Determination of residues of Compound X after one application of Product Y in bulb onion across 8 sites in Europe 2013-2014
1. 1
Jake Turner
University of Nottingham
F105 – Chemistry with a Year in Industry
Determination of residues of
Compound X after one application of
Product Y in bulb onion across 8 sites
in Europe 2013-2014
2015
Eurofins Agroscience Services
Matthew Allen
Alexander Blake
2. 2
Abstract:
The aim of this work was to evaluate the residues of Compound X in bulb onions after one
application of Product Y using HPLC/MS/MS. Compound X first had to be extracted from
the representative samples, a process that needed to be developed to reliably produce a 70-
110% recoveries for known concentrations of Compound X and Compound X’s ester (the
form of Compound X contained in Product Y).
The extraction method was substantially improved. Mean recoveries were increased from
55% to 101%. Statistical analysis of the decline curve indicates a root function first order
degradation mechanism.
7. 7
Introduction
Background Information
There are currently 129 herbicides approved for use in Europe under Reg. (EC) No 1107/2009
(EU Pesticide Database, 2015)1. No major new mode of action has been discovered for about
20 years. All 137 new herbicide active ingredients introduced from 1980 to 2009 had modes
of actions from other herbicides introduced before 19902. This apparent halt in herbicide
research comes from a shift the market took in 1996, when the first glyphosate-resistant (GR)
crop was introduced3. This now meant farmers could use glyphosate as a postemergance
herbicide against broadleaf and cereal weeds whilst leaving their genetically modified crop
unharmed. GR crops now account for more than 85% of transgenic crops grown worldwide4 5.
Farmers soon began using this technology year after year, placing immense selection pressure
on the weeds, exacerbating the already-present problem of the evolution of glyphosate
resistant weeds. They created an ecological hole, where any weed developing a resistance to
glyphosate can live in an unoccupied environment, devoid of any competition. Fields in the
mid-western United States have Amaranthus tuberculatus plants with resistances to ALS
(acetolactate synthase inhibitors), PPO (protoporphyrinogen oxidase inhibitors) and
glyphosate in every possible combination6. The worrying trend of increased evolution and
spread of glyphosate resistant weeds has caused an increase in investment for herbicide
discovery, as it is clear that a new mode of action is needed to which weeds have not evolved
a resistance to. The route from discovery to market, however, is not simple.
After determining that a herbicide is effective, it must be determined safe for use in Europe by
the European Commission before it can be marketed. Several properties of the product must
be evaluated at length, including the residues of the active compound in the final commercial
product. The data requirements for regulation are detailed in the official document
8. 8
“COMMISSION REGULATION (EU) No 284/2013 of 1 March 2013; setting out the data
requirements for plant protection products, in accordance with Regulation (EC) No 1107/2009
of the European Parliament and of the Council concerning the placing of plant protection
products on the market”. It is stated in Section 3 that all tests and analysis shall be conducted
in accordance with GLP (Good Laboratory Practice), a set of regulations detailed in
“Statutory Instrument 1999 No. 3106: The Good Laboratory Practice Regulations” and
“Statutory Instrument 2004 No. 994: The Good Laboratory Practice (Codification
Amendments etc) Regulations”.
This means that a residue decline study must be carried out. A crop must be grown,
sprayed, harvested, and analysed over several time points.
The whole process is by no means cheap or particularly quick. Figure 1 shows the cost of
developing a new plant protection product through the years 1995-20087.
Figure 1. The cost of developing a new plant protection product has increased steadily every
year.
9. 9
Although the cost of the research has stayed fairly constant, the cost of the field trials and the
developmental chemistry has increased dramatically, partly due to an increase in the data that
must be presented when the product comes to be registered. I have been told, somewhat
anecdotally, that the amount of paperwork produced for a single new compound could easily
fill the office I work in, which I conservatively estimate to be 150 m3. Wolfram Alpha tells
me this equates to approximately 24 million sheets of A4 paper.
Although residue analysis is a very small part of the data that must be presented, therefore a
very small part of the cost, it is essential that the industry continues to develop quicker and
less expensive methods for conducting studies to help reduce the cost of the whole process if
the glyphosate resistance menace is to be stopped.
Even with no new mode of action to develop and study, new plant protection products are
constantly being formulated, whether it be new molecules or existing ones. These still need to
go through the process detailed above, but have the advantage of years of research and
experience to help speed it up.
In this work, a new herbicide (Product Y) has been formulated using an existing active
ingredient (Compound X).
If the results of this project are to support (or oppose) the eventual marketing of Product Y,
every piece of raw data generated had to be traceable and reported under the GLP guidelines.
10. 10
Compound X
As Product Y is not yet registered, it would be a breach of confidentiality to disclose
structural information on Compound X. Although Compound X is an existing herbicide that is
widely used, Product Y is of new formulation.
Discussion can take place around the class of herbicides Compound X belongs to, as well as
general unspecific information about its functional groups that influence its chemistry.
Compound X is a synthetic auxin. Auxins are a group of plant hormones that have central
roles in regulating growth and behaviour in all stages of a plant’s life cycle. In high
concentrations, auxins are toxic to the plant, although they display more toxicity to
dicotyledons (plants who’s seeds have two embryotic leaves) than to Monocotyledons (plants
who’s seeds have one embryotic leaf)8. This can be exploited, as many crops of interest (such
as onion) are monocots, and many weeds are dicots.
In high doses, auxins stimulate a production of ethylene, another plant hormone. Ethylene
inhibits elongation growth, causes leaves to fall off, and can even cause plant death9.
Structurally, all auxins are aromatic rings with carboxylic acid groups. Compound X is a
pyridine- carboxylic acid auxin. As well as a carboxylic acid group, there are halide
substitutes and an amine substitute on the pyridine ring that serve to decrease electron density
on the ring.
Compound X does not have a significant vapour pressure below 250 oC, and exists almost
entirely in the ionized form (pKa < 3), making it suitable for LC/MS/MS analysis and
unsuitable for GC/MS analysis without derivatization.
11. 11
Product Y
Below is the product label, with sensitive information (such as product number) deleted.
Test Item 1
Name PRODUCT Y
Appearance / colour
clear faint yellow
Intended usage
herbicide
Active ingredient 1 COMPOUND X
Content of active
ingredient / Purity
nominal
200 g/L
Content of active
ingredient / Purity
analysed
201 g/L
Density (20 °C) analysed 0.970 g/cm³ Risk symbol(s)
Harmful
Certificate / Date of
analysis 17 Nov 2011
Expiry date
17 Nov 2013
Stability in spray solution
sufficient for the test
purpose (at least 1h)
Storage conditions
room temperature (> 5
°C, < 30°C)
Field Trials
In order to register PRODUCT Y, data must be presented on how much of COMPOUND X is
present in the crop at normal commercial harvest (NCH), and how the concentration of
COMPOUND X varies as the crop grows. This data needs to be gathered in a range of
growing conditions in different climates.
