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Crop Protection 35 (2012) 1-14

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Crop Protection
journal homepage: www.elsevier.com/locate/cropro

Pesticide residues in individual versus composite samples of


apples
after fine or coarse spray quality application
Mette E. Poulsena' *, Marcel Wennekerb, Jacques Withagenc, Hanne B. Christensena
National Food Institute, Technical University of Denmark, Moerkhoej Bygade 19, 2860 Soeborg, Denmark
Applied Plant Research, Wageningen University and Research Centre, P.O. Box 200, 6670 AE Zetten, The Netherlands
c
Biometris, Wageningen UR, Plant Sciences Group, P.O. Box 167, 6700 AD Wageningen, The Netherlands
a

Crop Protection 35 (2012) 3-14

A R T I C L E
I N F O

A B S T R A C
T

Article history:
Received 27 May 2011
Received in revised form
29 November 2011
Accepted 11 December
2011
Keywords:
Pesticides
Droplet size
Variability factor
Degradation
Composite samples
Fruit

In this study, field trials on fine and coarse spray quality application of pesticides on apples were
performed. The main objectives were to study the variation of pesticide residue levels in individual
fruits
versus composite samples, and the effect of standard ine spray quality application versus coarse
spray
quality application on residue levels. The applications included boscalid, bupirimate, captan,
fenoxycarb,
indoxacarb, pirimicarb, pyraclostrobin and thiophanate-methyl. Apples were collected from four
zones
in
the tree and pesticide residues were detected in the individual apples. None of the results for the
pesticides
residues measured in individual apples exceeded the EU Maximum Residue Levels (MRLs). However,
there
was a large variation in the residues levels in the apples, with levels from 0.01 to 1.4 mg kg -1 for
captan,
the
pesticide with the highest variation, and from 0.01 to 0.2 mg kg -1 for pyraclostrobin, the pesticide
with
the
lowest variation. Residues of fenoxycarb and indoxacarb were only found in a few apples, probably
due
to
the early application time of these two compounds. The evaluation of the effect of spray quality did
not
show any major difference between fine and coarse spray quality, except for carbendazim, the
degradation
product of thiophanate-methyl, where ine spray quality resulted in higher carbendazim residue
levels
than coarse spray quality. To examine the relationship between individual results and average
results
from
ten apples, 20 composite samples were statistically constructed from sets of ten of the individual
results.
The variability factors for the individual samples (n = 80) at the 97.5 percentile were calculated for
both
standard and air induction nozzle application and were in the range of 0.99.4. The variability factor
of
seven used when EU member states calculate possible exceeding of Acute Reference Dose (ARfD)
was
adequate to encompass almost all the average results from the analyses of ten individual apples.
However,
for captan up to 9% of the results were not covered depending on which of the mathematically
constructed
composite concentrations was chosen. The variability factor of three, recommend by Codex, seems
to
be
too low, because up to 30% of the apple samples for captan were not covered if the worst case
scenario
was
chosen. The factor of three seems was also too low for thiophanate-methyl.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
* Corresponding author. Tel.: +45 3588 7463; fax: +45 3588 7448.
E-mail address: mpou@food.dtu.dk (M.E. Poulsen).
0261-2194/$ see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cropro.2011.12.013

Pesticides are widely used in the production of fruit. In


apple
production fungicides and insecticides are used pre harvest to
protect the apples from a range of pests and diseases and to
provide
quality preservation. Due to the potential risk of pesticide
residues
to human health the use of pesticides is strictly regulated,
e.g.
by
the
establishment of Maximum Residue Levels (MRLs). Many
countries
have monitoring programmes for pesticide residues in food to
ensure that pesticide residues do not exceed their MRLs.
Monitoring

programmes have shown that fungicide and insecticide


residues
are
often found in apples. In the EC coordinated monitoring
programme
for 2007, pesticide residues were detected in 47% of the 3454
apple
samples, collected and analyzed by the Member States,
Norway
and Iceland (European Food Safety Authority, 2009). The EU
MRLs
(EU MRL database 2009) were exceeded in 2.7% of the
samples.

Pesticide residues per fruit weight unit are dependent on


three
processes, i.e. variation in initial spray deposit, physical decay
due
to
weather factors and growth dilution. Variability in residues
between

Crop Protection 35 (2012) 5-14

individual samples is inevitable, partly because it is


impossible
to
achieve a uniform spray deposition of pesticides. Application
technique, crop architecture and growth stage have all been

shown
to
affect variability in initial deposit. Canopy structure is one of
the
most important factors influencing pesticide residues. Many
studies

showed the importance of canopy structure in affecting initial


deposit concentrations (e.g. Xu et al., 2006; Rawn et al.,
2007);
usually fruits in the top and outside regions are likely to
receive
more
deposits than those inside the canopy. Also, in many practical
situations the initial deposit is influenced by spray technology
(i.e. sprayer type, sprayer settings and nozzle type). Large
variability
in the level of residues exists between individual sample units
or
composite samples. There are internationally agreed
standards
for
monitoring pesticide residues and for assessing risks of
consumer
exposure. In general pesticide levels in EU fruit are below the
European MRLs (European Commission, 2010).
Protection of the environment, especially surface water,
during
the application of plant protection products (PPP) is of
increasing
public concern. For economic reasons fruit growers often
apply
low
spray volumes, and hence use fine spray nozzles that produce
small droplets (fine spray quality) that are particularly prone
to
drift.
A new option for drift mitigation in orchards is the use of air
induction
nozzles, which provide larger drop sizes (coarse spray
quality).
Coarser droplets reduce the air-borne drift losses signiicantly
compared to fine droplet spray applications (Koch et al., 2001;
Jaeken
et al., 2003; Wenneker et al., 2005). Coarse sprays, especially
at
lower
spray volumes, might produce a poorer spray coverage
compared
to fine sprays, and arouse growers' fears of reducing the
biological
eficacy of the treatment. The effect of coarse droplet
application
on the biological eficacy in fruit growing (mainly apples) has
been
assessed extensively by various institutions and companies in
Germany, Belgium, The Netherlands and France (Koch et al.,
2001;
Knewitz et al., 2002; Friessleben, 2003; Jaeken et al., 2003;
Loquet
et al., 2009). No significant differences between coarse
droplet
application and conventional ine droplet application were
found.
Finer droplets with a smaller diameter give a greater coverage
for
any
given level of spray deposit compared to coarse droplets. In
order
to
compensate for less coverage it is often advised to use higher
water
volumes for coarse droplet spraying applications with air
induction
nozzles (Jaeken et al., 2003). In several studies the coarse
droplet
sprays produced slightly greater mean deposits and smaller
spray
losses (Cross et al., 2001; Balsari et al., 2005; Jamar et al.,

2010).
In
practice coarse droplet applications might result in more
visible
residue spots on apples and pears (fruits). However, it is
unknown
if
this also affects the residue levels of plant protection products
(PPPs)
in fruits as well. Because of the high values of spray drift in
orchard
spraying (Zande et al., 2001) compared to arable ield
applications
(Huijsmans et al., 1997), the reduction of the emission of PPPs
in
fruit
growing is still of major importance. One of the measures is
the
use
of
drift reducing nozzles (Zande et al., 2008). It is a relatively
simple
and
cheap method. The use of drift reducing nozzles in fruit
growing
has been increased in many countries in Europe, e.g. Belgium,
France,
Germany and the Netherlands. Drift reducing nozzles are
often
accepted within legislation for fruit growing; e.g. Germany
(JKI,
2010),
United Kingdom (HSE, 2010), and the Netherlands (CTGB,
2008).
A ield treatment with pesticides is not uniform and consequently the distribution of pesticide residues may be very
wide,
from
areas receiving high amounts of pesticides to areas receiving
less
amount of pesticide. In 1994 research carried out in the UK,
indicated
that there might be occasional and random high residues of
pesticides found in individual samples of fresh fruit and vegetables
(Harris et al., 2000; Harris, 2000). The finding of high residue
variability is considered important in the assessment of acute
dietary
exposure, as it might lead to a higher intake of pesticide
residues
than previously believed. Field trial data derived from
commercial
use of pesticides indicates that the distribution of residues is
not
signiicantly inluenced by the 1) mean value of the residue, 2)
time
between pesticide application and sampling, 3) chemical and
physical properties of the active ingredient or formulation or
4)
the
volume application rate used for foliar application on fruit
trees.
On the other hand the distribution of residues is likely to be
inluenced by the size, shape and density of the plants and mode
of
application of the pesticide (Ambrus, 2000). Research on the
causes
of residue variability has been conducted by Carter et al.
(2000),
but
no conclusive evidence could be obtained which identify
particular
causes for the variability. Hill and Reynolds (2002)
demonstrated

that no relationship could be obtained between the variability


factor
and the 1) commodity, 2) country of origin, 3) residue
concentration
or 4) physiochemical characteristics of the pesticide including
the
systemic/non-systemic
properties
of
the
pesticide.
Furthermore,
it was seen that aging of the residue did not signiicantly affect
the
variability. Consequently, variability may only be reduced in
terms
of
more uniform application of the pesticide. When assessing the
variability it is relevant to look at the precision of the
analytical
method, as variation might contribute to the variability of the
single
commodities. Investigations conducted by Harris and Davis
(1998)
and Hill and Reynolds (2002) showed that the analytical
variation
did not signiicantly contribute to the overall variation. Thus
the
main contributor to the variation must be the difference in
residue
content between the individual units.
Explanation of the variability in pesticide residues is
essential
for the development of methods to minimize residues in fruits.
In
this study, residue variability associated with individual apples
and
composite samples was studied for two application
techniques;
i.e.
fine and coarse spray quality application. It was hypothesized
that
variation among individual fruits is the most important factor
of
residue variation.
Field trials were performed in a commercial orchard in
2007
with application of pesticides routinely used for pest and
disease
management in apple production. Apples were collected from
4
zones in the tree.
The aims of this study were: (1) to determine residue levels
of
commonly applied pesticides in apple growing after ield treatment, (2) to study the variation of these residue levels in
individual
fruits and/versus (calculated) composite samples, and (3) the
effect of a standard ine spray quality versus coarse spray
quality
application on residue levels.
2. Materials and methods
2.1. Field work
A ield trial to evaluate the effect of spray quality (droplet
size/
nozzle type) was done in 2007 in a commercial orchard in the
Netherlands. The spray applications for the reference
treatment
(ine

spray quality) were performed with brown Albuz ATR hollow


cone
nozzles (7 bar spray pressure) and for the low-drift treatment
(coarse
spray quality) with air induction (Venturi) hollow cone nozzles
(Albuz TVI 80-015 at 6 bar spray pressure). Spray applications
were carried out in the same orchard, and with the same
spraying
machine; a Munckhof cross-low sprayer with eight operating
nozzles at each side. The power take-off (PTO) speed was ixed
at
430 rpm, and the driving speed was 8 km h -1. These settings
resulted
in a spray volume rate of approximately 200 l ha-1 for the
brown
Albuz ATR nozzles and 300 l ha -1 for the air induction nozzles.
The
standard advice to growers is to use a higher water volume
for
drift
reducing air induction nozzles (Jaeken et al., 2003). The spray
quality
of the nozzles was quantiied using a Phase Doppler Particle
Analyser
(PDPA, Aerometrics). These measurements showed that the
brown
Albuz ATR nozzle had a Volume Median Diameter (VMD) of
143
mm
and the Albuz TVI 80-015 had a VMD of 493 mm (Zande et
al., 2008).
The orchard was divided in two experimental plots. Each
plot
consisted of 8 apple tree rows; i.e. 4 times 2 alternating rows
of
the
apple varieties Elstar and Jonagold of 150 m length (with an
average
tree height of 3.00 m, 3 m row spacing, and 1.25 m tree
separation in

