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107
085
THS

31% is“. fit

 

This is to certify that the

thesis entitled
Effects of herbicides Paraquat and Atrazine
upon Collembola. Folsomia candida (Willem) and
Tullbe rgia Lranulata Mills

 

presented by

Jusup Subagja Dwidjasatmoko

has been accepted towards fulfillment
of the requirements for

M. S. degree in Zoom

 

0-7639

EFFECTS OF HERBICIDES PARAQUAT AND ATRAZINE UPON
COLLEMBOLA, FOLSOMIA CANDIDA (NILLEM) AND
TULLBERGIA GRANULATA MILLS

 

By

Jusup Subagja Dwidjasatmoko

A THESIS

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

MASTER OF SCIENCE

Department of Zoology

1978

 

ABSTRACT

EFFECTS OF HERBICIDES PARAQUAT AND ATRAZINE UPON
COLLEMBOLA. FOLSOMIA CANDIDA (NILLEM) AND
TULLBERGIA GRANULATA MILLS

 

By
Jusup Subagja Dwidjasatmoko

Folsomia candida (Willem) and Tullbergia granulata Mills were

 

fed on powdered brewer's yeast treated with herbicide. Three concen-
trations of Paraquat and Atrazine (600 ppm, 1000 ppm, and 5000 ppm)
were tested to evaluate the effects on Collembola. Cultures were
reared at 60° F and observed daily for 22 weeks.

Collembola fed on herbicides significantly decreased their egg
production and delayed the hatching time of their eggs. Feeding on
Atrazine also prolonged instar duration. Only 5000 ppm of Paraquat
lengthened the instar duration of f, candida, and was not significant
at lower doses. However, the instar duration of I, granulata was
significantly extended by feeding on Paraquat. Results suggest that
feeding on herbicides affect the reproductive system. Food treated
with 5000 ppm of Atrazine may be less palatable to Collembola; in
addition high mortalities were observed at this concentration

The possibility of direct lethal effects were also tested.
The Collembola were reared on soil sprayed with herbicide at field
application dosage. The concentrations were 600 ppm for Paraquat,

and 5000 ppm for Atrazine. Apparently, Paraquat does not have directly

Jusup Subagja Dwidjasatmoko

lethal effects on Collembola. However, Atrazine shows notable
effects.
The results of these experiments indicate that Atrazine pro-

duces more severe effects as compared to Paraquat.

ACKNOWLEDGMENTS

I am deeply indebted to Dr. Richard J. Snider, my major profes-
sor, for his valuable suggestions and assistance in the preparation of
this thesis. I wish to express my appreciation for his patient guidance
and encouragement in my graduate program.

I would also like to express my appreciation to Drs. T. Wayne
Porter, Lynn S. Robertson and James M. Tiedje, my guidance committee
members, for their suggestions in the preparation of this thesis and
participation in my graduate program.

Special thanks are extended to Mrs. Renate M. Snider for her
assistance and making the facilities in laboratory available for this
research, and to Dr. Roger R. Neitzel for his assistance in statistical
analysis.

I express gratitude to Gadjah Mada University, my alma mater,
and the Midwest Universities Consortium for International Activities

(MUCIA Inc.) for financial support and assitance of my study.

ii

TABLE OF CONTENTS

LIST OF TABLES .
LIST OF FIGURES.

INTRODUCTION.
REVIEW OF LITERATURE .
Effect on Density
Effect on Diversity. . .
Some Aspects of Direct Effects .
EXPERIMENT I.
Materials and Methods .
RESULTS
Mortality .
Instar Duration .
Egg Production
Egg Viability.
Incubation Period
DISCUSSION
EXPERIMENT II
Materials and Methods .
RESULTS AND DISCUSSION
SUMMARY

LITERATURE CITED

Page

vi

CD 00 050100 to

APPENDICES
Appendix
I. Folsomia Candida, Instar Duration in Days, at 60° F.
11. Tullbergja Granulata, Instar Duration in Days,
at 60° F . . . .
III. Folsomia Candida, Egg Production per Instar, at 60° F
IV. Tullbergia Granulata, Egg Production per Instar,
at 60° F . . . . . . . .
V. Folsomia Candida, Time Required for Hatching in Days
at 60° F . . . . . . . . . . . . .
VI. Tullbergia Granulata, Time Required for Hatching in
Days at 60° F. . . . . . . . . .
VII. Results of Soil Test .

 

 

 

 

 

 

iv

Page

37

42
45

47

50

51
52

Table

II.

III.
IV.

VI.

LIST OF TABLES

Experiment I--Number of Treatment Replicates .

 

Folsomia Candida, Cumulative Percent Mortality, at
60° F . . . . . . . .

Tullbergia Granulata, Cumulative Percent Mortality,
at 60° F . . . . . . . . . . .

 

Folsomia Candida, Egg Viability (in Percent) per Ovi-

position, at 60° F.

Tullbergia Granulata, Egg Viability (in Percent) per

 

Oviposition, at 60° F.

Experiment II--Number of Treatment Replicates.

 

Folsomia Candida, Percent Survival Reared on Soil, at
60° F . . . . . .

Tullbergia Granulata, Percent Survival Reared on Soil,

 

at 60° F

Folsomia Candida, Instar Duration in Days, at 60° F.

 

 

Tullbergia Granulata, Instar Duration in Days, at
60° F . . . . . . . .

Folsomia Candida, Egg Production per Instar, at 60° F .

 

Tullbergia Granulata, Egg Production per Instar,
at 60° F . . . . . . .

 

Folsomia Candida, Time Required for Hatching in Days
at 60° F . . . . . . . . .

 

Tullbergia Granulata, Time Required for Hatching in
Days at 60° F. . .

Page
10

l2

12

21

21
29

31

32
37

42
45

47

50

51

LIST OF FIGURES

 

 

 

 

 

Figure Page
1. Folsomia Candida, Instar Duration at 60° F . . . . . . l4
2. Tullbergia Granulata, Instar Duration at 60° F. . . . . 15
3. Folsomia Candida, Egg Production at 60° F . . . . . . l7
4. Tullbergia Granulata, Egg Production at 60° F . . . . . 19
5. Folsomia Candida, Incubation Period at 60° F . . . . . 22
6. Tullbergia Granulata, Incubation Period at 60° F . . . . 23

 

vi

INTRODUCTION

Herbicides are destructive chemicals commonly applied to con-
trol weeds in agricultural situations. Application of these chemicals
may contaminate plant remains, and as a result, may be toxic to soil
animals. Furthermore, elimination of the plants could reduce the
source of food for much of the soil fauna. Therefore, application of
herbicides may influence the soil community.

In 1952, Baudissin made an investigation on Collembola and
Acari in a field treated with 2.4-0. He found no effect of the herbi-
cide on the populations of Collembola and Acari. Since that time, the
influence of herbicides on soil animals has attracted much interest
among scientists. Effects caused by herbicides on soil fauna still
are not completely understood, even though many studies have been con-
ducted.

Among the arthropods, the insect order Collembola has the
greatest number of soil species besides Acari. They play an important
role in the breakdown of dead plants, and are among the most important
producers of humus (Schaller, 1968). Field investigations have shown
that some herbicides affect the populations of Collembola (Edwards &
Thompson, 1973). Some herbicides are indeed directly toxic to these
animals. However, in the field, the influence is more indirect than

direct (Edwards, 1969; 1970; Edwards & Thompson, 1973).

