %co. var. 3.8 Thurs 1:08 pm
1533488 5167070 3.37
Thurs 1:30 pm
1502368 5300467 3.53
Thurs 1:53 pm
1567992 5709143 3.64
Thurs 2:15 pm
1488677 5420032 3.64
mean 3.545 st.dev. 0.13 %co. var. 3.6
To determine the stability of the response a 2µg/mL standard was run to check the
consistency of the response areas of the analytes. The following Table 5 lists the times
and results of repeat injections.
14
Table 5. Peak Areas and Ratios for Folicur and Endosulfan - Repeat Time Endosulfan Folicur Ratio
area area Fri 4:32 pm 1390830 1530854 1.10 Fri 4:55 pm 1460547 1769263 1.21 Fri 8:00 pm 1568475 2572507 1.64 Fri 10:39 pm 1185207 1519919 1.28 Sat 9:09 am 1323779 2078456 1.57 Sat 11:47 am 951445 1537837 1.62 Sat 2:24 pm 1033634 1639207 1.59 Sat. 5:26 pm 2009933 3441277 1.71 mean 1.46 st.dev. 0.23 %co. var. 15.7
24 hour interval
Sun 5:04 pm 1743289 3328705 1.91 Sun 7:41 pm 1947554 4087547 2.10 Sun10:18 pm 2344345 4768814 2.03 Mon 1:19 am 2449763 5708950 2.33 Mon 3:34 am 1794702 3653934 2.04 Mon 5:27 am 1928334 3816721 1.98 Mon 2647791 5475146 2.07 mean 2.07 st.dev. 0.13 %co. var. 6.4
Response was very consistent within each analysis time frame. When analysis was
interrupted by a 24 hour period the ratio, calculated relative to the standard, increased but
remained constant through the remainder of the experiment. It was determined that
endosulfan is suitable as an internal standard.
2.1.2 Extension of Analytical Techniques Optimisation of Monocrotophos Detection Limit Using GC FPD Monocrotophos is thermally unstable and analysis by GC often results in poor peak
resolution and response. An experiment was conducted to optimise analytical conditions
by adjusting the oven temperature programs gradient and the injector temperature.
Experimental
Analytical grade monocrotophos (13.8 mg), supplied by Sigma Aldrich, was dissolved in
100mL of acetone. This was used to prepare a second 13.8µg/mL solution by diluting
15
1:100. Four GC vials were spiked with 25µL, 2.5µL of the 1.38 mg/mL solution and
25µL and 2.5µL of the 13.8 µg/mL solution. Each vial was spiked with 2µg of
chlorpyriphos, a phosphate containing chemical, included as an internal standard. All
were dissolved in 1mL of acetone.
The samples were analysed using the GC FPD. Basic conditions are listed below.
Analytical Conditions
Equipment: HP5890 GC coupled to flame photometric detector Injection: 1 µL, split automatic injections Column: SGE 30m BPX5, ID 0.32, film thickness 0.25m Head pressure: 12 psi. Temp. program: 60°C (held for 10 min), ramped to 80°C at 1°C/min and then at 2.5°C/min to 120°C Injection temp: 250°C Detector: 280°C
16
Several temperature gradients were trialed:
1. isothermal 150°C
2. 60°C (held for 2 min) 5°C/min to 250°C
3. 60°C (held for 2 min) 10°C/min to 250°C
4. 60°C (held for 2 min) 20°C/min to 250°C
To determine the detection limit of monocrotophos within the matrix of boronia concrete
4 x ~ 25mg of boronia concrete were dissolved in 1mL of acetone and spiked as
described above. A range of injector temperatures were trialed to minimise the
degradation of the analyte in the injection chamber.
Results
On an isothermal program at 150°C monocrotophos eluted at 11.911 mins, with poor
peak resolution. The 0.138 µg/mL solution gave a peak area of 69584, whilst the area of
the internal standard, chloropyriphos, was 229311. Temperature programs of 10 and 20
°C/min ramps were trialed. However, the 5°C/min ramp proved to be the program which
resulted in the best peak resolution and response. Table 6 shows the peak areas obtained
for the four solutions prepared.
Table 6. Peak Areas of Analytes using GC Temperature Gradient 5°C/min Peak area 138 µg/mL 13.8µg/mL 1.38µg/mL 0.138µg/mL monocrotophos 315897 33237 4981 not detected internal standard 878258 840260 756951
Table 7 lists the areas obtained for monocrotophos and the internal standard when
analysed within the matrix of boronia concrete.
17
Table 7. Peak Areas of Analytes in Boronia Concrete Injector
temperature
monocrotophos
peak area
int. std.
peak area
peak shape
150 150272 354028 poor
190 373733 437309 poor
200 459098 675330 good
210 338123 523065 good
225 423130 844443 good
250 315897 878258 poor
280 250454 849380 poor
Results indicate that the gas chromatography of monocrotophos is optimised by using a
slow temperature gradient. Peak shape and response is improved with the injector
temperature set between 200 and 225°C.
