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thesis entitled

FACTORS AFFECTING THE EFFICACY OF
DIPHENYLAMINE FOR APPLE
SCALD CONTROL

presented by

Lorenzo George Wilson

has been accepted towards fulfillment
of the requirements for

Ph oD 0 degree in HOPtiCUlture

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ABSTRACT

FACTORS AFFECTING THE EFFICACY OF
OTFHENYLAMINE FOR APPLE
SCALD CONTROL
By

Lorenzo George Wilson

This work concerns the postharvest application of
Ctipknenylamine (DPA) to apples for the control of storage
scetld, an important physiological disorder affecting the
(quetlity of the stored fruit. Investigations were con—
dtuited.to determine the need for renewing DPA solutions,
‘to Joelate the DPA content of the treatment solutions to
:resxidues and scald control, and to ascertain the effects
of‘.applying DPA at elevated hydrostatic pressures for
valfiious time durations. Factors studied included solution
agxa, temperature and contamination, plus the effect of
fiuiit submergence at several depths and durations for two
forumilations of DPA on three apple varieties. DPA resi-
<iues on the fruit, scald control and fruit injury were
evaluated.

Reliable residue values of 0-25 ppm DPA for whole
apples could not be obtained by gas chromatographic pro-
cedures. A colorimetric analytical procedure was adapted
whereby DPA extracted from blended apple tissue with
acetone was reacted with vanadium pentoxide and quanti-

tatively determined spectrophotometrically at 605 mu.

 

 

 

Lorenzo George Wilson

TPhixs basic procedure was employed also for estimating the
DFVX content of solutions. A simplified modification of
tdie rnethod for monitoring the DPA content of commercial
apgxlicator systems was examined.

Satisfactory scald control was obtained for all
vauoieties with 1000 ppm DPA, which gave residues of
apqxroximately 2 ppm. There was no significant improve-
Inerrt in control by treatment with 2000 ppm when employed
:foxe the Delicious variety, even though fruit residues
inerwe doubled. Untreated McIntosh apples developed 95%
SCiild, Delicious 59%, and Home Beauty 79%. No fruit
irrjuries from DPA treatments were observed.

DPA solutions contaminated with organic matter
5L1elded greater DPA residues on apples than clean solu-
txions. Aging of clean solutions yielded greater resi-
chies upon aging up to the maximum of four weeks, expec-

;ially on McIntosh as compared to Delicious. Apples

C"
*3

eated in solutions regulated to temperatures of 50,
6C), or 70CF had similar DPA residues. Residues were
ixicreased by submerging the apples to depths of 6

.ft and by prolonging the submergence treatment from 0 to
53 minutes. Although residues varied by fruit treatments,
:nean values remained within the 10 ppm tolerance estab-
lished by the FDA.

Autoradiography of Delicious apples submerged for

10 minutes at A.0 psi hydrostatic pressure in luC-DPA

Lorenzo George Wilson

solutions showed the DPA residues to be primarily confined
to, or adjacent to the epidermal areas of the fruit.

It was concluded that the variable practices used
for commercial scald control on apples with DPA yield
fruit residues within an acceptable range and result in

good scald control .

 

 

 

FACTORS AFFECTING THE EFFICACY OF
DIPHENYLAMINE FOR APPLE

SCALD CONTROL

By

Lorenzo George Wilson

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Horticulture

1969

'P‘

 

ACKNOWLEDGMENTS

The author wishes to express his sincere thanks
and gratitude to Dr. D. H. Dewey for his constant super-

'ViJSiJDn, guidance, aid, and understanding throughout these
studie&

The writer is also indebted to Dr. D. R. Dilley,

A.. ll. Kenworthy, N. C. Leeling, and C. J. Pollard who

:xrcnlided technical assistance, reviewed the manuscript,
and served as members of the committee.

Grateful acknowledgment is also accorded to Dr.

NI. J’. Zabik for his technical assistance; and to the

Monhigan apple growers who cooperated in several phases

of‘ the work.

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TABLE OF CONTENTS

ACKNOWLEDGMENTS
LIST OF TABLES.
LIST OF FIGURES

LI ST OF APPENDICES

INTRODUCTION
LITERATURE REVIEW.
MATERIALS AND METHODS

DPA Analyses For Residues on Apples.
Solution Analyses in the Laboratory.
Solution Analyses in the Field

Aging Solutions . .

Artificial Scald Induction.

Submersion (Hydrostatic Pressure)
Commercial DPA Applications

Synthesis of Radioactive Diphenylamine.
Autoradiography . . . . . . . . .
Statistical Procedures

RESULTS

DPA Analytical Technique .

DPA Residues on Apples as Affected by Aging
Solutions and Hydrostatic Pressure

Factors Affecting the DPA Content of
Solutions. .

Storage Scald Control as Influenced by DPA
Solution Age, Sanitation and Hydrostatic

Pressure .
Autfiradiography of Apples Submerged in.
C- DPA . . . . .
iii

Page

ii

vii

viii

15

15
17
l8

19
21
22

23
2A

25
26
27
27
27

38

A3
A6

Page
DISCUSSION . . . . . . . . . . . . . . 51
CONCLUSIONS. . . . . . . . . . . . . . 61
SUMMARY . A. . . . . . . . . . . . . . 63
LITERATURE CITED . . . . . . . . . . . . 66

APPENDIX. . . . . . . . . . . . . . . 72

iv

Table

l.

10.

11”

12h

LIST OF TABLES

Analysis of variance of DPA fruit residues
in the 1967 aging solution study.

Analysis of variance of DPA fruit residues
in the 1968 aging solution study.

Mean DPA residues (ppm) for apples as
affected by organic matter. .

Mean DPA residues (ppm) for apples as
affected by solution age . .

Analysis of variance of DPA residues in the
1967 submersion study

Analysis of variance of DPA residues in the
1968 submersion study . . . .

Mean DPA residues (ppm) for apples as
affected by duration of submersion in DPA
in 1967

Mean DPA residues (ppm) for apples as
affected by submersion in DPA solution
(hydrostatic pressure)

Analysis of variance for DPA solution con-
tent in the 1967 aging solution study

Analysis of variance for DPA solution con—
tent in the 1968 aging solution study

Mean DPA content (ppm) of solutions as
affected by solution age in 1967.

Analysis of variance of scald reduction data
from the 1968 aging solution study

Page

28

29

3O

32

37

37

39

39

Al

A2

“3

AA

Table Page

13. Analysis of variance of apple scald in the
1968 submersion study . . . . . . . . A5

1A. Apple scald control in submersion solutions
in 1968 as affected by DPA concentration . . A6

15. Commercial DPA solutions used in Michigan--
1968 . . . . . . . . . . . . . . A9

vi

iFigure

1.

LIST OF FIGURES

DPA residues (ppm) as affected by the
interaction between solution sanitation
and technical formulation for apples
dipped in 1000 ppm DPA solution for one
minute in 1967 . . . . . . . .

DPA residues (ppm) as affected by the
interaction between solution age and
apple variety for apples dipped in 1000
ppm DPA solution for one minute in 1967

DPA residues (ppm) as affected by the
interaction between solution age and
technical formulation for apples dipped
in61000 ppm DPA solution for one minute
19 7 . . . . . . . . . .

DPA residues (ppm) as affected by the

in

interaction between solution age and sani-
tation for apples dipped in l000 ppm DPA

solution for one minute in 1967.

DPA residues (ppm) as affected by the

interaction between solution age and sani-
tation for apples dipped in 1000 ppm DPA

solution for 30 seconds in 1968.

DPA residues (ppm) as affected by the

interaction between hydrostatic pressure

and DPA solution concentration for apples

submerged in DPA solutions for 0,2 or A

minutes in 1968

Apple scald control in submersion solutions

as affected by the interaction between

hydrostatic pressure and DPA treatments for

0, 2 or A minutes' duration . . .

Autoradiograph of cross section of Delicious

apple submerged in lL‘C-DPA solution for
minutes at A psi pressure. . . .

vii

10

Page

31

33

3A

35

36

A0

A7

A8

'Table

II.

III.

IV.

VI.

‘VII.

\IIII.

IX.

LIST OF APPENDICES

DPA residues (ppm) on McIntosh and
Delicious apples as influenced by DPA
formulations used over a A week period
in l967.

DPA residues (ppm) on McIntosh and

Delicious apples as influenced by the
presence of organic matter and the age
of the DPA treatment solution in 1967.

DPA residues (ppm) on McIntosh,
Delicious and Rome Beauty apples as
influenced by the presence of organic
magger and treatment solution age in
19 .

DPA residues (ppm) on apples resulting
from treatment in a simulated submersion
system. 1967. . . . . . . .

Effect of DPA solution concentration, dur-
ation of treatment and depth of submersion
of DPA residues on Delicious apples in
1968. o o o o o o o o o o

Apparent diphenylamine residues (ppm)
observed on Delicious apples treated in
a submersion system containing only
water - I968 . . . .

DPA (ppm) in clean and contaminated treat-
ment solutions of various ages in 1967

DPA (ppm) in clean and contaminated treat—
ment solutions of various ages in 1968

Effect of DPA solution age and sanitation

on scald reduction for McIntosh, Delicious
and Rome Beauty apples in 1968

viii

Page

73

7A

75

76

77

77

78

78

79

Table Page
X. Effect Of DPA concentration, submergence

depth and duration of treatment on scald
development on Delicious apples in 1968 . . 80

ix

INTRODUCTION

Scald, a physiological disorder of apples which
dcavelops during storage, is controlled by post—harvest
trweatment of the fruit with diphenylamine (DPA). Label
rwecommendations for use of this material encourage Opera-

‘tors to change solutions as they become dirty or at least

 

conce every A8 hours, but there is widespread reluctance
to comply with these precautions. There is a lack of
eexperimental evidence supporting the need for frequently
:renewing the solutions. Therefore, experiments were
designed to ascertain the effects of DPA solution age and
the presence of organic matter in the applicator systems
<on DPA residues on apples. Furthermore, there is a need
.for information on the relationship of DPA content of
‘treatment solutions to residues, and of both solution con-
<3entration and residue on scald control.

