SOME MORPHOLOGICAL Anni . *
PHYSIOLOGICAL STUDIES ON THE
EFFECTS‘OF CARBARYL. ON THE

EARTHWORM’, LUMBRlCUS TERRESTRES.

Thesis for the Degree of Ph. D,
MCI-“GM STATE UNIVERSITY
BENNY (WHEY.

1973

 

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ABSTRACT

SOME MORPHOLOGICAL AND PHYSIOLOGICAL STUDIES
ON THE EFFECTS OF.CARBARYL ON THE
EARTHWORM, LUMBRICUS TERRESTRIS

 

BY

Benny Cathey

The earthworm, Lumbricus terrestris was treated

 

topically with various concentrations of carbaryl in
distilled water suspension. At 21 days an LD50 of approx—
imately 20 ugs of carbaryl per gm of wet buss-bedding was
established for the earthworm under controlled laboratory
conditions.

Many non-lethal effects were seen at concentrations
of 0.3 to 6.0 ugs of carbaryl per gm of wet bedding. Some
of the behavioral responses to the treatment were: (1)
reactions to light and chemical stimuli, resulting in a
"withdrawal response"; (2) extensive coiling and constrict-
ing of the body; (3) swellings and redness in the anterior
region of the body of the worm; and (4) formation of
blisters.

Oxygen consumption in worms treated with non-
lethal concentrations of carbaryl ranged from 115 to 155 ul

02 per gm per hour and the rates in the controls ranged

Benny Cathey

from 82-101 pl 0 per gm per hour. Increases in oxygen

2
consumption were seen in treated animals even though there
were no external effects observed.

Histological studies have shown even with non-
lethal concentrations of carbaryl that the body wall is
damaged. There is extensive proliferation of epidermal
cells, and the circular muscle layer shows degenerative

effects with epidermal cells replacing the tissues in this

area.

SOME MORPHOLOGICAL AND PHYSIOLOGICAL STUDIES

ON THE EFFECTS OF CARBARYL ON THE

EARTHWORM, LUMBRICUS TERRESTRIS

 

BY

Benny Cathey

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Zoology

1973

ACKNOWLEDGMENTS

I wish to express my gratitude and appreciation to
Dr. Ralph A. Pax for his guidance and valuable criticism
throughout the course of this project.

I also wish to thank Drs. S. G. Wellso, M. J.
Zabik, and J. R. Shaver for their advice and critical
reading of this manuscript.

I wish to give special thanks to my colleagues,
Fred Divers and Charles Drewes, for their assistance in
the laboratory and Joseph Cameron for his advice and
friendship.

The writer wishes to thank Lonnie Eiland for his
advice on histological techniques.

Finally, I wish to thank my parents, Mr. and Mrs.
Cecil Cathey, for their moral support over the past four

years.

ii

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . . . . . . . . . V
LIST OF FIGURES O O O O O C O O O O O O O O O O O O 0 Vi
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . l

LDSO: Field and Laboratory Studies of Insecticides,

other than Carbaryl on Earthworms . . . . . . . . 1

Effects of DDT on Earthworms . . . . . . . . . . . 1
Effects of Insecticides, Other than

DDT on Earthworms . . . . . . . . . . . . . . . 4
Effects of Carbaryl on Earthworms . . . . . . . . . 6
OBJECTIVES OF THE STUDY . . . . . . . . . . . . . . . 8

MATERIALS AND METHODS . . . . . . . . . . . . . . . . 10

Source and Maintenance of Earthworms . . . . . . . . lO
Buss-Bedding Preparation . . . . . . . . . . . . . . 10
Pharmacological Procedures . . . . . . . . . . . . . 11
Source and Preparation of Carbaryl . . . . . . . . . 12
Injection of Carbaryl . . . . . . . . . . . . . . . 12
Topical Application of Carbaryl . . . . . . . . . . l4
Respiration Apparatus and Procedures . . . . . . . . 15
Histological Techniques and Light Microscopy . . . . 15

RESULTS . . . . . . . . . . . . . . . . . . . . . . . l7
LD50 Studies . . . . . . . . . . . . . . . . . . . . 17

Injection of Carbaryl: Preliminary Findings . . . l7
Topical Application of Carbaryl: LD Results . . 27

50
Morphological and Physiological Studies . . . . . . 29
External Symptoms After Treatment . . . . . . . . 29

Respiration Studies: Respiration Rates
Before Treatment . . . . . . . . . . . . .'. . . 34

iii

Respiration Studies: Respiration Rates
After Treatment . . . . . . . . . . .

Histological Studies . . . . . . . . . .
Data and Observations . . . . . . . . .
DISCUSSION . . . . . . . . . . . . . . . .
Toxicity of Carbaryl for Earthworms . . .
Comparison of Carbaryl Effects on Other
Animals 0 O O O O O O O C O O O O O O 0
Mode and Site of Ac ion of Carbaryl . . .

S UMMARY . C O O O O O O O O O O O I

LIST OF REFERENCES 0 O O I O O I O O O O .

iv

Page

36
4O
40
55
55

58
59

61

62

LIST OF TABLES

Table Page

I Mortality in earthworms in the 21 days
following injections with 10 ul of
corn oil-carbaryl mixture . . . . . . . . . . 18

II Mortality in earthworms in the 6 days
following injections with 10 ul of
acetone-carbaryl mixture . . . . . . . . . . 20

III Mortality in earthworms in the 6 days
following injection with 10 ul of
carbaryl-acetone mixture . . . . . . . . . . 22

IV Mortality in earthworms in the 5 days
following injection with 10 ul
carbaryl-distilled water suspension . . . . . 23

V Mortality in earthworms in the 30 days
following injections with 5 pl
carbaryl-distilled water suspension . . . . . 25

VI Mortality in earthworms in the 25 days
following injections with 5 ul
carbaryl-distilled water suspension . . . . . 26
VII Mortality in earthworm following topical
application of carbaryl: 81
earthworms/concentration . . . . . . . . . . 28

VIII Oxygen consumption for untreated animals:
(ul Oz/gm/hr) . . . . . . . . . . . . . . . . 37

Ix Oxygen consumption of earthworms . . . . . . . 39

X Data analysis of oxygen consumption . . . . . . 41

LIST OF F IGURES

Figure Page
1. LDSO: Carbaryl on earthworms . . . . . . . . . 3l
2. Oxygen consumption curve:

(p102 per gm per hour) . . . . . . . . . . . 43
3-A. Photomicrograph of normal earthworm

body wall X40 . . . . . . . . . . . . . . . . 46

3-8. Photomicrograph of earthworm body wall,
after treatment with 3pgs carbaryl/gm
of bedding X40 . . . . . . . . . . . . . . . 46

4-A. Photomicrograph of earthworm body wall,
after treatment with 6 pgs carbaryl
per gm bedding X40 . . . . . . . . . . . . . 49

4-B. Photomicrograph of earthworm body wall,
after treatment with 15 pg carbaryl
per gm bedding X40 . . . . . . . . . . . . . 49

5-A. Photomicrograph of normal (control)
earthworm body wall X430 . . . . . . . . . . 52

5-B. Photomicrograph of earthworm body wall,
after treatment with 3 pgs carbaryl
per gm of bedding X430 . . . . . . . . . . . 52

6-A. Photomicrograph of earthworm body wall,
after treatment with 6 pgs carbaryl
per gm of bedding X430 . . . . . . . . . . . 54

6-B. Photomicrograph of earthworm body wall,

after treatment with 15 pgs carbaryl
per gm of bedding X430 . . . . . . . . . . . 54

vi

INTRODUCTION

In recent years economic entomologists have become
increasingly concerned as to what effect various agricultural
chemicals such as insecticides may have on earthworms.