12. 12
The length of time that the crop takes to grow depends on a lot of environmental
factors, and hence will be different in different countries, or even different regions in the same
country. It is therefore impossible to conduct every field trial over the same length of time.
Instead, the BBCH (Biologische Bundesanstalt, Bundessortenamt und CHemische Industrie)
scale10 must be used to determine when the onion is at a certain phenological development
stage. The onion is therefore sampled at the same stage in its growth, regardless of how long
it has been growing.
The application of PRODUCT Y and the sampling occurred at the following BBCH stages:
For trials TRIAL-01,-02,-03,-09: Field Phases
Visit timing Activity Timing Phenological Stage:
1 Application 16 BBCH 6 leaves clearly visible
2 Sampling NCH (49 BBCH) Leaves dead, bulb top dry
13. 13
For trials TRIAL-05,-06,-07,-10: Field Phases
Visit timing Activity Timingb Phenological Stage:
1
Application 16 BBCH 6 leaves clearly visable
Sampling 1
0 DAA1 (0 days after
application 1)
Leaves dead, bulb top dry
2 Sampling 2 41 – 42 BBCH
Leaf bases begin to thicken or
extend
3 Sampling 3 45 BBCH
50% of the expected bulb or
shaft diameter reached
4 Sampling 4 47 – 48 BBCH
Bolting begins; in 10% of the
plants leaves bent over 70%
of the expected shaft length
and diameter reached
Leaves bent over in 50% of
plants
5 Sampling 5 NCH (49 BBCH) Leaves dead, bulb top dry
Eurofins has teams in many different European countries capable of conducting the field
trials. To obtain a variety of different climates and conditions, different sites were selected in
the UK (three in Wilson), Germany (Stade), France (Elne, Alsace and Meauzac), and Spain
(two in Seville).
Trial code Place of test
TRIAL-01 EAS UK, Wilson
TRIAL-02 EAS Germany, Stade
TRIAL-03 EAS France, Elne
TRIAL-04 EAS Spain, Seville
TRIAL-05 EAS UK, Wilson
TRIAL-06 EAS France, Alsace
TRIAL-07 EAS France, Meauzac
TRIAL-08 EAS Spain, Seville
14. 14
As is usually the case with such a large international study, various setbacks gave cause for
relocation mid-way through. Setbacks included trials being destroyed by storms and farmers
accidentally harvesting the crop before they could be collected as samples. Hence, the final
table of trial codes and their places of testing is as followed:
Lab phase / Trial
code(s)
Place of test
TRIAL-01 EAS UK, Wilson
TRIAL-02 EAS Germany, Stade
TRIAL-03 EAS France, Elne
TRIAL-05 EAS UK, Wilson
TRIAL-06 EAS Germany, Stade
TRIAL-07 EAS Spain, Canals
TRIAL-09 EAS Spain, Seville
TRIAL-10 EAS Spain, Seville
At the time of this project, trials 1-9 had been completed, with trial 10 awaiting normal
commercial harvest.
15. 15
The field sites for the trials were selected under the following requirements:
Crop variety Typical of those grown commercially in the region
Minimum distances between field
sites
Trials within the same study must be at least 15
kilometres apart or must differ significantly:
At least a different crop variety and another parameter
(soil, perennial age, TRV parameters, period of
application). Trials must be conducted in typical crop
growing regions in each country
Plot identification / replicates /
suggested plot size
U1 = untreated / 1 / suggested 30 m² for NCH studies /
120 m2 Residue Decline Studies
2 = treated/ 1/ suggested 30 m² for NCH studies / 120
m2 Residue Decline Studies
Minimum distance to edge of field 5 m (10 m if drift from neighbouring fields is an issue)
Minimum distance between control
and treated plot(s)
10 m
Minimum buffer zone to next use
of competing chemistry
10 m
Soil characterisation required Indication of soil type (non-GLP accepted). Laboratory
characterisation is not required.
Weather data (kind of data and
origin) required
Rainfall totals and air temperature minimum, maximum
and averages (daily and historical) obtained from the
nearest weather station (non-GLP accepted)
Limitation on competing chemistry
on the trial site (plots and buffer
zone)
No other formulations containing COMPOUND X to be
applied during trial and 12 months before start of trial
Information on pesticide history
required
Yes, current growing season and previous 3 years (non-
GLP accepted)
Information on maintenance history
required (cultivation, irrigation)
Yes, current growing season (non-GLP accepted)
Disposal of harvest Test Item is NOTregistered and harvest should be
destroyed
Special requirements Ensure plot size is large enough to obtain sufficient
specimen material
16. 16
Once the field site was ready, PRODUCT Y needed to be dispensed according to the
following standards:
Preparation of application
solution
Test item should be measured using a balance, if density is
available.
The amount of active ingredient applied will be calculated based
on the nominal values.
Application mode Foliar with plot sprayer
Type of nozzles Appropriate to growth stage and according to good agricultural
practice
Spray tolerance 10 %
Dose verification Measuring prepared and remaining spray solution after
application using calibrated equipment
Conditions on site Record in raw data : air temperature, soil temperature at 10 cm,
wind speed, relative humidity, cloud cover, at each day of
application (GLP data)
Record in raw data rainfall on the day of application
The boom used to spray the trial was calibrated. The evidence of calibration can be found in
Appendix 4.
The goal of the application procedure was to spray the same amount of PRODUCT Y per area
of onion crop.
17. 17
Discussion
Establishing the Limit of Quantification
The limit of quantification (LOQ) is the lowest concentration of the analyte that can be
quantified with certainty. In this study, the LOQ had been set to 0.01 mg/kg, based off
previous work done at the company with Compound X.
The limit of detection (LOD) is the lowest concentration of the analyte that can be
distinguished from the absence of the analyte. In this work, the LOD is set at 25% of the
LOQ.
If we are to measure residues, we need to compare instrument response to known
concentrations of Compound X. We do this by making calibration standards from the stock
solution of Compound X. The calibration range will stretch from the LOD to 250% of the
LOQ.