M.E. Poulsen et al. / Crop Protection 35 (2012) 5-14

the row). Both plots were sprayed for the whole season with
the
prescribed nozzle type, according to a standard commercial
spraying
scheme with insecticides and fungicides (Table 1), following
label
directions. Dose rates were therefore kept equal for the Albuz
ATR
and Albuz TVI nozzles.
The distribution in different zones of the canopy was
evaluated
by dividing the apple tree (spindle type) into four distinct
zones:
top, middle, bottom outside and bottom inside (Fig. 1). Four
apples
were collected from five trees per nozzle type and tree zone.
Selection of the trees and apples per zone was randomized
from
each tree row (variety Elstar) in the middle of the
experimental
plot. In summary, the levels in the ield sampling used were:
(1)
fruits within a zone, (2) zone within a tree, (3) trees within a
nozzle
type. Two days before the start of commercial harvest, single
fruit
samples were taken from ive trees for each zone and nozzle
type.
The total weight of each individual apple was determined
before
the residue analysis.
2.2. Chemicals
The pesticide standards of boscalid, bupirimate, captan,

Fig. 1. Four zones within the apples trees from where apples were sampled. T: Top,
M: Middle, BU: Bottom Outside, BI: Bottom Inside.

carbendazim, fenoxycarb, indoxacarb, pirimicarb, pyraclostrobin and


thiophanate-methyl (with certified purities >96%) were
purchased
from Dr. Ehrenstorfer (Augsburg, Germany). The acetonitrile
p.a.
was
from Fisher Scientific (Leichestershire, UK). Acetic acid and
ammonium acetate were from Merck (Darmstadt, Germany). The
magnesium sulphate was purchased from Mallinckrodt Baker
(Phillipsburg, NJ, USA), sodium chloride from Merck & Co.
(Whitehouse Station, NJ, USA), sodium citrate dehydrate and sodium
citrate
sesquihydrate
from
SigmaAldrich
Chemie
GmbH
(Taufkirchen,
Germany) and the clean/up sorbent PSA from Varian (Palo
Alto,
CA,
USA). Stock solutions of pesticides of 1 mg ml -1 were prepared
in
methanol or toluene and kept at -18 C. A standard mixture of
10 mg ml-1 in acetonitrile was prepared from the stock
solutions.
Matrix-matched standards in the concentration range from
0.0015

M.E. Poulsen et al. / Crop Protection 35 (2012) 5-14

Table 1
Application date, pesticide formulations and active ingredients.

M.E. Poulsen et al. / Crop Protection


10
35 (2012) 5-14

Application date
8 May 2007
14 May 2007
19 May 2007
25 May 2007
2 June 2007
6 June 2007
11 June 2007

Pesticide product
Merpan
Merpan
Merpan
Merpan
Merpan
Merpan
Merpan
Nimrod

Active ingredients
Captan
Captan
Captan
Captan
Captan
Captan
Captan
Bupirimate

Insegar

Fenoxycarb

Merpan
Nimrod

Captan
Bupirimate

Insegar

Fenoxycarb

19 June 2007

Merpan
Steward

Captan
Indoxacarb

25 June 2007
30 June 2007
8 July 2007
15 July 2007
19 July 2007
27 July 2007
04 August 2007
13 August 2007

Merpan
Merpan
Merpan
Merpan
Bellis
Merpan
Merpan
Bellis
Merpan

Captan
Captan
Captan
Captan
Boscalid + Pyraclostrobin
Captan
Captan
Boscalid + Pyraclostrobin
Captan

19 August 2007

Merpan
Pirimor

Captan
Pirimicarb

25 August 2007

Merpan
Topsin-M

Captan
Thiophanate-methyl

16 June 2007

Harvest 4 September 2007

* : Pirimor was sprayed to control aphids in the lower parts of the trees. The top
nozzles of the sprayer were closed in order to avoid unnecessary residues.

to 0.05 mg ml-1 ( ~ 0.0070.24 mg kg-1 matrix) were


prepared
by
diluting the 10 mg ml-1 mixture with sample extracts of
apples
bought as organically grown.
2.3. Analytical method
The analyses were carried out according to Anastassiades
et
al.
(2003) and modified by Paya et al. (2007). The apples were
homogenized individually without removing peel or apple
core.
Precisely 15.0 g of apple homogenate was extracted with 15.0
ml
of
acetonitrile and 6.0 g magnesium sulphate, 1.5 g sodium
chloride,
1.5 g sodium citrate dihydrate and 0.75 g sodium citrate
sesquihydrate were added. After 1 min of shaking and centrifugation
for
10 min, 1.0 ml of extracts were transferred to small centrifuge
tubes
with containing 0.05 g PSA and 0.15 g magnesium sulphate.
After
shaking for 30 s and centrifugation for 5 min, the extracts
were
analyzed by LC/MS/MS and GC/MS/MS in MRM mode with two
transitions (Table 2). The recovery samples analyzed together
with
the apple samples (n = 10) were between 76 and 101% (Table
2).
2.4. Statistical analysis
In statistical terms the whole experiment can be described
as
follows: Block structure (random model) was 'plot/tree/(zone
within tree)' (with 5 apples from each zone). The treatment
structure (fixed model) was '(nozzle type)*(zone within tree)'. Since
the

11
Table 2

M.E. Poulsen et al. / Crop Protection 35 (2012) 5-14

MRM transitions, cone voltages, collision energies and recoveries for LC/MS/MS and GC/MS/MS analysis of boscalid, bupirimate, carbaryl 13-C6, carbendazim, captan,
chlorpyrifos D10, fenoxycarb, indoxacarb, pirimicarb, pyraclostrobin, thiophanate-methyl and tetrahydrophthalimide. Carbaryl 13-C6 and chlorpyrifos D10 were used as
injection standards and consequently no recoveries are listed for these compounds.
GC/MS/MS
LC/MS/MS
Instrument Compound
Transistion1 Cone Voltage Collision energy Transistion2 Cone Voltage2 Collision Energy2 Recovery at 0.02 mg kg
Boscalid
343 >307 57
20
343 > 140 57
20
93%
Bupirimat

317

Carbaryl 13-C6

225

>166
>151

27

23

317

25

14

208

> 108
> 151

25

50

50

14

Carbendazim

192

Fenoxycarb

>160
>88

80

15

192

> 132

80

15

302

25

20

>

Indoxacarb

529

>218

65

20

>

Pirimicarb

239

>72

25

16

239

Pyraclostrobin

388

24

11

388

Thiophanate-methyl

343

50

12

343

Bupirimate

316

>194
>151
>208

15

273

> 163
> 93
> 193

Captan

149

>70

12

149

Chlorpyrifos D10

326

>262

15

292

Pirimicarb

238

>166

10

Tetrahydrophthalimide

151

>80

76%
85%
95%

> 182

25

14

24

25

98%

50

12

101%

10

92%

> 105

90%

> 260

10

166

> 96

15

87%

123

> 80

97%

nozzle-type treatment was carried out on the plots, its effect


was tested against variance amongst trees within plots. The
amount
of residue on the fruits appeared to have a lognormal
distribution,
all data were analyzed as a GLMM (Generalized Linear Mixed
Model)
based on a lognormal distribution using GenStat Release 9.2
statistical package (Lawes Agricultural Trust, Rothamsted
Research,
UK). Significant F-tests (p < 0.05) were followed by a Least
Significant Difference (LSD)-test for pair wise comparison of
treatment
means using LSD p-0.05 values. Treatment means were
estimated
on the transformed scale and later back transformed to the
original
scale.
3. Results and discussion
3.1. The effect of standard fine spray quality versus coarse
spray
quality application
The effects on pesticides residues in the apples will be
presented
by focusing on general characteristics of pesticide residues in
individual apples, nozzle effect (spray quality), zone effect
and
the
effects on the individual pesticides. The weight of the
individual
apples was in the range of 114252 g with a median value of
168
g.
No statistically signiicant correlation between the fruit weight
(size) and residue level was observed, showing that the
residue
levels were independent of the fruit size. Consequently, the
following results are based on the residue levels in the
individual
fruits expressed in mg kg-1
-1
.
3.1.1. General characteristics of pesticide residues