There also exists the possibility of incorporating
the DPA application process into apple hydrohandling sys-
‘tems so as to provide savings in time and reduce the
requirements for space and equipment in pre—storage opera—

‘tions. Such a procedure would possibly be hazardous

because the increased pressure may enhance penetration of
the DPA solutions. Fruit residues, possible injury, and
scald control under these circumstances of commercial

usage of DPA were evaluated.

LITERATURE REVIEW

Storage scald of apples was recognized as physio-
Llogical in origin and nature as early as 1903 (Powell and
iFulton). It has been studied by many workers and the
subject has been thoroughly reviewed by Pentzer and
IHeinze (195A), Ulrich (1958) and Smock (1961), in communi-
cations from The World Meteorlogical Organization (1963),
and by the USDA Agricultural Research Service (1965). The
following factors are recognized as tending to enhance
scald: immaturity, hot or dry weather during harvest
(particularly warm nights), delayed cold storage or slow
cooling of fruit, high storage temperatures, large fruit,
;poorly colored fruit, fruit high in nitrogen and poor
storage ventilation.

Methods for its control have been studied for many
years and discussed extensively by Smock and Southwick
(l9A5), Smock (l96l) and Lutz and Hardenburg (1968). An
important factor for effective control is fruit treatment
within the first four weeks after harvest. Since Smock's
original findings on the scald inhibiting prOperties of
diphenylamine (DPA) in 1955, others (Hardenburg and Ander-

son, l960 and 1962; Mattus, 1963; Smock, l96l) have

confirmed that this chemical provides dependable control
xihen prOperly used. It is now extensively used as a
.liquid dip or drench for direct application to the apples,
‘but Ginsburg (l968a) reports that DPA-impregnated wraps
are equally effective.

Recent ideas for hydrohandling of apples in bulk
<3ontainers (Dewey, 33*al.,l966; Stout, gt_al.,l966;
(Einsburg, 1967) suggest that chemicals may be incorporated
:into the handling liquids. Although very little has been
:reported on the subject (Daines, 1967; Porritt and
IWeheriuk, 1968), bulk handling of apples in water could
conceivably lead to the consolidation of sorting, sizing
and.grading operations with pre-storage treatment with a
suitable storage scald inhibitor. Considering fungicides,
IDaines (1967) reports increased rot control due to in-
creased hydrostatic pressures.

Diphenylamine, because of its wide use for control-
ling storage scald in Michigan, was of particular interest
.in this study. The Merck Index (1952) shows its formula
as C12H11N with a molecular weight of 169.22. It is made
loy heating aniline with hydrochloride. DPA crystals
goossess a floral odor and have a density of l.l6, a melt-
:ing point of 53—A°C, a boiling point of 302°C and a flash
pxoint of 153°C. Diphenylamine discolors in light and is
iJlSOlUble in water, but freely soluble in a variety of

cxrganic solvents. As a weak base, diphenylamine forms

salts with strong acids. Yatsu (1956) explains that in
combination with a strong oxidizer (i.e. vanadium pent-
<oxide in 10N sulfuric acid) DPA will form a blue oxida-
txion product; first being oxidized to diphenylbenzidine

Efl1d then to diphenylamine violet, as follows:

QEQQQAMEQ

diphenylbenzidine

QNHNQ

diphenylamine violet

 

Snell and Snell (l9A9) described a technique for
xnaking nitrate estimations using DPA in sulfuric acid.
The Merck Index (1952) states that this chemical is used
in the manufacture of dyes, stabilizing nitrocellulose
explosives and celluloid and the detection of several
oxidizing substances, with which, in the presence of
sulfuric acid, it gives a deep blue color. Incorporated
into the Dische reagent, DPA is used in an improved method
for the estimation of DNA in plant tissue (Giles and
Myers, 1965) and in the diagnosis of leafroll infection
in young potato leaves (Wenzl, 1965).

Small quantities of diphenylamine can be estimated
using potassium dichromate in sulfuric acid as an oxidizer

(Barnes, 19AA). Snell and Snell (19A9) suggest that an

aqueous solution of DPA produces a violet color with
vanadium, detecting 2.5 ppm. Bruce, et_al. (1958) blended
apple samples with 90% methanol and extracted the slurry
with petroleum ether. They then determined DPA by coupl—
ing with diazotized 2,A—dinitroaniline in phosphoric acid
and measuring the absorption at three wavelengths in the
ultraviolet or at 530 mu on a spectrophotometer. Kennett
(1961) refluxed the tissue sample with water and continu-
ously extracted the DPA from the aqueous condensate with
hexane. DPA was then partitioned into 18N sulfuric acid,
reacted with a vanadium pentoxide reagent and read at

600 mu on the spectrophotometer. Yatsu (1956) extracted
the DPA from apple tissue with 70% acetone and partitioned
it into pentane, adding KOH and alcohol and finally react-
ing it with a vanadium pentoxide reagent and reading
absorbance on a spectrophotometer at 600 mu within A min-
utes. Harvey (1958) extracted the peeling tissue with
petroleum ether and eluted the DPA from a magnesium oxide
column with 1:1 benzene-petroleum ether. The.pulp was
extracted with 70% acetone and the DPA partitioned into
petroleum ether. Both were reacted with the sulfuric
acid-vanadium pentoxide reagent and read at 590 mu within
5 to 7 minutes. Even though Bache, et_al.(l962) extracted
with 50% methanol, partitioned into petroleum ether and
then acidified with HCl, their reaction and wavelength

used were the same as Harvey's. Padfield and Clark (1963)

also used the same method as Harvey, except peelings were
placed in petroleum ether, brought to boiling under
reflux, cooled and then an aliquot of the petroleum ether
was chromatographed. In 1963, Gutenmann and Lisk des-
cribed a gas chromatographic technique in which they used
direct bromination of an acetone-hexane extract of apple
tissue containing DPA to yield, presumably, the ortho-,
para-hexabromo derivative of diphenylamine. This electro-
philic compound was chromatographed and determined by
electron affinity detection

Huelin and Coggiola (1968) presented evidence for a
role of a—farnesene in scald and showed that DPA reduced
its production in apples. Bull's eye rot (LOpatecki and
Burdon, 1966), core flush (Padfield, 1959) and internal
breakdown (Padfield, 1959 and Stevenson and Blake, 1961)
are diseases reportedly inhibited by DPA. Daines (1962)
showed that DPA markedly reduced soft rot or blue mold

rot (Penicilium expansum) and controlled Stayman Spot

 

(Daines, 1967). Ginsburg (l968b) found that DPA wrappers
reduced apple bitter pit significantly and that penicil-
lium rot of pears was controlled in storage by these
wrappers (Ginsburg, 1968).

Chemical injury of apples by DPA has been reported
by Dilley and Dewey (1963) on McIntosh and Rome Beauty

with this damage occurring on the non-cavity surface and

in the stem and calyx cavities, respectively. Similar injury
was observed on the cheeks and shoulders of Golden Delicious
(Pierson and Schomer, 1968). Daines (1962), Rep. East Malling
Res. Sta. (1967), Padfield (1959, 1961), Padfield and Smock
(1960) and Smock (1961) showed DPA accumulations at the bottoms
of containers and on fruits could result in injury to the
skin of several apple varieties. Padfield (1961) suggested
that complete drainage from the fruit is necessary to avoid
chemical burning. Hardenburg and Anderson (1962) related
similar injury to alcohol solutions of DPA. Damage to the
lenticels on the exposed side of the fruit that received the
impact of spray jets increased in severity with increased
duration of spraying (Rep. East Mall Res. Sta., 1967).
Padfield and Clark (1963) observed that a naturally occur-
ring browning of lenticels on Rome Beauty was aggravated

by DPA treatment. Hall, gt_a1, (1961) reported a similar
response on Granny Smith with accompanying injury appear-
ing as dense, shiny, black areas on the skin. They showed
that dipping in DPA caused senescent blotch to be more

severe and that DPA-treated fruit appeared greener than
untreated fruits after storage. Field applications by

spray caused early leaf drop and some stronger preparations
caused some marginal leaf scorching and brown spotting of

apples on the tree.

Hall, §t_al. (1961) reported that DPA has a very
limited mobility in apples and residues are low after
cold storage. When DPA-impregnated sheets are placed
between layers of apples in a container, DPA is effective
at a distance from the sheets, but this effective dis-
tance depends on the amount of DPA available in the paper.
They also observed, ”no apparent movement of DPA from
treated to untreated areas of the apple" and found a
significant difference in scald between treated and un—
treated halves. DPA penetrates the fruit skin, since
approximately equal amounts were found on the surface and
in the pulp (Bruce, et_al.,l958). Ginsburg observed in
1961 that the amount of DPA remaining on the surface was
negligible and the following year (Ginsburg, 1962) he
noted that the amount in the pulp was consistently higher
than in the peel. However, Harvey and Clark (1959) indi-
cated that about 8A% of the residual DPA on apple fruits
was removed from the surface with solvents and 9A% was
found in the outer 2-A mm of tissue. According to Den-
mead, §t_al. (l96l) DPA disappeared from the apple fruit
surface at an exponential rate which differs by variety.
These loss rates were sufficiently high to require that
estimations be made very soon after treatment to avoid
errors. Bache, gt_§l. (1962) stated that if they are to
be analyzed within 7 days it was acceptable to store

samples at 32°, but if stored for longer periods they

10

should be frozen at 0°F or lower to avoid excessive loss
of DPA by volatilization. Accordingly, the blended
samples should be analyzed immediately or stored in vapor-
tight containers, but for no longer than one week without
serious loss of DPA.