Laboratory studies are very helpful in understanding the direct
toxic effects. Study on the direct exposure of 2,4,5-T to_0nychiurus

aquadriocellatus Gisin, has provided valuable information on the

 

poisoning process of herbicides on Collembola. The Collembola are
paralyzed before dying (Eijsackers, 1975).

The present study was conducted to ascertain some biological
aspects of Collembola reared under direct feeding on herbicides, and
if field applications of herbicides have lethal effects on the soil
animals. This type of study is, at present, not commonly undertaken.

Therefore, this study is viewed as a basis for future investigations.

REVIEW OF LITERATURE

Effects of herbicides on soil animals have been investigated
primarily in field situations, at the "group" level. Only a few
laboratory experiments were conducted at the species level.

In fields, effects are highly variable and depend on a large
number of factors, i.e.: the diversity of soil fauna, soil condition,
weather and time of application, method of applications, and persis-
tence of their residues on soil (Edwards & Thompson, 1973). This
review is mainly concerned with the influence of herbicides on
Collembola. Under some circumstances, however, discussion of arthro-

pods and other soil animals are given in this review.

Effect on Density

Studies by Baudissin (1952), Rapoport & Canglioli (1963), Fox
(1964) and Prasse (1975) concluded that Collembola populations were
not affected by application of 2,4-D. Application of MCPA (4-chloro-
2-methy1phenoxyacetic acid) did not change the populations of
Collembola (Rapoport & Canglioli, 1963; Edwards & Arnold, 1964). This
was confirmed by Davis (1965) with a study in a field that had been
treated annually for a ten out of thirteen year period with MCPA. He
concluded that there was no immediate or long term effect on Collembola
populations. The same results were also concluded when Edwards and

Arnold (1964) tested Linuron and Triallate. Later, Edwards et a1.

(1974; 1975) found that the herbicide benzol-prop-aethyl slightly
decreased the number of Collembola; however, there was no effect on
the total soil fauna. Recent studies with Banvel-D applied in paddy
fields and grassland concluded that there was no direct effect on
Collembola, but it decreased the total number of soil arthropods
(Bhattacharya & Joy, 1977).

There are discrepancies as to the influence of Paraquat and
Dalapon. Curry (1970) reported that application of Paraquat in grass-
land decreased populations of Collembola and Acari, while Edwards
et a1. (1972) found more arthropods and fewer predatory Acari in the
treated plots. Earlier, Edwards (1970) observed that Paraquat had no
apparent effect on small invertebrates. Although Fox (1964) found
significant increases of Collembola in grassland treated with Dalapon.
Curry (1970) noted the opposite effect.

Ostensibly, TCA (trichloroacetic acid) allowed an increase of
Collembola populations (Fox, 1964). On the other hand, DNOC (2-methy1-
4,6-dinitrophenol) and Bladex decrease the number of Collembola and
Acari (Johnson et al., 1955; Edwards et al., 1961; 1971; 1974).
Decrease in number of isotomid Collembola was found after application
of Shell NL 19805 (Edwards, 1970).

Fox (1964) observed soil fauna populations in grassland for
three and four months after application of Atrazine, while Popovici
et a1. (1977) studied corn field for four months after application.
Both studies agreed that Atrazine decrease collembolan populations.

A series of observations at Rothamsted Experimental Station showed
numbers of Collembola and other soil animals diminished greatly by

application of Simazine. The effects were still obvious after six

years (Edwards et al., 1964; 1965; 1970). In arable soil, significant
lessening of Collembola after application of Simazine was noted by
Prasse (1975).

The possibility of using herbicides for controlling collembolan
pests as well as weeds was attempted by Marshall and Ilnytzky (1976).
They applied herbicide mineral oil, Shell ANK, in Sitka spruce and

western hemlock nurseries, for controlling weeds and Bourletiella

 

hortensis (Fitch), a collembolan pest in some agricultural crops.
Their study found that this herbicide only temporarily suppressed the
pest and concluded that as a pest control, it is not effective.
Obviously, application of herbicides affects populations of
Collembola. Although other herbicides prolong the effect, this is
generally considered as temporary. Persistence of residues in soil is
the most important factor in lengthening influence on animals (Edwards,
1973). Organisms that live in soil with more organic matter will
suffer potentially drastic effects, because organic matter absorbs
much of the herbicides. In this case, residual effect will last for a
longer period (Bhattacharya & Joy, 1977). Discrepancies between
results of observers, therefore, is not surprising since soil type

and experimental conditions may vary.

Effect on Diversity

 

Observations on multiformity of Collembola showed that 2,4-0
and MCPA did not affect diversity (Baudissin, 1952; Rapoport &
Canglioli, 1963; Davis, 1965). Evidently, reduction of population

numbers does not always change diversity. Application of Paraquat

and Dalapon decreased numbers of Collembola; however, it did not affect
diversity (Curry, 1970).

Studies on Collembola in arable soil treated with 2.4-0 and
Simazine yield distinct results. Prasse (1975) found that after appli-
cation of 2,4-D, there was more positive shifting dominance than
negative shifting. This means that more species increased in number

than decreased. Among them were Tullbergia krausbaueri (Borner),

 

Folsomia sp,, and Isotoma notabilis Schaffer. After application of
Simazine. 0n the other hand, there was more negative shifting dominance.
Lessening of total Collembola was followed by emergence of some

species; they were Isotomodes productus Linnaniemi, Pseudonurophorus

 

 

isotoma Borner, Lepidocyrtus cyaneus Tullberg, and Onychiurus armatus

 

 

(Tullb.) Gisin. The most drastic reduction occurred with the species

Pseudosinella alba (Packard), while Folsomia s3. decreased in all

 

layers.

Atrazine may suppress hypogastrurid and symphypleonid Collem—
bola in corn field, although they are able to reestablish after several
months (Popovici et al., 1977). Apparently, the effect of herbicides
on diversity of Collembola is not obvious. Edwards (1968) recognized
that, in general, the more active species are more susceptible to

herbicides.

Some Aspects of Direct Effects

 

Field studies have dealt primarily with changes in animal
populations. Laboratory studies on species level are very scarce.
Those that have demonstrated various aspects of direct effects of

herbicides on Collembola.

By exposing Onychiurus quadriocellatus Gisin to 2,4,5-T,

 

Eijsackers (1975) found this herbicide had a lethal effect on Collem-
bola. The higher the dose, the higher the mortality rate. There are
two phases in the poisoning process. First is intensification of
general activity, and second is lack of activity. At low doses,
Collembola began to increase their movements. By increasing the dose,
they showed irregular movements of legs or tremor-like contractions.
At last, in the highest dose, they became completely immobile. Doses
tested were 1.25, 2.5, and 5 cc/mz.

Further experiments showed that Collembola were able to escape
from the contaminated location, but in high dose, most were paralyzed
and not able to escape. From these results, since field concentrations
are generally low, there is a possibility for Collembola to escape
from the contaminated areas. Further, when patchy distribution of
herbicide occurs, the mortality will tend to decrease (Eijasackers,

1975).