94%

90%

in individual apples
Residues of bupirimate were detected in almost all the
apple
samples but less than 15% of the residues were above the
Limit
of
Quantiication (LOQ) of 0.005 mg kg -1. Pirimicarb was not
sprayed
by
the top nozzles (see Table 1), because only the lower parts of
the
trees
were infested by aphids, and consequently pirimicarb was not
found
in 36 apples picked from the top of the trees. Residues of
fenoxycarb
and indoxacarb were only found in few apples probably due to
the
early application time for those two compounds. Statistical
evaluation
of bupirimate, pirimicarb, fenoxycarb and indoxacarb is
therefore
not
included in this presentation. For carbendazim, residues were
detected above the LOQ (0.01 mg kg-1) in 156 of the 160 apples. All
four
apples with no detectable residues were sprayed by the air
induction
nozzle; two were picked from the top and two from the
bottom
inside.
Thiophanate-methyl residues were not detected above the
LOQ
(0.01 mg kg-1) in three apples, i.e. one air induction nozzle
application
top and two standard application bottom outside. Boscalid,
pyraclostrobin, captan and the degradation product
tetrahydrophthalimide were found in all 160 samples (LOQ = 0.01 mg kg -1).
This
is
in accordance with Xu et al. (2008) who found that initial
captan
deposits and subsequent residues on fruits and leaves are
generally
closer to a lognormal than to a normal distribution.
None of the results for the pesticides residues measured in
the
apples exceeded the MRLs (European Commission, 2010).
However,

12
Table 2

M.E. Poulsen et al. / Crop Protection 35 (2012) 5-14

there was a high variation in the residues with levels from


0.01
to
1.4 mg kg-1 for captan, the pesticides with the highest range,
and
from 0.01 to 0.2 mg kg -1 for pyraclostrobin, the pesticide with
the
lowest range.
3.1.2.
Spray quality (nozzle) effect
The mean values for the two applications were statistically
evaluated to see if one of the application modes resulted in
higher
residues. Fig. 2 shows the mean residue values from the 4
zones
of
both application modes. The evaluation showed that the
mean
residue levels for all apples sprayed with standard nozzles
(ine
spray quality) versus air induction nozzles (coarse spray
quality)
were not different for boscalid, pyraclostrobin, thiophanatemethyl
and the degradation product tetrahydrophthalimide. For
captan
the
mean value for air induction nozzle application was higher
and
for
the degradation product of thiophanate-methyl, carbendazim,
the
standard application gave higher mean values. Looking at the
mean
values for the 4 zones, only few differences were seen.
Captan
and
thiophanate-methyl had for air induction nozzle application

higher
mean values at the middle zone, while tetrahydrophthalimide
had
higher levels for the bottom inside zone. For carbendazim the
standard application resulted in higher mean values at all
zones.
More details are described below under the active
substances.
3.1.3.
Zone effect
The mean values for the four zones have been statistically
evaluated to see if one of the application modes gave a more
even
distribution (Fig. 3). The evaluation for boscalid and
pyraclostrobin
showed that the pesticides seem to be more evenly
distributed
by
the
air induction nozzle application, while it was opposite for
carbendazim. However, no major differences were seen. Xu et al.
(2008)
showed that the unit-to-unit variation contributes most to the
observed variability in captan residues, not only in the initial
spray
deposition but also over the subsequent period following the
application. More details are described below under the active
substances.
3.1.4.
Boscalid and pyraclostrobin
Boscalid and pyraclostrobin were sprayed simultaneously
(as they were active ingredients of the fungicide product
Bellis)

13

0.00
b

Boscalid
mg/kg

Whole tree BI

BU
Zone

Whole tree

0.10
mg/kg

Captan

BI

0.25
0.20

BU
Zone

0.08

BU
Zone

Thiophanate-methyl

0.15

BI BU
Zone

Whole tree BI

0.06
0.04
0.02

Pyraclostrobin

0.00
ME. Poulsen et al. /
Crop
Protection
35
(2012)
5-14

standard
mg/kg
air induction nozzle
0.10
0.35
0.10

Whole tree
Zone

BI

BU

0.08
0.30
0.08
0.25
0.06

0.00

0.06
0.20
0.04
0.15
0.04

mg/kg

0.10
0.02
0.02
0.05
0.00
0.00

0.35

'

0.05

0.30
0.25
0.20
0.15
0.10
0.05

Carbendazim
Tetrahydrophthalimide
mg/kg

0.10
b
b

14
Whole tree

BI

BU

M
T

15

Zone
Fig. 2. Effect of nozzle types (spray quality); Mean values of pesticide residues of boscalid, pyraclostrobin, captan and its degradation compound tetrahydrophthalimide,
carbendazim and thiophanate-methyl from analysis of individual apples picked at four zones within the tree. BI: bottom inside, BU: bottom outside, M: middle, T: top. The
mean
values for all apples are also shown. White bars are standard application Albuz ATR nozzle (fine spray quality); black bars are air induction nozzle application (coarse spray
quality).
Bars labeled with the same letter do not differ significantly p = 0.05.

twice towards at the end of the growing season to prevent


storage
rots (Table 1). No differences were observed between the
residue
levels per zone between the two nozzles types. The residue
patterns per zone for boscalid and pyraclostrobin were the
same
for
the two nozzle types, indicating that the spraying technique
did
not
influence the residues levels.
3.1.5. Captan
Captan was the most frequent applied fungicide (Table 1).
Captan contains an unstable trichloromethyl 'moiety' and
leads
to
degradation
to
tetrahydrophthalimide
and
tetrahydrophthalimide
acid. Huo et al. (2007) have reported that daily loss of captan
was
very small, the main loss of captan being due to wash-off by
rain
(Smith and MacHardy, 1984; Frank et al., 1985, 1987;

Northover
et al., 1986). The monograph on captan by FAO/WHO (1970)
also
reports that captan does not appear to undergo any chemical
change on the plant surface. However, Ticha et al. (2007)
showed
captan residues in apples successively decreased until
harvest.
None of the above quoted studies had measured the
degradation
product tetrahydrophthalimide. In this study tetrahydrophthalimide was found in higher levels than captan. The results
showed that approximately 80% of the sum of captan and
tetrahydrophthalimide was found as tetrahydrophthalimide, when
taking the differences in molecular weights into account. The
results are in line with results from Poulsen et al. (2009),
where
7
different spraying strategies were performed in commercial
orchards. However, part of the degradation could occur in the
laboratory during the chemical analysis. Captan is rapidly
degraded

16

Pyraclostrobin

0.00
0.15
0.20
0.25
0.10

Boscalid

BI BU
standard

BI BU
M
air induction

TM.E. Poulsen et al. / Crop


BI BU
standard
Protection 35
(2012) 5-14

mg/kg

T BI BU M
air induction

17

Standard
mg/kg
0.35
0.30

Tetrah yd roph th al i m ide

Captan

0.2
b

5
0.2
0
0.1
5
0.1

- 1
F
Iii
IX
M

BI

BU

air induction

BI

BU

BI BU
standard
T

BI BU M
air induction

18

BU
mg/kg
M
0.10
standard

Th ioph an ate-methyl
0.08

---------c

0.06

0.04
0.02

BU M
standard

BI BU M
air induction

Carbendazim
bb

Al

0.00 Bl

I
Bl

BU

air induction

19

Fig. 3. Effect of the zone within the tree; mean values of pesticide residues of boscalid, pyraclostrobin, captan and its degradation compound tetrahydrophthalimide,
thiophanatemethyl and carbendazim, from analysis of individual apples picked at four zones. BI: bottom inside, BU: bottom outside, M: middle, T: top. Results for standard application
(fine spray quality) and air induction nozzle application (coarse spray quality) are shown bars labeled with the same letter do not differ significantly p = 0.05.

in high temperatures e.g. present in the injector of the gas


chromatograph. However, the injection mode used for the
analysis
has been shown by Bailey (2007) to minimize the degradation
of
captan. As described in Materials and methods, the recovery
samples analyzed together with the apple samples (n = 10)
gave
a
mean
recovery
for
captan
at
90%
and
for
tetrahydrophthalimide
97% (spike level 0.02 mg kgmg/kg
a

-1

). This indicates the any possible


degradation in the injector was equal for calibration standards
and
samples. However, degradation may have occurred during
sample
preparation, when the apples were chopped.
Fig. 3 shows that the residue levels for captan and tetrahydrophthalimide have the same patterns for both nozzle types.
There was a general trend of higher residue levels for the
coarse
droplet application; with significant higher levels for the
middle
zone for captan and inner zone for tetrahydrophthalimide.

3.1.6. Thiophanate-methyl and carbendazim


Thiophanate-methyl and carbendazim are broad spectrum
systemic fungicides. However, carbendazim is also a
degradation
product of thiophanate-methyl. Thiophanate-methyl is quickly
absorbed by the plants surfaces and transported to the
various
parts as carbendazim. Mandal et al. (2010) have studied the
dissipation on grapes and found that the half-life of thiophanatemethyl
is between 4.74 and 6.52 day irrespective of location and
doses.
According to FAO/WHO (1974) the transformation to a great
extent
is light-catalyzed.
Thiophanate-methyl showed the same residue patterns for
the
zones and nozzles as captan/tetrahydrophthalimide did; i.e.
highest
residues in the bottom outside and the middle zone for both
nozzles types. Between the nozzles, residues were in higher
for
the
air induction nozzle (coarse spray quality), while the
degradation

20
Table
3

M.E. Poulsen et al. / Crop Protection 35 (2012) 5-14

mg/kg

Acceptable Daily Intake (ADI) and Acute Reference Dose (ARfD) in mg kg -1 body
weight for boscalid, pyraclostrobin, captan, tetrahydrophthalimide, carbendazim
and thiophanate-methyl.
Acceptable Daily Intake,

Acute Reference Dose,

ADI (mg kg-1 b.w.)


Boscalid

0.04

none

Bupirimate

0.05

0.05

Captan

0.1

Carbendazim

0.02

0.02

Fenoxycarb

0.04

none

Indoxacarb

0.006

0.02

Pirimicarb

0.035

Pyraclostrobin

0.03

Tetrahyd

ARfD (mg kg-1 b.w.)

rophthal
imide

0.3

0.1
0.03

Tetrahydrophthalimide

none found

Thiophanate-methyl

0.08

none found
0.2

product carbendazim shows the complete opposite deposition


pattern for the two nozzles types. With the standard nozzles
(fine
spray quality) the carbendazim level was doubled, in contrast
the
carbendazim levels for the air induction nozzles. The small
droplets
have a bigger total surface area and thereby more
susceptible for
degradation due to sunlight.
No correlations were seen between thiophanate-methyl
residue
level and carbendazim residue levels for the two nozzle types.
In general fruits in the inner and top zones received less
residue
(or deposit) than the outside and middle zones (for both
nozzle
types). This is in contrast to observations of Xu et al. (2008).
However, the trials of Xu et al. (2008) were performed with an
axial
fan sprayer and not with a cross-flow sprayer, as in this
experiment.
These two types of sprayer types give different deposition
proiles
(Michielsen et al., 2008).