Most of the investigations demonstrating the effects
of insecticides on soil invertebrates have been done from a
comparative viewpoint: i.e. (1) studies of various inver-
tebrates, involving a specific insecticide, such as carbaryl
and (2) studies of various insecticides involving a specific
invertebrate, such as the earthworm.

Earthworms occupy a central position in the economy
of the soil. Edwards (1968) has estimated that up to 60%
of the initial breakdown of plant material is brought about
by the earthworm. He has reported many large reductions
in populations of various soil invertebrates using various

chemicals, including a reduction in populations of earthworms.

LDSO: Field and Laboratory Studies

 

of Insecticides, other than
Carbaryl on Earthworms

 

 

Effects of DDT on Earthworms

 

There have been many investigations made showing

various effects of the insecticide DDT on invertebrates.

However, most of these investigations have been limited to
field observations.

Baker (1946) conducted a field study in which he
sprayed trees in several plots with DDT. One month later
after the foliage had been consumed by earthworms, he
observed a sharp decrease in the earthworm population in
the sprayed areas, compared to little or no change in the
number of earthworms in unsprayed areas.

Fleming and Hadley (1945) applied 50 lb of DDT
per acre. Observations made after 26 days showed that
partially grown worms were all killed. After repeating
this test using full grown worms, no lethal effects were
observed, even after more than two months. It was evident
that the full grown worms were very resistant to the DDT.
In another of their experiments 100 lbs of DDT per acre
was used on full grown worms for the same period of time,
however, the results were essentially the same.

Hoy (1955) reported a similar study using 40 lbs
of DDT per acre and observed no lethal effects. However,
Doane (1962) reported a two-year field study in which he
showed that earthworms were mildly affected by the applica-
tion of DDT in concentrations of 25 lb per acre. He also
reported that there are some changes that occur in the
normal activity and numbers of earthworms following applica-
tion of the insecticide. The reason for these changes has

not been thoroughly examined, especially over large areas.

He suggested that a problem involved in studying the lethal
effects of DDT on earthworms is.that the earthworms are.able
to accumulate large amounts of DDT without being killed.

Thompson (1971) found that when DDT concentrations
ranging from 1-5 lb. were applied per 10' x 6' plots, that
some effects on earthworms were seen, even 5 years after
treatment.

Hopkins (1957) reported a field study on English
redworms (species undefined) treated with 10% DDT at 15 lb
per acre. He found that with this treatment mortality of
only 7% was observed after 2 months. He concluded from
these data that it would require many years of consistent
application of this chlorinated hydrocarbon to endanger
the English redworm population.

Martin and Wiggins (1959) determined the tolerance
of Hybrid Red Wigglers (species undefined) to various insec-
ticides, including DDT in laboratory studies. They used
insecticide concentrations of 0.1, 1.0, 10, 100 and 1,000
ppm (parts per million). The worms were immersed in each
concentration for a period of 2 hours. After 24 hours the
percent survival was determined. They found that DDT, at
concentrations of 0.1, 1.0 and 10 ppm caused no mortality
in the worms; 100 ppm caused a 20% mortality and 1,000 ppm

caused a 60% mortality.

There are a number of generalizations which may be
made from field and laboratory studies on the effects of
DDT on earthworms.

DDT seems to have little or no effect on earthworms
probably because of their ability to accumulate large
quantities of the DDT without any damaging effects. A
second generalization is that laboratory studies of related
species of worms tend to show no damaging effects of DDT
treatment, also suggesting high tolerance levels for the
DDT and a third generalization is that length of time of
treatment is not critical to the worms, because some exper-
iments were conducted over a period of several years.

Effects of Insecticides Other
than DDT on Earthworms

Hoy (1955) reported in addition to the effects of
DDT on earthworms, the effects caused by a variety of other
insecticides. He found that the chlorinated hydrocarbons,
gamma BHC and crude BHC at 2—10 lb active isomer per acre
caused significant mortalities of earthworms (species
undefined). However, in soil with low organic matter con-
tent, crude BHC killed all the earthworms after only 8 weeks
of exposure.

Doane (1962) found that when he used chlordane and
dieldrin at 10 lb per acre, he had eliminated most of the

earthworms, Lumbricus terrestris from a field plot one year

 

after treatment.

Edwards (1965) made a field study of various insec-
ticides on various soil invertebrates. In this study he
used aldrin, dieldrin, telodrin, BHC, diazinon, and menazon
in average dosages of l—3 lb. per acre and for periods of
several weeks to several years. He observed no effects;
however, when heptachlor, chlordane, parathion, DNOC and
simazine were applied at the same dosages, moderate to
large reductions in the numbers of earthworms were noted.

In a more recent field study, Thompson (1971)
reported the effects of several insecticides on the numbers

of earthworms (Lumbricus sp.). The insecticides used were,

 

dursban, BAYER-37289, endrin, BUX, dasanit, carbofuran and
Stauffer N-2596 in various dosages of 1-5 lb. per 10' x 6'
plots. The effects of the eight insecticides on the numbers
of earthworms were observed after 3 weeks. Dursban and
BAYER-37289 had very little effect, but dasanit, carbofuran
and Stauffer N—2596 drastically lessened numbers of earth-
worms. BUX and endrin did not kill more than 60%.

Ruppel gt_al. (1973) in a study yet unpublished,
have investigated some effects on earthworms caused by
prophos, aldricarb, Tirpate and carbofuran. They used
dosages ranging from several ounces to several pounds.

Their results showed that those pesticides proved to be
toxic in the laboratory; however, other pesticides such as
dyfonate and phyrate caused more than 50% mortality only at
the highest dosage, while the other pesticides caused little

mortality.

After making these observations, it may be concluded
that while DDT seemingly caused little or no damage to the
earthworm, other types of pesticides even in small doses
may cause more damaging or even lethal effects to the earth-

worm.

Effects of Carbaryl on Earthworms

 

Edwards (1965) reported some effects of various
chemical residues, including carbaryl on the earthworm.

His results showed large reductions in the numbers of
earthworms, after 6 months in the soil at average doses of
1.5-2.0 1b of carbaryl per acre.

Thompson (1971) treated earthworms with carbaryl in
doses ranging from 1-5 lb per acre for 3 weeks. He noted
that the numbers of earthworms were drastically reduced in
the treated plots.

An Der Lan and Aspock (1962 and 1963) applied 0.3%
carbaryl suspensions to the epidermis of the earthworm and
noted a number of histopathological effects. They found
that there were growths of epithelial cells into other
regions of the body wall. Further investigations of
histOpathological effects of carbaryl showed that there was
a breakdown of the cell membrane and the formation of a
"syncytium". These earthworms showed the same or similar
changes as those treated with carcinogenic hydrocarbons as

shown by Gersch et a1. (1951 and 1957). An Der Lan and

Aspock (1962) concluded that the histopathological effects
caused by DDT, carbaryl, chlorinated hydrocarbons or
carcinogenic hydrocarbons generally show the same or a
similar effect on the epidermis of the oligochaetes.