To convert the LOQ residue into an equivalent concentration, we multiply it by the
final sample concentration:
0.01 𝑚𝑔 𝑘𝑔−1
= 0.01 µ𝑔 𝑔−1
0.01 µ𝑔 𝑔−1
∗
𝐹𝑖𝑛𝑎𝑙 𝑆𝑎𝑚𝑝𝑙𝑒 𝑊𝑒𝑖𝑔ℎ𝑡 ( 𝑔)
𝐹𝑖𝑛𝑎𝑙 𝑆𝑎𝑚𝑝𝑙𝑒 𝑉𝑜𝑙𝑢𝑚𝑒 ( 𝑚𝐿)
= 𝐿𝑂𝑄 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 (µ𝑔 𝑚𝐿−1
)
The final sample weight will be 10 g, as at no point an aliquot of extract is taken
(hence the full initial ‘weight’ of matrix will present in the final sample), and the final sample
volume is 10 ml. Hence, the LOQ equivalent standard is 0.01 µg/mL.
The calibration range used consisted of standards at 0.0025 µg/ml, 0.005 µg/ml, 0.025
µg/ml, 0.05 µg/ml, 0.125 µg/ml, and 0.25 µg/ml.
18. 18
Calculating Residues
To calculate the residues, a linear (R > 0.995) calibration curve must be constructed with a
minimum of 5 matrix match standards. Matrix match standards are used to account for any
matrix effects that might suppress or enhance the signal, leading to incorrect residue results
with solvent standards. Six matrix match standards were used in every run in case a more
linear response could be generated by dropping a standard from the curve.
It is important to prove that the curve is linear as high concentrations of the analyte
may overload the detector, and any further increase in concentration will not increase the
response, resulting in a flat topped curve. Therefore, any sample analysed to be over the linear
range cannot be quantified with confidence.
Once the range of concentrations is proved linear, the concentration of the sample can
be calculated. This can be done by calculation from the curve, or with bracketing standards.
Calculation from the curve calculates concentration from the sample’s peak area, and
the equation of the regression line. This is utilized when a change in instrument response
throughout the run is not a concern.
The bracketing standards method involves injecting a small amount of samples, in this
case three, bracketed by a single standard. For example, the sequence would read:
0.05µg/ml Standard
Sample-001
Sample-002
Sample-003
0.05 µg/ml Standard
The peak area of the two standards is averaged, and used in the following equation to
calculate concentration:
19. 19
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑢𝑔 𝑚𝑙−1
)
=
𝑃𝐴 𝑠𝑎𝑚𝑝𝑙𝑒 ∗ 𝐹𝑉 ( 𝑚𝐿) ∗ 𝐷𝐹 ∗ 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (µ𝑔 𝑚𝑙−1)
𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑃𝐴 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑𝑠 ∗ 𝐹𝑊( 𝑔)
PA = Peak Area
FV = Final Volume of Extract (mL)
DF = Dilution Factor
FW = Final Weight of Extract (g)
This technique is used when the response of the instrument drifts over the length of
the run. Seen as it is proven that the range is linear, now each sample peak area is considered
as a ratio to a changing peak area of a known concentration. Individual standard peak areas
must not differ from the mean of the two peak areas by more than 10%, as this indicates a
response change of significance in the time between the two standards being injected.
Batch Requirements
Experimental work was divided into batches. Two fortified recoveries (0.01 mg/kg and a level
concomitant with residues expected in the samples) were required per batch, per analyte. For
a batch to be successful, the mean residue in these recoveries needed to be within 70-110% of
their fortified values, with the relative standard deviation of these recoveries not exceeding
20%. This ensured that the extraction process was effective. In addition, a reagent blank (R-
Blank) that is essentially just the extraction solvent with no matrix or analyte must be
extracted alongside the samples to prove that the residues were not inherent in the reagents.
Compound X recoveries were used to test the extractability, and Compound X Ester
recoveries were used to evaluate the extent of hydrolysis.
The recoveries were fortified using standard solutions. For the making of these
solutions, please refer to Appendix 1.
20. 20
Experimental Procedure
The general procedure of extraction was a twice repeated 15 min. shake in solvent followed
by a hydrolysis then a liquid-liquid partition.
75 ml of Methanol/0.25N KOH was added to 10 g of the sample, which was then
shaken for 15 min. The sample was centrifuged, and the supernatant filtered off into a round
bottomed flask. The extract was then hydrolysed.
The aim was to hydrolyse the ester into the potassium salt, disallowing the reverse
reaction of the alcohol with the carboxylic acid back into the ester. The methanol and alcohol
could then be removed with a rotary film evaporator (RFE). At this point, I deviated from the
supplied method and used an additional 10 ml of solvent to wash the filter through. The
cotton wool used was reduced from a dark green colour to a light brown. Although this
addition to the method mildly increases the cost of the extraction, I thought it was justified as
it had the potential to save many re-extractions by ensuring none of Compound X was lost to
the cotton wool. As I found out later on in the study, this addition may have been the reason
behind the ‘jellification’ of certain samples, which caused a lot of re-extractions, as I have
discussed below in the batch by batch walk-though.
The hydrolysis was done under a reflux condenser. Original conditions were 1 hr at
80°C. This was increased to 2 hrs at 100°C. Thermal decomposition of Compound X occurs
at >350°C, and the autoignition temperature of the solvent is >400°C. Although it was
possible to gain high recoveries without this increase in hydrolysis time and temperature, the
step guaranteed full hydrolysis and did not elongate the method, as the samples were naturally
left cooling for an hour too long due to the schedule of the working day.
Once cooled, the extracts were filtered again through cotton wool. I again deviated
from the method by using an additional 10 ml of methanol to wash the filter. The alcohols
21. 21
were then removed via a RFE, leaving Compound X in an aqueous phase. Care was taken to
not take the extract to dryness to prevent loss of Compound X in aqueous solvent clusters.
The concentrated extract is then acidified to convert the Compound X potassium salt
into the protonated carboxylic acid, reducing its polarity and thus its affinity for the aqueous
phase.
This extract is transferred to a 50 ml centrifuge tube. A twice repeated liquid-liquid
partition was performed in ethyl acetate. Before adding the ethyl acetate to the tube, I used it
to wash the round bottomed flask to guarantee complete transfer of Compound X.
The organic phase was transferred and combined into a small round bottomed flask,
taken to near dryness on the RFE, and then gently taken to dryness with a stream of air. The
extract was then re-dissolved in acidified methanol. This acidity ensures the carboxyl acid
remains protonated through the column. Although protonated, Compound X is still polar due
to all the electron-withdrawing groups on the aromatic ring. This is essential to the
chromatographic process in reversed phase chromatography.
Batch-by-batch walk-through
Initially, two extraction batches were planned, one for the ‘whole plant’ matrix and one for
the ‘bulb’ matrix. This proved to be optimistic, as eight batches ended up being extracted.
Table 1 shows the batch numbers, their size, and their fate.
22. 22
Batch
No.
Size (Samples +
Recoveries)
Matrix Batch
successful?