EXPERIMENT I

Materials and Methods

 

This study used commercial formulations of Atrazine and Para-
quat which are now being applied in till/no till corn fields on the
Soil Research Plots, Michigan State University. Two species of Collem—

bola, Folsomia candida (Willem) and Tullbergia granulata Mills, were

 

selected as tested animals. 5, candida are laboratory specimens which
are widely used in soil arthropods experiments, while I. granulata
were collected from an old sod field at the Soil Research Plots. Both
species are parthenogenetic and all are females. The soil of collection
area is classed as a Spink loamy sand (Whiteside, 1976) and a member
of the 4a soil management group (Robertson et al., 1976). The area
was sampled, and the samples were analyzed in the Soil Testing
Laboratory, Michigan State University. The test results are presented
in Appendix VII.

Laboratory rearing techniques basically followed methods
described by Snider et a1. (1969), utilizing small plastic jars of
27 mm high by 34 mm in diameter. A mixture of plaster-charcoal in an
8:5 ratio was prepared for the rearing substrate. Cultures were main-
tained at approximately 100 percent relative humidity by dropping
distilled water to the substrate daily or as required, with a pipette.
Cultures were kept in controlled temperature incubator at 60° F in

total darkness.

Eggs of both species were transferred into experimental jars
from stock cultures with a needle, one egg into each jar. 0n the day
of hatching, newly hatched Collembola were fed on yeast treated with
herbicide. Food was renewed every four days or as required with the
same treated yeast to prevent it from being attacked by fungal mycelium.

The treated yeast was prepared as follows: 350 grams powdered
brewer's yeast was suspended in 100 m1 of a desired concentration of
herbicide. The suspension was then filtered, and the filtered yeast
was dried at room temperature. Because solubility of Atrazine is
limited, there were crystals of Atrazine left on the filter when dried.
The crystals and the dried yeast were mixed up together as food
treated with Atrazine.

This experiment was run with three concentrations of each
herbicide (Paraquat or Atrazine). The concentrations were 600 ppm,
1000 ppm, and 5000 ppm, since the solution which is applied to field
for Paraquat is about 600 ppm and for Atrazine is approximately 5000
ppm. Values were calculated from field application suggested by the
Cooperative Extension Service, Michigan State University (Chase &
Meggit, 1976; Nelson et al., 1976).

A total of 140 individuals of f. candida and 140 individuals
of I. granulata were reared individually for this experiment. Obser—
vations were conducted daily and terminated after 22 weeks. Table 1

summarizes this experiment.

10

Table l.--Experiment I--Number of Treatment Replicates.

 

Paraquat Atrazine

 

 

Species C°"tr°‘ 600 1000 5000 600 1000 5000
ppm ppm ppm ppm ppm ppm

 

l'n

. candida 20 20 20 20 20 20 20

I. granulata 20 20 20 20 20 20 20

 

RESULTS

Mortality

During the 22 week period, one individual of the controls,

5, candida, died at the fifteenth week. Only 3 individuals of E.
candida fed on Paraquat died; 2 individuals at 1000 ppm and one indi-
vidual at 5000 ppm. The same mortalities occurred in feeding Atrazine
at 600 ppm and 1000 ppm. However, there were 6 individuals or 30
percent of F, candida fed on 5000 ppm of Atrazine that died during the
22 week period (Table 2).

Mortalities of I. granulata occurred only at 5000 ppm of
Paraquat and Atrazine. At 5000 ppm of Paraquat, one individual died,
as opposed to 11 individuals at 5000 ppm of Atrazine. As in f,
candida, the mortalities began in the early weeks, and those that did
not die, survived until the end of the experiment (22 weeks) (Table 3).
At 5000 ppm of Atrazine, both species were found to be repelled by
food and were rarely discovered feeding. This situation did not
happen at lower concentrations of Atrazine and at all concentrations

of Paraquat.

Instar Duration

 

Data presented by Snider (1971) suggests that instar duration
can be determined by the presence of exuvia. Exuvia were recorded and

then removed. Since 5, candida tend to eat their own exuvia (Snider,

11

12

Table 2.--Folsomia Candida, Cumulative Percent Mortality, at 60° F.

 

 

 

 

Paraquat Atrazine

”99k C°"tr°‘ 600 1000 5000 600 1000 5000
ppm ppm ppm ppm ppm ppm

6 o o o '0 o o 5
9 o o o o o 10 1o
11 o o o o o 10 10
12 o o o o' 0 1o 15
15 5 o o o o 10 30
18 5 o 5 o 0 1o 30
21 5 0 1o 5 5 10 30
22 5 0 1o 5 5 10 30

 

Table 3.--Tullbergia Granulata, Cumulative Percent Mortality, at 60° F.

 

 

 

 

 

Paraquat Atrazine

“99k C°"tr°‘ 600 1000 5000 600 1000 5000
ppm ppm ppm ppm ppm ppm

2 o o o o 0 0 15

3 o o 0 o o o 35

4 o o o o o o 50

5 o o o o o o 55

22 o o o 5 o o 55

 

13

1971), daily observations overcame difficulties in finding exuvial
remains. However, in f, candida fed on Atrazine, especially at 5000
ppm, the Collembola ate their own exuvia soon after ecdysis. This
resulted in only a very small part being left that could have been
overlooked. Through careful observations, the remaining part was
usually found. Although I. granulata does not eat its own exuvia,
identical observational difficulties arose in determining first moults
because these Collembola are very small.

Mean instar duration of E, candida and I. granulata are
illustrated in Figures 1 and 2. In general, beginning at the sixth
instar, the treated f, candida had a longer instar duration than con-
trols. Significant extension of duration was shown by species fed on
5000 ppm of Paraquat and at all concentrations of Atrazine (Appendix I).
As compared to lower concentrations, feeding on 5000 ppm of Atrazine
had a significantly longer instar duration.

In I, granulata, all treated animals exhibited a longer instar
duration than controls (Figure 2), and this was significant at every
stadia (AppendiprI). As in f, candida, instar duration of I} SEEQET
lata_fed on 5000 ppm of Atrazine was significantly longer when compared
to lower concentrations.

Besides high mortalities in both species treated at 5000 ppm
of Atrazine, not all of those surviving reached the same instar. Only
6 individuals of E. candida out of 14 individuals surviving for the
22 week period had reached the thirteenth instar. In I, granulata,
among 11 individuals surviving, only 10 had reached the seventh instar;
9 reached eighth; 4 reached ninth; and 3 reached tenth. Both species

at this treatment concentration were considerably smaller than controls

FOLSOM M
141

12‘

DURATION In DAYS

 

Figure 1. Folsomia Candida,

 

14

C ANDIDA

 

— 5000 ppm Atrazine

—.--— 1000 ppm Atrazine
- . -. 600 ppm Atrazine
_.-.- 5000 ppm Paraquat
........ mm Paraquat
_ -... 600 ppm Paraquat
— CONTROL

INSTAB

Instar Duration at 60° F.

l5

 

 

 

Figure 2.

W

a We.

16

at the same instar. Although there were no measurements taken during

the experiment, size differences were obvious.

Egg Production

 

.5. candida begin to lay eggs shortly after the fifth moult is
completed and then, more or less regularly, at every two instars
(Snider, 1971). In this experiment, almost all the controls laid eggs
regularly at every two instars beginning at the sixth instar. Only one
individual was found to lay eggs at the seventeenth, nineteethn, and
twenty-first, instead of at the sixteenth, eighteenth, and twentieth
instar. In treated_§. candida, irregularities in ovipositions were
commonly found, and not all of them began to lay eggs at the sixth
instar.