Fine spray quality

Fine sprayquality

Coarse sprayquality

Coarse spray quality

X
Boscali
d nX

mg/k
0.60g

0.30
0.50

0.20
0.40
0.10
0.00

Captan
Composite

Individual

Pyraclostrobin
X

mg/k
0.25g

Composit

Individua

0.20

0.15

0.10
0.05

i
I

Composite
Individual
Composite
-----------------------------------------1----------------------------------------------------------------------------,-------------------------------------1

M.E. Poulsen et al. / Crop Protection


21
Table
3 35 (2012) 5-14

mg/k
1.5 g
0

Coarse spray qualityx

Fine sprayquality

0.80
0.60

Fine sprayquality
v

1.2

0.7
5

0.5
0

x
1.0
0

Coarse spray quality

I
i

0.25
0.00

Composite

Composite

Individual

1.00

Composite

Individual

Composite

22
Table
3

M.E. Poulsen et al. / Crop Protection 35 (2012) 5-14

Thiophanate-methyl

mg/k
0.30g

xx

0.25

Fine spray quality

0.15

0.10
0.05

Composite

K
Individual

0.20
0.15

1
Composite

Individua
l

Coarse spray quality

0.25

0.20

0.00

Carbendazim

mg/k
0.30g

0.10

i
i

'

0.05

0.00
Composite

Individual

Composite

Individual

Fig. 4. Results for composite and individual samples for pesticide residues of boscalid, pyraclostrobin, captan and its degradation compound tetrahydrophthalimide,
carbendazim
and thiophanate-methyl. Results are shown for both standard application Albuz ATR nozzle (fine spray quality) and air induction nozzle application (coarse spray quality).

23
Table 4

M.E. Poulsen et al. / Crop Protection 35 (2012) 5-14

Variability factor for standard and air induction nozzle application for boscalid,
captan,
carbendazim, tetrahydrophthalimide, thiophanate-methyl and pyraclostrobin.

3.2. Composite samples

Standard
application
Boscalid
Captan
Carbendazim
Tetrahydrophthalimide
Thiophanate-methyl
Pyraclostrobin

2.0
9.4
1.8
4.0
3.9
2.2

Air induction
nozzle application
2.7
4.3
0.9
2.7
3.8
2.6

The EU MRLs and CODEX MRLs for apples are set for the
mean
residue value of one kg or a minimum of ten apples (European
Commission, 2002; CODEX, 1999). Originally, the toxicological
evaluation of pesticides was performed for chronic effects
only
by
setting an Acceptable Daily Intake (ADI) (Table 3). For control
purposes of ADI an average residue value are more suitable in
order
to smooth out the variation in residue levels, because a
person
eating a high pesticide residue content apple one day will
most
likely eat an apple with at lower content the next day. Consequently, the control for pesticide residues in apples was
decided
to
be carried out for composite samples. Later on, the pesticides
were
evaluated for acute effects and Acute Reference Doses (ARfD)
were
set. To control the ARfDs it is important to know the pesticide
residue in the individual apple, because a person will be
exposed
to
acute effects from a speciic apple, if the residues are high
enough.
However, it would be costly to control the ARfD, if the apple
samples should be analyzed individually as ten apples should
be
analyzed. Instead, the pesticide residue levels in the
composite
samples are calculated back to the assumed highest possible
level
in the individual apples, by applying a variability factor to the
average concentration of ten apples. In EU member states the
variability factor used is seven (EU Commission, 2004),
whereas
CODEX has lowered the factor to a default value of three until
suficient data on single unit is available to calculate a more
realistic
variability factor (WHO, 2010).
To investigate the distribution of the pesticides between
the
apples from the two application modes, all apples were
individually
analyzed. The results of from the analysis of the individual
apples
showed large variations in residue levels, where the apples
with
the
highest concentration had between 12 and 200 times higher
residue
levels than in the apple with the lowest level. To examine the
rela-

Table 5
The minimum and maximum number of individual results not covered by the
variability
factor
3
and
7,
for
boscalid,
pyraclostrobin,
captan,
tetrahydrophthalimid,
carbendazim and thiophanate-methyl, obtained by standard and air induction
nozzle application. The number before the arrows refers to the highest concentration of the 20 mathematically constructed composite samples and the number
after
the arrow refers to the lowest concentration of 20 the mathematically constructed
composite samples. The mathematically constructed composite samples represent
a sample of ten apples used for control of MRLs.
Standard application

Boscalid
Pyraclostrobin
Captan
Tetrahydrophthalimid
Thiophanate-methyl
Carbendazim

Factor 3
0/1
0/1
7 / 24
0/8
2/8
0/0

Factor 7
0/0
0/0
1/4
0/0
0/0
0/0

Air induction
application
Factor 3
1/4
0/0
0/7
0/4
1/6
0/0

Factor 7
0/0
0/0
0/0
0/0
0/1
0/0

tionship between individual results and average results from


10 apples that are currently used for pesticides control of
MRLs,
composite samples were statistically constructed from ten of
the
individual results chosen at complete random. These
mathematically
constructed composite samples would represent the result if
ten
apples were homogenized together and then analyzed. The
results
from analysis of the individual apples and the calculated
pesticide
level for the composite samples are seen in Fig. 4. The
composite
samples showed less variability since they were average
concentrations of 10 results.
The variability factors at 97.5 percentile of the 80
individual
apples were calculated for both standard and air induction
nozzle
application as:
Variability factor = Concentration of the individual sample
at
97.5 percentile/back transformed mean.
The calculation showed variability factors in the range of
0.99.4 (Table 4). The variability factors for captan were
highest,
9.4
and 4.3. Xu et al. (2006) calculated variability factors for
captan
to
be approximately 2, which is much lower than in this study.
The results from the analysis of the individual apples and
results
from the mathematically constructed composite samples can
show
if the variability factor of three or seven would be adequate to
cover
the highest results from the analysis of the individual apples.
Table 5 shows the number of high results from the analysis of
individual apples that would not be covered by a variability
factor
of either three or seven, if applied to the results
mathematically
constructed composite samples.

24
Table 4

M.E. Poulsen et al. / Crop Protection 35 (2012) 5-14

The figures before the arrows are the number of individual


results
that are above three or seven times the lowest average
concentration
of the 20 mathematically constructed composite samples and
the
igures after the arrows are likewise the number of individually
results that is above three or seven times the highest
concentration
of the 20 mathematically constructed composite. The
minimum
and
maximum number of results not covered by the variability
factors
of
three and seven were calculated by counting the number of:
Results of analysis of individual apples >3 (or 7)*lowest (or
highest) result of 20 mathematically constructed composite
samples.
For example, for boscalid and standard application (ine
spray
quality) Table 5 shows that the variability factor of 7 is
adequate
because even if the lowest result of composite samples was
used.
However, for the variability factor of three one result (the
highest)
of the individual results would be above 3 times the lowest
(or highest) result of 20 mathematically constructed
composite
samples. Likewise for the air induction application (coarse
spray
quality) the variability factor of seven were adequate, while
for
the
factor of three, the four results (the four highest) would be
above
3
times the lowest (or highest) result of 20 mathematically constructed composite samples, if the lowest result of the

composite
samples was used and one result (the highest) if calculated
with
the
highest result for the composite samples.
As seen from Table 5 the variability factor of seven seems
to
be
adequate to include almost all the results for the pesticides.
For
captan up to 9% of the results were not covered depending on
the
composite concentration chosen. However, the variability
factor
of
three seems to be too low, because up to 30% of the apple
samples
for captan were not covered if the worst case scenario was
chosen.
Also for thiophanate-methyl the factor of three seems to be
too low.
In conclusion, no statistically significant correlation
between
the
fruit weight (size) and residue level was observed, showing
that
the residue levels were independent of the fruit size. None of
the
results for the pesticides residues measured in the apples
exceeded
the EU MRLs. For boscalid and pyraclostrobin no differences
were
seen in the residue levels for the two spraying techniques.
The
coarse spray quality (air induction nozzle) resulted in higher
levels
for captan and thiophanate-methyl at the middle zone and for
tetrahydrophthalimide at the bottom inside zone. However,
with

25

M.E. Poulsen et al. / Crop Protection 35 (2012) 5-14

the ine spray quality (Albuz ATR nozzle), the carbendazim


levels
were doubled in contrast to the coarse spray quality. The
small
droplets had in total a bigger surface area and thereby
thiophanatemethyl was supposed to be more susceptible for degradation
to
carbendazim due to sunlight. Pesticide residues levels of the
apples
were affected by the zone of the fruits within the tree. There
were
no major differences in residue levels of the apples between
nozzle
types for the same zone within the tree. The unit-to-unit
variation
seems to contribute most to the observed variability. The
variability
factors calculated for both fine spray quality (Albuz ATR
nozzle)
and
coarse spray quality (air induction nozzle) application (n = 80)
and
were in the range of 0.99.4. The use of a variability factor of
seven
in the risk assessment calculation of acute effects based on
control
composite samples seems to be adequate to include almost
all
the
individual pesticide residues results in this study.
Acknowledgments
The authors wish to thank the European Commission and
the Dutch Ministry of Agriculture, Nature & Food Quality for
financial
support of the ISAFRUIT project. The ISAFRUIT project is
funded
by
the
European Commission under the Thematic Priority 5-Food
Quality
and Safety of the 6th Framework Programme of RTD (Contract
no.
FP6FOOD-CT-2006-016279). We also thank fruit grower Mr. M.
Koning
for
making his orchard available for the experiments and Mr. R.
Anbergen
for technical assistance.
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Zande van de, J.C., Heijne, B., Wenneker, M., 2001. Spray Drift Reduction in
Orchard
Spraying (State-of-the-Art December 2001). Institute of Agricultural and Environmental Engineering/PPO-fruit. IMAG Report 2001-19, Wageningen. 38 p. (in
Dutch with English summary).
Zande van de, J.C., Holterman, H.J., Wenneker, M., May, 2008. Nozzle classification
for drift reduction in orchard spraying: identification of drift reduction class
threshold nozzles. In: Agricultural Engineering International: The CIGR Ejournal
Manuscript ALNARP 08 0014, vol. X.