Reason for
failure
1 19 Bulb No
Mean recoveries
outside the range
70-110%
2 19 Whole Plant
Yes, upon further
investigation
Initial ester
recoveries were
determined to be
outside acceptable
range
3 19 Bulb No
Mean recovery for
Compound X
R0.01 52%
4 6 Whole Plant No Samples jellified
5 12 Bulb Yes N/A
6 12 Bulb Yes N/A
7 4 Whole Plant Yes N/A
8 6 Whole Plant Yes N/A
Table 1. A table to showthe size ofeach batch, along with their matrix, whether they passed or
failed, and their reasons for failure if applicable.
When I started work on the project, batches 1 and 2 had been extracted and analysed. The
study was handed to me with both of these batches having failed.
My first intention was to re-extract a batch, implementing the improvements I had planned
and detailed above. I soon found that a large batch of 19 samples was difficult to extract in a
day. Under GLP regulations it is ideal that all samples in a batch be extracted at the same
time. This removes the possibility of a change in conditions during the batch that could affect
the results.
23. 23
The main problem lay in the RFE stages. Due to a lack of automatic RFE units, the vacuum
pressure had to be adjusted manually, with no accurate indication of the pressure reading.
This lead to a lot of trial and error in getting the pressure correct, often times the pressure was
assessed to be too low, only to have the mixture bump and boil over into the RFE upon a
slight opening of the valve.
Once a sample had been sucked into the RFE unit, it was considered lost, as the
potential for contamination and/or loss of Compound X was considered too high.
I assessed the length of time between the current date and the date of the last study
involving Compound X. Over 12 months had passed, and thus the possibility of
contamination was considered low. This did not apply to the units that had already been used
in the study, as samples had already been lost. To decrease the likelihood of contamination, I
cleaned the RFE unit’s with DECON® 90. To assess whether this was sufficient, I flushed an
R-blank through the cleaned unit, then took the condensed solvent through the extraction
process. The resulting chromatograph showed no contamination, as seen in Figure 2.
Figure 2. The R-blank sample proves there is no Compound X residues inside the RFE after a
DECON®
Clean
24. 24
Next, it had to be assessed whether the full amount of Compound X could be retrieved
from the RFE unit once it had been sucked through. I avoided washing with solvents, as this
would increase the volume of the mixture that needed putting through the RFE again, further
increasing the chance of losing the sample again. To collect as much as possible from the
RFE, the unit was pivoted until all the liquid in the condenser flowed into the condensate-
collecting flask, then left to drain for 5 mins.
Analysis of one recovery at a level of 0.01 mg/kg was performed. The recovery was
deliberately left to boil over to a degree seen in previous batches. There was approximately 20
ml of solvent collected in the flask.
The peak area of this recovery was compared to an equivalent standard of 0.01 µg/ml.
The comparison was run in triplicate. Figure 3 shows the comparison of a single set of
chromatographs, whilst Table 2 shows the average difference of peak area.
Figure 3. The 0.01µg/ml Standard (left) compared with the R0.01 mg/kg recovery (right)
0.01 µg/ml Standard Peak Area R0.01 mg/kg Recovery Peak Area Recovery %
214303 193380 90
215576 192005 89
216134 198921 92
Average: 90
Table 2. Proof that samples taken through the RFE can be recovered with no impact.
25. 25
The results of this test were conclusive, a sample could be taken through the REF and
the results would not be affected.
To further prove this, a recovery could be extracted that was not deliberately flushed
through the RFE alongside one that was. A comparison of these recoveries would determine
how much was lost to the RFE, and how much was lost to the extraction itself.
Batch 3
Due to the difficulties of extracting a full sized batch in a day, I chose to reduce the number of
samples per batch from 14 (recoveries and R-blanks not included) to 7. This does increase
the total number of samples that need extracting (as every new batch requires an R-Blank and
4 recoveries), but reduces the risk that batches will need re-extracting.
Whilst I was extracting batch 3, I noticed that particular samples, including the recovery that
ultimately failed low, thickened into a jelly in the hydrolysis step. I hypothesized that this
must be the starch becoming a gel during the heating process. It appeared that in the previous
filtration step, if too much of the plant cellular material passed through the filter, the resulting
filtrate would thicken upon heating.
When entering the results of batch 3 into the calculation spreadsheet, I discovered that batch 2
had been calculated incorrectly.
In the case of the ester recoveries, a conversion must take place to factor in the
difference in molecular masses of the Compound X ester and Compound X.
This was performed in batch 2, but was complicated by the contamination of the
untreated sample used for the recovery. It was necessary to correct the residue of the
recoveries by the amount found in the untreated.
26. 26
In this case, the conversion for the ester was done before subtracting the residue in the
untreated, leading to subtracting a Compound X residue from a Compound X ester residue.
This lead to a much lower recovery. I corrected the order of conversion, and recoveries
increased to acceptable levels.
Batch 4
Before the bulb could be re-extracted, another issue needed to be addressed.
The recoveries used should be concomitant with the levels of residue expected in the samples.
In the now-passed second batch, residues were found well above the level of 0.1 mg/kg. This
meant that I could not be certain that I could fully extract residues at the level I was seeing in
my results.
Proof of this was to be found by extracting very high level recoveries. As I did not know
whether the results of the high residue samples were correct, or were actually much higher, it
would be useless to extract recoveries just higher than the levels I was seeing. Instead, I chose
to extract recoveries at 50 mg/kg. If these samples could be extracted, the levels in the field
samples certainly could too.
Batch 4 consisted of an R-blank, an untreated, a pair of 0.01mg/kg Compound X and
Compound X ester recoveries, and a pair of 50 mg/kg Compound X and Compound X ester
recoveries.
After the hydrolysis step, all the samples except the blank were found to be jellified, despite
the extra care I had taken during the filtration. I concluded that it must be the additional
flushing of the cotton wool with the extra solvent that caused the excess starch to be present. I
removed this additional step, and encountered no further jellification thereafter.
27. 27
As the jellification process was already shown to reduce recoveries, I discarded the
batch as not to waste valuable instrumental run time.
Batch 5
This batch was the first of two that covered the onion bulb matrix, previously extracted in
batch 1.
Batch 5 was carried out successfully. I encountered no problems aside from the occasional
loss of sample through the RFE, which were swiftly recovered with no impact on the results.
The average recovery for the batch was 94% with a relative standard deviation of 10%.
Batch 6
This was the second of the two batches that covered the onion bulb matrix, previously
extracted in batch 1.
Batch 6 was also carried out successfully.
The average recovery for the batch was 96% with a relative standard deviation of 14%.