Data in Appendix III and Figure 3 show that the treated E,
candida significantly reduced their egg production at every oviposition
as compared to controls. Feeding on Atrazine also had significantly
lower egg production than feeding on Paraquat. Apparently, there was
no significant difference between feeding on 600 ppm and 1000 ppm of
either Paraquat or Atrazine. Feeding on 5000 ppm of Atrazine
inhibited their fecundity. Among them, only 4 individuals were found
to lay eggs with less than 4 eggs at every oviposition.

I, granulata begin to lay eggs at the fourth instar, and then
at every instar thereafter. As in E. candida, the oviposition occurs
not more than 24 hours after moulting. Eggs are very small, soft
and colorless. As they develop, the eggs increase in size and become
more opaque. In contrast to f, candida, this species lays eggs

singly, not in clumps or clusters. A cluster of 2-3 eggs might be

1601

FOLSOMIA CANDIDA

. 5000 ppm Atrazine

a 1000 ppm Atrazine
600 ppm Alrozino
. 5000 ppm Paraquat

E

140*

D 1000 ppm Paraquat

8

120‘

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Folsomia Candida, Egg Production at 60° F.

 

Figure 3.

18

found, but this was uncommon. Laying on wall of jar also happened
rarely. Because they are colorless, of small size, and widely dis-
persed over substrate, observations were made with difficulty.

I, granulata lay few number of eggs. They produce 1-4 eggs
at the first oviposition, and then increase numbers in following
ovipositions until reaching a peak of 4-11 eggs at eighth to tenth
instar. In the following instar, they decrease their egg production
(Figure 4 and Appendix IV).

All treated I. granulata significantly reduced their egg
production as compared to controls. However, there was no difference
between feeding on Paraquat and Atrazine. At 5000 ppm of both herbi-
cides, egg productions were significantly different from the lower
concentrations. No difference appeared between 600 ppm and 1000 ppm
(Appendix IV). Treated I, granulata also showed irregularities in
ovipositions, and some of them did not begin to lay eggs at the fourth

instar.

Egg Viability

 

For determination of egg viability, only eggs which were laid
on the substrate were counted. Eggs laid on wall of jar were counted
for egg production and then removed, because these eggs would soon dry.
Therefore, these eggs were not included in egg viability determination.
After chorionic rupturing, eggs were transferred into fresh jars and
were incubated until hatching. Eggs which were attacked by molds were
also excluded. Some treated f, candida were found to lay "incomplete"

eggs, analogous to a mass of jellylike substance, and these were also

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é ' l I N! I

v
8993 W USBWDN

12

'—
'-

O
,—

O)

(D

In

INSTAR

Figure 4.

20

excluded from egg production and egg viability determinations. Egg

viability was calculated as follows:

total hatching of eggs at same instar
X 100%

 

total number of eggs at same instar

Results indicate that,in general, there is no difference
between controls and treated Collembola. However, egg viability of
f, candida fed on 1000 ppm and especially 5000 ppm of Paraquat, by the
fifth oviposition, drastically decreased (Table 4). Feeding I,
granulata on 5000 ppm of Paraquat showed only slightly lower viability
as compared to controls (Table 5). There is an indication that high
concentrations of Paraquat may affect egg viability. Feeding on
Atrazine showed no effect on egg viability. Small egg production at
5000 ppm of Atrazine resulted in high percentage of egg viability
(Table 5).

Incubation Period

A number of eggs from every oviposition were taken randomly
and were transferred into other jars for recording the incubation
period. Days needed for hatching in relation to numbers of eggs in
percent are illustrated in Figures 5 and 6. Statistical analysis'
showed that eggs of treated Collembola had a longer incubation period
as compared to controls (Appendices V & VI). Time needed for incuba-
tion of f. candida at 60° F is 13-22 days, while I. granulata needs
25-31 days.

21

Table 4.--Folsomig_Candida, Egg Viability (in Percent) per Oviposition,

 

 

 

 

 

at 60° F.

. Paraquat Atrazine

211135 C°"tr°‘ 500 1000 5000 500 1000 5000

ppm ppm ppm ppm ppm ppm

1 95.3 95.4 95.3 93.9 100 93.0 --
2 98.3 97.7 97.0 95.0 98.2 98.8 100
3 95.5 95.3 95.1 92.1 99.0 88.7 100
4 89.7 95.1 75.4 90.9 99.0 89.9 100
5 95.8 95.2 73.5 81.3 98.0 78.3 100
5 95.1 95.2 72.5 50.7 95.5 91.7
7 89.1 89.0 85.9 54.5 89.4 90.0
8 90.3 89.2 89.0

 

Table 5.--Tullbergia Granulata, Egg Viability (in Percent) per Oviposi-
tion, at 60"F2

 

 

 

 

. Paraquat Atrazine

21113; C°"tr°‘ 500 1000 5000 500 1000 5000

ppm ppm ppm ppm ppm ppm

1 100 100 100 85.2 92.3 95.8 100
2 95.2 91.1 95.0 93.9 88.9 94.3 100
3 98.2 98.7 97.4 98.3 89.9 93.1 100
4 95.5 97.8 94.3 92.1 98.0 95.3 100
5 95.4 99.0 95.5 98.8 95.3 95.1 100
5 96.8 95.7 95.9 94.4 97.2 95.8 100
7 95.5 95.1 95.3 95.9 95.9 95.8 100
8 95.7 95.5 95.1 89.7 95.9 94.1
9 95.9

 

 

22

 

 

 

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.E 20.

hzmocma >02m30m¢u

(Relation

between number of eggs and time needed for hatching.)

Folsomia Candida, Incubation Period at 60° F.

 

Figure 5.

600 ppm Paraquat

1000 ppm Paraquat
5000 ppm Paraquat

—- CONTROL

O—o—o
1.1-...

o.--
I—..

23

TULLIERGIA GRANULATA

600 ppm Atrazine

1000 ppm Atrazine
"— 5000 ppm Atrazine

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35-
30-
10-

(Relation

DAYS
between number of eggs and time needed for hatching.)

TIME
Tullbergia Granulata, Incubation Period at 60° F.

 

 

Figure 6.

DISCUSSION

Observations concerning life history of E, candida as given
by Marshall and McE Kevan (1962) and Green (1964) are in some circum-
stances, different from the present study. Here, rearing methods and
food supplies are different from previous studies. However, in many
respects, these observations were conducted under conditions as those
of Snider (1971) and Snider and Butcher (1973). Therefore, results
are practically similar.

Up to the present time, the life history of I. granulata
has never been reported. Breeding biology and post-embryonic develop-

ment of Tullbergia krausbaueri (B6rner) were observed by Hale (1965a,b),

 

and later Petersen (1971) found parthenogenesis in the species. Hale's

records state that I, krausbaueri begin to lay eggs at the third

 

instar, although only a single specimen was found to do so from among
23 individuals observed. Maximum egg production is reached at the
seventh and eighth instars with averages of 7.6 and 7.25. The present
study shows that parthenogentic female I, granulata attains sexual
maturity at the fourth instar, and maximum egg production at the

eighth and tenth instar with means of 7.5, 7.95 and 7.2, respectively
(Appendix IV). These data indicate that there is no great variation
between the two species. Egg viability of I, granulata is considerably

high at every oviposition, while Petersen (1971) reported low

24

25

viability in parthenogenetic I. krausbaueri due to injuries during

 

transfer from one chamber to another.
Instar duration of I. granulata also has not been specially

observed. Observations on I. krausbaueri and other species show that

 

instar duration gradually increases with progressing age (Hale, 1965b).
Reports on f, candida state that the first instar to be of longer
duration than following immature instars (Green, 1964; Snider, 1971;
Snider & Butcher, 1973). The present study clearly shows that first
instar of I. granulata is longer than subsequent instars, at least up
to the eleventh. First instar is followed by the shortest duration

of second and third instar, and then duration increases gradually
(Figure 2).