28

M.E. Poulsen et al. / Crop Protection 35 (2012) 5-14

Contents lists available at SciVerse ScienceDirect

Crop Protection
journal homepage: www.elsevier.com/locate/cropro

Crop Protection 35 (2012) 29-14

Pesticide residues in individual versus composite samples of


apples
after fine or coarse spray quality application
Mette E. Poulsena' 1, Marcel Wennekerb, Jacques Withagenc, Hanne B. Christensena
National Food Institute, Technical University of Denmark, Moerkhoej Bygade 19, 2860 Soeborg, Denmark
Applied Plant Research, Wageningen University and Research Centre, P.O. Box 200, 6670 AE Zetten, The Netherlands
c
Biometris, Wageningen UR, Plant Sciences Group, P.O. Box 167, 6700 AD Wageningen, The Netherlands
a

1 Corresponding author. Tel.: +45 3588 7463; fax: +45 3588 7448.
E-mail address: mpou@food.dtu.dk (M.E. Poulsen).
0261-2194/$ see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cropro.2011.12.013

30

A R T I C L E
I N F O

M.E. Poulsen et al. / Crop Protection 35 (2012) 5-14

A B S T R A C
T

Crop Protection 35 (2012) 31-14

Article history:
Received 27 May 2011
Received in revised form
29 November 2011
Accepted 11 December
2011
Keywords:
Pesticides
Droplet size
Variability factor
Degradation
Composite samples
Fruit

In this study, field trials on fine and coarse spray quality application of pesticides on apples were
performed. The main objectives were to study the variation of pesticide residue levels in individual
fruits
versus composite samples, and the effect of standard ine spray quality application versus coarse
spray
quality application on residue levels. The applications included boscalid, bupirimate, captan,
fenoxycarb,
indoxacarb, pirimicarb, pyraclostrobin and thiophanate-methyl. Apples were collected from four
zones
in
the tree and pesticide residues were detected in the individual apples. None of the results for the
pesticides
residues measured in individual apples exceeded the EU Maximum Residue Levels (MRLs). However,
there
was a large variation in the residues levels in the apples, with levels from 0.01 to 1.4 mg kg -1 for
captan,
the
pesticide with the highest variation, and from 0.01 to 0.2 mg kg -1 for pyraclostrobin, the pesticide
with
the
lowest variation. Residues of fenoxycarb and indoxacarb were only found in a few apples, probably
due
to
the early application time of these two compounds. The evaluation of the effect of spray quality did
not
show any major difference between fine and coarse spray quality, except for carbendazim, the
degradation
product of thiophanate-methyl, where ine spray quality resulted in higher carbendazim residue
levels
than coarse spray quality. To examine the relationship between individual results and average
results
from
ten apples, 20 composite samples were statistically constructed from sets of ten of the individual
results.
The variability factors for the individual samples (n = 80) at the 97.5 percentile were calculated for
both
standard and air induction nozzle application and were in the range of 0.99.4. The variability factor
of
seven used when EU member states calculate possible exceeding of Acute Reference Dose (ARfD)
was
adequate to encompass almost all the average results from the analyses of ten individual apples.
However,
for captan up to 9% of the results were not covered depending on which of the mathematically
constructed
composite concentrations was chosen. The variability factor of three, recommend by Codex, seems
to
be
too low, because up to 30% of the apple samples for captan were not covered if the worst case
scenario
was
chosen. The factor of three seems was also too low for thiophanate-methyl.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
Pesticides are widely used in the production of fruit. In
apple
production fungicides and insecticides are used pre harvest to
protect the apples from a range of pests and diseases and to
provide
quality preservation. Due to the potential risk of pesticide
residues
to human health the use of pesticides is strictly regulated,
e.g.
by
the
establishment of Maximum Residue Levels (MRLs). Many
countries
have monitoring programmes for pesticide residues in food to
ensure that pesticide residues do not exceed their MRLs.
Monitoring
programmes have shown that fungicide and insecticide
residues
are
often found in apples. In the EC coordinated monitoring
programme

for 2007, pesticide residues were detected in 47% of the 3454


apple
samples, collected and analyzed by the Member States,
Norway
and Iceland (European Food Safety Authority, 2009). The EU
MRLs
(EU MRL database 2009) were exceeded in 2.7% of the
samples.
Pesticide residues per fruit weight unit are dependent on
three
processes, i.e. variation in initial spray deposit, physical decay
due
to
weather factors and growth dilution. Variability in residues
between
individual samples is inevitable, partly because it is
impossible
to
achieve a uniform spray deposition of pesticides. Application
technique, crop architecture and growth stage have all been
shown
to

32

M.E. Poulsen et al. / Crop Protection 35 (2012) 5-14

affect variability in initial deposit. Canopy structure is one of


the

most important factors influencing pesticide residues. Many


studies

showed the importance of canopy structure in affecting initial


deposit concentrations (e.g. Xu et al., 2006; Rawn et al.,
2007);
usually fruits in the top and outside regions are likely to
receive
more
deposits than those inside the canopy. Also, in many practical
situations the initial deposit is inluenced by spray technology
(i.e. sprayer type, sprayer settings and nozzle type). Large
variability
in the level of residues exists between individual sample units
or
composite samples. There are internationally agreed
standards
for
monitoring pesticide residues and for assessing risks of
consumer
exposure. In general pesticide levels in EU fruit are below the
European MRLs (European Commission, 2010).
Protection of the environment, especially surface water,
during
the application of plant protection products (PPP) is of
increasing
public concern. For economic reasons fruit growers often
apply
low
spray volumes, and hence use fine spray nozzles that produce
small droplets (fine spray quality) that are particularly prone
to
drift.
A new option for drift mitigation in orchards is the use of air
induction
nozzles, which provide larger drop sizes (coarse spray
quality).
Coarser droplets reduce the air-borne drift losses signiicantly
compared to fine droplet spray applications (Koch et al., 2001;
Jaeken
et al., 2003; Wenneker et al., 2005). Coarse sprays, especially
at
lower
spray volumes, might produce a poorer spray coverage
compared
to fine sprays, and arouse growers' fears of reducing the
biological
eficacy of the treatment. The effect of coarse droplet
application
on the biological eficacy in fruit growing (mainly apples) has
been
assessed extensively by various institutions and companies in
Germany, Belgium, The Netherlands and France (Koch et al.,
2001;
Knewitz et al., 2002; Friessleben, 2003; Jaeken et al., 2003;
Loquet
et al., 2009). No signiicant differences between coarse droplet
application and conventional ine droplet application were
found.
Finer droplets with a smaller diameter give a greater coverage
for
any
given level of spray deposit compared to coarse droplets. In
order
to
compensate for less coverage it is often advised to use higher
water
volumes for coarse droplet spraying applications with air
induction
nozzles (Jaeken et al., 2003). In several studies the coarse
droplet
sprays produced slightly greater mean deposits and smaller
spray
losses (Cross et al., 2001; Balsari et al., 2005; Jamar et al.,
2010).
In

practice coarse droplet applications might result in more


visible
residue spots on apples and pears (fruits). However, it is
unknown
if
this also affects the residue levels of plant protection products
(PPPs)
in fruits as well. Because of the high values of spray drift in
orchard
spraying (Zande et al., 2001) compared to arable field
applications
(Huijsmans et al., 1997), the reduction of the emission of PPPs
in
fruit
growing is still of major importance. One of the measures is
the
use
of
drift reducing nozzles (Zande et al., 2008). It is a relatively
simple
and
cheap method. The use of drift reducing nozzles in fruit
growing
has been increased in many countries in Europe, e.g. Belgium,
France,
Germany and the Netherlands. Drift reducing nozzles are
often
accepted within legislation for fruit growing; e.g. Germany
(JKI,
2010),
United Kingdom (HSE, 2010), and the Netherlands (CTGB,
2008).
A ield treatment with pesticides is not uniform and consequently the distribution of pesticide residues may be very
wide,
from
areas receiving high amounts of pesticides to areas receiving
less
amount of pesticide. In 1994 research carried out in the UK,
indicated
that there might be occasional and random high residues of
pesticides found in individual samples of fresh fruit and vegetables
(Harris et al., 2000; Harris, 2000). The finding of high residue
variability is considered important in the assessment of acute
dietary
exposure, as it might lead to a higher intake of pesticide
residues
than previously believed. Field trial data derived from
commercial
use of pesticides indicates that the distribution of residues is
not
signiicantly inluenced by the 1) mean value of the residue, 2)
time
between pesticide application and sampling, 3) chemical and
physical properties of the active ingredient or formulation or
4)
the
volume application rate used for foliar application on fruit
trees.
On the other hand the distribution of residues is likely to be
inluenced by the size, shape and density of the plants and mode
of
application of the pesticide (Ambrus, 2000). Research on the
causes
of residue variability has been conducted by Carter et al.
(2000),
but
no conclusive evidence could be obtained which identify
particular
causes for the variability. Hill and Reynolds (2002)
demonstrated
that no relationship could be obtained between the variability

factor
and the 1) commodity, 2) country of origin, 3) residue
concentration
or 4) physiochemical characteristics of the pesticide including
the
systemic/non-systemic
properties
of
the
pesticide.
Furthermore,
it was seen that aging of the residue did not signiicantly affect
the
variability. Consequently, variability may only be reduced in
terms
of
more uniform application of the pesticide. When assessing the
variability it is relevant to look at the precision of the
analytical
method, as variation might contribute to the variability of the
single
commodities. Investigations conducted by Harris and Davis
(1998)
and Hill and Reynolds (2002) showed that the analytical
variation
did not signiicantly contribute to the overall variation. Thus
the
main contributor to the variation must be the difference in
residue
content between the individual units.
Explanation of the variability in pesticide residues is
essential
for the development of methods to minimize residues in fruits.
In
this study, residue variability associated with individual apples
and
composite samples was studied for two application
techniques;
i.e.
ine and coarse spray quality application. It was hypothesized
that
variation among individual fruits is the most important factor
of
residue variation.
Field trials were performed in a commercial orchard in
2007
with application of pesticides routinely used for pest and
disease
management in apple production. Apples were collected from
4
zones in the tree.
The aims of this study were: (1) to determine residue levels
of
commonly applied pesticides in apple growing after ield treatment, (2) to study the variation of these residue levels in
individual
fruits and/versus (calculated) composite samples, and (3) the
effect of a standard ine spray quality versus coarse spray
quality
application on residue levels.
2. Materials and methods
2.1. Field work
A ield trial to evaluate the effect of spray quality (droplet
size/
nozzle type) was done in 2007 in a commercial orchard in the
Netherlands. The spray applications for the reference
treatment
(fine
spray quality) were performed with brown Albuz ATR hollow