These data show that carbaryl seems to be a toxic
compound for the earthworm and tends to cause various
changes in the epidermis of the earthworm, especially when

applied directly to the earthworm in low concentrations.

OBJECTIVES OF THE STUDY

There have been a considerable number of field
studies studying the effects of various insecticides on
various soil invertebrates. Only a few investigations
showing the toxic action of carbaryl on earthworms have
been reported. Of the investigations which have been con-
ducted, none involves the use of carbaryl in behavioral and
metabolic studies and little data have been reported showing
histological effects on earthworms.

There are several reasons for using the earthworm,
Lumbricus terrestris and carbaryl in this research study:
(1) carbaryl is a chemical which is widely used as an agri-
cultural insecticide; (2) according to An Der Lan (1962),
carbaryl effects earthworms at very low concentrations;

(3) earthworms are easy to maintain; (4) large samples of
worms can be studied in a short period of time; (5) earth-
worms possess a relatively simple organ-system and are
therefore easier to study than many other higher inverte-
brates, from both a gross morphological and histological
standpoint.

With these considerations in mind, the major objec-
tives of this investigation were to determine: (1) under

controlled condition, what the LD (lethal dose at which

50

50% of the animals die) is for the earthworm, treated with
various concentrations of carbaryl; (2) whether the normal
behavioral patterns (such as reactions to external mechani-
cal stimuli, coiling ability, locomotion and responses to
light) are altered significantly, as a result of exposure
of the worms to non-lethal doses of carbaryl; (3) what
histological changes occur as a result of exposure of worms
to lethal and non-lethal dosages of carbaryl over a speci-
fied period of time; (4) what metabolic changes, such as
changes in respiratory rates, if any, may be observed in
the treated animals as a result of exposure of the worms

to various sub-lethal concentrations of carbaryl over a

period of time.

MATERIALS AND METHODS

Source and Maintenance of Earthworms

 

The earthworm, Lumbricus terrestris was used in all

 

experiments. The worms were purchased from the Wholesale
Bait Company, Hamilton, Ohio. The animals weighed between
2.0-5.0 gms and measured 15-20 cm in length. They were
maintained at a constant temperature of 15°C. in buss—
bedding in wooden boxes measuring 90 x 45 x 60 cm. Approx-
imately 250 worms were kept in each box.

The maturity of the earthworms was established by
the presence of a clitellum and only mature worms were
used. Animals were always used within 3 weeks of their

purchase.

Buss-Bedding Preparation

 

Buss-bedding is an artificial soil, prepared by
Don Buss Manufacturing Company; Lanark, Illinois. It is a
fully nutrient food for the earthworms and only water is
needed in the preparation of the bedding for the earthworms.
The buss-bedding mixture was prepared in a ratio of
432 gm dry bedding per liter of tap water. This bedding

was thoroughly mixed by hand until evenly moistened.

10

ll

Pharmacological Procedures

One drawback in the use of carbaryl is the lack of
a commercially acceptable emusifiable concentrate (Reeves,
1962). Louloudes gt_al. (1962) reported the use of a
hydroxy-lecithin compound as a solvent for carbaryl and
others have suggested acetone or alcohol as solvents for
carbaryl (Metcalf, 1961; Dorough and Casida, 1964).

In preliminary tests, acetone and corn oil were
tested as solvents for carbaryl, but the results (explained
more fully in the "results" section) obtained showed neither
of these solvents to be acceptable. There are two reasons
why these solvents could not be used: (1) with acetone as
the solvent many of the controls died, suggesting that ace-
tone in itself is toxic to the worms; (2) corn oil proved
to be a difficult solvent to use in these experiments. It
was necessary to use a microliter syringe to make injections
and the viscosity of corn oil is such that it cannot be
easily forced through the needle of the syringe.

As a result of these preliminary tests the proce-
dures used in the following studies were based primarily
on two methods: (1) injections of a carbaryl-distilled
water suspension into animals and (2) exposure of worms to
buss-bedding which had been wetted with the carbaryl-

distilled water suspension.

12

Source and Preparation of Carbaryl

 

Carbaryl (l-naphthyl-N-Methylcarbamate) has the
trade name Sevin. This synthetic insecticide is a product
made by Union Carbide Corporation; Salinas, California and
the samples used in these studies were 99% pure and were
donated by Dr. Norman Leeling, Pesticide Research Center,
Michigan State University, East Lansing, Michigan.

The carbamate, carbaryl is a synthetic derivative
of the alkaloid physostigmine or eserine found in the

Calabar bean, Physostigma venonsum, of French West Africa.

 

Carbaryl is a white crystalline compound, melting point
142°C, relatively insoluble in water (about 0.1%) and some—
what soluble in organic solvents (Metcalf, 1961). However,
the 99% pure carbaryl was added to distilled water in various
concentrations and suspensions were formed by vigorously
agitating the mixture with a magnetic stirrer. This sus-
pension was then added to the prepared buss-bedding mixture

or used for injection of animals.

Injection of Carbaryl

 

The carbaryl for the experiments was prepared using
three different solvents: (l) carbaryl dissolved in corn
oil; (2) carbaryl dissolved in acetone and (3) carbaryl

suspended in distilled water.

13

The first group of experiments which was performed
involved the injection of known amounts of various concen-
trations of carbaryl into the worms.

There were several experiments conducted using a
corn oil-carbaryl mixture. A total of 20 worms per concen-
tration were injected. The total amount injected was:

1 x 10's, 5 x 10‘s, 1 x 10'4, 5 x 10'4, 1 x 10’3, 5 x 10‘

3
and 1 x 10-2 pgs of carbaryl per worm.

There were several experiments conducted using an
acetone-carbaryl mixture. A total of 50 worms per concen-
tration were injected. The total amount injected ranged

2 to 1.6 x 10_1 pgs of carbaryl per worm.

from 1 x 10‘
Prior to injections the animals were narcotized
for a few minutes in a 0.1% solution of chloretone. A
microliter syringe was used for these injections. The
controls were injected with 10p1 of the solvent. The next
group of treated worms were injected with 10p1 of the
carbaryl-distilled water suspension, in dosages of 0.10,
0.12, 0.14 and 0.16 pgs of carbaryl per worm. The injec-
tions were made under the dorsal surface of the animal,
just posterior to the clitellum. Careful observations
were made for 1-2 minutes to see that the carbaryl was not
immediately eliminated. After the injections, the worms
were placed in individual milk cartons (9 x 19 x 9 cm.) at

10 worms per box. These milk cartons had been prepared by

cutting one side open and sealing the other openings with

l4

freezer tape. After the worms and bedding were placed in
the boxes, the boxes were covered with porous, clear plastic
wrap. During the next 3 weeks, daily observations were made

of the treated and control animals.

Topical Application of Carbaryl

 

To study the effects of topical application of
carbaryl to earthworms, the worms were exposed to buss-
bedding to which various concentrations of carbaryl had
been added.