The method of application of DPA affects residues

(Hall, et al.,l969). Apraying fruit in crates gave rela-

i“.

tively high residues (Bache, et al.,l962a). Flooding
with DPA suspensions for 30 minutes in a hydrocooling

system (Yatsu, 1956) resulted in very high residues,

 

therefore he suggested that hydrocooler applications .
should be limited to solution concentrations of 100-200 5
ppm. Dipping apples in DPA solutions resulted in immedi-
ate values of 7-8 ppm, but after 3-7 months of storage,
residues were 3 ppm or smaller. Ginsburg (1961, 1962)
found that dip treatments of 1 min duration resulted in
the apples taking up far less DPA than from wraps which
were constantly in contact with the fruit. The higher
residues found with wraps were due to vaporization onto
the fruit (Bache, et_§l,,l962a). Wiping treated apples
with a dry cloth resulted in 50% more DPA being adsorbed
by the fruits (Denmead, g£_al,,1961), but the mechanical
removal of excess DPA deposit left by wettable powder
sprays did not impair scald control (Padfield and Smock,
1960). Insufficient residues for effective control were

observed by Martin (1959) when DPA was vaporized with

11

steam and applied to apples in a tunnel. Preharvest
sprays resulted in low residues and poor scald control
(Bache, g£_§l,,1962a). Harvey and Clark (1959) reported
residues of one ppm which dropped to 0.5 ppm with a delay
of 3 days between spraying and harvesting.

As the duration of DPA post-harvest spray treatments
increased for all varieties, residues increased almost f
linearly up to a high of 18 ppm for Dougherty sprayed for
A minutes (Denmead, gt_al.,l961). They postulated that

this may have been due to permanent changes occurring in

 

the apple's wax coating and reported 1-2 ppm residues for I
all varieties after 1 minute of treatment. Padfield and
Clark (1963) also observed considerable increase in DPA
uptake with increase of treatment time and suggested that
1-2 minutes is adequate.

Diphenylamine residues are related to the amount of
the chemical applied (Hall, et_al,,l961). Denmead, gt_al.
(1961) observed that DPA residues on the fruit increased
about 9 ppm over a 500—3000 ppm range of DPA solution con-
centration, but uptake was not proportional to the concen-
tration of the emulsion, which gradually fell off as a
result of uptake by apples and dilution of the emulsion.
They also pointed out that the oil emulsion agent alone,
without DPA, afforded some protection against scald.

This would be expected with varieties known to respond to

oiled wraps.

l2

Varieties of apples vary widely in DPA uptake
(Harvey and Clark, 1959; Padfield, 1961; Padfield and
Clark, 1963 and Smock, l96l). Denmead, g£_§l. (1961)
reported that the level of DPA uptake from a particular
treatment appeared to be negatively correlated with
scald susceptibility; for instance, Granny Smith showed
the lowest residues, but was rated as the most susceptible
to scald of those examined. Other varieties, not con-
sidered to be susceptible, were found to have exception-
ally high residues that ranged up to 1A.5 ppm. In stat—
ing that varieties influence inhibitor coverage, Mattus
(1963) reported that the best coverage was obtained on
Stayman, Winesap and York; lesser, but still good coverage
occurred on Rome Beauty; and the least on Delicious,
Jonathan and Grimes Golden, which were about equal.

Fruit maturation increased DPA coverage (Mattus,
1963); however, Padfield and Clark (1963) reported that
residues differ in relation to maturity by variety, with
no general trend toward greater residues with either
immature or senescent fruits. Denmead, g§_§l. (1961)
observed that storing apples at room temperature before
treatment increased DPA uptake by about 100% after four
weeks. In this period of time there was a noticeable
increase in the "greasy" feel of the apple surface and an
expected increased absorption of oil from the DPA emul-

sion.

13

The effectiveness of scald inhibitors is readily
and easily evaluated using Martin and Grassia's (1965)
half-fruit method. Hall, gt_§l. (1961) reported that
about 0.1 mg of DPA would be needed per average sized
apple to control scald, but observed that some residual
scald was present even with the use of DPA, especially at
badly bruised areas and in the calyx cavity. They also
reported that dipping in DPA gave commercial scald con-
trol of fruits from early harvests and showed a quanti-
tative relationship between severity of scald and the
concentration of DPA required for control. For instance,
1500 ppm was adequate for severe scald, but less was
required in years of slight scald. Hilkenbaumer (1961)
showed a marked decrease in scald after DPA treatment,
the effect being proportional to the strength of the
solution. The initial DPA concentration on the apple's
surface was positively correlated with the extent of
the scald protection during storage and holding apples
at room temperature prior to DPA treatment reduced the
susceptibility to scald (Denmead, g£_§l., 1960). Pad-
field (1961) indicated that reasonable control was
achieved with 250 and 500 ppm, and under all conditions,
1000 ppm DPA gave effective scald control. He also
stated that 5 ppm residual DPA on the fruit surface gave
effective control. Smock (1961) suggested that 3 to 5

ppm on the fruit at harvest time was adequate and Huelin

 

1A

(1968) indicated that 0.2 ug/cm2 in apple peelings
appeared to be the minimum required to inhibit scald-
inducing reactions.

Artificial induction of scald symptoms was effected
by holding apples in an unsealed, 150 gauge polyethylene
liner (Smock, 1961). This method served as a rough pre-
diction procedure, but tended to underestimate the amount
of scald developing in storage. Brooks, et a1. (1919)
and Huelin (l96A) promoted typical scald development by
exposure to fruit volatiles. Dilley, gt_al. (1963) pro-
posed scald to be a two stage phenomenon, the first
induced by anaerobiosis (as for example, 72 hours in a
100% nitrogen atmosphere) the second by actual symptom

development under aerobic conditions at 70°F.

 

MATERIALS AND METHODS

DPA Analyses For Residues on Apples. The gas chroma-

 

tographic technique outlined by Gutenmann and Lisk (1963)
for the rapid determination of DPA in apples seemed suit-
able to the requirements of these experiments. DPA was
extracted from chopped apple tissue with acetone and the
extracts stored at -10°C by this procedure. Great diffi-
culties were encountered in obtaining consistent and
accurate results. Standardized concentrations of DPA
finally were accurately analyzed after the halogenation
step was altered by the use of chlorine instead of bromine.
Analysis of all fruit samples from the 1967-68 harvest
season treatments were completed using this modified tech-
nique. However, the determined DPA residues did not agree
with the projected values according to the applied treat-
ments. Additional extensive and prolonged efforts to
accurately detect DPA by this method did not yield results
of acceptable reliability.

The procedure was further modified to use the spec-
trophotometric technique described by Yatsu (1956) as
modified by Bruce, gt_§l. (1957) and Harvey (1958). Since

acetone absorbs light at similar wavelengths to DPA, it

15

 

16

was necessary to transfer the DPA into hexane. One
hundred milliliters of a mixture of hexane isomers were
added to 100 m1 of acetone extract of apple tissue in a
1000 ml separatory funnel. A highly purified grade of
hexane was needed to reduce the interference caused by
the impurities found in most grades. Five ml of 1.0 M
calcium chloride was added along with 600-700 ml of 1
deionized water to break the emulsion formed in partition-
ing the DPA into the hexane. These liquids were shaken

for two minutes then allowed to stand for two minutes

 

before drawing off the lower layer containing acetone, I
water and CaC12. The hexane phase, containing the DPA

was then transferred to a second 1000 m1 separatory funnel
and excess water and 5 ml of CaCl2 were again added. Shak-
ing for two minutes was followed by a 2-minute standing
period before the DPA-hexane layer was drawn off into a

125 m1 separatory funnel. Five ml of vanadium pentoxide
reagent (100 ug/ml of vanadium pentoxide in 10 M sulfuric
acid) were added to the DPA-hexane phase and this mixture
was shaken for 2 minutes, allowed to stand 2 minutes and
then the reacted product (bottom layer) was vacuum-
filtered through a Teflon-coated fiber glass disc into a
small test tube. This filtered product was transferred

to a cuvette and placed in the light path of a Beckman,
Model DU spectrophotometer. The absorbancy of this pro-

duct was read using a tungsten light source with a

l7

wavelength setting of 605 mu. The color of this product
was very unstable so all readings were made at exactly

6 minutes from the time the chemicals were first com-
bined. The reaction of the DPA extracted from apple
tissue plus the vanadium pentoxide reagent resulted in
the formation of an opaque, non-uniform sludge. Passing
this viscous product through the filter cleared the solu-
tion and increased the accuracy of this procedure. Exper-
imental samples were read on the spectrophotometer using
acetone, which was partitioned in the manner described,
as a blank. Known concentrations of DPA in acetone were
partitioned into hexane and reacted with the vanadium
pentoxide reagent to prepare a standard curve.