cone
nozzles (7 bar spray pressure) and for the low-drift treatment
(coarse
spray quality) with air induction (Venturi) hollow cone nozzles
(Albuz TVI 80-015 at 6 bar spray pressure). Spray applications
were carried out in the same orchard, and with the same
spraying
machine; a Munckhof cross-low sprayer with eight operating
nozzles at each side. The power take-off (PTO) speed was ixed
at
430 rpm, and the driving speed was 8 km h -1. These settings
resulted
in a spray volume rate of approximately 200 l ha-1 for the
brown
Albuz ATR nozzles and 300 l ha -1 for the air induction nozzles.
The
standard advice to growers is to use a higher water volume
for
drift
reducing air induction nozzles (Jaeken et al., 2003). The spray
quality
of the nozzles was quantiied using a Phase Doppler Particle
Analyser
(PDPA, Aerometrics). These measurements showed that the
brown
Albuz ATR nozzle had a Volume Median Diameter (VMD) of
143
mm
and the Albuz TVI 80-015 had a VMD of 493 mm (Zande et
al., 2008).
The orchard was divided in two experimental plots. Each
plot
consisted of 8 apple tree rows; i.e. 4 times 2 alternating rows
of
the
apple varieties Elstar and Jonagold of 150 m length (with an
average
tree height of 3.00 m, 3 m row spacing, and 1.25 m tree
separation in

35

M.E. Poulsen et al. / Crop Protection 35 (2012) 5-14

the row). Both plots were sprayed for the whole season with
the
prescribed nozzle type, according to a standard commercial
spraying
scheme with insecticides and fungicides (Table 1), following
label
directions. Dose rates were therefore kept equal for the Albuz
ATR
and Albuz TVI nozzles.
The distribution in different zones of the canopy was
evaluated
by dividing the apple tree (spindle type) into four distinct
zones:
top, middle, bottom outside and bottom inside (Fig. 1). Four
apples
were collected from five trees per nozzle type and tree zone.
Selection of the trees and apples per zone was randomized
from
each tree row (variety Elstar) in the middle of the
experimental
plot. In summary, the levels in the field sampling used were:
(1)
fruits within a zone, (2) zone within a tree, (3) trees within a
nozzle
type. Two days before the start of commercial harvest, single
fruit
samples were taken from ive trees for each zone and nozzle
type.
The total weight of each individual apple was determined
before
the residue analysis.
2.2. Chemicals
The pesticide standards of boscalid, bupirimate, captan,

Fig. 1. Four zones within the apples trees from where apples were sampled. T: Top,
M: Middle, BU: Bottom Outside, BI: Bottom Inside.

carbendazim, fenoxycarb, indoxacarb, pirimicarb, pyraclostrobin and


thiophanate-methyl (with certified purities >96%) were
purchased
from Dr. Ehrenstorfer (Augsburg, Germany). The acetonitrile
p.a.
was
from Fisher Scientific (Leichestershire, UK). Acetic acid and
ammonium acetate were from Merck (Darmstadt, Germany). The
magnesium sulphate was purchased from Mallinckrodt Baker
(Phillipsburg, NJ, USA), sodium chloride from Merck & Co.
(Whitehouse Station, NJ, USA), sodium citrate dehydrate and sodium
citrate
sesquihydrate
from
SigmaAldrich
Chemie
GmbH
(Taufkirchen,
Germany) and the clean/up sorbent PSA from Varian (Palo
Alto,
CA,
USA). Stock solutions of pesticides of 1 mg ml -1 were prepared
in
methanol or toluene and kept at -18 C. A standard mixture of
10 mg ml-1 in acetonitrile was prepared from the stock
solutions.
Matrix-matched standards in the concentration range from
0.0015

36

M.E. Poulsen et al. / Crop Protection 35 (2012) 5-14

Table 1
Application date, pesticide formulations and active ingredients.

M.E. Poulsen et al. / Crop Protection


37
35 (2012) 5-14

Application date
8 May 2007
14 May 2007
19 May 2007
25 May 2007
2 June 2007
6 June 2007
11 June 2007

Pesticide product
Merpan
Merpan
Merpan
Merpan
Merpan
Merpan
Merpan
Nimrod

Active ingredients
Captan
Captan
Captan
Captan
Captan
Captan
Captan
Bupirimate

Insegar

Fenoxycarb

Merpan
Nimrod

Captan
Bupirimate

Insegar

Fenoxycarb

19 June 2007

Merpan
Steward

Captan
Indoxacarb

25 June 2007
30 June 2007
8 July 2007
15 July 2007
19 July 2007
27 July 2007
04 August 2007
13 August 2007

Merpan
Merpan
Merpan
Merpan
Bellis
Merpan
Merpan
Bellis
Merpan

Captan
Captan
Captan
Captan
Boscalid + Pyraclostrobin
Captan
Captan
Boscalid + Pyraclostrobin
Captan

19 August 2007

Merpan
Pirimor

Captan
Pirimicarb

25 August 2007

Merpan
Topsin-M

Captan
Thiophanate-methyl

16 June 2007

Harvest 4 September 2007

*: Pirimor was sprayed to control aphids in the lower parts of the trees. The top
nozzles of the sprayer were closed in order to avoid unnecessary residues.

to 0.05 mg ml-1 ( ~ 0.0070.24 mg kg-1 matrix) were


prepared
by
diluting the 10 mg ml-1 mixture with sample extracts of
apples
bought as organically grown.
2.3.Analytical method
The analyses were carried out according to Anastassiades
et
al.
(2003) and modified by Paya et al. (2007). The apples were
homogenized individually without removing peel or apple
core.
Precisely 15.0 g of apple homogenate was extracted with 15.0
ml
of
acetonitrile and 6.0 g magnesium sulphate, 1.5 g sodium
chloride,
1.5 g sodium citrate dihydrate and 0.75 g sodium citrate
sesquihydrate were added. After 1 min of shaking and centrifugation
for
10 min, 1.0 ml of extracts were transferred to small centrifuge
tubes
with containing 0.05 g PSA and 0.15 g magnesium sulphate.
After
shaking for 30 s and centrifugation for 5 min, the extracts
were
analyzed by LC/MS/MS and GC/MS/MS in MRM mode with two
transitions (Table 2). The recovery samples analyzed together
with
the apple samples (n = 10) were between 76 and 101% (Table
2).
2.4. Statistical analysis
In statistical terms the whole experiment can be described
as
follows: Block structure (random model) was 'plot/tree/(zone
within tree)' (with 5 apples from each zone). The treatment
structure (fixed model) was '(nozzle type)*(zone within tree)'. Since
the

38
Table 2

M.E. Poulsen et al. / Crop Protection 35 (2012) 5-14

MRM transitions, cone voltages, collision energies and recoveries for LC/MS/MS and GC/MS/MS analysis of boscalid, bupirimate, carbaryl 13-C6, carbendazim, captan,
chlorpyrifos D10, fenoxycarb, indoxacarb, pirimicarb, pyraclostrobin, thiophanate-methyl and tetrahydrophthalimide. Carbaryl 13-C6 and chlorpyrifos D10 were used as
injection standards and consequently no recoveries are listed for these compounds.
GC/MS/MS
LC/MS/MS
Instrument Compound
Transistionl Cone Voltage Collision energy Transistion2 Cone Voltage2 Collision Energy2 Recovery at 0.02 mg kg
Boscalid
343 >307 57
20
343 > 140 57
20
93%
Bupirimat

317

Carbaryl 13-C6

225

>166
>151

27

23

317

25

14

208

> 108
> 151

25

50

50

14

Carbendazim

192

Fenoxycarb

>160
>88

80

15

192

> 132

80

15

302

25

20

>

Indoxacarb

529

>218

65

20

>

Pirimicarb

239

>72

25

16

239

Pyraclostrobin

388

24

11

388

Thiophanate-methyl

343

50

12

343

Bupirimate

316

>194
>151
>208

15

273

> 163
> 93
> 193

Captan

149

>70

12

149

Chlorpyrifos D10

326

>262

15

292

Pirimicarb

238

>166

10

Tetrahydrophthalimide

151

>80

76%
85%
95%

> 182

25

14

24

25

98%

50

12

101%

10

92%

> 105

90%

> 260

10

166

> 96

15

87%

123

> 80

97%

nozzle-type treatment was carried out on the plots, its effect


was tested against variance amongst trees within plots. The
amount
of residue on the fruits appeared to have a lognormal
distribution,
all data were analyzed as a GLMM (Generalized Linear Mixed
Model)
based on a lognormal distribution using GenStat Release 9.2
statistical package (Lawes Agricultural Trust, Rothamsted
Research,
UK). Significant F-tests (p < 0.05) were followed by a Least
Significant Difference (LSD)-test for pair wise comparison of
treatment
means using LSD p-0.05 values. Treatment means were
estimated
on the transformed scale and later back transformed to the
original
scale.
3. Results and discussion
3.1. The effect of standard fine spray quality versus coarse
spray
quality application
The effects on pesticides residues in the apples will be
presented
by focusing on general characteristics of pesticide residues in
individual apples, nozzle effect (spray quality), zone effect
and
the
effects on the individual pesticides. The weight of the
individual
apples was in the range of 114252 g with a median value of
168
g.
No statistically signiicant correlation between the fruit weight
(size) and residue level was observed, showing that the
residue
levels were independent of the fruit size. Consequently, the
following results are based on the residue levels in the
individual
fruits expressed in mg kg-1
-1
.
3.1.1. General characteristics ofpesticide residues