Development of New Methods for the Detection of Herbicides in Boronia Harvests Communications with the essential oil industry identified a range of herbicides which
may reduce production costs. Before these pesticides could be used in a commercial
crop, information on the possible hazards introduced by way of contamination of oil
products, was sought. Little to no data was available on many of the chemicals of
interest. It was devised that basic field trials would be established to assess the efficacy
of the herbicides and establish which of the chemicals present as residues in the final
extract of boronia plants.
As these trails were established as a preliminary experiment, the test parameters did not
follow those detailed in the guidelines published by the NRA. In addition, the large
number of herbicides to be investigated limited the degree of validation undertaken for
each of the analytical methods developed. As such analytical grade standards were not
purchased for the majority of chemicals investigated. For some, the active ingredient was
purified from the commercial product. Where time constraints made this unfeasible, the
commercial product was used to establish methods and standard curves, with the %ai
composition used to calculate the concentrations.
18
Initially, pesticides were divided into two groups. Those that were amenable to GC and
pesticides whose ai incorporated an acidic moiety into their molecular structure.
Standard solutions of the GC amenable pesticides were prepared. Table 8 lists the
solutions prepared.
Table 8. Standard Solutions of Pesticides Pesticide Active ingredient Weight
(mg) Volume
chloroform Goal oxyflurofen 3.1 25 Linuron 4.1 25 Stomp pendimethalin 5.8 50 Sertin sethoxydim 6.4 50 Krovar diuron & bromacil 104.4 50 Sinbar terbacil 62.7 50 Allicide chlorpropham 98.8 50 Solicam norflurazon 81.3 50 Flexidor isoxaben 95.7 50 Gesaguard prometryn 5.8 50 Orthene acephate 16.9 100 Lorsban chlorpyriphos 9.6 100 Simazine 23.7 100 Sulfan oryazalin 24.2 100 Frontier dimethenamid 21.7 100 Verdict haloxyfop 2.5 25
The first step of method development involved the analyses of mixtures of the solutions
by GC MSD to determine the elution orders, response values and mass spectra. These
parameters could then be used to establish analytical protocols on the high resolution MS
GC.
For convenience the pesticides were dealt with in four groups. Group 4 chemicals did not
undergo the first step of GC MSD analyses before transposing the methodology to GC
HR MS. These were the active ingredients: simazine, dimethenamid, acephate,
chlorpyriphos and oryzalin.
The standard solutions of linuron, pendimethalin. oxyflurofen and sethoxydim were
combined to produce a solution which contained approximately 30µg/mL of each ai.
19
Method Development for the Analyses of Residues of Linuron, Stomp, Goal and Sertin Standards for the ai of Stomp (pendimethalin) was supplied by the Australian
Government Analytical Laboratories (AGAL). Linuron and the ai of Goal, oxyflurofen
had been purified previously (see RIRDC Final Report 1996). Sethoxydim is present at a
concentration of 186.8g/L in the commercial product, Sertin. Sufficient quantities of
these products were weighed into 50mL volumetric flasks to give solutions of 1mg/mL.
Actual weights sub-sampled are listed in Table 8. The four solutions were subsampled
and combined to produce a mixed solution containing approximately 30µg/mL of each ai.
Samples were analysed by GC MSD under the conditions listed below in the total ion
monitoring mode.
20
Analytical conditions
Equipment: HP5890 GC coupled via an open split interface to a HP 5970B mass selective detector (MSD)
Injection: 1 µL, split automatic injections Column: 25 m HP5, 0.22 mm id, 0.25 µm film thickness Head pressure: 25 psi. Temp. program: 50°C (held for 1 min), ramped to 290°C at 10°C/min (held for 5
minutes) Injection temp: 250°C Detector: 290°C
Figure 1 shows the total ion trace recorded. Figures 2, 3, 4 and 5 record the mass spectra
recorded for linuron, pendimethalin, oxyflurofen and sethoxydim respectively.
21
Figure 1. Total Ion Chromatogram for a Solution of Linuron, Pendimethalin,
Oxyflurofen and Sethoxydim
Figure 2. Mass Spectrum of Linuron
22
Figure 3. Mass Spectrum of Pendimethalin
Figure 4. Mass Spectrum of Oxyflurofen
Figure 5. Mass Spectrum of Sethoxydim
23
Method Development for the Analyses of Residues of Allicide, Krovar, Sinbar and Solicam The same process was followed for the remaining GC amenable pesticides.
Chloropropham was present in the commercial herbicide, Allicide, at 800g/L. Diuron
and bromacil were present at levels of 400g/Kg each in Krovar. Terbacil (800g/L) and
norflurazon (800g/L) were the ai of Sinbar and Solicam respectively. The weights
subsampled are also recorded in Table 8.
The four solutions were also subsampled and combined to produced a mixed chloroform
solution containing approximately 30µg/mL of each ai. The herbicides were analysed
24
under the same conditions listed above. Figure 6 shows the total ion chromatogram
(TIC) of the solution, followed by the MS of each ai in Figures 7 to10.