Solution Analyses in the Laboratory. Solutions pre—
pared from liquid emulsion formulations of DPA can be
analyzed with greater uniformity than those made from
wettable powders. For both, technical grade DPA was mixed
with water to prepare treating solutions according to
manufacturers' specifications. Before sampling, the solu-
tion was well mixed in order to insure a representative
1 ml aliquot which was diluted with 9 m1 of absolute etha-
nol. From this diluted solution 0.1 ml was removed with
a micro-syringe and reacted in a test tube with the
vanadium pentoxide reagent. The degree of blue color
development indicated the amount of DPA. The stability

of the color of this product was greater than that of DPA

18

residue analyses, but still had to be read on the spectro—
photometer within 5 to 7 minutes after the initial combi—
nation Of the chemicals. These colored products were
compared with the vanadium pentoxide reagent combined

only with ethanol and read at 585 mu wavelength (Kraght,
1966). Standard curves were prepared from known concen—
trations of technical grade DPA dissolved in absolute r
ethanol.

Solution Analyses in the Field. A test was devised

 

for relatively simple estimations of DPA content in solu-

 

tions in use in storages and packing houses. The same (
vanadium pentoxide reagent, previously described for DPA
residue and DPA solution analyses, was carefully taken to
the field in a tightly-sealed, glass bottle. A plastic
or glass eyedropper calibrated to 1 ml was used to take
an aliquot from a well—mixed solution. Preferably a
pipette or syringe should be used for greater accuracy,
but for coarse estimations the eyedropper suffices. One
ml of the solution was diluted with 9 ml of alcohol, if
available, or water and shaken well. The same sampling
device could be used to remove a 1 m1 aliquot of the
diluted sample for reaction in a separate vessel (test
tube) with 5 m1 of the vanadium pentoxide reagent. The
intensity of blue color development was directly corre-
lated with the amount of DPA (up to 100 us) which, upon

reaction with vanadium pentoxide, formed a blue color

19

complex that was near the upper limits of the spectro-
photometer at the wavelength used. However, for field
estimations, this deep blue color was readily distin-
guished from the less intense blue color of solutions
which had become weakened by usage. Two solutions were
compared for field estimations; therefore, an unused
sample of the solution was taken and held for this pur-
pose. The solution in question was treated as des-
cribed above and at the same time a fresh solution,
which was prepared according to the manufacturers'
specification, was also diluted and reacted with the
reagent. By comparison of colored products the relative
concentration of DPA in solution could be adequately
estimated. Such a test should indicate whether or not
an operator should replace his treating solution. The
test could be further improved by preparing in advance
solutions of known concentrations of DPA and removing 1
ml aliquots for reaction and comparison with unknowns.
This would provide a range of concentrations to fit the
solution in question.

Aging Solutions. In 1967—68 treatments were delayed

 

while DPA analytical techniques were studied in an effort
to improve their reliability. Delicious and McIntosh
apples were harvested and stored until February, when
this study was conducted. The samples consisted of ran—

domly selected fruit representing all harvests and all

20

trees from which the apples were obtained. At the start

of the experiment, 12 solutions were prepared. Two for-
mulations of liquid emulsions were provided by commercial
sources; "A," Deccoscald 25,1 and "B," No-Scald DPA
Liquid,2 both of which were prepared to 1000 ppm accord-
ing to label directions. About 6 grams of chopped organic
matter consisting of orchard trash, soil and rotten fruits,
were added to half of the solutions.

At A weekly intervals, lO—fruit samples of McIntosh
and Delicious apples were dipped for one minute in each
of the above solutions and allowed to dry at room tempera-
ture. Once dried, all apples were chopped and the DPA
extracted and quantitatively determined by previously
described procedures.

In 1968 the treating solutions were prepared at
intervals prior to the harvest of each variety. For ex-
ample, McIntosh apples harvested at the Graham Experiment
Station on September 12th were treated within A8 hours in
solutions prepared 1, 2 or A weeks earlier or in a solu-
tion freshly prepared the day of treatment. As in the
previous year, there was a corresponding solution that
contained orchard trash, rotting apples and soil for each
clean solution. All solutions were held at room tempera-

ture in covered plastic containers of 10 gallons capacity.

 

l
Calif.
2

Product of Wallace and Tiernan, Inc., Monrovia,

Product of Chemley Products, Chicago, Ill.

 

 

 

21

Fruits were harvested earlier than of optimum maturity in
order to be favorable for the development of storage
scald. A sample consisted of randomly selected apples
encompassing all the trees included in the harvest of
each variety. Two 50—apple replicates of each of the
eight treatments were dipped for 30 seconds each. After
allowing the apples to dry at room temperature, 10 fruits
were randomly selected for DPA residue analysis, placed
in a plastic bag and frozen immediately. When time per-
mitted, these were thawed slightly, chopped and extracted
in the manner described previously. The remaining A0
apples of each sample were carefully placed in an open
plastic bag (2 samples per field crate) and held at 32°F.
After all varieties had been treated and stored, they
were transferred on October 22 to 38°F and held until it
was established that scald could be artificially induced
by nitrogen treatment in the laboratory.

Artificial Scald Induction. The apples with storage

 

scald potential were artificially induced to scald by the
procedure described by Dilley, e£_al. (1963). A sample of
20 apples was placed in a closed container through which
nitrogen gas was passed continuously at the rate of 250 to
300 ml per minute. The apples were maintained in this
oxygen—free atmosphere for 72 hours at 20°C, then trans-
ferred to air and held another 72 hours at 20-25°C. Apple

scald was classified as (a) none (non-existent or only in

22

association with bruising), (b) slight if present but not
Obvious to the average consumer, or (c) severe if it was
obvious to even the untrained person that the fruit was
defective. To place McIntosh, Delicious and Rome Beauty
varieties on common ground, scald reduction values were
used: the difference in the number of scalded apples
between DPA-treated and untreated fruits. {
Submersion (Hydrostatic Pressure). In 1967 the
physical factors involved in treating apples under water

were examined, whereas in 1968, both physical and physio-

 

logical effects of hydrostatic pressure were studied. In
1967, hydrostatic pressures up to A psi were developed
using a 6-quart pressure cooker vessel containing 1000
ppm DPA treating solution and 10 apples. Treatment time
varied from 0 to 8 minutes, plus one minute which was
required to place the apples in solution, establish the
desired pressure (via application of compressed air to
vessel) and remove the fruits from the treating system.
Each sample was prepared randomly (as in the aging solu-
tion studies) and handled following treatment in a similar
manner to previous tests. Both Delicious and McIntosh
apples were studied.

In 1968, Delicious apples were treated with two con-
centrations of DPA (1000 and 2000 ppm) plus one treatment
of water alone. Hydrostatic pressures of 0, 1.5 and 3.0

psi were developed by submerging the apples just below the

23

surface of the solution, 2.5 feet and 5 feet beneath the
surface, respectively. A lOO-gallon treatment tank was
constructed from two 55 gallon drums welded together end
to end. Each 50-apple sample was lowered to the speci-
fied depth in a weighted metal container. Total treat-
ment time, including submersion and removal from the
system, was 30 seconds plus the designated treatment r
time. The fruits were drained before packing 10 apples

and A0 apples in previously described containers for

 

residue analyses or physiological determinations, respec-
tively. The treating solutions were sampled and analyzed
for DPA content.

Commercial DPA Applications. The concentration of

 

DPA in commercial treating systems, both fresh and after
varying periods of usage, and as affected by varying
amounts of orchard trash accumulation were obtained from
seven cooperators. Each operator was asked to collect a
70 ml sample of his solution at the start and finish of
each day's run. It was also suggested that samples be
taken whenever the solution was fortified or altered in
any other way. The screw top vials containing these
samples were stored in a cool place at the storage until
collected by MSU Horticulture Department personnel near

the end of the season.

2A

Synthesis of Radioactive Diphenylamine. Aniline

 

hydrochloride-luCWL)l was converted to aniline-luC(UL)
and the synthesis of Mueller (1959) for DPA was subse-
quently followed, since yields of 65% were reported,
using aniline as the precursor. Labeled aniline hydro-
chloride in ethanol was combined with 0.A M NaOH and
shaken thoroughly to remove all the radioactive material
from its ampoule. Aniline hydrochloride was then parti—
tioned into chloroform and this fraction transferred to
a 100 m1 boiling flask to which a condenser was attached.
Heating proceeded very slowly until the chloroform was
distilled Off. A capillary tube attached to a nitrogen
cylinder was placed in the condenser during the final two
hours of distillation and for the remaining steps in the
synthesis. This nitrogen atmosphere was used to reduce
the possibility of oxidation of the diphenylamine. Re-
fluxing was discontinued after 8 hours and the resulting
liquid had a total volume of 2—3 ml to which 0.3 gram of
iodine was added to act as a catalyst for the final reac-
tion. Heat was applied continuously until only a white
crystalline product and black char (iodine) remained in
the boiling blask. All reamining materials were dis—
solved in chloroform and steam distilled (Sigal §t_§l.,

1966) collecting a total volume of 800 ml distillate,

 

1Lot No. 212-266 from New England Nuclear, Boston,

Mass.

 

25

which was dried in a separatory funnel with excess anhy-
drous sodium sulfate. The remaining 600 m1 of pink-
colored distillate, was very slowly distilled on a spin-
ning band column to a volume of 5 ml. This product,
diphenylamine, was diluted in 100 m1 absolute ethanol
and used in tracer studies. The Amax of this product
was the same as that for analytical grade DPA.