94%

90%

in individual apples
Residues of bupirimate were detected in almost all the
apple
samples but less than 15% of the residues were above the
Limit
of
Quantification (LOQ) of 0.005 mg kg -1. Pirimicarb was not
sprayed
by
the top nozzles (see Table 1), because only the lower parts of
the
trees
were infested by aphids, and consequently pirimicarb was not
found
in 36 apples picked from the top of the trees. Residues of
fenoxycarb
and indoxacarb were only found in few apples probably due to
the
early application time for those two compounds. Statistical
evaluation
of bupirimate, pirimicarb, fenoxycarb and indoxacarb is
therefore
not
included in this presentation. For carbendazim, residues were
detected above the LOQ (0.01 mg kg-1) in 156 of the 160 apples. All
four
apples with no detectable residues were sprayed by the air
induction
nozzle; two were picked from the top and two from the
bottom
inside.
Thiophanate-methyl residues were not detected above the
LOQ
(0.01 mg kg-1) in three apples, i.e. one air induction nozzle
application
top and two standard application bottom outside. Boscalid,
pyraclostrobin, captan and the degradation product
tetrahydrophthalimide were found in all 160 samples (LOQ = 0.01 mg kg -1).
This
is
in accordance with Xu et al. (2008) who found that initial
captan
deposits and subsequent residues on fruits and leaves are
generally
closer to a lognormal than to a normal distribution.
None of the results for the pesticides residues measured in
the
apples exceeded the MRLs (European Commission, 2010).
However,

39
Table 2

M.E. Poulsen et al. / Crop Protection 35 (2012) 5-14

there was a high variation in the residues with levels from


0.01
to
1.4 mg kg-1 for captan, the pesticides with the highest range,
and
from 0.01 to 0.2 mg kg -1 for pyraclostrobin, the pesticide with
the
lowest range.
3.1.2.Spray quality (nozzle) effect
The mean values for the two applications were statistically
evaluated to see if one of the application modes resulted in
higher
residues. Fig. 2 shows the mean residue values from the 4
zones
of
both application modes. The evaluation showed that the
mean
residue levels for all apples sprayed with standard nozzles
(ine
spray quality) versus air induction nozzles (coarse spray
quality)
were not different for boscalid, pyraclostrobin, thiophanatemethyl
and the degradation product tetrahydrophthalimide. For
captan
the
mean value for air induction nozzle application was higher
and
for
the degradation product of thiophanate-methyl, carbendazim,
the
standard application gave higher mean values. Looking at the
mean
values for the 4 zones, only few differences were seen.
Captan
and
thiophanate-methyl had for air induction nozzle application

higher
mean values at the middle zone, while tetrahydrophthalimide
had
higher levels for the bottom inside zone. For carbendazim the
standard application resulted in higher mean values at all
zones.
More details are described below under the active
substances.
3.1.3.Zone effect
The mean values for the four zones have been statistically
evaluated to see if one of the application modes gave a more
even
distribution (Fig. 3). The evaluation for boscalid and
pyraclostrobin
showed
that
the
pesticides
seem
to
be
more
evenlydistributed
bythe
air induction nozzle application, while it was opposite for
carbendazim. However, no major differences were seen. Xu et al.
(2008)
showed that the unit-to-unit variation contributes most to the
observed variability in captan residues, not only in the initial
spray
deposition but also over the subsequent period following the
application. More details are described below under the active
substances.
3.1.4.Boscalid and pyraclostrobin
Boscalid and pyraclostrobin were sprayed simultaneously
(as they were active ingredients of the fungicide product
Bellis)

40
Boscalid

Pyraclostrobin
M
.
E
.

0.00

P
o
u
l
s

mg/kg

Whole tree BI

BU
Zone

Whole tree

0.10
mg/kg

Captan

BI BU
Zone

T
n
e
t

0.08

BI

0.25
0.20

BU
Zone

Thiophanate-methyl

0.15

Whole tree BI

0.02
0.00

0.05

Whole tree
Zone

0.00

mg/kg

BI

BU

T
.
/

0.06
0.04

0.10

BU
Zone

C
r
o
p
P
r
o
t
e
c
t
i
o
n
3
5
(
2
0
1
2
)
5

1
4

0.35
0.30
0.25
0.20
0.15
0.10
0.05

s
t
a
n
d
a
r

41
Boscalid

Pyraclostrobin
d
mg/kg
air induction nozzle
0.10
0.35
0.08
0.30
0.25
0.06
0.20
0.04
0.15
0.10
0.02
0.08
0.05
0.00
0.00
0.06
0.04
0.02
0.00
'
a

Tetr
ahy
dro
pht
hali
mid
e
mg/kg
0.10
b
b

W
h

Carbendazim
b

M
T

42
Boscalid

Pyraclostrobin
Zone
Fig. 2. Effect of nozzle types (spray quality); Mean values of pesticide residues of boscalid, pyraclostrobin, captan and its degradation compound tetrahydrophthalimide,
carbendazim and thiophanate-methyl from analysis of individual apples picked at four zones within the tree. BI: bottom inside, BU: bottom outside, M: middle, T: top. The
mean
values for all apples are also shown. White bars are standard application Albuz ATR nozzle (fine spray quality); black bars are air induction nozzle application (coarse spray
quality).
Bars labeled with the same letter do not differ significantly p = 0.05.

twice towards at the end of the growing season to prevent


storage
rots (Table 1). No differences were observed between the
residue
levels per zone between the two nozzles types. The residue
patterns per zone for boscalid and pyraclostrobin were the
same
for
the two nozzle types, indicating that the spraying technique
did
not
influence the residues levels.
3.1.5. Captan
Captan was the most frequent applied fungicide (Table 1).
Captan contains an unstable trichloromethyl 'moiety' and
leads
to
degradation
to
tetrahydrophthalimide
and
tetrahydrophthalimide
acid. Huo et al. (2007) have reported that daily loss of captan
was
very small, the main loss of captan being due to wash-off by
rain
(Smith and MacHardy, 1984; Frank et al., 1985, 1987;

Northover
et al., 1986). The monograph on captan by FAO/WHO (1970)
also
reports that captan does not appear to undergo any chemical
change on the plant surface. However, Ticha et al. (2007)
showed
captan residues in apples successively decreased until
harvest.
None of the above quoted studies had measured the
degradation
product tetrahydrophthalimide. In this study tetrahydrophthalimide was found in higher levels than captan. The results
showed that approximately 80% of the sum of captan and
tetrahydrophthalimide was found as tetrahydrophthalimide, when
taking the differences in molecular weights into account. The
results are in line with results from Poulsen et al. (2009),
where
7
different spraying strategies were performed in commercial
orchards. However, part of the degradation could occur in the
laboratory during the chemical analysis. Captan is rapidly
degraded

43
Boscalid

Pyraclostrobin
0.00
0.15
0.20
0.25
ME.
0.10Poulsen et al. / Crop Protection 35 (2012) 514

mg/kg

BI BU
standard

T BI BU M
air induction

44
Boscalid

Pyraclostrobin
Standard
mg/kg
0.35
0.30

Tetrah yd roph th al i m ide

Captan

0.2
b

5
0.2
0
0.1
5
0.1

- 1
F
Iii
IX
M

BI

BU

air induction

BI

BU

BI BU
standard
T

BI BU M
air induction

45
Boscalid

Pyraclostrobin
BU
mg/kg
M
0.10
standard

Th ioph an ate-methyl
0.08

---------c

0.06

0.04
0.02

BU M
standard

BI BU M
air induction

Carbendazim
bb

Al

0.00 Bl

I
Bl

BU

air induction

46
Boscalid

Pyraclostrobin
Fig. 3. Effect of the zone within the tree; mean values of pesticide residues of boscalid, pyraclostrobin, captan and its degradation compound tetrahydrophthalimide,
thiophanatemethyl and carbendazim, from analysis of individual apples picked at four zones. BI: bottom inside, BU: bottom outside, M: middle, T: top. Results for standard application
(fine spray quality) and air induction nozzle application (coarse spray quality) are shown bars labeled with the same letter do not differ significantly p = 0.05.

in high temperatures e.g. present in the injector of the gas


chromatograph. However, the injection mode used for the
analysis
has been shown by Bailey (2007) to minimize the degradation
of
captan. As described in Materials and methods, the recovery
samples analyzed together with the apple samples (n = 10)
gave
a
mean
recovery
for
captan
at
90%
and
for
tetrahydrophthalimide
97% (spike level 0.02 mg kgm g /k g
a

-1

). This indicates the any possible


degradation in the injector was equal for calibration standards
and
samples. However, degradation may have occurred during
sample
preparation, when the apples were chopped.
Fig. 3 shows that the residue levels for captan and tetrahydrophthalimide have the same patterns for both nozzle types.
There was a general trend of higher residue levels for the
coarse
droplet application; with significant higher levels for the
middle
zone for captan and inner zone for tetrahydrophthalimide.

3.1.6. Thiophanate-methyl and carbendazim


Thiophanate-methyl and carbendazim are broad spectrum
systemic fungicides. However, carbendazim is also a
degradation
product of thiophanate-methyl. Thiophanate-methyl is quickly
absorbed by the plants surfaces and transported to the
various
parts as carbendazim. Mandal et al. (2010) have studied the
dissipation on grapes and found that the half-life of thiophanatemethyl
is between 4.74 and 6.52 day irrespective of location and
doses.
According to FAO/WHO (1974) the transformation to a great
extent
is light-catalyzed.
Thiophanate-methyl showed the same residue patterns for
the
zones and nozzles as captan/tetrahydrophthalimide did; i.e.
highest
residues in the bottom outside and the middle zone for both
nozzles types. Between the nozzles, residues were in higher
for
the
air induction nozzle (coarse spray quality), while the
degradation

47
Table
3

M.E. Poulsen et al. / Crop Protection 35 (2012) 5-14

mg/kg

Acceptable Daily Intake (ADI) and Acute Reference Dose (ARfD) in mg kg -1 body
weight for boscalid, pyraclostrobin, captan, tetrahydrophthalimide, carbendazim
and thiophanate-methyl.
Acceptable Daily Intake,

Acute Reference Dose,

ADI (mg kg-1 b.w.)