In these experiments milk cartons prepared as
described previously were used. Each box contained 50 gm
of buss-bedding. To each of the boxes of buss-bedding
100 m1 of tap water was added and was thoroughly mixed
with the bedding.

Using a torsion balance 1.5 mgs of the 99% pure
carbaryl were weighed and mixed with 10 ml of distilled
water. This mixture was added to 150 gm of wet buss-
bedding. After this stock of carbaryl and buss-bedding
had been thoroughly mixed, several concentrations were
obtained from the stock mixture. The concentrations which
were obtained from the stock mixture were: 15, 30, 60, 150,
300, 600 and 1,500 pgs of carbaryl per gm wet bedding.
Twenty worms were added to each box which contained the
various concentrations of bedding mixture. The boxes were

then covered with clear, porous plastic wrap, so that:

l5

(1) moisture would be maintained in the bedding and (2) so
worms could not escape from the boxes. During the next
three weeks, daily observations were made on the number of

dead worms, or any obvious symptoms the worms displayed.

Respiration Apparatus and Procedures

The respiration rates of earthworms were determined
by using a Scholander Microvolumetric Respirometer, Model
VR—300 (The Mark Company, Randolph, Mass.).

Some of the preliminary procedures used for deter-
mination of the oxygen consumption are noted in the results.
The worms were weighed and one worm was placed in each vial
with 5 gm of bedding. After 15 minutes equilibration time,
the vials were closed and the data recorded every 10 minutes
for one hour. A vial containing 5 gm of bedding was used

as a control.

Histological Techniques and Light Microscopy

 

After the three week period of observation of the
control and topically treated worms, the worms were removed
from the bedding and placed in boxes containing shredded
paper for two days so that the worms would clear their
digestive tract of materials.

The intact worms were placed in Bouin's fixative
for 1-2 days. The worms were then removed and 1-1/2 cm
cross sections were cut from each worm at the area just

posterior to the clitellum.

16

The following procedures were used for preparing
the tissues for light microscopy.

1. Washing: the tissues were washed for several
hours in tap water to remove the excess Bouin's fixative.

2. Dehydration: the tissues were carried through
a dehydration series (using various concentrations of
alcohol) which involved the removal of water from the
tissues. The following series of ethyl alcohol were used
in the dehydration: 30, 50, 70, 80, 90, and 100%.

3. Clearing: the tissues were removed from the
alcohol and placed in xylene for 30 minutes.

4. Infiltration: the tissues were infiltrated
with paraffin for 24 hours at a temperature of 50-52°C.

5. Embedding and Sectioning: the tissues were
embedded with paraffin and 12p thick sections were made.

6. Staining: the tissues were stained with
Harris' hematoxylin and double stained with eosin.

7. Mounting: the tissues were mounted using a
permanent mounting medium.

The procedures for clearing, infiltrating, section-
ing, staining and mounting are modified procedures taken
from Humanson's (1972) "Histological Techniques of Animal

Tissues".

RESULTS

LD Studies

50

 

Few studies have been conducted showing the effects
of carbaryl on earthworms and most of the studies which
have been conducted are those which show only lethal dosages
of insecticides on earthworms. The only controlled labora-
tory study was that of An Der Lan and Aspock (1962 and 1963)
in which a 0.3% suspension of carbaryl in water was applied
to the cuticle. No experiments in which carbaryl was in-
jected directly into worms have been reported.

Injection of Carbaryl:
Preliminary Findings

 

 

The injected animals were observed daily for a
period of 21 days. (See Table I). As can be seen from
this table there occurred a comparatively low mortality in
the worms at all concentrations. Most deaths occurred
within the first two weeks of treatment, and there was no
significant increase in the numbers of mortalities from
days 13 to 21. These data suggest the following: (1) that
the concentrations were too low to cause any large percent-
age of deaths in 21 days; (2) that since a very small

percentage of deaths occurred in the worms after about two

17

18

Table I Mortality in earthworms in the 21 days following
injections with 10 p1 of corn oil—carbaryl mixture.

 

 

 

Concentration (pg/Worm) No. Worms % Mortality
Controls 20 20%
1 x 10'5 20 40%
5 x 10'5 20 20%
1 x 10’4 20 40%
5 x 10'4 20 30%
1 x 10"3 20 30%
5 x 10'3 20 30%
1 x 10'2 20 50%

 

19

weeks, the worms were going to survive the effects of the
carbaryl and (3) that since 20% of the controls died and
50% is the largest number of mortalities to occur in the
treated animals, the solvent used was insufficient for
future studies.

As a result of these initial experiments higher
concentrations of carbaryl were used in the subsequent
experiments. A shorter period of time for treatment and
a different solvent were also used.

Several experiments were conducted which involved
the injection of a carbaryl-acetone mixture into the
earthworms. A group of 20 worms was injected with 10 p1
of carbaryl per worm per concentration. The total amount
injected at each concentration was 0.01, 0.02, 0.03, 0.04,
0.05 and 0.10 pgs of carbaryl per worm.

The earthworms were observed for a period of 6
days and their mortality rates were recorded. Table II
shows that 40% of the controls died within 6 days and that
a 100% mortality was achieved at concentrations of 0.04
and 0.10 pgs of carbaryl per worm, after only 6 days of
treatment.

To support the previous findings of the carbaryl-
acetone experiments, a second group of experiments
involving the use of the carbaryl-acetone mixture was
conducted with a total of 30 worms per concentration and

injected with 10 p1 per concentration per worm. In this

20

Table II Mortality in earthworms in the 6 days following
injections with 10 p1 of acetone-carbaryl mixture.

 

 

 

Concentration (pg/Worm) No. Worms % Mortality
Controls 20 . 40%
0.01 20 40%
0.02 20 60%
0.03 20 80%
0.04 20 100%
0.05 20 40%

0.10 20 100%

 

21

group of experiments, however, the concentrations were
increased to 0.10, 0.12, 0.14, 0.16 pgs of carbaryl per
worm. After observations for 6 days, the mortality rates
were recorded.

As can be seen from Table III, a 30% mortality
occurred in the controls, after only 6 days. This table
also shows that 0.12 and 0.16 pgs of carbaryl per worm gives
a 100% mortality.

There was a significant percentage of deaths in
the control worms in this experiment as well as the previous
one. This suggests that the acetone may be toxic to the
worms, thus it appears that acetone is an unsuitable sol-
vent for these experiments.

In a further attempt to find a more suitable sol-
vent, several experiments were conducted which involved the
use of a carbaryl-distilled water suspension. A total of
30 worms per concentration were used. The concentrations
of carbaryl used in these tests were 0.10, 0.12, 0.14, and
0.16 pgs per worm, the same as those used in the previous
experiments. Observations were made for a period of 5 days.

As can be seen from Table IV no mortality occurred
in the controls over a period of 5 days, while in all the
carbaryl treated groups there was significant mortality.
This suggested that distilled water was a suitable solvent

to use in the application of carbaryl to earthworms.

22

Table III Mortality in earthworms in the 6 days following
injections with 10 pl of carbaryl-acetone

 

 

 

mixture.