Autoradiography. Apple tissue discs (l-A mm thick)

 

for autoradiography were cut from the center of the
luC—DPA treated apple with a sharp knife. Each free—hand
section was then placed on a no. 3 Buchner funnel mounted
on a 500 m1 vacuum filter flask and connected to a Virtis
freeZe—drying system. A 12 cm disc of Teflon—coated
fiberglass filter membrane was placed beneath and adja-
cent to the apple disc. Between this and the funnel was
a 9.5 cm Whatman No. 1 filter paper. To increase the
vacuum on the tissue, a sheet of Saran film was laid over
the top of these materials and pulled tightly against the
apple. Within 2A hours, the apple disc was dehydrated,
but leathery and pliable. This was separated from the
membrane and Saran film sheets and placed in a desiccator
until used in autoradiography. Kodak Royal Blue X—ray
film (Wang & Willis, 1965) provided good sensitivity and

acceptable resolution upon exposure of the discs for 28

days.

26

Statistical Procedures. Analysis of variance was
applied to the data of these experiments. Means of fac-
tors found to be significant by the F test were further
compared by Tukey's w procedure (Tukey's test) at the 5

percent level (Steel and Torrie, 1960).

RESULTS

DPA Analytical Technique. The technique described

 

for analyzing for DPA residues in apple tissue was
periodically checked to ascertain its reliability as a A

method of recovering maximum residues. In one such test,

 

known quantities of DPA were added to the blended apple
tissue and recovered by extraction. Fortification with
quantities of DPA which would produce theoretical re-
coveries of 3.2 and 6.A ppm resulted in average recoveries
of 103% and 96.6%, respectively. Apparent residues aver-
aged 3.37 ppm higher than the values which could be attri—
buted to DPA. This interference was probably due to pre-
harvest spray materials remaining on the apples and the
chemical components of the apples themselves. Corrected
DPA residues are reported as total apparent DPA minus
values representing interference from non-DPA sources.

DPA Residues on Apples as Affected by Aging Solu-

tions and Hydrostatic Pressure. The analyses of variance

 

of data obtained from the aging solution studies in 1967
and 1968 are summarized in Tables 1 and 2, respectively.
The effect of varieties was significant in 1967, with the

mean for McIntosh being 3.93 ppm and Delicious, 2.8A ppm.

27

28

TABLE l.--Analysis of variance of DPA fruit residues in
the 1967 aging solution study (see Appendix

Table I or II for data).

 

 

Source df
Blocks 2
Total for variety 5
Blocks 2
Variety 1 A2.78 *
Error a 2
Total for formulation 11
Total for variety 5
Formulation l 71.18 **
Variety x formulation 1 6.2A
Error b A
Total for sanitationl 23
Total for formulation 11
Sanitation 1 31.75 **
Sanitation x variety 1 3.28
Sanitation x formulation 1 16.85 **
Sanitation x formulation x variety 1 A.22
Error c 8
Total for age 119
Total for sanitation 23
Age A 8.07 **
Age x variety A 3.A3 *
Age x formulation A 6.61 **
Age x sanitation A 3.31 *
Error d 80

 

lClean DPA solutions vs. those containing organic

matter (OM).

29

TABLE 2.--Ana1ysis of variance of DPA fruit residues in
the 1968 aging solution study (see Appendix
Table III for data).

 

Source df F

 

I...)

Blocks

Total for variety
Blocks

Variety

Error a

l\)|\)l—’U‘l

l—'

HJ:
.cuaowOqu u3m+4U1H

Total for sanitation
Total for yariety
Sanitation
Sanitation x variety
Error b

Total for age

Total for sanitation
Age

Age x variety

Age x sanitation
Error c

.35 **
.28
.50 **

U'll—‘ON

[\J

 

 

lClean DPA solutions vs. those containing organic
matter (OM).

30

No varietial differences occurred in the 1968 trials.

The source of technical DPA was a factor, with mean resi-
due values of A.59 ppm for formulation Al and 2.18 ppm
for formulation B2 in 1967. Only one material (Formula-
tion A) was used in the 1968 studies. The addition of
organic matter to DPA solutions increased fruit residues

in both years, being significant at the 1% level in 1967

—"'=L L

and at the 5% level in 1968 (Table 3). In 1967 there was

TABLE 3.--Mean DPA residues (ppm) for apples as affected
by organic matter.

 

 

 

Control Organic Matter
1967 2.33 A.A3
1968 1.38 8.07

 

a highly significant interaction between the sanitation
of the DPA solution and DPA formulation (Figure l). A

rather gradual increase in fruit residues occurred with
aging of the solutions in 1967, whereas, res1dues almost
doubled between the 2— and A-week old solutions in 1968

(Table A). Tukey's test revealed differences between the

 

lDeccoscald 25, product of Wallace and Tierman, Inc.,
Monrovia, Calif.

2No-Scald DPA Liquid, product of Chemley Products,
Chicago, Ill.

31

 

E
a.
a.
‘
as
C:
CONTROL
FORMULATION
Figure 1.

DPA residues (ppm) as affected by the inter»
action between solution sanitation and technical

formulation for apples dipped in 1000 ppm DPA solution
for one minute in 1967.

32

TABLE A.--Mean DPA residues (ppm) for apples as affected
by solution age.

 

Age (weeks)
0 l 2 3 A

 

1967 2.11 a 3.55 be 3.81 be 3.05 ab ' A.39 c
1968 1.77 a 3.8A b A.A9 b --- 8.80 c

 

lMeans not followed by the same letter are signif-
icantly different by Tukey's test.

means of DPA residues for apples treated in solutions of
different ages in 1967, especially when fresh and A—week
old solutions were compared. Similar differences between
means were detected in the 1968 trials. Figure 2 illus-
trates the significant interaction between apple varieties
and solution age in the 1967 trials. The interaction
between DPA solution age and the technical formulation of
DPA in 1967 was significant at the 1% level (Figure 3).
Results in both seasons revealed significant interactions
(1967, P=.05; 1968, P=.01) between DPA solution age and
the presence of organic matter in the solutions, as shown
in Figures A and 5 for 1967 and 1968, respectively.

The analyses of variance of data obtained in the
submersion studies conducted in 1967 and 1968 are summar—
ized in Tables 5 and 6, respectively. Apple varieties
were significantly different in 1967 with residues for

McIntosh being A.29 ppm and Delicious being 5.63 ppm of

33

IMcINl1ISH

DPA (ppm)

DELICIOUS

 

o 1 2 3 4
TIME (weeks)

Figure 2. DPA residues (ppm) as affected by the inter—
action between solution age and apple variety for apples
dipped in 1000 ppm DPA solution for one minute in 1967.

 

3A

DPA (ppm)

 

11ME (weeks)

Figure 3. DPA residues (ppm) as affected by the inter-
action between solution age and technical formulation

for apples dipped in 1000 ppm DPA solution for one
minute in 1967.

35

DPA (ppm)

CONTROL

 

TIME (weeks)

Figure A. DPA residues (ppm) as affected by the inter-
action between solution age and sanitation for apples
dipped in 1000 ppm DPA solution for one minute in 1967.

36

DPA (ppm)

CONTROL

 

O 1 2 3 4
TIME (weeks)

Figure 5. DPA residues (ppm) as affected by the interaction
between solution age and sanitation for apples dipped in
1000 ppm DPA solution for 30 seconds in 1968.

37

TABLE 5.--Analysis of variance of DPA residues in the
1967 submersion study (see Appendix Table IV

 

 

for data).

Source df F
Total 39
Varieties l 7.78 *
Time (Duration) 3 13.A6 **
Pressure A 12.18 **
Time x Pressure 12 —-—
Error 19

 

TABLE 6.-—Analysis of variance of DPA residues in the
1968 submersion study (see Appendix Table V
for data).

 

 

Source df F

 

H

Blocks

Total for concentration
Blocks

Concentration

Error a

7036.20 **

HPJFH»

l—’
.E'NNUUH
UL)

Total for time

Total for concentration
Time (Duration)

Time x concentration
Error b

.82

[.4
tNI’UUOI-J
LU
U")

Total for pressure

Total for concentration
Pressure

Pressure x concentration
Error b

.A5 **
1A.85 *

 

38

DPA. The duration of the hydrostatic pressure applica-
tion had a significant effect on DPA residues of apples
in 1967 (Table 7), but not in 1968. The data presented
in Table 8 shows for both seasons that as the hydrostatic
pressure on the apples in DPA solution increased the DPA
residues on the apples increased.

It may be observed (Table 6) that residues were
significantly affected by DPA solution concentration in
1968, having means of 2.10 and A.08 ppm DPA residues for
apples treated in 1000 and 2000 ppm DPA solutions,
respectively. Figure 6 illustrates the significant inter—
action observed between hydrostatic pressure and DPA solu-
tion concentration in the 1968 trials. In addition to
submersion treatments in 1000 and 2000 ppm DPA solutions,
a third treatment of submerging apples in water alone
resulted in apparent DPA residue values of 1.28 to 3.0A
ppm (Appendix Table VI). The mean interference value of
2.10 ppm was subtracted from the treatment values to give
the corrected DPA residues.

Factors Affecting the DPA Content of Solutions. The
analyses of variance for the DPA concentrations of solu-
tions used in the aging solution studies are summarized
in Tables 9 and 10. The presence of organic matter
significantly reduced the amount of measurable DPA in
solution in 1967, but not in the 1968 trials. In the

absence of organic matter the solutions contained an

 

39

TABLE 7.--Mean DPA residues (ppm) for apples as affected
by duration of submersion in DPA solution in

 

 

1967.1
Time (min.)2
1 2 3 A r
2.87 a A.AO ab 5.51 be 6.70 c

 

lMeans not followed by the same letter are signif—
icantly different by Tukey's test.