High mortalities were observed in feeding on 5000 ppm of
Atrazine. This was not clear whether they were caused by treated
food or by starvation. Repellent effects had been observed by
Eijsackers (1975) when Onychiurus quadriocellatus Gisin were given a
choice between untreated and treated quadrants with 2,4,5-T, a toxic
herbicide to Collembola. Obviously, the Collembola were found to
choose untreated quadrants. If 5000 ppm of Atrazine has repellent
effects, starvation could be the cause of mortality. Therefore, food
treated at 5000 ppm of Atrazine may be less palatable to Collembola.

Influence of herbicides on fecundity as well as instar duration
has not been recorded. However, modification in fecundity, life span
and egg viability in several insects due to sublethal effects of
insecticides have been known for several years (Knutson, 1955; Edwards
8 Thompson, 1973). Although the mechanism is still not understood,

apparently reproductive system is affected (Moriarty, 1969). Another

26

explanation, given by Duncan (1963), states that less feeding could
cause reduction of fecundity since Aedes aegypti Linn. treated with
sublethal dose of dieldrin did not eat as much as the untreated.
Here, reduction in feeding was only observed at 5000 ppm of Atrazine,
even so it was not observed in other treatments. Besides lessening
in fecundity, eggs were also retarded in hatching. In addition, some
of the treated f, candida were found to lay "incomplete" eggs as well
as reduction of egg viability. Therefore, feeding on herbicides may
affect the reproductive system. Further investigations are needed in
this area.

Based on argument that herbicides are applied over plants,
not to the soil, previous investigators believed that herbicides have
no direct effect on soil animals (Fox, 1964; Curry, 1970; Edwards &
Thompson, 1973). Accordingly, elimination of weeds could reduce food
sources for some soil animals, and indirectly affect soil animal
populations. Only Triazine herbicides, including Atrazine, have a
direct effect (Edwards, 1970). In addition to the above, foliar-
applied herbicides can also penetrate soil by rainfall, although con-
centrations are considerably lower. Results of the present experiment
indicate that herbicides, at least Paraquat and Atrazine, can exert
direct effects on Collembola.

Results reported here explain previous studies. Curry (1970)
found reduction of collembolan populations in grassland after being
sprayed with Paraquat, while Fox (1964) and more recently Popovici
et a1. (1977) reported the same results after application of Atrazine.
Fecundity, instar duration, hatching time, and mortality are among the

most important factors in population dynamics. Any distraction to

27

those factors will consequently affect populations. However, in the
field, we do not know if the animals actually eat the contaminated food

or are able to avoid it.

EXPERIMENT II

Materials and Methods

This experiment was set up to study the possibility of lethal
effects of herbicides to Collembola in soil sprayed at applied concen-
tration. Utilizing the same size of jars as in Experiment I, a soil
substrate was provided for rearing. The soil was obtained from the Ap
horizon of an old sod at the Soil Research Plots, and was dried in the
sun for weeks in order to eliminate any animals living in the soil.
The soil was described in Experiment 1. Before being used as sub-
strate, it was broken up and spread out, then was moistened with
distilled water. The moistened soil was then placed into experimental
jars until about 1 cm deep. Finally, substrates were sprayed with
herbicide at applied concentration, 600 ppm for Paraquat and 5000 ppm
for Atrazine (Chase & Meggit, 1976; Nelson et al., 1976). Spray tech-
nique utilized a compressed air sprayer with a distance of 50 cm from
the surface of substrates.

Fifteen eggs of f, candida and I, granulata were transferred
into each experimental jar, and cultures were kept in a controlled
temperature incubator at 60° F in total darkness. When eggs were
transferred, those of f, candida were one week after being laid,
while those of I. granulata were two weeks. This experiment had a

total of 45 jars for each species and consisted of three experiments.

28

29

The number of living animals were counted at three time inter-
vals, 5 jars at each count. First count was conducted in the one week
after hatching, and thereafter every week. Counting was made by a
simple floatation method, using a saturated solution of sodium
chloride. Soil substrate was poured into the saturated solution and
stirred. Animals would float and were counted under a binocular

microscope. The experimental method is summarized in Table 6.

Table 6.--Experiment II--Number of Treatment Replicates.

 

 

Species Counting Control Paraquat Atrazine
.F. candida I 5 5 5
II 5 5 5
III 5 5 5
.1. granulata I 5 5 5
II 5 5 5

III 5 5 5

 

RESULTS AND DISCUSSION

Percent survival of the Collembola observed weekly are pre-
sented in Tables 7 and 8. From those tables, it is apparent that
Collembola reared on soil sprayed with 5000 ppm of Atrazine suffered
lethal effects. There was no significant difference between Collembola
reared on soil sprayed with 600 ppm of Paraquat and the unsprayed.
Lethal effects of Atrazine significantly began at the second week, and
it continued into the following week.

Eijsackers (1975) reported lethal effects on Collembola by
exposing Q, quadriocellatus to 2,4,5-T. In accordance with Edwards
(1970), this study shows that Triazine herbicides have direct effect
on soil animals. Since 5000 ppm of Atrazine is the concentration of
the solution which is applied over the field, most hazardous effect is
on soil animals living in uppermost layers. Those animals have the
greatest chance of being in contact with the herbicide. But Eijsackers
(1975) suggested that repellent action as previously mentioned permits
Collembola to escape from the contaminated area. Ability of animals
to escape will tend to decrease mortality under field conditioned.
While direct application of Atrazine kills animals, obviously Paraquat

exhibits no direct lethal effect.

30

31

Table 7.--Folsomia Candida, Percent Survival Reared on Soil, at 60° F.

 

 

~ Sprayed Sprayed
Week Control Paraquat Atrazine F
600 ppm 5000 ppm
1 94.7 92.0 97.3 p' 3.05
:_5.57 :- 2.95 i, 3.67 NS
2 97.3 88.0 14.7 256.7**
1 3.67 :_ 5.56 1_ 8.68 P': .001
3 90.7 88.0 10.7 100.7**
:_l3.8 : 11.95 :_23.8 P.: .001
F 1.10 .59 54.62**
NS NS P's .001

:.= standard deviation.

32

 

Table B.--Tullbergia Granulata, Percent Survival Reared on Soil, at

 

 

60' F
Sprayed Sprayed
Week Control Paraquat Atrazine F
600 ppm 5000 ppm
1 82.7 77.3 58.7 4.40
i, 3.67 :_12.1 1 19.64 NS
2 86.7 80.0 49.3 9.06*
: 10.55 :_15.65 g: 17.38 P.: .05
3 82.7 80.0 21.34 13.19**
1, 9.91 :_16.33 g: 14.64 P :_.01
F .81 .02 6.33*
NS NS P < .05

 

:,= standard deviation

SUMMARY

1. Fecundity and instar duration of I, granulata are described
based on a 22 week period of laboratory observations at 60° F. The
species attains sexual maturity at the fourth instar, and maximum egg
production at the eighth to tenth, with means of 7.5, 7.95 and 7.2.
Duration of the first instar is the longest, followed by the shortest,
second and third instars, and then increases gradually thereafter.