Batch 7
This batch was the re-extraction of the 50 mg/kg recoveries needed to prove batch 2 was
successful. Between the time of batch 4 and batch 7, I had realised that I did not need to
extract low recoveries with this batch. The study plan did state that the batches needed 4
recoveries, but an exception could be made here as no field samples were being analysed. The
batch size was thus reduced to 4, with a R-blank, and untreated, and the two 50 mg/kg
recoveries.
The recoveries needed to be diluted once the final extract had been gathered to ensure they
were within the calibrated range of the detector, or the detector would become overloaded and
28. 28
the recoveries would fail low. I chose to dilute them by a factor of 500 to take them into the
range of 0.01 mg/kg.
The matrix match standards for this batch were made using the untreated sample diluted by a
factor of 500 to preserve the same matrix composition.
The batch was successful, with an average recovery of 101% with a relative standard
deviation of 14%.
Batch 8
At this point in the study, I thought I had completed the analytical phase of the project. That
was until I discovered that in batch 2 a sample had been lost to the RFE. This one sample was
the only remaining thing to be analysed. If I had realised this earlier, I could have place it
inside the high recovery batch 7.
The batch was successful with an average recovery of 92% with a relative standard deviation
of 9%.
Results
NCH Trials
It was found that in all trials, the residue in the onion at normal commercial harvest was
below the LOQ (>0.01mg/kg).
Trial-01
Timing Trt Sample code Commodity Compound X residue found (mg/kg)
NCH U1 01-001A Bulb <0.01
NCH T 01-002A Bulb <0.01
29. 29
Trial-02
Timing Trt Sample code Commodity Compound X residue found (mg/kg)
NCH U1 02-001A Bulb <0.01
NCH T 02-002A Bulb <0.01
Trial-03
Timing Trt Sample code Commodity Compound X residue found (mg/kg)
NCH U1 03-001A Bulb <0.01
NCH T 03-002A Bulb <0.01
Trial-09
Timing Trt Sample code Commodity Compound X residue found
(mg/kg)
NCH U1 09-001A Bulb <0.01
NCH T 09-002A Bulb <0.01
Decline Trials
Trials 6 and 7 looked like typical decline graphs, but trial 5 exhibits qualities that indicate a
spraying mistake, an analytical mistake, or a unique climate conditions effect.
It is easier to describe the mathematical properties of the decline curves if the results can be
plotted as a linear regression. In order to achieve this, time and/or residue values must be
transformed.
Six different descriptions were tested, 1st Order, 1.5th Order, 2nd Order, and their root
functions, shown in Table 3.
30. 30
Function Time modifier Residue modifier
1st
N/A log 𝑅
1.5th
N/A
1
√ 𝑅
2nd
N/A
1
𝑅
RF 1st
√ 𝑡 log 𝑅
RF 1.5th
√ 𝑡
1
√ 𝑅
RF 2nd
√ 𝑡
1
𝑅
Table 3. The various functions tested to fit the residue decline curves.
The decline curves were transformed, the regression line calculated, and then the estimated
values of residue (R) on the regression line were back-transformed into the original system.
The best fitting description was the one which produced the smallest sum of square residuals
(SSR) between the estimated values of Rmod in the back transformed system, and the real
values of Ri.
The goodness of fit is tested with the aid of a test quantity, D (see Appendix 2).
When taking the Horwitz Curve11 into consideration, weighting factors can be assigned to
account for the scatter of the analytical values in the original system. Horwitz showed that the
relative standard deviation (as a percentage of the measured value) increases by one power of
2 for every two powers of 10 by which the concentration falls. Confidence limits should then
be narrower at higher concentration ranges, i.e. at the start of the study. A weighting of R =
Rlog2 was applied in line with Horwitz’s original suggestion for a K factor of 1/√2.
Weighted confidence intervals are calculated from the data (see Appendix 2), then
plotted on the linearized decline curve before being back transformed into the original system.
From these intervals, it is possible to see what residues could be expected in the worst
31. 31
possible circumstance, information that is invaluable to predicting the maximum residue
levels that should be allowed in regulations, although this will not be explored here.
Trials 6 and 7 exhibited RF 1st order reaction mechanisms, whereas trial 5 was found to not fit
any of the models with 90% confidence.
Trial-06
Days after application Residue (mg/kg)
0 2.17
14 0.18
26 0.1
36 0.06
54 0.01
Table 4. Residue values found for the time points in Trial-06
Figure 4. Residue valuesplotted as a scatter graph. A clear decline can be seen, with a faster rate
initially and a more gradual decline after 14 days.
32. 32
Calculated SSR in the six back transformed systems:
1st 0.79904 1.5th 2517.72631 2nd 4.96535
RF 1st 0.08975 RF 1.5th 0.69375 RF 2nd 4.97329
This shows a RF 1st order linear decline is the best description, with a 773% difference
between the SSR of the best fitting description and the second best description.
This is an incorrect and misleading statistic. The values of the slope and intercept of the linear
regressions in each model must be taken into consideration alongside the modification
performed on the residue values. 1.5th order and 2nd order mechanisms and their root functions
both involve the inverse of the residue values. Hence, if at any point the linear regression line
is 0, a value of infinity will be generated in the back transformed system. We must therefore
discount any linearized system in which the slope and intercept have different signs.
With this in mind, the following SSR values are obtained:
1st 0.79904 1.5th N/A 2nd N/A
RF 1st 0.08975 RF 1.5th N/A RF 2nd N/A
The difference between the best fit and the second best fit is now 890%.
The test quantity D was tested at a 95% confidence level and shows a significant difference
from 0, proving a good fit:
𝐷 = 0.1816559
Hence, a RF 1st order transformation is performed and plotted:
33. 33
Figure 5. The plot ofthe RF 1st
order linear regression from Trial-06. The transformed residues
are show, with the solid regression line and its confidence limits,with the confidence limits ofthe
predicted values of R shown with dotted lines.
The weighting is applied:
Figure 6. The RF 1st
order linear decline curve with weighting applied to the confidence limits. It
can be observed that the confidence range is now narrower at the start of the trial.
34. 34
The goodness of fit is re-tested with the test quantity D, and was found to increase:
𝐷 = 0.19399727
The linear curve is back-transformed into the original curve. The graph below is shown with a
logarithmic residue axis to aid visibility.
Figure 7. The back transformed graph for Trial-06.
The T/2 time (time taken for the residue to fall to half the initial concentration) was calculated
to be 1.13 days using the formula
𝑇
𝐴
=
log 𝐴
−𝑠𝑙𝑜𝑝𝑒
.
Trial-07
Days after
application
Residue
(mg/kg)
0 4.45
30 0.11
44 0.05
64 0.02
103 0.01
Table 5. Residue values found for the time points in Trial-07
35. 35
Figure 8. Trial-07 mimics Trial-06, although has a much higher initial concentration.