 

2Treatment times included an additional 1 min. for
preparation.

TABLE 8.--Mean DPA residues (ppm) for apples as affected
by submersion in DPA solution (hydrostatic
pressure).+

 

Pressure (psi)

 

0 l 2 3 A 5
1967 3.76 a 2.62 a -— A.82 ab 6.29 bc 7.31 c
1968 2.76 a -- 2.57 a -- 3.95 b -—

 

lMeans not followed by the same letter are sig-
nificantly different by Tukey's test.

A0

   

I
2000 ,I
I
I
I
A I 1...
E I
2 ---------"
‘
a.
a:
O 1.5 3
PRESSURE (psi)
Figure 6. DPA residues (ppm) as affected by the inter-

action between hydrostatic pressure and DPA solution
concentration for apples submerged in DPA solutions for

O, 2, or A minutes in 1968.

Al

TUABIJE 9.--Ana1ysis of variance for DPA solution content
in the 1967 aging solution study (see Appendix
Table VII for data).

 

Source df F

 

Blocks (Formulations) 1

Total for temperature
Blocks (Formulations)

(\JIUI—‘U‘I

 

Temperature 0.53
Error a
TWital for sanitation 11
Tkytal for temperature 5
Sanitationl 1 15,114 *
Sanitation x temperature 2 2,Ao
Error!) 3
Tkytal for age 59
Tkotal for sanitation 11
Age A 7.86 **
Age x temperature 8 1.01
Age x sanitation II 1.89
Age x sanitation x temperature 8 0,18
jError c 2A

1

Clean DPA solutions vs. those containing organic
matter (OM) .

A2

TVUSLE 10.--Analysis of variance for DPA solution content
in the 1968 aging solution study (see Appendix
Table VIII for data).

 

 

Source df F

Blocks 2

Total for sanitation 5

Blocks 2
Sanitationl 1 7.95 (
Error a 2

Total for age 23

Total for sanitation 5

Age 3 1.12
Age x sanitation 3 -—
Error b 12

 

 

l
Inatter.

Clean DPA solutions vs. those containing organic

average of 899 ppm of DPA, but with organic matter, an
aAnerage of only 733 ppm of DPA was measured. The age of
tlie solutions significantly affected the amount of measur-
allle DPA only in 1967. The influence of solution age is
.pxnesented in Table 11. The mean values were significantly
djgfferent but not consistent with age.

Submersion solutions made up according to label
rwacommendations were analyzed for DPA content in 1968.
Tide 1000 ppm solution contained an average of 10A6 ppm
IDPVX- The 2000 ppm solution contained an average of 1912

:pgnn during the treatment period.

43

TVKBLE ll.—-Mean DPA content (ppm) of solutions as affected
by solution age in 1967.1

 

Age (weeks)
0 1 2 3 II,

 

1021 a 886 ab 7A8 bc 826 abc 600 c

 

lMeans not followed by the same letter are signifi-
canqtly different by Tukey's test.

Storage Scald Control as Influenced by DPA Solution
Age, Sanitation and Hydrostatic Pressure. Scald control
muss evaluated in 1968 to determine the efficacy of the
cfluemical scald inhibitors. It was recorded as the total
runnber of scalded apples for three solution ages compared
vvitfli the No DPA treatment, and analyzed as a randomized
tfilocku There were significant differences between treat-
rnerrts and between varieties for scald control. Consider-
jqu the scald reduction comparisons for the 1968 aging
sc>lution study (Table 12), the only significant effect was
airtributed to varietal differences, being 11.69, 9.75 and
1LI.88 scald reduction units1 for McIntosh, Delicious and
chnne Beauty, respectively.

Scald control differences between the three 1968
stibmersion treatments were evaluated and the analysis of

vsxriance of this data is shown in Table 13. The only

 

———

lScald reduction units are the difference between
ugrtreated and DPA—treated apples in the number of scalded

fruiits per 20.

 

AA

TULBLE 12.--Ana1ysis of variance of scald reductionl data

from the 1968 aging solution study (see
Appendix Table IX for data).

 

 

 

 

Source df F
Blocks 1
Total for variety 5
Blocks 1
Variety 2 29.36 *
Error a 2
Total for sanitation 11
Total for variety 5
Sanitation l --
Sanitation x variety 2 ——
Error b 3
Total for age A7
Total for sanitation 11
Age 3 _-
Age x variety 6 __
Age x sanitation 3 1.52
Error c 2A
1

Scald reduction units are the difference between
uxytreated and DPA-treated apples in the number of scalded

fxulits per 20.

A5

TABLE 13.--Analysis of variance of apple scale in the
1968 submersion study (see Appendix Table X

 

 

for data).
Source df F

Blocks 1
Total for concentration 5
Blocks 1
Concentration 2 229.58 **
Error a 2
Total for time 17
Total for concentration 5
Time 2 -—-
Time x concentration A
Error b 6
Total for pressure 53
Total for time 17
Pressure 2 -—-
Pressure x concentration A ———
Pressure x time A 3.03 *
Error c 26

 

 

factor which significantly affected scald control was DPA
concentration of the solutions (Table 1A). Scald control
observed in the 1968 DPA submersion studies was affected
by the significant interaction between hydrostatic pres-
sure and duration of DPA treatment. As the pressure
increased from 0 to 3 psi and as the duration of exposure
to this pressure increased from 0 to A minutes, scald was

reduced, as illustrated in Figure 7.

A6

TABLE lA.--App1e scald control in submersion solutions in
1968 as affected by DPA concentration.1

 

DPA Concentration (ppm)
O 1000 2000

 

(Average number of scalded apples / 20 fruits)

15.06 a 2.17 b 1.38 b

 

lMeans not followed by the same letter are signifi—
cantly different by Tukey's test.

Autoradiography of Apples Submerged inluC-DPA.

 

Exposure of apples to A psi pressure for 10 minutes in
1000 ppm DPA solution fortified with luC-DPA revealed

that most of the DPA remained at or near the Delicious
apple surface, with only a trace in the seed cavity. It
appeared that some DPA was forced through lenticels beyond
the hypodermal area (Figure 8).

Commercial Use of DPA. Table 15 summarizes the sur-

 

vey results from seven apple storage operators in Michigan
during the 1968 harvest season. According to the solution
analyses, none of the operators attained an average DPA
content in his system which approximated the desired con-
centration. The average range between high and low con-
centrations was about 700 ppm. In 1000 ppm preparations,
DPA concentrations dropped as low as one-tenth the desired
level; for 2000 ppm preparations concentrations dropped

to 60% of the initial content. The average concentrations

A7

’0----------

UP

SCAIDED (Ne EHHts)

 

O 1.5 3
PRESSURE (psi)

Figure 7. Apple scald control in submersion solutions as
affected by the interaction between hydrostatic pressure
and DPA treatments for 0, 2, or A minutes' duration.

A8

 

.
. "q.
.o
s.
. c
.3 .
v. ’
co’
Q.
\ . t l
'3 25'

Figure 8. Autoradiograph of or ss section of Delicious
apple submerged in 1 C-DPA solution for 10
minutes at A psi pressure.

A9

 

Edd

 

 

m.sds m.:me m.mfim dam Aooofiv
H.moo m.oHHH m.mam cocoon oustosa
m.maoa m.samm m.ooma sad ooom
m.moo o.mooa m.m@m and sees omssosa
m.smm ®.ame m.nam coca casonooooo condos am .Hoa .Ho: can A
A.Ams H.3mw o.mm oooa and oasom o: sesame mm .Hod dad a
m.oaca m.mafim c.0oma ooom and canon o: . I- ea .Hoa can m
0.0mm o.aeoa o.msm coca oasooooooa I- m .Hom .c -
Asauznmv

o.aooa m.memm o.amma ooom sacooooooa I- as .Hos cocoon :

moEom
H.9m: m.:moa F.0ma QQQH panomoOoo: cflooafim mm .Hoo .Hoz cones; m

mddmOCHE

amoEom

H.mem m.mmaa m.oss oooa ouconoooeo condos ma .Hoo .Hoz Eocene m
Adzv sonata Axostev
m.amm o.mam m.sm oooa ado canon o: .ccodso mm .Hoa .Hoz corona H
ommpo>< swam sou ooafimoo <do .opo moademm
.o Hoamcz .o mofip6fipm> Eoumzm scoopedo
coapoaom :fl AEQQV <mo o. . a z

 

.mwmallcmeCOHz CH pom: mcowpzaom <mo HmfionoEEooll.mH mqm<e

50

of 1000 and 2000 ppm applications were about 60% and 80%,
respectively, of the desired levels.

The average concentration of DPA in drench applica-
tor systems was about 15% higher than for systems in

which the apples were dipped in 1000 ppm of DPA solution.

DISCUSSION

Storage scald control is the ultimate test of DPA
application methods, provided the resulting DPA residues
on the apples do not exceed the established FDA tolerance I
of 10 ppm. Parameters of usage that neither impair scald

control nor result in unacceptable DPA residues on apples

 

should be of minimal concern to the industry and con-
sumers.