2. f, candida and I. granulata were fed herbicides Paraquat
and Atrazine at 600 ppm, 1000 ppm, and 5000 ppm. Feeding on herbi-
cides resulted in reduction of fecundity, extension of instar duration
and time needed for hatching of their eggs. These results suggest
that herbicides affect reproductive system.

3. There was no lethal effect on Collembola reared on soil
sprayed with 600 ppm of Paraquat. However, effect was pronounced on
Collembola reared on soil sprayed with 5000 ppm of Atrazine.

4. Results of these experiments suggest that Atrazine has

the most severe effects on Collembola as opposed to Paraquat.

33

LITERATURE CITED

Baudissin, F. G. von. 1952. Die Wierkung von Pflanzenschutzmitteln
auf Collembolen und Milben in verschiedenen. Boden. Zool.
Jahrb. (Syst.) 81:47-90.

Bhattacharya, T. & V. C. Joy. 1977. Effect of Banvel-D, a herbicide,
on the microarthropods population of soil. Indian Biologist 9:
47-51.

Chase, R. W. & M. F. Meggit. 1976. Weed Control. No till corn: 4.
Extension Bulletin E-907. Cooperative Extension Service,
MSU, January 1976.

Curry, J. P. 1970. The effects of different methods of sward estab-
lishment and the effects of herbicide paraquat and dalapon on
the soil fauna. Pedobiologia 10:329-61.

Davis, B.N.K. 1965. The immediate and long-term effects of herbicide
MCPA on soil arthropods. Bull. Ent. Res. 56:357-66.

Duncan, J. 1963. Post-treatment effects of sublethal doses of
dieldrin on the mosquito Aedes aegypti L. Ann. Appl. Biol.
52:1-6.

Edwards, C. A. & M. K. Arnold. 1964. The side effects of toxic
chemicals in the soil on arthropods and earthworms. Rothamsted
Exp. Stat. Report for 1963, p. 148.

Edwards, C. A., Arnold, M. K. & Fryer. 1965. Effects of insecticides
in the soil. Rothamsted Exp. Stat. Report for 1964, p. 185.

Edwards, C. A. 1969. Soil pollutants and soil animals. Sci. Amer.
220:88-99.

. 1970. Effects of herbicides on the soil fauna. Proc. 10th
Brit. Weed Control Conf., pp. 1052-62.

Edwards, C. A., J. K. Lofty & C. J. Stafford. 1971. Pesticides and
soil fauna. Rothamsted Exp. Stat. Report for 1970, p. 194.

Edwards, C. A., J. K. Lofty & A. E. Whitting. 1972. Paraquat and

slit-seeding. Rothamsted Exp. Stat. Report for 1971,
pp. 212-13.

34

35

Edwards, C. A. & A. R. Thompson. 1973. Pesticides and the soil fauna.
Residue Review 45:1-79.

Edwards, C. A. a C. J. Stafford. 1974. Pesticides and the arthropod
soil fauna. Rothamsted Exp. Stat. Report for 1973, pp. 205-6.

Edwards, C. A., J. K. Lofty & C. J. Stafford. 1975. Pesticides and
the soil fauna. Rothamsted Exp. Stat. Report for 1974, p. 113.

Edwards, C. A. & C. J. Stafford. 1976. Effect of a herbicide on the
soil fauna. Rothamsted Exp. Stat. Report for 1975, p. 129.

Eijsackers, H. 1975. Effects of the herbicide 2,4,5-T on Onychiurus
quadriocellatus Gisin (Coll.). In: Progress in Soil Zoology.
Jan Vanek (Ed.). Academia: Publishing House of the Czecho-
slovak Acad. of Sci. Prague, pp. 481-88.

 

Fox, W. B. 1948. 2,4,0 as a factor in increasing wireworm damage of
wheat. Sci. Agr. 28:423-24.

Fox, C. J. S. 1964. The effects of five herbicides on the numbers of
certain invertebrate animals in grassland soil. Can. J. Plant
Sci. 44:405-9.

Green, C. D. 1964. The life history and fecundity of Folsomia candida
(Willem) var. distincta (Bagnall) (Collembola: Isotomidaé).
Proc. R. Entomol. Soc. London (A) 39:125-28.

Hale, W. G. 1965a. Observation on the breeding biology of Collembola
(II). Pedobiologia 5:161-77.

. 1965b. Post-embryonic development in some species of
Collembola. Pedobiologia 5:228-43.

Johnson, C. G., R. M. Dobson, T. R. E. Southwood, J. W. Stephenson &
L. R. Taylor. 1955. Preliminary observation on the effect
of weedkiller on insect populations. Rothamsted Exp. Stat.
Report for 1954, pp. 129-30.

Knutson, H. 1955. Modifications in fecundity and life span of
Drosophila melanogaster Meigen following sublethal exposure
to an insecticide. Ann. Entomol. Soc. Amer. 48:35-39.

 

Marshall, V. G. & D. K. McE Kevan. 1962. Preliminary observation on
the biology of Folsomia candida Willem, 1902 (Collembola:
Isotomidae). The Can. Entomol. 94:575-86.

Marshall, V. G. & S. Ilnytzky. 1976. Evaluation of chemically con-
trolling the collembolan Bourletiella hortensis on germinating
Sitka spruce and western hemlock in the nursery. Can. J. For.
Res. 6:467-76.

36

Moriarty, F. 1969. The sublethal effects of synthetic insecticides on
insects. Biol. Rev. 44:321-57.

Nelson, L. V., L. S. Robertson, M. H. Erdman, R. G. White & D. Quisen-
berry. 1976. Guide Lines. No till corn: 1. Extension
Bulletin E-904. Cooperative Extension Service, MSU, March
1976.

Petersen, H. 1971. Parthenogenesis in two common species of Collem-
bola: Tu11bergia krausbaueri (Barner) and Isotoma notabilis
Schaffer. Rev. EcOTT'Biol. Sol 8:133-38.

Popovici, I., G. Stan, V. Stefan, R. Tomescu, A. Dumen, A. Tarta &
F. Dan. 1977. The influence of Atrazine on soil fauna.
Pedobiologia 17:209-15.

Prasse, J. 1975. The effect of herbicides 2,4-D and Simazine on the
coenosis of Collembola and Acari in arable soil. In: Progress
in Soil Zoology. Jan Vanek (Ed.). Academia: Publishing House
of the Czechoslovak Acad. of Sci. Prague, pp. 469-80.

Rapoport, E. H. & G. Canglioli. 1963. Herbicides and the soil fauna.
Pedobiologia 2:235-38.

Robertson, L. S., D. L. Mokma, D. L. Quisenberry, W. F. Meggit & C. M.
Hansen. 1976. Soils. No till corn: 3. Extension Bulletin
E-906. Cooperative Extension Service, MSU, January 1976.

Schaller, F. 1968. Soil Animals. The University of Michigan Press,
Ann Arbor, 144 pp.

Snider, R. J., J. Shaddy & J. W. Butcher. 1969. Some laboratory
techniques for rearing soil arthropods. Mich. Ent. 1:357-62.

Snider, R. 1971. Laboratory observations on the biology of Folsomia
candida (Willem) (Collembola: Isotomidae). Rev. Ecol. Biol.
Sol. 10:103-24.

