INFLUENCE OF H U M I D I T Y AND RAIN ON Ut AKE

AND M E T A B O L I S M OF '4C-AZINPHOS-METHYL IN
BEAN PLANTS 1
W. STEFFENS and J. WIENEKE
Arbeitsgruppe Radioagronomie cler Kernforsehungsanlage
duUeh GmbH, 517 Julieh, West Germany

In several experiments the influence of relative humidity and rain on uptake and
metabolism of carbonyl-14C-azinphos-methyl was examined in bean plants under
the following environmental conditions: Growth room with 35/80%, 65/85% and
95/95% (day/night) relative humidity and open field with and without rain.
Increasing relative humidity had an enhancing effect on the rate of uptake and
metabolism. A higher portion of water-soluble compounds was found in the bean
tissue, although the azinphos-methyl itself is relatively non-polar. Low relative
humidity and possible dry periods in summer will reduce the uptake and will leave
the azinphos-methyl relatively persistent on the leaf surface. Rain or spray
irrigation easily removed azinphos-methyl from the leaves. The rate of this removal
seemed to depend on the intensity and time of rainfall after application. However,
repeated wettings by rain may simultaneously stimulate uptake and metabolism of
azinphos-methyl by the leaves.

Pesticides applied to plants or soil are exposed to certain environmental influences

which may have an effect on their mode of action, uptake, and fate in plant and soil.
Primarily the climatic factors (also termed "weathering") such as light, temperature,
humidity, and wind seem to be important.

In numerous investigations it has been demonstrated that photodecomposition is of

importance for the degradation of certain pesticides (Crosby 1969 and 1973, Hull 1970,
Plimmer 1970). Light seems to stimulate or inhibit the uptake by plants depending on
the plant as well as on the chemical properties of the respective pesticide (Hull 1970).
High temperature and humidity generally improve the permeability of the cuticle re-
suiting in an increased uptake of certain pesticides. Dew, mist, or drizzle influence the
extent and intensity of uptake processes. On the other hand, pesticides are removed from
the leaf surface by rain or wind. In addition, leaching of pesticides or their metabolites
can occur (Crosby 1973, Hull 1970, Plimmer 1970).

With reference to the insecticide S-(3,4-dihydro-4-oxobenzo [d]-[1,2,3]-triazin-3-

ylmethyl) dimethyl phosphorothiolothionate, (azinphos-methyl), little is known about

1In collaboration with Bayer AG Leverkusen,Pflanzenschutzanwendungstechnik, BiologischeForschung

u. Isotopeninstitut, F. R. G. Presented at the Third International Congress of Pesticide Chemistry
(IUPAC), Helsinld, July. 3-9, 1974.

Archives of Environmental Contamination 364

and Toxicology Vol. 3,364-370 (1975)
9 1975 by Springer-Verlag New York Inc.
 Azinphos-methyl Metabolism in Bean Plants 365

the influence of environmental factors on persistence, degradation, uptake, and metabolism

in plants. Only the effect of light, temperature, pH, formulation and mode of application
has been investigated under more or less laboratory conditions (Crosby 1969, Schulz
et al. 1970, Liang and Lichtenstein 1972). Previous investigations with 14C-azinphos-
methyl showed differences in the metabolism of this insecticide, which seemed to be due
to climatic factors, especially to humidity (Steffens and Wieneke 1974, Wieneke and
Steffens 1975). Therefore additional experiments were conducted in a growth room and
under open field conditions to study the influence of relative humidity and of rain on the
fate of azinphos after application to bean leaves.