Figure 6. Total Ion Chromatogram of a Solution of Diuron, Chloropropham,
Terbacil, Bromacil and Norflurazon
Figure 7. Mass Spectrum of Chloropropham
25
Figure 8. Mass Spectrum of Terbacil
Figure 9. Mass Spectrum of Bromacil
Figure 10. Mass Spectrum of Norflurazon
26
All were suitable for analysis by GC, except sethoxydim, whose parent ion was not
evident in the peak identified as sethoxydim related.
As found previously, both linuron and diuron are thermally labile. The degradation
product may be monitored for a semi-quantitative analysis.
Method Development for the Analyses of Residues of Gesaguard and Verdict Finally, the active ingredients of Gesaguard (prometryn) and the parent molecule of the
active ingredient of Verdict (haloxyfop-etotyl), which had been supplied purified by
AGAL, were solvated in chloroform and made up in a volumetric flask to a concentration
of 30µg/mL and analysed under the same conditions listed above. The TIC for the GC
MSD run is illustrated in Figure 11, with the mass spectra of the two analytes shown in
Figures 12 and 13.
27
Figure 11. Total Ion Chromatogram of Solution of Prometryn and Haloxyfop-etotyl
Figure 12. Mass Spectrum of Prometryn
Figure 13. Mass Spectrum of Haloxyfop-etotyl
28
High Resolution Mass Spectrometry of Herbicides The mass spectra for the 11 herbicides detailed above were used to establish a analytical
method using high resolution mass spectrometry, gas chromatography. For convenience,
the herbicides were investigated in separate groups. A method for terbacil and
oxyflurofen was developed first.
Terbacil and Oxyflurofen
Experimental
A 0.1 µg/mL acetone solution of terbacil and oxyflurofen was prepared from stock
solutions detailed above. Boronia leaves collected from a farm on Bruny Island,
Southern Tasmania, which had never been treated with terbacil or oxyflurofen were
ground under liquid nitrogen. Subsamples of 3g of ground leaf were weighed into 6 x
30mL scintillation vials. A recovery experiment was established by fortifying the ground
leaves in 4 of the vials with 0.25µg of each analyte using the 100µg/mL solution of
terbacil and oxyflurofen. 5mLs of acetone was added to each of the vials and the
mixtures were placed on shaker bath at room temperature for 3 hours. 1mL of each of the
four fortified boronia leaf samples were quantitatively transferred to GC vials.
To establish a standard curve, 4 GC vials were spiked with 0.1, 0.01, 0.001 and 0.0005
µg of the 2 herbicides using the standard solutions. 1mL of the extract from the 2 ground
leaf samples which had not been fortified with analytes (blank) were used to provide a
matrix for the standard curve. 1mL of the blank extract solution was transferred to each
of the GC vials. 5µL of a 4.8mg/mL solution of the internal standard, endosulfan, was
added to all the GC vials.
Conditions for High Resolution Mass Spectrometry GC
Samples were analysed on a HP 5890 Gas Chromatogram directly coupled to a Kratos
Concept ISQ Mass Spectrometer. The GC was equipped with a BPX5 fused silica
capillary column (25m, 0.22mmi.d, o.25µm film thickness).
1µl splitless injections of samples were analysed using a carrier gas flow program of 30
29
psi/min from 25 to 40 psi, held for 0.1 min, then at 30 psi/min to 25 psi, then at 1 psi/min
to 35 psi. The GC injection temperature was 260°C and the oven temperature
programmed from 60°C to 290°C at 20°C/min. Ions monitored for terbacil was 161.0117
between 5:00 and 7:50 mins. Ion 194.9534 was monitored for internal standard,
endosulfan, between 7:50 and 8:12 minutes. For oxyflurofen, ion 252.0398, monitored
between 8:12 to 9:00 minutes was used for quantitative assessment. A dwell time of
300ms/ion and 50ppm voltage sweep were employed for all ions. Resolution of 10,000
(10% valley definition) and the ion m/z 242.9856 from perfluorokerosene was used as the
lock mass for all analytes and the internal standard. Electron ionisation was undertaken
at a source temperature of 210°C and an electron energy of 70eV, with an accelerating
voltage of 5.3kV. A representative chromatogram is shown in Figure 14.
Figure 14. GC High Resolution MS Trace for Oxyflurofen, Terbacil and
Endosulfan at Levels of 1ppm
30
Terbacil and oxyflurofen were detectable to levels of 2ppb of wet leaf weight.
Recoveries were 68.8% (st.dev. 8.8) and 104 (st.dev. 4.3) for terbacil and oxyflurofen
respectively.