The smallest SSR in the back transformed system is found in the RF 1st order mechanism:
1st 9.90839 1.5th 1.20636 2nd N/A
RF 1st 0.50650 RF 1.5th N/A RF 2nd N/A
The weighted RF 1st order mechanism is graphed with the confidence intervals:
Figure 9 The weighted RF 1st
order linear decline curve and confidence limits for the straight
line and predicted points.
36. 36
The test quantity D confirms a good fit at a 95% confidence interval:
𝐷 = 0.19357052
The curve is back transformed into the original system, again with a logarithmic scale for the
residues to aid visibility:
Figure 10. Trial-07 RF 1st
order mechanism back transformed into the original system with
confidence limits for the straight line and the predicted intervals.
The T/2 time was calculated to be 1.15 days, very similar to the T/2 time of Trial-06.
Trial-05
Days after
application
Residue
(mg/kg)
0 0.16
21 0.05
36 0.22
51 0.08
84 0.01
Table 6. Residues found in Trial-05
37. 37
Figure 11. The graph of the residue values found for the time points in Trial-05. There is no
clear pattern to the decline.
It is immediately clear that Trial-05 will not fit one of the six functions. The residue in the
onion appears to increase in the third time point, along with the initial time point being far
below the other two trials.
A number of things could have caused this result.
Although the paperwork suggests that the trial was sprayed correctly, I have no way of
knowing if it was performed exactly as detailed. This could explain how there is almost no
Compound X on the onion immediately after spraying.
Even if the right amount was sprayed on the crop, it by no means assures that the spray
solution reached the crop. A strong gust of wind is all it would take for the fine mist of spray
solution to be diverted off course. This to me seems unlikely, as the randomized selection
process for picking representative sub samples of the crop should eliminate such outliers, and
the paperwork details wind no stronger than that in Trials-06 and -07.
38. 38
Another possibility is that somewhere along the complex chain of custody, certain samples
were mixed up. Although there is no way I can scientifically verify this, it is interesting to
place the residues in descending order, then analyse the resulting decline curve.
After re-ordering the residues, it was found that the resulting decline curve was a 1st
Order decline, confirmed as a good fit with a 95% certainty with 𝐷 = 0.19357052. It makes
sense that the curve is not a RF 1st Order, as the root function models deal with ‘kinked’ 1st
Order declines which result from high initial concentrations. As the re-organized Trial-05 has
a low starting concentration of 0.22 mg/kg, there is no need for a root function.
The half-life of the molecule under these circumstances appears to disagree with the
half-life of Compound X in Trials-06, yet agree with Trial-07 (if the first data point in these
trials is removed and the resulting 1st order decline curve is analysed). Trial-05 suggests a
half-life of 19 days, whereas the 1st order curves for Trials-06 and -07 suggest 10 and 22 days
respectively. As these trials were carried out in different climates, different half-lives at these
low residue levels are expected, however Trials-05 and -06 were performed in similar
climates (UK and Germany), whereas Trial-07 was performed in Spain, a much hotter climate
where the half-life of Compound X is expected to be lower.
At this point, further speculation into possible reasons for these discrepancies would
deviate too far away from scientific reasoning. The discussion into whether the samples had
been mixed up or not will therefore be left at the conclusion that the event was possible, but
unlikely.
Another explanation for the odd results seen in Trial-05 is error on the analytical end,
although the values of the procedural recoveries indicate that there was no loss of Compound
X anywhere in the process.
39. 39
Re-analysis of the samples was impossible, as under company policy no additional work can
be done without authorisation from the sponsor. As the results presented here pass all the tests
for a ‘successful’ result, repeat analysis could not be justified.
Further Investigation
The modelling of the trials has proved enlightening, but so far have not given any insight onto
how the molecule degrades.
There are two factors at play during the decline curve; the degradation of Compound X and
the dilution by increased plant matter. Researching and documenting the growth of the plants
alongside these residue decline trials would open the door to statistical analysis of the dilution
of the plant, giving a much more correct picture of the fate of Compound X.
It would also allow investigation into the multiple pathways by which the true degradation of
the molecule occurs. Compound X does not remain solely on the surface of the plant. It is
absorbed under the plant surface. The rate of degradation on the surface due to environmental
factors will be different than the rate of internal biological degradation. Understanding these
rates as well as the rates of transport between the surface and plant interior will further
increase understanding of how Compound X interacts with the plant.
In terms of more immediate further research, work could be done using the new GC/MS/MS
instrument recently acquired by Eurofins Agroscience Services. A derivatization step could be
developed, and the sample could be injected with only a light clean up. The costs of this could
be compared to the current method described here.
40. 40
Experimental
Extraction Method
Chemicals
Information pertaining to the identity and source of the reagents used for this extraction
method is summarised in Table 7.
Reagent Grade Source
Ethyl Acetate Pesticide
Thermo Fisher Scientific UK Ltd, Loughborough,
England
Methanol HPLC Thermo Fisher Scientific UK Ltd
Acetonitrile HPLC Thermo Fisher Scientific UK Ltd
Anti-bumping granules N/A Thermo Fisher Scientific UK Ltd
Deionised Water HPLC Thermo Fisher Scientific UK Ltd
Conc. Hydrochloric
Acid
AR Thermo Fisher Scientific UK Ltd
Formic Acid 98/100% Thermo Fisher Scientific UK Ltd
pH indicator paper pH 0-7 Thermo Fisher Scientific UK Ltd
Potassium Hydroxide Analytical Thermo Fisher Scientific UK Ltd
Table 7. The chemicals used in the study along with their grade and source.
41. 41
Instrument and Apparatus
Information pertaining to the identity of instruments and apparatus used for analysis is
summarised in Table 8:
Equipment Type
High Performance Liquid Chromatograph Applied Biosystems – MDS/SCIEX API
4000 LC-MS/MS with a CTC Analytics
autosampler and Agilent 1200 LC pump
(Applied Biosystems –MDS/SCIEX, 120
Birchwood Boulevard, Warrington, England)
High Performance Liquid Chromatograph TSQ Quantum Classic triple quadrupole
mass spectrometer with a Surveyor LC pump
and a CTC Analytics autosampler
Analytical column Synergi Polar-RP, 100mm x 4.6 mm internal
diameter 2.5µm partical size (Phenomenex,
Hurdsfield Industrial Estate, Macclesfield,
Cheshire, England)
Preparation equipment Stephan Bowl UM212 (Redwood Food
Systems Ltd, Watford, England) / Retsch
Mill (Haan, Germany)
General laboratory glassware Round bottom flass, volumetric flasks,
measuring cylinders
Nalgene jars 250ml capacity (Thermo Fisher Scientific
UK ltd)
Centrifuge Herrich Rotina 48 (Sartorius Ltd, Epsom,
England)
Heating Mantle (6 place) M-Tops EAM9204-06 (P & R Lab Supplies)
Shaker Denley Orbital
Rotary Film Evaporator (RFE) Stuart RE300 (LAB3, Ross Road Business
Center, Northampton, England)
Ultrasonic Bath Ultrawave U500
Table 8. The equipment used in the project.