McIntosh, Delicious and Rome Beauty apples in these
tests developed varying amounts of scald without treat-
ment, whereas, the application of DPA reduced scald inci-
dence to commercially acceptable levels. The promotion
of scald development by the artificial procedure of Dilley,
gt_al.(1963) proved satisfactory, except for the develop-
ment of severe external suboxidation symptoms on McIntosh
which hindered the scald evaluations. Untreated Delicious
apples evaluated for scald 9 weeks after harvest in the
aging solution study developed 16 percent less scald than
those similarly treated at 10 weeks after harvest in the
submerging study. This response reflected Huelin's (1968)
observation that untreated apples usually are stored

about one—third of their normal storage life before there

51

52

is an onset of visible scald. Denmead, §t_§1.(l961) found
that DPA uptake was negatively correlated with varietal
susceptibility to scald and, generally, Delicious is more
susceptible to scald than McIntosh under Michigan condi-
tions. Therefore, the greater residues observed on
McIntosh after the initial two treatment periods (Figure
2) were expected and probably related to the more advanced
maturity of McIntosh, a variety of relatively short stor-
age life. Varietal differences in scald development and
DPA uptake could also be expected in view of similar find-
ings reported by Smock (1961) and Padfield and Clark
(1963).

The significant interaction between variety and
solution age observed in 1967, but not in 1968, may be
attributed to the differences in experimental procedures
followed in the two years. All solutions in 1967 were
prepared at the same time and aging of these solutions
occurred simultaneously with aging of the fruit which was
held in storage at 32°F. In 1968, the solutions were aged
prior to harvest so all apples could be treated at a com-
parable age within A8 hours of harvest. Fruit aging may
have increased the uptake and retention of DPA since
Mattus (1963) has shown that better DPA coverage was
obtained as fruit maturity advanced. McIntosh approached
senescence more rapidly than Delicious, a late storage

variety, thereby accounting for the varietal differences

 

53

that resulted in the significant interaction found in
1967.

Delicious had greater DPA residues than McIntosh
in the submergence tests. This may have been related to
the open calyx, a characteristic more common to Delicious
than to McIntosh. Hydrostatic pressures developed by
submergence may have caused DPA to be forced into the
cavities of Delicious apples. This was borne out by the
consistently and progressively higher DPA residues in
Delicious as the pressure increased and as the pressure
was sustained longer. This suggests that problems may
occur in practice when Delicious are subjected to hydro-
static pressure during handling operations.

Submerging Delicious apples in 1000 ppm DPA solu-
tion provided scald control equal to 2000 ppm. Scald
developed on apples submerged in water, but not on those
submerged in DPA, which accounted for scald control dif-
ferences attributed to concentration (Table 13). Scald
developed on 75 percent of the apples submerged in water
and on about one-tenth of the apples submerged in DPA.
These data do not support Hilkenbaumer's (1961) report
that the effect of DPA treatment on scald was propor-
tional to the DPA solution concentration. This may be
due to the method of application, since increased DPA
uptake in relation to greater hydrostatic pressures and

increased duration of application (Table 8) resulted in

5A

fewer scalded apples. DPA residues were twice as great
on apples treated in 2000 ppm as on apples treated in
1000 ppm solutions. A similar effect was observed by
Denmead, gt_§l (1961) and Hall, gt_§l (1961) in that DPA
residues on apples were related to the amount of the
chemical applied. Residues at both concentrations were
acceptable, but since there was no improvement in scald
control at 2000 ppm, DPA applications of 1000 ppm, as
recommended by Dewey and Dilley (196A) for Michigan would
appear to be adequate.

The gas chromatographic procedure for the determina-
tion of DPA residues in apples (Gutenmann and Lisk, 1963)
proved unsatisfactory. This was apparently due to the
acetone extraction of a wide spectrum of compounds from
the blended apple tissue, possibly including other pesti-
cide residues as well as naturally occurring compounds
present in apples. Detection of the chlorinated deriva—
tive was reproducible and quantitative using standard
preparations of DPA; however, the introduction of apple
tissue resulted in unreliable results which could not be
related to the fruit treatments. Extensive trials and
modifications of this procedure to overcome these diffi-
culties proved time consuming and inadequate. Even though
this procedure apparently has provided satisfactory
results in some laboratories, it was neither accurate

nor dependable for the apples employed in this study.

55

Many hours of subsequent experimentation provided a modi-
fication of the colorimetric technique (Yatsu, 1956,
Harvey, 1958) that enabled the salvation of experimental
materials collected in the 1967 trials. This colori-
metric procedure was used also to determine DPA residues
in 1968. Recoveries of DPA were satisfactory and com-
parable to those reported by Yatsu (1956) and Harvey
(1958).

Researchers or analysts desiring to detect DPA
residues in limited quantities of apple tissue should find
this modified colorimetric technique to be satisfactory.
To minimize interference from impurities, however, it is
essential to use dependably pure reagent grades of acetone
and hexane. Furthermore, all DPA residue values should be
corrected for interferences due to natural substances from
the apple tissue and other spray residues readily ex-
tracted by acetone and diffusible into hexane.

Such factors as age of the DPA solution, presence of
organic matter in the DPA solutions, depth of submergence
of the apples in DPA solutions and the resulting hydro-
static pressures, and duration of treatment at the
increased hydrostatic pressures did not affect scald con—
trol. However, all of these factors, as well as others,
increased fruit residues of DPA above the effective mini-
mum levels needed for scald control according to Huelin
(1968), yet within the 10 ppm residue tolerance estab-

lished as safe by the FDA.

56

One could expect that solution age and the presence
of organic matter in the solution would make DPA less
available so as to decrease fruit residues in comparison
to those from freshly prepared or clean solutions. The
combining of DPA with the organic matter, however, may
have a partitioning effect and provide a better means of
transfer of the chemical to the fruit surface. The sur-
face tension of the fruit cuticle treated with old, con—
taminated solutions might be sufficiently reduced to
increase the DPA uptake.

While at least partially responsible for increased
DPA uptake, the aging and contamination of solutions also
impaired the measurement of DPA in solution. Representa—
tive samples of the solution may not have been obtained
for analysis; if true, the results would suggest that
another effect of age and contamination was to promote
the formation of a mixture rather than a colloidal sus-
pension. This effect would make the DPA particles less
available for detection by the colorimetric procedure.
Although not investigated in these trials, the possibility
of breakdown products of DPA forming upon aging and in
the presence of organic matter should be considered.
Fresh solutions and solutions free of contamination from
orchard trash contained relatively consistent amounts of
measurable DPA. Therefore, if DPA breakdown products are
involved, they must be, apparently, less prevalent and

less problematic under optimum conditions.

57

Crystals were occasionally observed on the apples
treated with DPA solutions containing organic matter in
1967. In 1968, an oily deposition on fruits was some-
times associated with Old, contaminated solutions.
Therefore, even though treatment of apples in DPA solu-
tions which had foul odors and appeared dirty provided
acceptable residues and scald control, operators need to
use good judgment in replacing solutions in order to
avoid fruit rots and other problems.

DPA residue differences were noted in relation to
technical formulation of the two materials used, yet both
materials were acceptable in respect to scald control,
freedom from injury and residue tolerance. The differ-
ences may be associated with oil base composition varia-
tions in their manufacture.

DPA treatment solution temperatures of 50, 60 and
70°F did not affect residues and this permitted tempera-
tures to be used as replicates for analysis of the 1967
results. The lack of a statistically significant tem-
perature effect should be Of particular importance to
fruit handlers since they cannot afford to control the
temperatures within the DPA applicator systems. The
results indicated that residue or scald problems from
materials used in this temperature range should be of

little practical concern.

58

Previously mentioned experimental fruit handling
differences may have been responsible for the significant
effect of duration of submergence on DPA residues which
occurred in 1967, but not in 1968. Since duration of
treatment affected the DPA residues in the studies of
Denmead, gt_§l.(l96l) and Padfield and Clark (1963), they
suggested DPA applications should be limited to less than
2 minutes. Longer durations were herein studied since
fruit hydrohandling systems may purposely or inadvertantly
expose apples to DPA solutions at 3- to 5—foot depths for
at least several minutes.

Hydrostatic pressures developed by either air pres-
sure or water head yielded similar effects, therefore,
hydrostatic pressure must be responsible for physically
forcing additional DPA into the apples. Even though such
depths as employed in these tests exceeded those employed
in apple hydrohandling systems, DPA residues were within
FDA tolerances, fruit injuries were absent, and scald
was controlled.

Both hydrostatic pressure and duration of treatment
increased DPA residues on apples. Autoradiography
revealed that luC-DPA was primarily deposited at the fruit
surface with traces in the core cavity. One might expect
the open—calyx of Delicious to permit more DPA to enter
the interior of the apple when this variety is subjected

to such extreme conditions of hydrostatic pressure. The

59

predominance of lLAC-DPA at the surface, where scald occurs,
suggests a local effect of DPA in controlling scald. But,
if the observations of Padfield (1959) and Stevenson and
Blake (1961) that DPA reduces internal breakdown are true,
the effect of DPA may be realized within the fruit in

areas not directly in contact with the material during
treatment.

The survey of DPA usage by fruit handlers illustrated
the diversity of practices employed in Michigan. Appar-
ently there is a great range in suitable concentrations
that will control scald sufficiently. Dewey and Dilley
(l96A) considered that 1000 ppm DPA will control scald on
apples in Michigan, but some growers still use 2000 ppm
on some varieties to be certain of scald control. Also,
there appeared to be considerable lattitude in the pre—
cautions required for the use of DPA on apples. Operators
seem to be using very dirty appearing solutions which con-
tain far less DPA than recommended, apparently without
encountering scald control problems.

The higher concentrations of DPA found in drench
than in dip systems may be attributed to the constant
mixing which occurs in the former applicator due to the
circulation of the liquid which is required for applica-
tion. In the dip system, mixing is less certain, even
with mechanical stirring or circulating equipment within

the dip tanks.