Snider, R. M. & J. W. Butcher. 1973. The life history of Folsomia
candida (Willem) (Collembola: Isotomidae) relative to
temperature. The Great Lake Ent. 6:97-106.

Whiteside, E. P. 1976. Private communication.

Williams, J. H. 1970. Herbicides--their fate and persistence in
soils. NAAS Quart. Rev. 87:119-31.

APPENDICES

 

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mm. mm. pp. op. mF. NP. om. .n
aamm.o~ F.o mm.m mn.m p.m mu.m F.m .m a
mpum PP-¢ o1¢ cue m-e one mle .u
mm. we. mp. up. pm. up. mm. .3
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mm. pm. mm. em. mm. up. um. .n
emp.m am.“ m.o m~.o mm.m mm.m m~.o .m _
sag ooom Egg coop Egg coo Egg ooom Egg coop Egg coo
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H x~ozmmm<

37

38

 

 

 

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39

 

 

 

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«apm.~P «mm.m «wo.w «mm.~ m.m P.“ m.m .m _F
Eng ooom Egg coop sag com sag ooom sag coop Egg coo
Focucoo cmumcH
m=w~mga< panacea;

 

.oaacmpcou--.H a_amh

40

 

 

 

 

Np-o m_-m m-e .8
am. mm. mm. .3
,ko.m m.~ mm.“ .m om
m_-m F_-N m-m .u
we. _m. NN. .n
,Ne.m o.~ mm.o .m m_
pp-o m_-m op-m o,-m __-~ m-m .8
mm. «4. mm. mm. on. my. .3
*_~.m .F.m ,~.o_ mm.“ m~.~ op.~ .m mp
o.-m mp-m up-“ op-m NP-“ m-m .0
ea. mm. om. am. we. om. .n
«om.m P.m ,o.oP F_.m _.m mm.“ .m up
op-m N_-~ o_-~ .F-o op-“ m-o .8
mm. mm. mm. om. em. «N. .n
vem.m a..m amm.m m~.m _P.~ cu.“ .m 0_
Eng ooom Eng ooo_ sag coo sag ooom sag coop sag coo
Pogucou gmumcH
memngp< paaamcma

 

.uascwpcou--.n m_nmc

41

.mo..W a sum: .mcowumgpcmucou Lmzop ms“ mm Frwz mm Fogpcou ms“ EOL$ acmLm$$mU xpucmuw$wcmwm
.8. .v. a 5.; .338... 9: so: 2885 228:2?
mucus

Logcm unoccmpm

:me

.H m—nmh com

030*

mmuocpoou

42

 

 

 

ep-~p mP-F_ op-~P up-op c_-o_ wF-P_ m_-oF .8
mm. mm. mm. mm. oe. mm. Pm. .n
aapm.¢P a~.m_ o.m_ amm.mp B.NF m.m_ m.~F .m m
w~-- mp-o_ o_-~_ m_-m m_-m op-F_ mp-oP .u
m~.P _e. mm. mm. mm. mm. mp. .n
««~m.m_ amo.mp ao.m~ o.m_ mm.PF «m~.mp mN.P_ .a a
mmupp mplm mpum mpum mmlm oplo epuw .u
um. oe. mm. mm. «s. cm. em. .2
«am.¢_ w.~_ m.mp w.~— «m.m_ m.FF w.op .o m
mplm mpnm mpuop umum opuop mpuw mpum .u
em. mm. mm. mm. ow. ea. Pm. .n
«emm.¢P ~.~F m.PF «B.NF mm._F m~.NP o._~ .m N
m~-m_ m~-mp e~-m_ m~-mF om-m_ mm-~. e~-mp .8
mm. mm. mm. mm. Fo.P eF.P me. .n
m.- e.o~ m¢.mp o._~ me.o~ m.m_ N.- .a _
sag ooom sag ooo_ sag coo sag Doom sag ooo_ sag com
Pogucoo gmumcm
mewngp< umaomcma

 

 

.m coo pm .mxmo cw co_pmczo cmumcH .mumFacmcc mwmcwnppah--.mfi mpamh

HH x~ozmma<

43

 

 

 

mplmp mpump “Flop --op mP-FF o~1NP mplup .u
.1 mm. mm. Fm. _e. me. cm. .3
@m.n~ emn.ep mN.eF «pp.mp «mw.¢p a~.mp N.mp .o op
“pump mpump m—ump omump mpumF mpupp m~-~_ .u
.1 Pa. mm. we. mm. we. cm. .a
em.mp «mn.¢_ mm.mp «mm.mp «mm.ep ~.¢P m.~P .w m
mpuep “Pip" m~-op mmnmp mplpp mpump mF1n .u
1. mm. Pm. om. oe. oe. mm. .n
emm.op «o.ep m.mp «emm.mp «o.¢~ *~.¢P m~.- .m m
“_ump mpump N~-~— m_-pp mpuop “pup? N_-m .u
.1 mm. om. mm. mm. mm. mm. .3
em.¢~ «~.e_ amm.¢_ «o.¢p amn.m_ amm.mp ¢.~p .m m
mmuup mpum oF1PF mulo— o~-_F mFupp Npuop .0
mm. _e. em. on. me. me. m_. .n
aaoo.m_ «m.mp «mm.m~ «F.¢F «m.mp «mo.ep m.pp .m o
sag ooom Egg coop sag coo sag Doom sag coo, sag coo
_Pocucou cmpmcm
wcPNQLp< pmacmcmm

 

.uaacwucou--.HH b.5mc

44

mo..w a cum; .mcowpmcucmucou gmzop mg» mm ppm: mm Fogucou mcu socm pcmgmmmwc apwcmuwmpcm_m u ea

mo..w a cpwz .Pocpcoo mg“ sage pcmcmmwpu xppcmuwmwcmwm u

mcowum>cmmno mo Lassa: sop xgm> mo mmamuma mwmapmcm Fouwumpumum soc; covapuxm u
moans u

coccm ugmucmpm u

cums n

(0509'!

 

mpupp .0
mm. .3
F.mp .m pp

 

Ema ooom

Eng ooo_ Eng coo Ema ooom Eng coop Eng ooo

 

 

Pocucou cmumcH
mchmca< pmzomcma

 

.umzcwpcou--.cfi a_nac

45

 

 

 

mno oquo Fouo mppumm m~_lmm mN—uok muplmo .0
cm. m.~ mm.m om.m em.m ep.e om.m .n
««p~.F «up.mm «mo.o¢ «o.mm am~.mm «mn.mm m.mm~ .m mP-NP
muo mmlm Pm-o mo_-mm omF1~¢ mN_-mo pru—u .u
mm. mm.P ¢~.~ m.m om.v m.m w.e .a
««¢F.~ ym.¢F «F.Nm comm.mm «m.m~ am.mm o.nop .m _p-o~
~-o mmlm mule meum .n-~m o~-om mmlmm .0
mm. mm.p em.P ~¢.~ em.m no.~ m~.m .n
«em.p «mm.¢_ e~.mp «emm.om «mo.om «mm.om m.mm .m m-w
o~-_ npuo mpum ¢~-o mmspp mN-N_ .u
we. m~.F No. sm.p om. om.P .n
o «m¢.m «mn.m «amm.o_ _.mp mo.¢~ me._m .m “lo
0 o o o o o o m-P
Eng ooom Egg coop Egg com Egg ooom sag coop sag com
Pocpcou cmumcH
mcw~mcu< unacmcoa