Specimen Preparation
Excess adhering soil was removed, and the sample was homogenised with the aid of syphon
carbon dioxide to maintain homogeneity. Representative sub samples of 10 g were taken, then
the remaining sample stored as bulk.
42. 42
Preparation of standard solutions
Compound X stock solution in 0.1% formic acid in acetonitrile was prepared with the aid of
an ultrasonic bath by weighing 0.02000 g Compound X into a 28 ml glass vial, then adding
20.0 ml 0.1% formic acid acetonitrile to give a 1000 µg/ml solution.
Compound X ester stock solution in acetonitrile was prepared with the aid of an ultrasonic
bath by weighing 0.02000 g Compound X ester into a 28 ml glass vial, then adding 20.0 ml
acetonitrile to give a 1000 µg/ml solution.
These solutions were assigned the references COMP 1-03.12.14 and ESTER 1-03.12.14
respectively.
These standards were diluted via serial dilution for use as fortification standards in the
procedural recovery process and as calibration standards. Dilutions from the stock solution
were performed as shown in Appendix 1.
Extraction
The 10 g of sub sample was weighed into a nalgene® jar (250 ml). Methanol/0.25N KOH
(90/10v/v, 75 ml) was added, and then vigorously shaken for 15 min. on a flatbed shaker. The
jar was centrifuged (3500 rpm, 5 min.) and the supernatant was filtered through cotton wool
into a round bottomed flask (500 ml). Further methanol/0.25N KOH (90/10v/v, 75 ml) was
added to the nalgene® jar, and the above extraction was repeated, combining the two
75 ml extracts into the same round bottomed flask.
Hydrolysis
A pinch of anti-bumping granules was added to the combined extract. The extract was
refluxed for 2 hours at 180°C. The extracts at this point could be left to cool overnight.
43. 43
The refluxed extract was filtered through cotton wool into a second round bottom flask
(500 ml). The original flask was washed with methanol (10 ml) and transferred into the
second flask. The extract was then concentrated (RFE, 40°C) to a volume of less than 10 ml.
Liquid-liquid partition
The concentrated extract was quantitatively transferred to a graduated centrifuge tube (50 ml)
with deionised water, then the volume adjusted to approx. 15 ml. 6N hydrochloric acid (1 ml)
was added and the pH of the extract measured using pH indicator paper. If the extract’s pH
was greater than 2, it was lowered to below 2 with dropwise addition of 6N HCl.
The second round bottomed flask was rinsed with Ethyl Acetate (25 ml), which was then
added to the centrifuge tube. The tube was shaken vigorously by hand for 1 min., then
centrifuged (3000 rpm, 2 min.). The organic phase was transferred to a round bottom flask
(250 ml). The above partition was repeated, combining the two 25 ml organic phases into the
same round bottom flask.
The extract was concentrated (RFE, 40°C) to near dryness. The extract was then taken to
dryness using a gentle stream of nitrogen, and then redissolved in 0.1% formic acid in
methanol (10 ml) with the aid of an ultrasonic bath.
The extract was centrifuged (13000 rpm, 5min.) before being analysed by LC/MS/MS.
Analysis
HPLC Parameters
The samples were analysed via HPLC/MS/MS.
The column used was a Phenomenex Synergi™ 4 µm Polar-RP 80 Å, 100 x 4.6 mm. The
column features a polar endcapped, ether-linked phenyl phase on an ultra-pure silica
backbone. The phenyl groups on the column will retain Compound X through π-π
44. 44
interactions. As a consequence, Compound X will stay associated with the stationary phase
longer than other organic molecules.
A pneumatically assisted Electrospray Ionization (ESI) probe was used. As Compound X will
be found protonated due to the acidity of the extract and mobile phase, the negative ions are
not formed due to Compound X’s acidity. Instead, it is the potential difference between the
capillary tip and the counter electrode (3-6 kV) over the short distance (> 2 cm), producing
strong electric fields (106 V m-1) that pump electrons into the molecules in the solution.
Reduction occurs, and the negatively charged ions are forced away from the capillary tip.
Once the surface tension of the solvent is overcome, the spherical bulge snaps into a Taylor
cone and small charged droplets are ejected. These solvent droplets are desolvated along their
flight path through further mid-air Taylor cone formation. This continues until single ions
remain, which enter the mass spectrometer.
The mobile phases used were A = 0.1% Formic Acid in LCMS grade H2O, and B = LCMS
grade Acetonitrile. A 1 ml/min flow rate was used with a 2:1 rate post column splitter to
increase sensitivity.
A gradient program was run over 15 minutes, as seen in Table 9 and Figure 12.
Time (min) Composition
0.00 A=90% B=10%
1.50 A=90% B=10%
7.00 A=10% B=90%
8.00 A=10% B=90%
8.10 A=90% B=10%
15.00 A=90% B=10%
Table 9. The gradient program used for each run.
45. 45
Figure 12. The graphical representation of the gradient program.
The highly aqueous minute long period at the start of the run serves to flush all none retained
molecules off the column. The gradual increase of MeCN elutes Compound X at 7.1 min. The
switch to a 100% organic phase cleans the column ready for the next injection.
MS/MS Parameters
The MS/MS was run in selective reaction mode, monitoring two transitions. Both of these
involved the same loss of mass, but with different starting masses. The difference in starting
mass was due to the Cl35 and Cl37 isotopes. As expected, the ratio of the two signals was about
1.75:1.
The Q2 collision gas was set to 1.2 mTorr.
The collision energy was set to 18 eV.
46. 46
Acknowledgements
I would like to thank my placement supervisor Matthew Allen for the guidance and support
he has shown me throughout this project.
I also thank the study director for this study, Ashfaq Kahn, for allowing me to write about this
project and working alongside me on those days of endless RFE’ing.
I also thank Alexander Blake for the help and dedication in ensuring that my placement was
successful, and for pointing me down the right paths.
Thank you to Toby Vye, who rushed to the rescue when the instrument was making bad
noises.
47. 47
Appendix 1 – Fortificationand calibrationstandards
Fortification Standards
Dilution Solvent: 0.1% FA in MeOH
Standard Diluted Standard
Std. Ref. Conc.