Concentration No. % No. Days No. Days
(pg/Worm) Worms Mortality 50% Killed 100% Killed
Controls 30 30% -- --

0.10 30 67% 3 --
0.12 30 100% 3 4
0.14 30 67% 4 --

0.16 30 100% 4 5

 

23

Table IV Mortality in earthworms in the 5 days following
injections with 10 p1 of carbaryl-distilled water
suspension.

 

 

Concentration No. % No. Days No. Days!
(pg/Worm) Worms Mortality 50% Killed 100% Killed

 

Controls 30 0% -- -—
0.10 30 67% 4 --
0.12 30 100% 3 5
0.14 30 67% 3 --

0.16 30 83% 3 --

 

24

In a second group of experiments, using the same
solvent (distilled water), a total of 6 worms per concen-
tration were used and the concentrations used previously
were the same, except that the amount of carbaryl-distilled
water injected was decreased from 10pl per worm to 5p1 per
worm and the worms observed for a period of 30 days. The
results of these experiments are recorded in Table V. This
table shows that a mortality of 83% was achieved in all
concentrations between 8 and 12 days after treatment. No
mortalities occurred from day 12 to day 30, suggesting that
the animals which were living after day 12, would survive '
the treatment.

In a third group of experiments, the concentrations,
amounts injected and the number of animals used remained
the same as in experiment two; however, a test period of
25 days was used. The results are summarized in Table VI.
As can be seen from this table, thirteen days were necessary
to achieve a 100% mortality in most concentrations.

From these injection experiments several conclusions
seem warranted: (1) the type of solvent which seems to be
most applicable for the carbaryl is distilled water. No
mortalities were seen in the controls and there are few
inconsistent patterns in mortality rates at the various
concentrations: distilled water was the best solvent to

use in these tests; (2) using the carbaryl—distilled water

25

Table V Mortality in earthworms in the 30.days following
injections with 5pl of carbaryl-distilled water

 

 

 

suspension.

Concentration No. % No. Days No. Days
(pg/Worm) WOrms Mortality 50% Killed 100% Killed
Controls 6 0% -- --

0.05 6 83% 10 --
0.06 6 83% 12 --
0.07 6 83% 9 --

0.08 6 83% 8 --

 

26

Table VI Mortality in earthworms in the 25 days following
injections wity Spl of carbaryl-distilled water

 

 

 

suspension.

Concentration No. % No. Days No. Days
(pg/Worm) Worms Mortality 50% Killed 100% Killed
Controls 20 0% -- --

0.05 20 100% 6 13
0.06 20 90% 4 ~-
0.07 20 100% 4 13

0.08 20 100% 4 8

 

27

mixture, in the various concentrations allowed for longer
test periods to be used, i.e. longer time to observe the
development of symptoms in earthworms.

All of the remaining experiments in this study are
based on these preliminary findings.

Topical Application of Carbaryl:
LD50 Results

 

In four separate experiments, seven concentrations
of carbaryl were used to test the effects of topical appli—
cation of carbaryl to the earthworm. The worms used in
these tests were exposed to the following dosage of
carbaryl: 1.5, 3.0, 6.0, 15, 30, 60 and 150 pgs of carbaryl
per gm of wet buss-bedding. A total of 81 worms were used
for each dosage.

The treated worms were placed in a constant temper-
ature room at 15.5°C, and were observed for symptoms for
3 weeks. The mortality rates were recorded for each group
and for each concentration from the first day through the
twenty-first day after treatment.

The results of these experiments are presented in
Table VII. No mortalities were noted in the controls nor
in concentrations of 1.5, 3.0, and 6.0 pgs of carbaryl per
gm of wet bedding, therefore, these concentrations were not

included in Table VII.

28

Table VII Mortality of earthworms following topical appli-

 

 

 

cation of carbaryl: 81 earthworms/concentration.

Days No. Worms Dead/Concentration (pg/gm)

15pg 30pg 60pg 150pg
1-5 0 0 0 0
6 0 2 3 5
7 1 3 4 ll
8 2 3 4 12
9 3 4 ll 17
10 5 6 15 19
11 5 8 21 26
12 8 11 25 36
13 9 12 32 46
14 10 15 37 51
15 10 18 40 55
16 10 20 44 58
17 10 26 46 62
18 12 31 49 66
19 12 36 54 70
20 14 41 59 73

21 15 46 60 74

 

29

Figure I gives a probit plot of the data in Table
VII. From this figure the LD50 for the carbaryl on earth-
worms at 3 weeks is approximately 20 pgs of carbaryl per
gm of wet bedding. As can be seen in this table, there is
a direct correlation of the lethal effects on the worms to
increases in concentration of the carbaryl. The first line
at the bottom of the graph represents responses in the
worms at 7 days of treatment and each successive line,
represents odd days of treatment (9, ll, 13, 15, 17, 19
and 21). This graph also shows that at the dosages tested
that more than 3 weeks are necessary to show complete
mortality. Since the highest number of days of treatment
(21 days) were not sufficient to cause complete mortality,

longer time periods are required using these concentrations.
Morphological and Physiological Studies

External Symptoms After Treatment

The symptoms discussed here were noted during
the previous LD50 studies. Some symptoms were seen in the
animals at carbaryl concentrations of 15, 30, 60 and 150
pgs per gm wet bedding after only 1-2 days of treatment.
These primary symptoms were: (1) constriction of the body,
especially in the area of the clitellum and swellings at the
anterior region of the worm; (2) a slight redness throughout

the worm's body with a greater intensity in the region of

Figure l

30

Figure 1

Percent mortality for earthworms.treated.with
15, 30, 60 and 150 pgs of carbaryl per gm of
buss-bedding. These.probit plots.show the.re—
sponses to concentrations beginning on the 7th
day (lowest line) and going through odd days

to the let day of treatment (top line).

 

////// W M g

.////. /./ .m

 

 

 

>._._._<._.~_O<< «a

32

the clitellum; (3) coiling throughout the entire.length of
the animal; (4) decreased locomotive ability and (5) a
decreased "withdrawal ability", i.e. responses to external
mechanical stimulation were much less than in the controls.
After 10 days of observation, these same symptoms were seen
in the animals at the lower concentrations of 1.5, 3.0 and
6.0 pgs carbaryl per gm of wet bedding.

After 2 weeks many of the animals at the higher
concentrations had begun to die. Before death many secon-
dary changes occurred: (1) the worms lost their ability to
constrict and coil, and appeared in a totally relaxed state;
(2) there was a definite swelling in the area anterior to
the clitellum; (3) the discoloration of the body became
localized and only the anterior region of the body had a
reddish color; the color in the posterior end of the body
ranged from yellow to white or colorless; (4) the posterior
end of the body became hard and shrunken; (5) almost no
movement was seen in the animals, especially in the pos-
terior region of the body; (6) there were little or no
responses to external mechanical stimulation; (7) blisters
had formed on the surface of the worm and the epidermis
appeared to have become very thin and swollen and sometimes
these areas of the skin has burst open, resulting in the
expulsion of coelomic fluids and waste materials of the gut.
When this occurred, there was extensive bleeding in the

areas of the ruptures. Most of these lesions occurred in

33

the middle and anterior regions of the animal and (8) large
amounts of mucus were secreted from the surface of the body.