Bromacil, Diuron, Chloropham, Norflurazon and Pendimethalin
A range of solutions were prepared for the chemicals as described for the previous
experiment from which a standard curve could be constructed. The recovery experiment
was also conducted in a similar manner to that described, whereby untreated boronia
leaves were fortified with the analytes and extracted concurrently with the real leaf
samples. The basic parameters for the GC HR MS were as detailed above. The ions
monitored for the 5 analytes are listed below:-
Stomp: pendimethalin 252.0984 (C11H14N3O4), 281.1375 (C13H19N3)4) Krovar: diuron 186.9584, 188.9555 bromacil 204.9613, 206.9593 Allicide: chloropropham 215.0529 (C10H12ClNO2), 213.0557 Solicam: norflurazon 303.0386 (C12H9ClF3N3O), 305.0359
Results
Figure 15 displays an example trace. Below are the regression statistics for the standard
curves established for 4 of the analytes investigated. Diuron, which degraded when
analysed in organic solvents such as chloromethane, did not degrade when analysed
within the matrix of the boronia extract. The matrix is in some way protecting the diuron
chemical. A second method development experiment will be required using the ions
produced in the MS of the non degraded chemical. Table 9 lists the results for the
recovery experiment.
31
Figure 15. Example Trace of the Analyses of a Pesticide Standard
SOLCIAM-Norflurazon Regression Statistics
R Square 1.000 x1 0.175 STOMP-Pendimethalin Regression Statistics
R Square 1.000 x1 0.220 KROVAR-Bromacil Regression Statistics
R Square 1.000 x1 0.123 ALLICIDE-chloropropham Regression Statistics
R Square 0.999 x1 0.243
Table 9. Recovery Experiment Results ai % recovery st. dev.
norflurazon 85.7 10 pendimethalin 108.8 27 bromacil 63.7 5 chloropropham 88.8 7
32
Diuron, Bromacil, Chlorpyriphos, Acephate, Dimethenamid, Simazine, Sethoxydim and Oryzalin
Method work up for analytes previously dealt with are included in this experiment. The
parameters for Bromacil, for example, had been determined in previous experiments.
However, we need to analyse for Bromacil simultaneously with the new target chemicals.
The active ingredients of Orthene (acephate) and chlorpyriphos were available as purified
standards from AGAL. 16.9 and 9.6 mg of each were dissolved in 100mLs of
chloroform. Solutions of 10µL/mL were prepared by dissolving or diluting Frontier,
Simazine and Sulfan in chloroform as detailed in Table 8. These solutions were diluted
to give a solution containing 10µL/mL of each. Again standard solutions were used to
fortify leaf extract with a range of pesticide solutions to construct a standard curve.
Recovery experiments were also repeated by spiking ground untreated leaves with 10 µL
of the 10 µg/mL solutions.
33
GC HR MS Conditions
The basic parameters for the GC analyses was as previously detailed. A BPX5 column
was used. The pressure gradient was different, however, with the pressure rising from 25
to 35 psi at 1 psi/min.
Results
A full scan of the mixture of the 9 pesticides was conducted on the GC HR MS. Figure
16 show the chromatogram obtained. Figures 17 to 20 show the mass spectra of the
relevant analytes.
Figure 16. Total Ion Chromatogram of 9 Mixed Pesticides by GC HR MS.
34
35
Figure 17. Mass Spectrum of Chlorpyriphos
Figure 18. Mass Spectrum of Acephate
36
Figure 19. Mass Spectrum of Dimethenamid
Figure 20. High Resolution Mass Spectrum of Sethoxydim
From the information provided from the full scan, single ion monitoring (SIM) for each
37
pesticide used the ions listed.
diuron 186.9584, 188.9555 (artefact), retention time 4:59
acephate 136.0164, retention time 5:51
simazine 201.0781, 186.0546, retention time 7:44
dimethenamid 230.0406, 232.0327, retention time 8:23
bromacil 204.9613, 314, 316, retention time 8:53
chlorpyriphos 196.9202, 198.9172, retention time 8:54
norflurazon 303.0386, retention time 10:40
sethoxydim 191.0946, 219.1259, retention time 10:53
endosulfan (internal standard) 194.9534
Figure 21 records the simultaneous analyses of the herbicides by GC HR MS in the SIM
mode.
38
Figure 21. GC HR MS Chromatogram of Norflurazon, Bromacil, Chlorpyriphos,
Sethoxydim, Dimethenamid, Simazine, Acephate and the Artefact Produced by
Thermal Degradation of Diuron.
39
Oryzalin was not detectable. Diuron degradation was not consistent under the GC
conditions and the different matrixes made quantitative analyses of diuron unreliable.
Acephate background noise to signal ratio was very high such that the detection limit of
the analyte will be relatively high.
Bromacil and chlorpyriphos eluted as one peak and could only be distinguished when
separate ions were monitored.
Analyses of Herbicides with Acidic Moieties There are several complications in the analyses of the active ingredient of the herbicides
with acidic functional groups. In the commercial formulations, the parent chemicals are
presented in the ester form. When the esters come into contact with the soil, the ester is
cleaved so that a carboxylic acid is formed. It is the acidic form which is bioactive.
When monitoring for residues for these chemical types, consideration has to be given to
the detection of the parent chemical as well as the active ingredient. In addition, often
the parent chemicals come in several forms of the ester (i.e methyl and ethyl esters). The
acidic forms require derivatisation to their methyl ester to render the chemicals GC
amenable. This was done using diazomethane.