(µg/ml)
Aliquot
taken (ml)
Final vol.
(ml)
Conc.
(µg/ml)
Std. Ref. Prep Date Expiry
Date
COMP 1-
03.12.14
1000 2 20 100 COMP 2-
03.12.14
03.12.14 03.05.15
COMP 2-
03.12.14
100 2 20 10 COMP 3-
03.12.14
03.12.14 03.05.15
COMP 3-
03.12.14
10 2 20 1 COMP 4-
03.12.14
03.12.14 03.05.15
COMP 4-
03.12.14
1 2 20 0.1 COMP 5-
03.12.14
03.12.14 03.05.15
Dilution Solvent: MeCN
Standard Diluted Standard
Std. Ref. Conc.
(µg/ml)
Aliquot
taken (ml)
Final vol.
(ml)
Conc.
(µg/ml)
Std. Ref. Prep Date Expiry
Date
ESTER 1-
03.12.15
1000 2 20 100 ESTER 2-
03.12.15
03.12.14 03.05.15
ESTER 2-
03.12.15
100 2 20 10 ESTER 3-
03.12.15
03.12.14 03.05.15
ESTER 3-
03.12.15
10 2 20 1 ESTER 4-
03.12.15
03.12.14 03.05.15
ESTER 4-
03.12.15
1 2 20 0.1 ESTER 5-
03.12.15
03.12.14 03.05.15
52. 52
Appendix 2 – Calculationof statistical parameters
SSR in the back transformed system
The best fitting model was chosen by minimizing the following equation:
∑( 𝑅𝑖 − 𝑅 𝑚𝑜𝑑 )2
Where Ri is the measured residue value and Rmod is the estimated residue value in the back
transformed system.
The test of the goodness of fit
The coefficient of determination r2 is first calculated, again in the back transformed system:
𝑟2
= 1 −
∑( 𝑅𝑖 − 𝑅 𝑚𝑜𝑑 )2
∑( 𝑅𝑖 − 𝑅̅)2
Where 𝑅̅ is the mean of the measured Ri.
The test quantity D is then:
𝐷 = | 𝑟| −
𝑡
√𝑡2 + ( 𝑛 − 2)
With t being the t-value (two sides) with 3 degrees of freedom, 2.35336.
Weighted linear regression and confidence intervals
Terms for the slope and intercept of the weighted regression:
𝑆𝑙𝑜𝑝𝑒 ( 𝑚) =
𝑆 𝑤𝑥𝑦
𝑆 𝑤𝑥𝑥
𝐼𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡 ( 𝑐) = 𝑌̅𝑤 − 𝑚 ∙ 𝑋̅ 𝑤
Where
53. 53
𝑋̅ 𝑤 =
∑ 𝑤𝑋
∑ 𝑤
𝑌̅𝑤 =
∑ 𝑤𝑌
∑ 𝑤
𝑆 𝑤𝑥𝑥 = ∑ 𝑤𝑋2
− ∑ 𝑤( 𝑋̅ 𝑤)2
𝑆 𝑤𝑥𝑥 = ∑ 𝑤𝑋𝑌 − ∑ 𝑤𝑋̅ 𝑤 𝑌̅ 𝑤
Therefore the residual variance is:
𝑠 𝑅𝑒𝑠𝑡
2
=
1
3
[𝑆 𝑤𝑦𝑦 −
𝑆 𝑤𝑥𝑦
2
𝑆 𝑤𝑥𝑥
]
With 𝑆 𝑤𝑦𝑦 = ∑ 𝑤𝑌2
− ∑ 𝑤( 𝑌̅𝑤)2
The confidence interval for the straight line (CISL) and prediction interval (CIP):
𝐶𝐼𝑆𝐿 = 𝑌̅𝑤 + 𝑏( 𝑋 − 𝑋̅ 𝑤)± 𝑘5 ∙ ∆𝑠5
With 𝑘5 = √2 𝐹95%(2,3) and ∆𝑠5 = 𝑠 𝑟𝑒𝑠𝑡 ∙ √
1
∑ 𝑤
+
( 𝑋−𝑋̅ 𝑤)2
𝑆 𝑤𝑥𝑥
The value of F95% (2,3)is 9.5520945
𝐶𝐼𝑆𝐿 = 𝑌̅𝑤 + 𝑏( 𝑋 − 𝑋̅ 𝑤)± 𝑘6 ∙ ∆𝑠6
With 𝑘6 = 𝑡95%,𝑓=3 and ∆𝑠6 = 𝑠 𝑟𝑒𝑠𝑡 ∙ √
1
𝑤
+
1
∑ 𝑤
+
( 𝑋−𝑋̅ 𝑤)2
𝑆 𝑤𝑥𝑥
The value of t is 2.35336
234. 234
Appendix 4 – Boom Spraying Calibrations
Trial Test Q (g) R (g) VA (g) TS (s) Mean TS TOS (g/s) Mean TOS
1
1 2010 550 1460 25.19
27.03
57.95951
57.5793292 2010 570 1440 25.25 57.0297
3 2010 240 1770 30.65 57.74878
2 1 NA NA 2170 30.05 NA 72.21298 NA
5 1 2000 0.69 1999 30.3 NA 65.98383 NA
6 1 NA NA 2110 30.03 NA 70.26307 NA
3 1 NA NA 1780 60 NA 29.66667 NA
7 1 NA NA 1703 30.03 NA 56.70996
Q = Quantity of Water placed into the sprayer (g)
R = Quantity of water remaining after calibration test (g)
VA = Quantity applied (g) (VA = Q-R)
T = Duration of Spraying (s)
TOS = Total output per second (g/s)
Trial PA (m2
) PL (m)
PW
(m)
VHs
(L/ha)
V (L) T (s) R TR (s) S (m/s)
1
30 10 3 200 0.6 10.42041 1 10.42041 0.959655
2 60 20 3 200 1.2 16.61751 1 16.61751 1.20355
5 120 20 6 200 2.4 36.37255 2 18.18627 1.09973
6 120 40 3 200 2.4 34.15735 1 34.15735 1.171051
3 30 10 3 200 0.6 20.22472 1 20.22472 0.494444
7 120 20 6 200 2.4 42.32061 2 21.16031 0.945166
PA = Plot Area (m2)
PL = Plot Length (m)
PW = Plot Width (m)
235. 235
VHs = Volume per hectare (L/ha)
V =Volume of spray solution applied to plot V = (PA x VH)/10,000
T = Time of spraying (s) T= (V x 1000)/TOS
R = Rows
TR = Time available to spray each row TR = T/R
S = (PL x R)/T = Speed Calculation
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