During the second and third weeks after beginning
of the treatment, the animals treated with the lower con-
centrations of 1.5, 3.0 and 6.0 pgs carbaryl per gm of
bedding, were carefully observed and it was found that most
of the primary symptoms, such as coiling, twisting, con—
stricting of the body, especially in the region of the
clitellum and redness, had seemingly disappeared and these
animals now seemed to have recovered, at least to the extent
that it was not possible on external observations to see
any physical damage or even distinguish between the treated
animals and the control animals.

Some of the other types of behavioral responses of
the worms treated with carbaryl were: (1) the treated
animals (in all concentrations) after two days, responded
to various chemicals, such as 10% KOH, 10% HCl, 1% chloretone
and 10% NaCl solutions, which were applied to the epidermis
of the worms. In each case, these animals gave a "with-
drawal response". When these same chemicals were applied
to the treated animals on the tenth day, only the animals
in the lower concentrations (of 1.5, 3.0 and 6.0 pgs per
gm wet bedding) responded by withdrawing; (2) all treated
animals (at all concentrations) on the second day reacted
to intense light by showing withdrawal responses. When

these animals were stimulated with the light after the

34

tenth day, only the animals at the two highest.concentra-
tions of 60 and 150 pgs did not respond to the light;
however, on the twenty-first day, only animals at the lowest
concentration (1.5pgs) responded to the light.

Based on the data collected here, full evidence of
poisoning was not immediately obvious. Deaths continue to
occur for a period of time in excess of 3 weeks. The symp—
toms these treated animals showed seem to follow a definite
pattern; first there was an initial stage of degeneration
which I have called primary symptoms from which the animals
seem to have the ability to recover; secondly there were a
set of secondary symptoms from which the animals apparently
could not recover.

Respiration Studies: Respiration
Rates Before Treatment

In the previous set of experiments the LD was

50
established for the earthworms in a given population for
a period of 3 weeks. However, it was noted that at the
lower concentrations of 1.5, 3.0 and 6.0 pgs carbaryl per
gm wet bedding, worms though showing some symptoms of
poisoning were not killed.

Thus, mortality at these lower concentrations cannot
be used as an index of the toxicity of carbaryl for the

earthworms. Descriptions of external symptoms are not

easily quantifiable, and it seems likely that at

35

concentrations even lower than those used in the previous
experiments, deleterious effects may occur with no external
manifestations of symptoms.

In an effort to obtain a reliable measure of sub-
lethal effects of carbaryl treatments on earthworms, a
series of experiments were undertaken in which oxygen
consumption in carbaryl treated worms was measured.

As far as could be determined from the review of the
literature, no respiration studies had been conducted
showing respiration rates of earthworms after treatment
with an insecticide. Johnson g£_al. (1942) have shown the
respiration rates of untreated earthworms to be in the
range of 40-50 mm3 02/gm/hr under various laboratory
conditions for animals weighing from 2-5 gms and at a
temperature of 10°C.

In the initial set of experiments, an attempt was
made to establish the pattern of respiration rates in
untreated animals was established. In this group of exper—
iments, a total of 32 worms were used. The animals were
removed from the bedding and placed into the volumetric
respirometer which had a constant temperature of 16.5°C,
and the data from the experiments were recorded every ten
minutes for one hour.

The following results were obtained. Of the 32
worms used in these experiments, the average oxygen con—

sumption for the worms whose size ranged from 2-5 gm

36

(and whose temperature was 16.5°C) was approximately 93

pl 02/gm/hr, with the range being from 82-101 p1 02/gm/hr.
These data were collected from four different experiments,
using eight animals per experiment. See Table VIII. The
rates are higher than those reported by Johnson EE_El°
(1942). These experiments I have conducted are at a tempera-
ture nearer the Optimum for earthworms.

Respiration Studies: Respiration
Rates After Treatment

 

 

Respiration experiments were conducted in which

treated animals were used from prev1ous LD50 experiments.

Animals of these lower concentrations showed no lethal
effects, but did show external symptoms. Non-lethal con-
centrations of 1.5, 3.0 and 6.0 pgs of carbaryl per gm wet
bedding were used. A total of 26 worms per concentration
were used for each of the three dosages and 26 untreated
control worms were evaluated for each group of treated
worms (See Table IX). All animals were tested on the
twenty-first day after treatment. The respirometer temper-
ature for all of the experiments was always maintained at
16.5°C. These data were usually collected in the after—
noon and early evening. Most of these experiments were
conducted in the late spring and summer seasons and only

mature worms were used in these tests.

37

Table VIII Oxygen consumption for untreated animals,
pl OZ/gm/hr.

 

 

 

Animal No. Experiment No. l 2 3 4

l 86 101 96 101
2 87 87 83 91
3 96 92 84 96
4 101 99 90 99
5 99 98 98 89
6 89 98 98 100
7 94 88 96 83
8 _9_6 .17. -512 22

Average: 93 95 92 94

(mean)

 

38

 

 

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mo msoum mso nuw3 mEuo3nuHmm mo mmumu coaumfismcoo cmmwxo mo
mmsoum w m30£m magma mflne .maouusoo paw pmummuu “mauoznuumm

Ham MOM muflHHQMHHm> Hmaflsm mo mammamcm cm m30£m xH magma

mEHo3£uHmm mo coaumESmsoo cmmhxo xH magma

39

 

 

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0.00 0.00H 0.00 0.00H 0.00 0.0NH 0.00 0.00H 0.00 0.0MH 0.00 0.0mH
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0.00 0.00H 0.00 0.va 0.00 0.NOH 0.00 0.NOH 0.00 0.NmH 0.00 0.NMH
0.00 0.0HN 0.00 0.NOH 0.00 0.NOH 0.00 0.00H 0.00 0.0vH 0.00 0.0VH
0.00 0.0HN 0.v0 0.NMH 0.00 0.0MH 0.00 0.NMH 0.00 0.0NH 0.00 0.0VH
v.00 0.00a 0.00 N.v0 N.00H 0.HHH v.00 N.0HH 0.H0 0.00 0.00 0.00
N.0mH 0.MMH 0.00 N.05 0.00 N.v0 0.00 v.00 0.00 0.NOH 0.H0 0.05
v.00 0.H¢H ¢.N0 0.HHH 0.v0 0.50 0.vm 0.0Ha 0.50 0.00M 0.05 N.m0
0.5HH V.MVH 0.05 v.HMH 0.00 0.00H w.H¢ v.HOH v.00 0.00H 0.00H 0.00
0.v0 0.HHH 0.00 0.00 0.00 0.NMH 0.N0 N.00H 0.00 N.H0 N.0HH 0.50
0.0NH N.0HH 0.05 N.v0 0.00H v.00 N.Nm 0.00H 0.00 N.00H 0.05 0.00
0.00M 0.NMH 0.00 0.00 N.v0 0.NHH 0.00 v.00 0.00 0.05 0.00 v.00
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0.v0 N.00H 0.00 0.00 0.00 v.HOH v.5H v.00 v.50H v.m5H 0.00H v.0va
v.HOH 0.NVH N.Nm 0.NHH 0.00 N.0HH 0.5m 0.00 0.0MH 0.0VH N.05 N.05
«.55 v.50H 0.05 0.00H 0.00 v.HMH 0.50H N.0HH 0.05 0.55 N.H0 0.00H
0.00 0.00 N.N0 0.NHH N.v0 0.50 v.0HH 0.0MH 0.50 0.00H 0.0MH 0.va
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v.00 0.00H 0.00 V.MHH 0.H0 N.HNH n.00 w.vma 0.05 0.0MH 0.50 0.00H
0.0 0.0 m.H N.H 0.0 0.0
.usou .osou .usou .osoo .usoo .ocoo .usoo .osou .usoo .ocoo .ucoo .osoo