The herbicides investigated were Garlon (trichlopyr), Lontrel (clopyralid), Dicamba,
MCPA and Verdict (haloxyfop). The derivatisation of trichlopyr and the structures of the
active ingredients of the herbicides are shown below.
40
Again, leaves from the farm located on Bruny Island, which had not been treated with
pesticides, were ground under liquid nitrogen. Samples of ground leaves (3g) were
transferred to 6 x 30mL scintillation vials. A recovery experiment was established by
fortifying the ground leaves in 4 of the vials with 0.25µg of each analyte using the
100µg/mL solution of pesticides. 5mLs of acetone was added to each of the vials and the
mixtures were placed on shaker bath at room temperature for 3 hours. 1mL of each of the
4 fortified boronia leaf samples were quantitatively transferred to GC vials.
To establish a standard curve, 4 GC vials were spiked with 0.1, 0.01, 0.001 and 0.0005
µg of the acidic herbicides using the standard solutions. 0.75 mL of the extract from the
2 ground leaf samples which had not been fortified with analytes (blank) were used to
provide a matrix for the standard curve. 0.75 mL of the blank extract solution was
41
transferred to each of the GC vials. 5µL of a 4.8mg/mL solution of the internal standard,
endosulfan, was added to all the GC vials.
Diazomethane Solution
Potassium hydroxide,16g, was weighed into a 250mL conical flask. 40mL of distilled
water and 50mL of diethyl ether was added. The mixture was placed on ice. N-methyl-
N-nitrosourea (3g) was added slowly and the mixture was stirred for 20 minutes. The
mix was transferred to a separatory funnel and the aqueous layer drained and washed
with a further 25mLs of ether. The ether solutions were combined and stored at -4°C
until use.
Derivatisation Procedure
0.75mL of the diazomethane in ether solution was added to the 0.75 mL solutions in GC
vials containing various levels of the mixed herbicides for the standard curves and
recovery experiments. The vials were left at room temperature for 10 minutes. 200µL of
glacial acetic acid was added to each vial to quench the remaining diazomethane. GC
vials were crimp sealed, ready for analysis.
GC HR MS Conditions
As for previous experiments, a full scan was first completed to determine retention time
of the analytes and the mass spectra. GC conditions were as previously detailed. The
mass spectra of triclopyr parent ester, dicamba and clopyralid derivatised methyl ester
and the parent ester of haloxyfop are recorded in Figures 22 to 25.
42
Figure 22. Mass Spectra of Ethylene Glycol Butyl Ether Ester of Triclopyr
Figure 23. Mass Spectra of Methyl Ester of Dicamba
43
Figure 24. Mass Spectra of Methyl Ester of Clopyralid
Figure 25. Mass Spectra of 2-Ethoxy Ethyl Ester of Haloxyfop
The three pesticides Dicamba, fluroxypyr and clopyralid were in the acidic form when
44
standards were prepared. However, the active ingredients of Garlon and Verdict were
still present as esters in the standards prepared. Triclopyr (Garlon) was present as an
ethylene glycol butyl ether ester, whilst haloxyfop (Verdict) was present as the ethoxy
ethyl ester. Establishing a method for the monitoring of the methylated form, produced
by the derivatisation of the carboxylic acid forms of the chemicals could only be done by
estimating a retention time and the selecting ions based on the mass spectra. The
expected ions were monitored over a time range covering the estimated retention time.
A SIM sequence was established for the following ions:-
haloxyfop ethoxy ethyl ester 316.0352 retention time 10:47
endosulfan internal std. 194.9534 retention time 9:50
trichlopyr butoxy ethyl ester 209.9280 retention time 10:00
Trichlopyr methyl ester 211.9251, 20939280 retention time 7:25
MCPA methyl ester 214.0397 retention time 6:39
Dicamba methyl ester 202.9666 retention time 6:21
Clopyralid methyl ester 173.9513 retention time 5:58
Results
Figure 26 records the ion traces for the simultaneous analyses of the herbicides.
45
Figure 26. GC HR MS SIM of Herbicides with an Acidic Moiety
46
Verdict: The haloxyfop ester of verdict was detected down to the level of 2 ng in 1.5mLs.
This is related back to the 3g of leaf extracted such that the lower detection limit of the
parent ester in ground leaf is 0.07ppb relative to wet weight. The recovery experiment
yielded 84% (std. dev. 3) of the fortified haloxyfop parent ester. The derivatised ester of
the carboxylic acid was detected in the standard of highest concentration.