Materials and m e t h o d s

In all experiments carbonyl-X4C-azinphos-methyl was used. In a number of experiments

this labelling position proved to be highly resistant against complete mineralization or
respiration to 14CO2 by plants (Steffens and Wieaeke 1974, Wieneke and Steffens
1975). Bean plants (Phaseolus vulgaris 'Saxa') were used as standard plant material.
Details on standard cultivation, formulation, spray application, cleanup procedures, and
thin layer chromatography are described elsewhere (Steffens and Wieneke 1974,
Wieneke and Steffens 1975). Four hundred/ag (in the field experiments, 500/ag) of
azinphos-methyl with specific activities ranging from 3.7 to 8.0/2Ci/mg were applied to
the first trifoliate leaf. Experimental time was 14 days. Seven experiments were conducted
in a growth room under these standardized climatic conditions: 12 hr photoperiod with
one hr of adjustment each in the morning and evening, 14,000 Lux light intensity, day/
night temperatures, 23~176 The day/night relative humidity varied as follows: two
experiments at low humidity 35%/80%, two experiments at high humidity 95%/95%, and
three experiments at medium humidity 65%/85%.

In August and September, 1973, in addition, four experiments were run in the open
field, two of them in nutrient solution with rain or spray irrigation and two simultaneously
in nutrient solution and soil without rain. The maximum temperatures ranged from
16.5~ to 31.8~ during the daytime and the minimum temperatures at night were from
2.8~ to 15.3~ The respective values for the relative humidity were: 22 to 63%
(minimum) during daytime and 92 to 95% (maximum) at night. During the experiments
without rain one mm of precipitation was registered as dew. The water from rain or
irrigation was collected quantitatively.

The following fractions were investigated for x4C and azinphos-methyl or its
metabolites: Benzene strip from the leaf surface; rainwater; hexane, chloroform, and
water fractions; the non-extractable residue from the treated leaf; and the non-treated
plant parts.

Results and discussion

In table I the results of a number of experiments, carried out under different growth
room or field conditions, are compared with those obtained from experiments con-
ducted simultaneously under standardized environmental conditions. The trend of
366 W. Steffens and J. Wieneke

Table I. Distribution of radioactivity in several fractions of beans 14 days

after application of 14C-azinphos-methyl to the first
trifoliate leaf (% of total applied)

Various conditions Standard conditions

(I 1 experiments) (3 experiments)
Fraction Average SD Average SD

Benzene strip 61.6 -+ 13.9 63.0 + 4.0

Hexane 1.5 + 0.6 1.5 + 0.4
Chloroform 0.6 + 0.4 0.6 + 0.2
Water 20.3 -+ 9.6 22.9 + 3.5
Residue
(treated leaf) 4.7 + 2.9 3.8 + 0.2
Shoot 4.1 + 3.1 3.8 + 1.I
Root 0.8 +- 0.5 0.8 + 0.3
Other activitya 1.9 + 1.5 1.2 -+ 0.5

Recovery 95.4 + 3.8 97.7 -+ 3.3

aRadioactivity from cuffs used during application, nutrient solution and waste
during cleanup (precipitates, filters, glassware washing, etc.)

14C-azinphos-methyl degradation seems to be very similar in both cases. However,

going into more detail, there are apparently significant standard deviations (Table I,
column 1), especially for the benzene strip, water fraction, and solid residues. These
reveal that influences of the environment, primarily climatic factors, have occurred.

One other factor contributing to the great variation of the experiments in Table I
(column 1) may be the fact that these experiments were partly conducted subsequently,
and that the status of the plants varied although the method of plant growing generally
was standardized. Some indication in this respect may be derived from the data in Table
II. Both experiments without rain were conducted simultaneously under the same field
conditions except the nutrient medium (soil and nutrient solution). As a consequence
the nutritional status of the plants was different, which may have contributed to the
variations occurring between the individual fractions of the two experiments (benzene
strip, water fraction, and solid residue).

The main influence on processes of uptake and degradation of pesticides in experi-

ments with the same plant material can b'e expected from climatic factors. Besides the
effect of light and temperature on photodecomposition the relative humidity is very
important. High humidity is thought to increase the permeability of the leaf cuticle and
to maintain materials sprayed on the leaf in mobile condition for a longer time after
application or to induce rewetting processes especially when detergents or formulations
are used (Crosby 1973, Hull 1970).
 Azinphos-methyl Metabolism in Bean Plants 367

Table II. Distribution of radioactivity in several fractions of beans 14 days

after application of 14C-azinphos-methyl to the first trifoliate
leaf as related to field conditions with and without spray
irrigation or rain (% of total appliedJ