 

 

Aun\sm\~o any suflHHQMflum> Hmsaca mo mHmAHmca

msuo3nuumo mo sowugasmcoo smmaxo xH magma

mvm05000ammvm0
HHHHHHHNNNNNNN

HNMVLOKOmeOr-{N
r-lv-lt-i

°ou tequv

40

In a second set of experiments, using the same con-
trolled conditions and the same number of animals per con-
centration, three concentrations, lower than those previously
utilized were tested. The new concentrations used were
0.3, 0.6 and 1.2 pgs of carbaryl per gm of wet bedding. No
external symptoms were seen in animals treated with these
concentrations. All of the data from the previous two
groups of experiments are recorded in Table IX, the results
of the data analysis are shown in Table X and are graph-
ically shown in Figure 2.

There appears to be a definite relationship between
effects of carbaryl and respiration rates. The initial
conclusions of these data suggest: (1) that carbaryl
treated animals consumed oxygen at a faster rate than did
the untreated animals, and (2) that oxygen consumption
increased in treated animals even though there may be no
external signs of carbaryl poisoning. Thus, oxygen con-
sumption may be used as a measure to show effects of

carbaryl on earthworms.

Histological Studies

 

Data and Observations

 

An Der Lan and Aspock (1962 and 1963) reported the

general biological characteristics of Lumbricus sp. after

 

treatment with carbaryl. They noticed that after treating

the earthworm with a lethal concentration of 0.3% carbaryl,

41

 

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wosmuwmeo .0.0 H wmfismcou H£\E0\No H: :oHumuucmocou

 

 

cOHHQEdwsoo cm05xo mo mHmhamcm mama x magma

 

42

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man How mmumu coHumuHmmwn map musmmmummn mcHH UHHom map paw
maouucoo 03¢ 00 mmumu coHumuHmmmH map mucmmmummu msHH Umpuop
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mmumu coHumuHmme 00 ammuw m 0H50Hm

43

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0 \ 03.
0.0 0% N. — _ _ 1.4
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JH/B/ "o ITT

44

many blisters appeared on the epidermis and ruptures

occurred within only a few days after treatment. Based on
these observations, they conducted histological studies of
the earthworm body wall. The results of these investigations
showed that there was a change in the position of the
epithelial cells of the epidermis. It was also noted in
their investigations that various lesions or "skin cancers"
were present. These epidermal cancers were seen only in
worms treated with lethal concentrations of carbaryl.

In my experiments with sub-lethal concentrations
of carbaryl several non-lethal effects that were caused by
carbaryl were noted. Therefore it seemed likely that there
would also be some histological effects in earthworms
treated with sub-lethal concentrations of carbaryl.

In these histological studies earthworms were
treated and observed for three weeks after using four
concentrations of: 1.5, 3.0, 6.0 and 15 pgs of carbaryl
per gm of bedding. The first three concentrations were
non-lethal after three weeks of treatment, while 15 pgs
of carbaryl per gm of bedding resulted in some mortality.
A control group of animals was also observed for three
weeks, but no external changes were observed in this group.

The transverse section as seen in Figure 3-A shows
the body wall of a normal untreated worm, it shows '

longitudinal and circular muscle fibers, and the epidermis.

45

Figure 3

Photomicrograph of the norma1.earthworm.body wall

(12p thick in transverse section). In the normal or
control worm the longitudinal muscle layer (LM) con-
sists of pennate bundles, and several layers of circu-
lar muscle fibers (CM) which lie adjacent to the
longitudinal layer on one side and the epidermis (E)

on the other side. X40

Photomicrograph of earthworm body wall (12p thick in
transverse section), after treatment with carbaryl at

3 pgs of carbaryl per gm of bedding for 3 weeks. The
longitudinal muscle layer (LM) shows little or no change,
however, the circular muscle layer (CM) has been affected
by showing what appears to be degenerating tissues and

lesions in the epidermal layer (arrow). X40

 

46

 

Figure 3

47

At a concentration of 1.5 pgs of carbaryl per gm
of bedding, no gross histological changes were seen, the
treated animals appearing identical to the control animals.

At a concentration of 3 pgs of carbaryl per gm of

bedding, however, significant changes in the histology of

the body wall were seen. After three weeks, the epidermal

layer was broken at various places along its surface.

Increased cell division in these areas was also observed.

In addition, the circular muscle layer appeared to be

degenerating. Figure 3-B gives an example of an animal

treated with 3 pgs of carbaryl per gm of bedding for 3

weeks.

Animals were also treated with 6 pgs of carbaryl per

gm of bedding for three weeks. An example of the typical

results of this treatment is shown in Figure 4-A. This

section shows some very definite lesions and breaks in the
epidermal layer of tissues. There also appears to be a

degeneration of the circular muscle layer and a prolifera-
tion or extensive growth of epidermal cells into the areas
where the lesions and breaks have occurred. This degenera—
tion and proliferation of tissues is more extensive than at

the lower concentrations.

When animals were treated with 15 pgs of carbaryl
per gm of bedding for three weeks, a small percentage
(5-10%) of the total population of the earthworms were

killed. Even though most of the worms survived during this

 

48

Figure 4

Photomicrograph of earthworm body wall (12 p thick in
transverse section) after treatment with carbaryl at

6 pgs of carbaryl per gm of bedding for 3 weeks. This
section shows that the longitudinal muscle layer (LM)
appears to have minor changes from the control. There
are lesions in the epidermis (E) and circular muscle
layer (CM). There is a proliferation of epidermal cells

where the lesions have occurred (arrows). X40

Photomicrograph of earthworm body wall (12 p thick in
transverse section) after treatment with carbaryl at

15 pgs of carbaryl per gm of bedding for 3 weeks. The
longitudinal muscle layer (LM) does not exist in bundles
as in the controls, but appears to be very irregular in
shape. The circular muscle layers (CM) have degenerated
and there is a proliferation of epidermal cells in these

affected areas. X40

 

 

 

49

   

 

50

period of time, very extensive damage was seen in the histo-
logical sections of almost all of the worms (Figure 4-B).

In particular areas there is extensive degeneration and
almost complete loss of the circular muscle layer.. In

addition there is a great proliferation of epidermal cells

 

in the same area. This extensive growth of cells and
degeneration of muscle fibers occurs at random throughout
the entire circumference of this anterior region of the worm.

Figure 5-A, 5—B, 6-A and 6-B shows similar cross—

 

sections of body wall tissues as seen in Figures 3-A, 3-B,
4-A and 4-B respectively at a much higher magnification
(X430).