Verdict-Haloxyfop Regression Statistics
R Square 1.000 x1 0.105
The regression statistics calculated for the haloxyfop parent ester cannot directly apply to
the response of the methyl ester of the carboxylic acid. However, as a standard curve for
the methyl ester cannot be established without purifying the carboxylic acid form, it is
necessary to use the regression statistics of the haloxyfop parent ester by relating the
relative abundances of the ions monitored for each analyte. The relative abundances of
mass to charge ratio (m/z) of ions monitored by HR MS was assumed to be equal. The
regression statistic of the ion monitored for the parent ester was related to the ion
monitored for the carboxylic acid ester by multiplying the slope of the graph of the
standard curve by the ratio of the relative abundances of the 2 ions monitored. It must be
emphasised that this is not a standard quantitative method. The experiment is designed to
only give us an indication of the contamination of boronia crops after the application of
herbicides. Method development would require the isolation of the acidic form of the
pesticide to establish standard curves, recoveries and repeatability of the analyses.
Garlon (triclopyr): The parent ester of triclopyr was detected to 0.001µg in 1.5mL,
equivalent to 0.3 ppb of ground weight (wet). The recovery experiment yielded 85%
(std. dev. 8) of the fortified analyte. The regression statistics of the parent ester were
again related to the carboxylic acid ester by multiplying the slope of the standard curve
by the relative abundance of the 2 ions monitored.
Garlon-Trichlorpyr ester Regression Statistics
R Square 1.000 x1 0.317
47
Dicamba, MCPA and Lontrel (clopyralid): The purified carboxylic acid form of these
three herbicides were available such that standard curves could be established for each.
The regression statistics are listed below.
MCPA Regression Statistics
R Square 0.989
x1 0.098
Dicamba Regression Statistics
R Square 0.992
x1 0.082
Lontrel-Clopyralid Regression Statistics
R Square 0.989
x1 0.617
The detection limit for MCPA was 0.002 µg in 1.5mL or 0.4ppb of ground leaf wet
weight. Recoveries yielded 63.2 % (std. dev. 0.8) of the fortified standard.
The detection limit for Dicamba was 0.2ng in 1.5mLs or 0.07 ppb of ground leaf wet
weight. recoveries yielded 60% (std. dev. 5) of the fortified analyte.
The detection limit of clopyralid was 12 ng in 1.5 mLs or 4ppb of ground leaf wet
weight. Only 46 % (std. dev. 0.3) of the analyte was recovered from the fortified
sample.
2.1.3 Storage Experiments Stability of the Active Ingredients of Folicur, Nuvacron and Tilt in Boronia under Standard Storage Conditions Field trials, monitoring the degradation of pesticides in boronia crops, had been
established in 1995 and 1996. Samples were collected at the relevant time periods after
pesticide application, sealed in plastic bags and frozen at -18°C. Samples were stored for
periods of 2 weeks to over one year. It was necessary to determine if the analytes were
48
stable under storage conditions, to validate the experimental method and to fulfil the
requirements of the NRA for the registration of pesticides in essential oil crops.
Experimental
Boronia, which had not been treated with Folicur, Tilt or Nuvacron, were obtained from a
commercial boronia farm situated on Bruny Island in Southern Tasmania. The leaves
were separated from the woody stems and ground under liquid nitrogen. 2g of each was
weighed into 33 x 20mL scintillation vials. Each vial was spiked with 2µL of 0.2mg/mL
solution of propiconazole and tebuconazole. Each vial was further spiked with 50µL of
0.1mg/mL solution of monocrotophos.
The vials were labelled NFB 1 to 33. Vials 4 to 33 were placed in a -18°C freezer. 5
mLs of acetone were added to samples NFB 1 to 3 were sonicated for 10mins and left to
stand for 10mins.
A standard curve was constructed by extracting 4g of untreated boronia with 15mLs of
acetone. 1mL aliquots were transferred to GC vials and spiked with 5µL, 1µL and 0µL
of 0.2mg/mL tebuconazole and propiconazole solutions and 10µL, 2µL and 0µL of a
0.1mg/mL solution of monocrotophos.
The samples were analysed by high resolution GCMS under the following conditions:-
Injection: 1 µL, split automatic injections Column: 25 m HP1, 0.22 mm id, 0.25 µm film thickness Gas Flow: 25 psi - 30 psi/ min - 40 psi (0.1 min) - 30 psi/min - 25
psi - 1 psi/min - 35 psi Temp. program: 60°C-20°C/min-290°C Injection temp: 230°C Detector: Kratos high resolution mass spectrometer Ions monitored: 254.2973 (C18),252.0714, 250.0743 (Folicur), 261.0262,
259.0291(Tilt), 97.0527, 127.0160 (Nuvacron)
Table 10 lists the dates on which the frozen samples were thawed, extracted and
analysed.
49
Table 10. Analysis Dates of Pesticide Storage Experiment - Boronia Date Days in storage Sample Names 19-Feb-97 0 NFB 1-3 11-Mar-97 20 NFB 4-6 26-Mar-97 35 NFB 7-9 13-May-97 83 NFB 10-12 13-Nov-97 269 NFB 13- 15 28 Mar. 98 402 NFB 16-18
Tables 11, 12 and 13 show the results of the storage experiment involving tebuconazole,
propiconazole and monocrotophos in boronia samples.