Without rain With rain

Nutrient Spray Spray irrig.
Fraction solution Soil irrigation + nat. rain

Rainwater strip - - 57.0 68.3

Benzene st~p 65.3 52.8 23.5 3.8
Hexane 1.9 2.2 0.8 0.2
Chloroform 0.4 0.7 0.2 0.3
Water 12.8 19.9 5.5 10.2
Residue
(treated leaf) 4.9 9.5 2.1 2.9
Shoot 1.3 1.7 1.4 1.3
Root 0.3 - 0.3 0.2
Other activitya 3.6 3.0 4.8 3.0

Recovery 90.5 89.8 95.6 90.2

aRadioactivity from cuffs used during application, nutrient solution and waste
during cleanup (precipitates, filters, glassware washing etc.).

The effect of humidity with respect to t4C-azinphos-methyl is illustrated in Figure

1. The results clearly demonstrate that with increasing relative humidity the radio-
activity in the benzene strip, which still was related by more than 90% to azin-
phos-methyl, will decrease while simultaneously the amount of polar radioactive
substances in the water fraction and the radioactivity in the residue of the treated leaf
will increase. In agreement with previous results (Wieneke and Steffens 1975) the
14C.activity in the water fractions was mainly related to two metabolites and totally to
more than ten. In addition the chromatographic distribution of these metabolites was
not influenced by the relative humidity; only the radioactivity of the individual
metabolites expressed as percentage of total applied varied. The relatively low values of
the hexane and chloroform fractions were not influenced. The chromatographic distribu-
tion of radioactive substances determined in these fractions corresponded to that found
in previous experiments (Wieneke and Steffens 1975).

The radioactivity in the non-treated plant parts (shoot and root) increased to 11.5% of
total applied, when the relative humidity amounted to 95%. Tissue extraction and analyses
revealed that more than 70% of the radioactive substances found in the remaining non-
treated shoot was water soluble, only 4% was organic soluble and 26% of the radioactivity
was unextractable. These results are in agreement with findings reported elsewhere
 368 W. Steffens and J. Wieneke

%
80 Total applied

70

60 \
\
\
50 \
\
\
40 "o
J J"~
30

20

10
~-................. . . ~ ~
0 i i
D

35 65 95%
Humidity

Fig. 1: Influence of relative humidity on the distribution of radioactivity in different

fractions of beans 14 days after application of 14C.azinphos.methy 1 to the first trifoliate
leaf (% of total applied), o Benzene strip, 9 Water, [] Shoot + root, 9 Residue (treated
leaf, A Hexane + chloroform.

(Steffens and Wieneke 1974) suggesting that polar derivatives from azinphos-methyl
have been partially circulating within the whole plant.

For the evaluation of the behavior and fate of a pesticide standardizing and repeating
of experiments as well as experiments carried out close to practical conditions are of
great importance. One considerable effect in field investigations is due to rewetting
processes and rainfall. For 14C-azinphos-methyl this is demonstrated in Table II.
Azinphos-methyl is a relatively non-polar substance with a maximum solubility in water
of 33 ppm. Nevertheless up to 68% of the radioactivity determined to be about 90%
still as azinphos-methyl was removed by irrigation or rain from the leaf surface. This
high degree of removal by water could mainly be due to the use of a formulation
(effect of wetting agents, etc.)

Under field conditions, azinphos-methyl will be washed from the plant easily by
natural rain and may be thus masking the apparent degradation obtained by residue
analysis. The amount of azinphos-methyl removed from the plants by rain will drip upon
the soil. This suggests also investigations to find whether this compound or possibly its
daughter products formed in the soil may be able to reenter the plant.
 Azinphos-methyl Metabolism in Bean Plants 369