Boscalid

0.04

none

Bupirimate

0.05

0.05

Captan

0.1

Carbendazim

0.02

0.02

Fenoxycarb

0.04

none

Indoxacarb

0.006

0.02

Pirimicarb

0.035

Pyraclostrobin

0.03

Tetrahyd

ARfD (mg kg-1 b.w.)

rophthal
imide

0.3

0.1
0.03

Tetrahydrophthalimide

none found

Thiophanate-methyl

0.08

none found
0.2

product carbendazim shows the complete opposite deposition


pattern for the two nozzles types. With the standard nozzles
(fine
spray quality) the carbendazim level was doubled, in contrast
the
carbendazim levels for the air induction nozzles. The small
droplets
have a bigger total surface area and thereby more
susceptible for
degradation due to sunlight.
No correlations were seen between thiophanate-methyl
residue
level and carbendazim residue levels for the two nozzle types.
In general fruits in the inner and top zones received less
residue
(or deposit) than the outside and middle zones (for both
nozzle
types). This is in contrast to observations of Xu et al. (2008).
However, the trials of Xu et al. (2008) were performed with an
axial
fan sprayer and not with a cross-flow sprayer, as in this
experiment.
These two types of sprayer types give different deposition
proiles
(Michielsen et al., 2008).

Fine spray quality

Fine sprayquality

Coarse sprayquality

Coarse spray quality

X
Boscali
d nX

mg/k
0.60g

0.30
0.50

0.20
0.40
0.10
0.00

Individual

Captan
Composite

Pyraclostrobin
X

mg/k
0.25g

i
Composit

Individua

0.20

0.15

0.10
0.05

i
I

Composite
Individual
Composite
-----------------------------------------1----------------------------------------------------------------------------,-------------------------------------1

M.E. Poulsen et al. / Crop Protection


48
Table
3 35 (2012) 5-14

mg/k
1.5 g
0

Coarse spray qualityx

Fine sprayquality

0.80
0.60

Fine sprayquality
v

1.2

0.7
5

0.5
0

x
1.0
0

Coarse spray quality

I
i

0.25
0.00

Composite

Composite

Individual

1.00

Composite

Individual

Composite

49
Table
3

M.E. Poulsen et al. / Crop Protection 35 (2012) 5-14

Thiophanate-methyl

mg/k
0.30g

xx

0.25

Fine spray quality

0.15

0.10
0.05

Composite

Individual

X
x

0.20
0.15

Composite

Individua
l

Coarse spray quality

0.25

0.20

0.00

Carbendazim

mg/k
0.30g

0.10

i
i

'

0.05

0.00
Composite

Individual

Composite

Individual

Fig. 4. Results for composite and individual samples for pesticide residues of boscalid, pyraclostrobin, captan and its degradation compound tetrahydrophthalimide,
carbendazim
and thiophanate-methyl. Results are shown for both standard application Albuz ATR nozzle (fine spray quality) and air induction nozzle application (coarse spray quality).

50
Table 4

M.E. Poulsen et al. / Crop Protection 35 (2012) 5-14

Variability factor for standard and air induction nozzle application for boscalid,
captan,
carbendazim, tetrahydrophthalimide, thiophanate-methyl and pyraclostrobin.

3.2. Composite samples

Standard
application
Boscalid
Captan
Carbendazim
Tetrahydrophthalimide
Thiophanate-methyl
Pyraclostrobin

2.0
9.4
1.8
4.0
3.9
2.2

Air induction
nozzle application
2.7
4.3
0.9
2.7
3.8
2.6

The EU MRLs and CODEX MRLs for apples are set for the
mean
residue value of one kg or a minimum of ten apples (European
Commission, 2002; CODEX, 1999). Originally, the toxicological
evaluation of pesticides was performed for chronic effects
only
by
setting an Acceptable Daily Intake (ADI) (Table 3). For control
purposes of ADI an average residue value are more suitable in
order
to smooth out the variation in residue levels, because a
person
eating a high pesticide residue content apple one day will
most
likely eat an apple with at lower content the next day. Consequently, the control for pesticide residues in apples was
decided
to
be carried out for composite samples. Later on, the pesticides
were
evaluated for acute effects and Acute Reference Doses (ARfD)
were
set. To control the ARfDs it is important to know the pesticide
residue in the individual apple, because a person will be
exposed
to
acute effects from a speciic apple, if the residues are high
enough.
However, it would be costly to control the ARfD, if the apple
samples should be analyzed individually as ten apples should
be
analyzed. Instead, the pesticide residue levels in the
composite
samples are calculated back to the assumed highest possible
level
in the individual apples, by applying a variability factor to the
average concentration of ten apples. In EU member states the
variability factor used is seven (EU Commission, 2004),
whereas
CODEX has lowered the factor to a default value of three until
suficient data on single unit is available to calculate a more
realistic
variability factor (WHO, 2010).
To investigate the distribution of the pesticides between
the
apples from the two application modes, all apples were
individually
analyzed. The results of from the analysis of the individual
apples
showed large variations in residue levels, where the apples
with
the
highest concentration had between 12 and 200 times higher
residue
levels than in the apple with the lowest level. To examine the
rela-

Table 5
The minimum and maximum number of individual results not covered by the
variability
factor
3
and
7,
for
boscalid,
pyraclostrobin,
captan,
tetrahydrophthalimid,
carbendazim and thiophanate-methyl, obtained by standard and air induction
nozzle application. The number before the arrows refers to the highest concentration of the 20 mathematically constructed composite samples and the number
after
the arrow refers to the lowest concentration of 20 the mathematically constructed
composite samples. The mathematically constructed composite samples represent
a sample of ten apples used for control of MRLs.
Standard application

Boscalid
Pyraclostrobin
Captan
Tetrahydrophthalimid
Thiophanate-methyl
Carbendazim

Factor 3
0/1
0/1
7 / 24
0/8
2/8
0/0

Factor 7
0/0
0/0
1/4
0/0
0/0
0/0

Air induction
application
Factor 3
1/4
0/0
0/7
0/4
1/6
0/0

Factor 7
0/0
0/0
0/0
0/0
0/1
0/0

tionship between individual results and average results from


10 apples that are currently used for pesticides control of
MRLs,
composite samples were statistically constructed from ten of
the
individual results chosen at complete random. These
mathematically
constructed composite samples would represent the result if
ten
apples were homogenized together and then analyzed. The
results
from analysis of the individual apples and the calculated
pesticide
level for the composite samples are seen in Fig. 4. The
composite
samples showed less variability since they were average
concentrations of 10 results.
The variability factors at 97.5 percentile of the 80
individual
apples were calculated for both standard and air induction
nozzle
application as:
Variability factor = Concentration of the individual sample
at
97.5 percentile/back transformed mean.
The calculation showed variability factors in the range of
0.99.4 (Table 4). The variability factors for captan were
highest,
9.4
and 4.3. Xu et al. (2006) calculated variability factors for
captan
to
be approximately 2, which is much lower than in this study.
The results from the analysis of the individual apples and
results
from the mathematically constructed composite samples can
show
if the variability factor of three or seven would be adequate to
cover
the highest results from the analysis of the individual apples.
Table 5 shows the number of high results from the analysis of
individual apples that would not be covered by a variability
factor
of either three or seven, if applied to the results
mathematically
constructed composite samples.

51
Table 4

M.E. Poulsen et al. / Crop Protection 35 (2012) 5-14

The figures before the arrows are the number of individual


results
that are above three or seven times the lowest average
concentration
of the 20 mathematically constructed composite samples and
the
igures after the arrows are likewise the number of individually
results that is above three or seven times the highest
concentration
of the 20 mathematically constructed composite. The
minimum
and
maximum number of results not covered by the variability
factors
of
three and seven were calculated by counting the number of:
Results of analysis of individual apples >3 (or 7)*lowest (or
highest) result of 20 mathematically constructed composite
samples.
For example, for boscalid and standard application (ine
spray
quality) Table 5 shows that the variability factor of 7 is
adequate
because even if the lowest result of composite samples was
used.
However, for the variability factor of three one result (the
highest)
of the individual results would be above 3 times the lowest
(or highest) result of 20 mathematically constructed
composite
samples. Likewise for the air induction application (coarse
spray
quality) the variability factor of seven were adequate, while
for
the
factor of three, the four results (the four highest) would be
above
3
times the lowest (or highest) result of 20 mathematically constructed composite samples, if the lowest result of the

composite
samples was used and one result (the highest) if calculated
with
the
highest result for the composite samples.
As seen from Table 5 the variability factor of seven seems
to
be
adequate to include almost all the results for the pesticides.
For
captan up to 9% of the results were not covered depending on
the
composite concentration chosen. However, the variability
factor
of
three seems to be too low, because up to 30% of the apple
samples
for captan were not covered if the worst case scenario was
chosen.
Also for thiophanate-methyl the factor of three seems to be
too low.
In conclusion, no statistically significant correlation
between
the
fruit weight (size) and residue level was observed, showing
that
the residue levels were independent of the fruit size. None of
the
results for the pesticides residues measured in the apples
exceeded
the EU MRLs. For boscalid and pyraclostrobin no differences
were
seen in the residue levels for the two spraying techniques.
The
coarse spray quality (air induction nozzle) resulted in higher
levels
for captan and thiophanate-methyl at the middle zone and for
tetrahydrophthalimide at the bottom inside zone. However,
with

52

M.E. Poulsen et al. / Crop Protection 35 (2012) 5-14

the fine spray quality (Albuz ATR nozzle), the carbendazim


levels
were doubled in contrast to the coarse spray quality. The
small
droplets had in total a bigger surface area and thereby
thiophanatemethyl was supposed to be more susceptible for degradation
to
carbendazim due to sunlight. Pesticide residues levels of the
apples
were affected by the zone of the fruits within the tree. There
were
no major differences in residue levels of the apples between
nozzle
types for the same zone within the tree. The unit-to-unit
variation
seems to contribute most to the observed variability. The
variability
factors calculated for both fine spray quality (Albuz ATR
nozzle)
and
coarse spray quality (air induction nozzle) application (n = 80)
and
were in the range of 0.99.4. The use of a variability factor of
seven
in the risk assessment calculation of acute effects based on
control
composite samples seems to be adequate to include almost
all
the
individual pesticide residues results in this study.
Acknowledgments
The authors wish to thank the European Commission and
the Dutch Ministry of Agriculture, Nature & Food Quality for
financial
support of the ISAFRUIT project. The ISAFRUIT project is
funded
by
the
European Commission under the Thematic Priority 5-Food
Quality
and Safety of the 6th Framework Programme of RTD (Contract
no.
FP6FOOD-CT-2006-016279). We also thank fruit grower Mr. M.
Koning
for
making his orchard available for the experiments and Mr. R.
Anbergen
for technical assistance.
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