Table 11. % Tebuconazole Remaining in Boronia After Storage Date Storage µg tebuconazole % tebuconazole
days remaining 19-Feb-97 0 1.06 ± 0.008 100.0 11-Mar-97 20 0.92 ± 0.04 86.7 26-Mar-97 35 1.00 ± 0.04 94.1 13-May-97 83 0.92 ± 0.03 86.5 13-Nov-97 269 0.95 ± 0.04 89.8 28 Mar. 98 402 1.06 ± 0.03 100.2
Table 12. % Propiconazole Remaining in Boronia After Storage Date Storage µg propiconzole % propiconazole
days remaining 19-Feb-97 0 1.18 ± 0.004 100.0 11-Mar-97 20 1.00 ± 0.03 84.8 26-Mar-97 35 1.21 ± 0.12 102.8 13-May-97 83 1.03 ± 0.03 87.4 13-Nov-97 269 0.95 ± 0.03 80.7 28 Mar. 98 402 1.20 ± 0.03 101.9
Table 13. % Monocrotophos Remaining in Boronia After Storage
Date Storage µg. % monocrotoph. days monocrotophos remaining
19-Feb-97 0 1.36 ± 0.16 100.0 11-Mar-97 20 1.36 ± 0.05 99.8 26-Mar-97 35 1.39 ± 0.36 102.0 13-May-97 83 1.39 ± 0.12 102.2 13-Nov-97 269 Analyses failed 28 Mar. 98 402 1.43 ± 0.07 105.3
50
Stability of the Active Ingredients of Folicur and Tilt in Peppermint under Standard Storage Conditions Field trials, monitoring the degradation of pesticides in peppermint crops, had been
established in 1995 and 1996. Samples were collected at the relevant time periods after
pesticide application and sealed in plastic bags and frozen at -18°C. Samples were stored
for periods of 2 weeks to over one year. It was necessary to determine if the analytes
were stable under storage conditions, to validate the experimental method and to fulfil the
requirements of the NRA for the registration of pesticides in essential oil crops.
Experimental
Peppermint, which had not been treated with Folicur or Tilt over a period of 8 months
prior to collection, was obtained from a commercial peppermint farm situated in the
Meander Valley in Northern Tasmania. The leaves were ground under liquid nitrogen.
2g of each was weighed into 33 x 20mL scintillation vials. Each vial was spiked with
2µL of 0.2mg/mL solution of propiconazole and tebuconazole.
The vials were labelled FTP 1 to 33. Vials 4 to 33 were placed in a -18°C freezer. 5 mLs
of methanol were added to samples FTP 1 to 3 which were sonicated for 10mins and left
to stand for 10mins.
A standard curve was constructed by extracting 4g of untreated peppermint with 15mLs
of methanol. 1mL aliquots were transferred to GC vials and spiked with 5µL, 1µL and
0µL of 0.2mg/mL tebuconazole and propiconazole solutions.
The samples were analysed by high resolution GCMS under the following conditions.
51
Injection: 1 µL, split automatic injections Column: 25 m HP1, 0.22 mm id, 0.25 µm film thickness Gas Flow: 25 psi - 30 psi/ min - 40 psi (0.1 min) - 30 psi/min - 25
psi - 1 psi/min - 35 psi Temp. program: 60°C-20°C/min-290°C Injection Temp: 230°C Detector: Kratos high resolution mass spectrometer Ions monitored: 254.2973 (C18),252.0714, 250.0743 (Folicur), 261.0262
Table 14 lists the dates on which the frozen peppermint samples were thawed, extracted
and analysed.
Table 14. Analysis dates of Pesticide Storage Experiment - Peppermint Date Days in storage Sample Names 19-Feb-97 0 FTP 1-3 11-Mar-97 20 FTP 4-6 26-Mar-97 35 FTP 7-9 13-May-97 83 FTP 10-12 13-Nov-97 269 FTP 13- 15 28 Mar. 98 402 FTP 16-18
The results for the storage experiment involving tebuconazole and propiconazole in
peppermint samples are shown in Table 15.
Table 15. % Tebuconazole Remaining in Peppermint After Storage Date Storage µg tebuconazole % remaining
days detected 19-Feb-97 0 0.95 ± 0.09 100.0 11-Mar-97 20 1.00 ± 0.06 105.3 26-Mar-97 35 0.5 ± 0.2 52.6 13-May-97 83 0.74 ± 0.09 77.9 13-Nov-97 269 0.87 ± 0.04 91.6 28 Mar. 98 402 0.87 ± 0.08 91.6
Table 16. % Propiconazole Remaining in Peppermint After Storage Date Storage µg propiconazole % remaining
days detected 19-Feb-97 0 1.03 ± 0.08 100.0 11-Mar-97 20 1.07 ± 0.08 103.9 26-Mar-97 35 0.7 ± 0.1 68.0 13-May-97 83 1.10 ± 0.08 106.8 13-Nov-97 269 0.91 ± 0.01 88.3 28 Mar. 98 402 1.1 ± 0.1 106.8
52
2.2 Field Trials Degradation of Folicur and Tilt Residues in Peppermint In the RIRDC Final Report 96, the field trials to determine the degradation of residues of
Folicur and Tilt in peppermint were reported. The results are re-presented here relative to
the dry weight of peppermint analysed. More importantly, the results listed below are
required in this report for the comparison of degradation profiles of pesticides in different
crops. The basic outline of the experiment was as follows.