60

The DPA analysis results for commercial solutions
demonstrated the need of a means for rapidly testing the
DPA content of commercially employed drench and dip solu-
tions. Such a field test would provide the necessary
information to adjust the DPA solution concentration to
compensate for the daily variations that are constantly

encountered.

CONCLUSIONS

The colorimetric technique as modified for

these studies is a satisfactory method of

determining DPA residues of 0-25 ppm in apple

tissue, and suitable for the determination of

DPA in relatively small quantities of material.

Under the conditions of these studies, scald

control was influenced by apple variety and DPA

concentration, but not by aging, the presence

or organic matter, increased hydrostatic pres-

sure, and duration of treatment at increased

hydrostatic pressures of the solutions employed

for treating the apples.

It was found that the following treatments

increased DPA residues in apples:

a. dipping in 2000 ppm compared with 1000 ppm
solutiOns,

b. dipping in progressively older DPA solutions,

c. dipping in DPA solutions contaminated with
orchard trash and soil,

d. submersion to depths as great as 6.5 feet, and

e. submersion periods prolonged up to 8 minutes.

61

62

Solution temperatures of 50, 60 or 70°F did not
affect DPA residues on apples.

Within the limits of these trials, the amount
of measurable DPA in solutions decreased as a
result of the presence of organic matter and
upon aging of the solution.

There is a need to monitor the DPA content of
solutions in applicator systems. For this
purpose an analytical procedure, useful to

operators as a guide for adjustment of the DPA

 

content of their solutions, was developed. ,

SUMMARY

DPA residues for apple fruit in the range of 0 to
25 ppm could not be satisfactorily determined by gas
chromatographic procedures. A colorimetric method was
adapted and satisfactorily employed. Further modifica-
tion permitted the colorimetric method to be used for
ascertaining the DPA content of solutions prepared
according to label recommendations for treatment of
apples with DPA for scald control. A simplified version
of the colorimetric procedure was tested and found to be
of limited value for the estimation of DPA content of
solutions in commercial applicators.

McIntosh had greater residues than Delicious when
treated in DPA solutions aged for up to A weeks, but in
the submerging studies Delicious, commonly characterized
by open-calyx, had greater residues than McIntosh.

Both technical formulations of DPA employed pro-
vided satisfactory DPA residues on apples.

The presence of organic matter in DPA solutions
resulted in greater DPA residues on apples than when
treated in relatively clean solutions, but storage scald

control was not impaired nor were injuries attributed to

63

 

6A

organic matter. This factor resulted in less measurable
DPA in solutions. Aging DPA solutions maintained free
of extraneous material for up to A weeks had no effect
on DPA residues for apples treated in these solutions.

No difference in scald control was observed between
apples submerged in 1000 ppm and 2000 ppm solutions, even
though the latter produced DPA residues twice as great as
at 1000 ppm.

The method of artificially producing scald symptoms
was satisfactorily used for these experiments and re-
vealed that DPA controlled scald in comparison to un-
treated apples, which developed commercially significant
scald.

Although DPA residues on apples varied in response
to treatment conditions, they were within the 10 ppm
tolerance established as safe by the Food and Drug Admin-
istration.

Autoradiography of Delicious apples submerged in
luC-DPA solution confirmed others' findings that the

majority of DPA residues are in or adjacent to the apple

peel.

LITERATURE CITED

LITERATURE CITED

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Barnes, H. 19AA. Estimation of small quantities of
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APPENDIX

73

APPENDIX TABLE I.--DPA residues (ppm) on McIntosh and
Delicious apples as influenced by DPA
formulations used over a A week period

 

 

 

 

 

in 1967.
Formulation
A B
(3E2) McI Del McI Del Y
DPA (ppm) DPA (ppm)

0 2.28 1.73 1.95 2.51 2.11
l 5.11 A.79 2.19 2.12 3.5A
2 6.36 A.58 2.32 2.01 3.81
3 5.36 3.16 2.19 1.51 3.05
A 8.38 A.18 3.22 1.82 A.39
I 5.50 3.69 2.37 1.99 3.38

 

7A

APPENDIX TABLE II.--DPA residues (ppm) on McIntosh and
Delicious apples as influenced by
the presence of organic matter and
the age of the DPA treatment solu-
tion in 1967.

 

 

 

 

 

Control Organic Matter
(3&2) McI Del McI Del 7
DPA (ppm) DPA (ppm)
O 2.09 1.97 2.1A 2.27 2.11
l 2.07 2.19 5.22 A.71 3.5A
2 2.56 2.28 6.12 A.32 3.81
3 2.16 1.61 5.39 3.06 3.05
A 3.83 2.A3 7.76 3.A0 A.39
Y 2.5A 2.09 5.33 3.55 3.38

 

75

APPENDIX TABLE III. DPA residues (ppm) on McIntosh,
Delicious and Rome Beauty apples
as influenced by the presence
of organic matter and treatment
solution age in 1968.

 

 

 

Free of With
Organic Matter Organic Matter
Solution McI Del Rome McI Del Rome Age
Age (wks.) X

 

DPA (ppm) DPA (ppm)

0 (fresh) 1.17 0.01 1.16 2.32 2.15 1.96
A.12 0.37 0.55 3.39 2.30 1.79 1.7

l 0.58 1.75 2.10 A.78 7.52 12.98
1.82 2.15 0.59 3.18 A.87 3.73 3.8

2 0.95 0.82 0.17 13.86 7.66 1.97
0.8A 1.23 2.21 1A.15 6.78 3.22 A.u

A 2.87 0.33 1.58 3A.83 15.63 7.58
2.66 0.53 2.A8 11.22 18.00 7.85 8.8

Variety E 1.87 0.89 1.36 10.97 8.11 5.1A A.72

Sanitation 7 1.38 8.07

 

76

APPENDIX TABLE IV.-—DPA residues (ppm) on apples result-
ing from treatment in a simulated
submersion system. 1967.

 

Duration of Treatment (min)

 

HydrOStatiC l 2 u 8

 

 

 

 

 

Pressure (psi) X
McIntosh
0 1.67 3.83 A.67 5.90 A.02
l A.99 1.99 2.37 6.A6 3.95
2 3.66 A.AA 3.71 5.18 A.2A
3 3.33 3.5A A.76 7.09 A.68
A 2.38 2.8A 8.91 9.63 5.9A
E 3.21 3.33 A.88 6.85 A.29
Delicious
0 1.71 3.92 A.21 7.A3 A.32
1 0.80 2.95 3.30 3.70 2.68
2 .69 5.65 A.32 8.87 5.38
3 A.A5 7.A0 8.08 11.70 7.90
A 5.63 8.8A 11.2A 8.51 8.55
Y 3.05 5.75 6.23 8.0A 5.63

 

77

APPENDIX TABLE V.--Effect of DPA solution concentration,
duration of treatment and depth of
submersion on DPA residues on Deli-
cious apples in 1968.

 

DPA Solution (ppm)

 

 

 

 

 

 

 

DZEERtITSI) szggaiga7881) Duration of treatment (min)
0 2 A mean

1000
0 0 1.89 1.89 2.A2 2.07
2 5 1.5 2.16 0.95 2.A2 1.8A

5 3 2.70 l.A5 3.06 2.140 LILL-

2.10

2000
0 0 3.57 3.39 3.37 3.AA
2 5 1 5 2.17 A.32 3.AA 3.31
5 3 A.22 A.03 8.23 5.A9
A.08

 

APPENDIX TABLE VI.--Apparent diphenylamine residues (ppm)
observed on Delicious apples treated
in a submersion system containing
only water - 1968.

 

Duration of Treatment (min)

 

 

Treatment Hydrostatic
Depth (ft.) Pressure (psi) 0 2 A X
0 0 1.28 1.77 1.70 1.58
(surface)
2.5 1.5 2.10 3.0A 2.02 2.38
5.0 3.0 1.38 2.97 2.66 2.3A

1.25 2.59 2.13 2.10

X)

 

78

APPENDIX TABLE VII.—-DPA (ppm) in clean and contaminated
treatment solutions of various ages

 

 

in 1967.
Age (wks) Control Organic Matter (0M)
DPA (ppm) DPA (ppm)
0 (fresh) 972 1069
1 1017 755
2 835 622
3 927 725
A 7AA A56
3? = 899 ppm Y = 733 ppm
All Treatments §'= 816 ppm

 

 

contaminated
various ages

APPENDIX TABLE VIII.——DPA (ppm) in clean and
treatment solutions of

 

 

 

in 1968.
Control Organic Matter (0M)
Age (Weeks) I II III I II III
McI Del Rome McI Del Rome
DPA (ppm) DPA (ppm)

0 (fresh) 1068 1132 1068 9A9 325 A9A
l 830 112A 10A5 53A 525 905
2 789 1068 109A 72A 6A3 A05
A 1979 7A0 887 199A A89 869

Y = 1069 ppm Y = 738 ppm

. All Treatments Y = 816 ppm

 

79

APPENDIX TABLE IX.--Effect of DPA solution age and sani-
tation on scald reduction1 for
McIntosh, Delicious and Home Beauty
apples in 1968.

 

Solution McIntosh Delicious Home Beauty
Age (wks)

 

 

 

Control 0M Control 0M Control 0M

 

(Scald reduction units)

 

 

O 10 13 10.5 10.5 15.5 15.5 -.wufi“
1 10 11 8.5 9 15.5 15.5
2 15 11 10.5 9 15 15
A 8 15.5 10.5 9.5 1A.5 12.5
1

Scald reduction units are the difference between
untreated and DPA—treated apples in the number of scalded
fruits per 20.

80

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