The extent of removal of a pesticide from the leaf surface is dependent on a number
of parameters such as intensity and amount of rain and time of rainfall after application
(Hull 1970). As far as azinphos-methyl is concerned some hints can be derived from data
depicted in Table III. Nearly 50% of the activity applied was removed two-days after
treatment by a relatively small amount of rain applied as spray irrigation. The subsequent
spray irrigations or natural rainfalls had little effect. It seems that the total amount of rain
is less important than the intensity and the time of rainfall after treatment. An analysis
of the solution containing the compounds removed from the leaf surface by the first
spray irrigation revealed that about 90% of the activity found in this solution was azin-
phos-methyl; in the solution of the second irrigation this portion was less than 80%.
However, there was also a substantial amount of water-soluble compounds which might
partially result from leaching processes (Hull 1970, Tukey 1970), especially since the
irrigation was repeated.

Since a considerable portion of azinphos-methyl was removed a few days after applica-
tion the amount of activity (Table II) found in the benzene strip of the plant at harvest
time 14 days after application was small. Furthermore due to the high portion of azinphos-
methyl removed from the leaf surface by spray irrigation or rain the total amount of
activity found in the other fractions was relatively low as compared to the activity
determined in the respective fractions of the experiments without rain. However, there
are distinct deviations between the activity values found in the benzene strips of the two
experiments with spray irrigation or rain, probably due to the number of rains and the
total amount of rainfall.

Table 11I. Radioactivity removed from bean leaves by spray

irrigation or natural rain after application of
14C~zinphos-methyl to the first trifoliate
leaf (% of total applied)

Spray irrig.
Rainfall Days after Spray irrig. Activity Days after or rainfall Activity
no. application mm % application mm %

1 2 6.9 48.3 2 3.2 45.2

2 5 5.0 6.1 5 3.5 12.2

3 8 5.0 2.6 10 4.1 a 7.1

4 - - - 12 0.6 a 0.8

5 - - - 13 6.3a 3.0

Total 16.9 57.0 17.7 68.3

anatural rain
370 W. Steffens and J. Wieneke

Although most of the pesticide was removed from the leaf surface by repeated spray
irrigation and rainfall, a greater percentage of radioactivity was determined in the water
fraction of experiment II than in experiment I with rain. This can be explained by the
fact that frequent rewetting of the leaf surface stimulated the absorption of azinphos-
methyl resulting in an increased metabolism.

References
Crosby, D. G.: Experimental approaches to pesticide photodecomposition. Residue Re-
views 25, 1 (1969).
Crosby, D. G.: The fate of pesticides in the environment. Ann. Rev. Plant. Physiol. 24,
467 (1973).
Hull, H. G.: Leaf structure as related to absorption of pesticides and other compounds.
Residue Reviews 31, 1 (1970).
Liang, T. T., and E. P. Lichtenstein: Effect of light, temperature, and pH on the
degradation of azinphos-methyl. J. Econ. Entomol. 65, 315 (1972).
Plimmer, J. R.: Principles of photodecomposition of pesticides. Degradation of synthetic
organic molecules in the biosphere. ISBN 0-309-02046-8. Nat. Acad. Sci. Washing-
ton, D. C., 279 (1970).
Schulz, K. R., E. P. Lichtenstein, T. T. Liang, and T. W. Fuhrmann: Persistence and
degradation of azinphos-methyl in soils, as affected by formulation and mode of
application. J. Econ. Entomol. 63,432 (1970).
Steffens, W., and J. Wieneke: Untersuchungen zu Aufnahme und Stoffwechsel yon
14C-markiertem Methyl- und ~thyl-Gusathion in Kulturpflanzen. I. Extraktion,
Fraktionierung und 14C-Bilanz. Pflanzenschutz-Nachrichten Bayer, accepted for
publication (1974).
Wieneke, J., and W. Steffens: Untersuchungen zu Aufnahme und Stoffwechsel yon 14C-
markiertem Methyl- und .~thyl-Gusathion in Kulturpflanzen. II. Chromatograph-
isehe Auftrennung und quantitative Bestimmung der Metaboliten und Schritte zu
ihrer Identifizierung. Pflanzenschutz-Nachrichten Bayer, accepted for publication
(1975).

Manuscript received September 23, 1974; accepted October 15, 1974