At two selected sites in Southern Tasmania, 12 plots of 36m2 areas were selected. There
were 2 application rates/pesticide. 1 and 2 times the normal application rates of 500mL
/ha. Each site required 3 applications of all pesticides, at approximately 2 week intervals.
Samples were taken before and after each application. Re-sampling occurred from
thereon at 1 day, 4 days, 2 week, and 4 week intervals. Samples were collected by
harvesting 1m2 at random from each block, ensuring no sample was taken from within
1m from each block boundary. The samples were transferred to plastic bags and sealed.
Each sample was weighed and frozen until the day of analysis.
Leaf Extraction
5g of each leaf sample was placed in a large test tube. 25mLs of redistilled methanol
were added and the samples were homogenised. 1mL of extract was quantitatively
transferred to GC vials. 25µL of 0.22mg/mL of octadecane, the internal standard, was
added and the vials sealed for analysis for residues of Tilt and Folicur. Repeatability
studies and recovery experiments are detailed in RIRDC Report 96.
The peak areas obtained for the standard curves were related to pesticide residue
concentration by linear regression. The slopes of the standard curves were used to
calculate the residue mass in the subsample of peppermint analysed by applying the
formula:
µg of propiconazole = slope x peak area
The results for the field trial are presented in this report relative to the dry weight of
53
peppermint harvested in Tables 17, 18 and 19.
Table 17. The Degradation of Tebuconazole & Propiconazole in Peppermint at Site
2 Treatment 0.5 L/ha Tilt 0.5 L/ha Folicur
Days mean st. dev. mean st. dev. mg/Kg mg/Kg
post application 1 0 48 13 31 7 prior application 2 14 1.9 0.8 3 3 post application 2 14 56 9 43 29 prior application 3 24 1.9 0.8 2.4 0.5 post application 3 24 41 13 30 19
25 33 9 27 5 28 23 1 21 10 38 1.9 0.4 1.7 0.1 47 0.7 0.5 1.5 0.3 113 0.2 0.2 0.2 0.2
Table 18. The Degradation of Tebuconazole in Peppermint at Site 1 & 2 Treatment 0.5 L/ha Foli. 1.0 L/ha Folicur
Days mean std. mean std. µg/Kg dev. µg/Kg dev.
post application 1 0 54 27 55 25 prior application 2 17 1.9 0.9 4 4 post application 2 17 268 119 prior application 3 32 1.2 0.1 11 7 post application 3 32 38 6 142 73
33 17 5 65 6 36 28 18 45 15 45 4 2 4.9 0.7 64 0.26 0.06 1.3 0.5 96 0.3 0.2 0.8 0.4 oil 0.011 0.003 0.04 0.01
54
Table 19. The Degradation of Propiconazole in Peppermint at Site 1 & 2 Treatment 0.5 L/ha Tilt 1.0 L/ha Tilt
Days mean std. mean std. µg/Kg dev. µg/Kg dev.
post application 1 0 51 18 62 36 prior application 2 17 0.45 0.04 0.5 0.1 post application 2 17 83 30 168 20 prior application 3 32 1.6 0.3 3.9 0.9 post application 3 32 52 6 90 10
33 37 8 79 6 36 22 4 49 10 45 1.31 0.07 2.1 0.9 64 0.3 0.1 0.6 0.2 96 0.06 0.04 0.09 0.12
Field Trials of Folicur and Tilt in Boronia Tilt is used to combat rust in boronia. Previous studies (RIRDC Final Report 96) showed
that residues of propiconazole, the ai of Tilt, is of concern for the essential oil industry.
An alternative fungicide, Folicur, when used in alternate years to Tilt, may reduce the
accumulation of propiconazole in boronia. Folicur, is not registered for use in boronia.
As such, field trials were established, not only to determine the expected residue levels
of the ai, tebuconazole, but to gather data for presentation to the NRA with view to
obtaining formal registration.
Field Trial Establishment Folicur in Boronia
Two sites were selected for the trial of Folicur in boronia crops. These were located in
Lower Longley, owned by R. McEldowney, and South Bruny Island, owned by Gregg,
both in Southern Tasmania.
1 x 10 meter rows were marked with pegs and sprayed with applications of 20mL of
Folicur, 100g of Dithane and 20mLs of Bond wetting agent in 20L of water. Dithane is
applied in conjunction to the systemic fungicides to all boronia crops in Tasmania to
achieve adequate control of fungal spores. Buffer zones of 4 m were left at the end of
each row. The Site 1 application was with a automated, tractor driven spraying unit with
timing adjusted to deliver 500mL of Folicur and 1Kg of Dithane per hectare as
55
recommended. The application was repeated to give 3 replicate blocks. To comply