 

 

.a com “a .cmumcH can cowuuauoaa mam .mu_u=mo eweompoa--.HHH a_aah

HHH x~ozmaa<

.mo..w a sup: .mcowumgucmucou cmzop on» ma —sz mm Pocucou ms» soc; ucmcmmmwu apucmuwwwcmwm u ea
mo..w a saw: .Focucou as» scat acacmtccu apocau_cv=mcm

meowum>cmmno mo Lassa: zap asm> mo mmsmumn mwmxpmcm Pmuwumwumpm soc; uwuapuxm u a

maze; u u “cogcm ucmucmum u a “cums u a

II
-R

46

 

 

 

 

Neumm ompuem maples ampumm oomuo .u
F~.~ mm.P_ Fm.PN m¢.m_ mm._P .n
am~.~m am~.~m «mo.mpp a~o.oo~ mp.omp .m quom
mule Nuuo mmpuo mmp-o mmpno eomlem .u
em.m mm.m -.m cm.“ m~.op mn.~ .n ,
«m~.¢m am~.mm «mw.mm new.Fm *Nm.wm PN.F¢_ .m mp-mp
“muo mm1o empno meF-mm oepno pr-ou .o
mm.m -.m oo.m mo.m em.m ou.m .n
«mm.~e «am.me amu.Pm amm.FoF «_.~o_ Ne.~mp .o m_-m_
m-o smuo mmuo om_-~P ~e_-mm mmpnmm mF~1mm .u
.. ep.m m~.m mm.o mN.m ww.m _o.o .n
9pm. am~.m¢ «mm.om «m.mm «m.¢o_ «mm.mop m.emp .m mF1¢P
Egg ooom sag coop sag coo Egg ooom sag coop Egg com
pocucou cmumcm
mewngp< pmaamgma

 

.umaccpcou--.HHH mFDMF

 

47

 

 

muo “so Fpuo ~10 muo m-o mum .u

No. Ne. mm. mm. mm. om. me. .n
aamm.~ «m~.m o.¢ «emo.m m~.m mm.e mm.m .m N

mlo o-o m-o e-o mlo 01o mum .u

me. me. mm. om. me. mm. mm. .3
rtom.F im.~ ¥m¢.m «cw.~ «mw.m «m.m mo.m .m m

«.0 muo m-o m-o m-o m-o onm .0

mm. em. um. um. ow. mm. mm. .5
aamo.~ amm.~ mp.m eemo._ o.m mm.m N.e .m m

Nuo m-o m-o m-o m-o m-o cup .8

mm. mm. mm. om. mm. Fm. mm. .n
seem. «~.. mm._ «mm.p m._ mm.p m.~ .m a
o o o o o o o m-p

Ea 88 58 82 ea 08 can 88 ea 82 8a 08
_ocucoo cmpmca
mamecu< panacea;

 

 

.u com um .cmpmcH Lma cowuuauocm mum .mwmpzcmcu armcmappzhuu.>H upon»

>H x~ozmaa<

48

 

 

 

 

mum .0
Fe. .5
mo.m .m N_
mno Bio muo muo muo opne .0
pm. mm. mm. mm. om. me. .n
#m.~ *mo.¢ *«wm.p uN.¢ «mm.m mm.o .m pp
#10 «no mno mic mic opno P—ne .u
I: Pm. om. Fm. we. we. Fe. .a
eo.m km.m «mm.¢ «um¢.~ «o.¢ km.m N.m .w op
#10 mno mic muo muo mno Ppno .u
nu ¢m. on. cm. «0. mm. mm. .2
®¢.~ «m.m «¢.m ««N.N «m.¢ *m.¢ mm.m .w m
euo muo muo muo mno opuo Ppnm .o
1: cm. Nm. om. No. mm. mm. .a
©0.m «mp.e m¢.m «ap.m «m.¢ *o.m m.m .m m
Eng Doom Ema coop Ema ooo Eng ooom San coop Sun 000
Focpcou cmumcH
mcw~mgu< panamcma

 

.uazcvpcoo--.>H «_nap

49

.mo..w a :pwz .mcowpucpcmocou gmzop ms» mm _sz mm pocucou mg» seem pcmcmmmwn xpucmowmwcmwm u
.mo..w a cup; .Pocucou mg» Eocw pcmcmmmwu apucmumwwcmwm u

.mcomum>cmmno we Lungs: zap xcm> mo mmsmuwn mwmxpmcm _muwumwumum soc; cmuapuxm u

mmcmg u

Loccm ugmczmum u

some n

.>H «Pack now

IUDUQJ'R

mmuocpoom

cmmzpwn mucmgmmmwu unmovmwcmwm oz

..o.o w.a gov: ”Focucou as» soc» «cacaccwo »_»=auwcwemwm

.muwuwacm; sumo cwzuwz mcowumcucmucou

i

 

 

 

 

.m:o_um>cmmno mo Lanes: sop acm> mo mmamumn mwmxpmc< qu_umwumum soc» umuapuxm u e
_N-mp F~-¢_ o~-ep NN-4P oN-¢F P~-¢_ NN-m_ amcam
Noom._ mmpm.p mmum.. mmm,._ mace._ mmom._ .>ao .upm
Amy ANPPV Aokv Amopv Ao~_v A__FV A.~PV “.mno .ozv
em~.mp 440.5, a¢_.~P «mm.~. amm.o_ aem.~_ N_.GF cam:
sag ooom sag coop gag com sag ooom sag ooo_ sag com
Focpcoo
ocwumcu< panacea;

 

.m com um when cw mcwguum: so» umc_:cmm msvh

> x~azwaa<

 

.mumucau a_sompoa--.> apnah

50

:mmzumn mucmgmmmwo

acmupmwcmwm oz

.1 .mupuwncog comm cwnuwz mcopumcucoucou
.mo.o v a saw: «Focucou on» scum acmsmmmwu xpucmummwcmwm u «

 

 

 

 

_m-- mm10~ mm-m~ mm-- mm1NN mm1- ~m1mm magma
mono._ mom¢.F amm_._ NMNF._ mpm_._ omoo.F mooo._ .>aa .ecm
Aemv Ao~_v Ammpv Ao~_v Ammpv AN¢_V Aaepv A.mno .ozv
,mm.m~ «Fe.m~ 14m.m~ o~.a~ 1m¢.m~ aem.mm mm.m~ cam:

sag Doom sag coop sag coo sag ooom sag ooo_ sag com
Fogucou
mcw~mcu< aazcmcma

 

 

.m com um mama cw acmzuum: com vmcwacmm mswh .mum—scmcw mwmcmnppz 11.~> «page

H> x~ozwaa<

51

APPENDIX VII

RESULTS OF SOIL TEST*

Soil pH : 5.6

Lime index : 6.4

Lbs. per acre of:

Phosphorus : 79
Potassium : 96
Calcium : 720
Magnesium : 177

Percent of total exchangeable bases:

Potassium : 4.6
Calcium : 67.7
Magnesium : 27.7

Soil texture : loamy sand

Manure : no

Plowing depth (in.) ' : 6

 

*Recommended Chemical Soil Test Procedure for the North
Central Region, North Dakota Agric. Exp. St., North Dakota St. Univ.,
North Central Pub. No. 221, Bull. No. 499, February 1975.

52

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