From these histological observations it is evident
that even at non-lethal concentrations of carbaryl, exten—
sive hist0pathological changes occur. These changes may be
due to increased mitotic cell divisions. Thus, from these
histological studies as well as the respiration studies, it
would appear that simple utilization of mortality data by
itself to assess the impact of carbaryl treatment on worms
does not give an accurate account of the potential changes
carbaryl may cause in earthworm populations. Because the
earthworms were exposed to the carbaryl via the bedding
and the pathological changes which occurred affected the
epidermal and circular muscle layers first it appears likely
that the earthworms are killed or affected via topical
application of the insecticide rather than by ingestion of

the insecticide in their food.

 

51

Figure 5

Photomicrograph of a normal (control) earthworm body
wall (12 p thick in transverse section) showing the
epidermis (E), circular muscle layer (CM) and a por-

tion of the longitudinal muscle layer (LM). X430

Photomicrograph of an earthworm body wall (12 p thick

in transverse section), which has been treated with

3 pgs of carbaryl per gm of bedding. This section shows
several changes in the epidermis (E) and circular muscle
layer (CM). Here minor changes can definitely be
observed in the epidermal layer (arrow) which differ

remarkably from the epidermis of the controls. X430

 

52

 

 

Figure 5

 

53

Figure 6

6-A is a photomicrograph of an earthworm body wall (12 p
thick in transverse section) which has been treated with
6 pgs of carbaryl per gm of bedding. This section (arrow)
shows the beginning degeneration of the circular muscle

V.)

layer (CM) and growth of the epidermal layer of cells (E).
X430

6—B is a photomicrograph of an earthworm body wall (12 p
thick in transverse section), which has been treated with
15 pgs of carbaryl per gm of bedding. This section (arrowfl
shows an extensive proliferation of the epidermal cell (E)

in the regions of the circular muscle layer (CM) and the

epidermis (E). X430

 

 

 

 

 

54

 

 

 

Figure 6

DISCUSS ION

The results obtained in this study show that the
earthworm, Lumbricus terrestris, is affected both morpho-
logically and physiologically by carbaryl. Both injection
and topical applications cause effects. Histological
evidence indicates that one major site of action of carbaryl

on earthworm is the body wall (cuticle, epidermis, circular

and longitudinal muscle layer).

Toxicitonf Carbaryl for Earthworms

There have been few previous studies of the toxicity
of carbaryl on earthworms. Edwards (1965) in field studies
reported effects of carbaryl residues (at doses of 1.5 to
2 lbs per acre) up to 6 months after treatment. The effect
was a large reduction in the number of earthworms. Thompson
(1971) also reported data from field studies on the effects
of carbaryl on earthworms. He found that when he used
various concentrations ranging from 1 to 5 lbs per 10' x 6'
plots and 3 weeks for treatment, caused a 60% reduction in
the number of earthworms in the treated plots. An Der Lan
and Aspock (1962) in controlled laboratory experiments,
tested the effects of a 0.3% suspension of carbaryl brushed

onto the cuticle of the worms. They found that this

55

 

 

 

56

txeatment was lethal to the earthworms and in addition,

caused a variety of histopathological changes in the worms.

My studies show an LD50 for carbaryl of approximately 20 pgs

per gm of wet bedding after 21 days of treatment.

Because the methods of application have been so
different in all of the previous studies conducted up to
the present time, it is impossible to make direct compari-
sons between by results and those of other workers; however,
as a result of my studies several points should be made
about the toxicity of carbaryl for earthworms. First, any
determination of the toxicity of carbaryl for earthworms
is heavily dependent on the dosage time used, i.e. my

results show that more than 3 weeks were needed for complete

mortality in the earthworm population tested. Thus,

mortality studies involving only a few hours or days of
treatment will not give a good measure of the full effects
on earthworm populations that long term exposure to carbaryl
might have.

A second point is that the use of mortality of
worms in laboratory studies may not be an accurate indicator

<3f the impact of carbaryl applications in the field on

earthworm populations. My data do indicate that at concen-

'tnations of carbaryl considerably below those at which

Imxrtalities occur, the worms are so adversely affected,

euui their survival in the field might be considerably

lessened.

 

 

u ‘ 5

57

The results I have obtained on respiration rates

have indicated a consistent increase in rates at doses

ranging from 0.3 to 6.0 pgs of carbaryl per gm of wet

bedding at 3 weeks. These are concentrations considerably

beltnv those at which lethal effects were shown in my labor-

atory studies. My results show growths of epithelial cells

frtnn the epidermis into the area of circular muscles,
(iegeneration of the circular muscle fibers and a change in

the normal position of the longitudinal muscles of the body

wall at carbaryl concentrations below the lethal level.

A third point which should be made is that extra-

polation from my data to expected results in the field must

be done with considerable caution. Several factors may

change the ways in which worms are affected in field studies

(1) since the breakdown of carbaryl will vary greatly with

time and soil composition, it is difficult to know if

carbaryl has been changed to its various metabolities

before, during or after exposure; (2) the worms in field

studies are not labeled, therefore, there is no accurate

way make sure that the worms initially exposed to the

carbaryl.are the same as those observed days, weeks or even

monUuslater; (3) the application of carbaryl is usually

in Unaform of a wettable powder, sprayed or dusted on the

soil surface. Using this technique. it is difficult to

lqmwlmm many worms are actually being exposed to the

carbaryl.

 

‘j _,._. n. .,'.
I

I

I

Fr
r.‘

 

58

Comparison of Carbaryl Effects
on Other Animals

The toxicity of carbaryl as seen in the present
study for earthworms is similar to that Sugiyama (1971 and

1972) and his co-workers determined for the silkworm,

 

Bombyx mori and the black-tipped leafhopper, Bothrogonia
japonica. The LD

 

50 for the silkworm was calculated to be

12.6 pgs carbaryl per gm of body weight for the 2nd day of

tine 4th instar and 15.2 pg carbaryl per gm body weight for

the 4th day of the 5th instar with topical application.

LD50 value of carbaryl to the black-tipped leafhopper was
determined with topical application to be equivalent to

0.99 pg per gm body weight for the nymph and 1.46 pg per gm
body weight for the adult.

The 20 pg per gm wet bedding
LD

50 I have determined for earthworms is in the same

general area. Reeves (1962) reported laboratory studies

of acute oral toxicity of carbaryl. He reported an LD50
for the rat of 510-560 mg per kg; for the rabbit 710 mg

perquand for the guinea pig 280 mg per kg of body weight.
An Der Lan and Aspock (1962) reported an LD50 of

5401mgper gm body weight for the flatworm, Crenobia alpina,

 

ushmgtopical application of the carbaryl. My LD50 value

of approximately 20 pgs carbaryl per gm of wet bedding

indicates a much different toxicity to earthworms, suggest-

:hw'akmoad spectrum of effectiveness of carbaryl to
invertebrates .

 

‘1.

59

Mode and Site of Action of Carbaryl

My studies, as well as those of An Der Lan and

Aspock (1962) suggest that one site of action of carbaryl

is time body wall. The results of their investigations as

‘welJ.aas mine showed extensive growths of epithelial cells,
degenerating circular muscle fibers, a change in the posi-
ticnlcaf the longitudinal muscle bundles and breaks in the

(article throughout the body surface of the animal.

The symptoms observed in the carbaryl treated

 

earthworms do not appear to be specific to carbaryl.

Gersch (1951 and 1957) reported effects of various
dyes and stains on the body wall of the earthworm. His
results showed growths of epithelial cells and degenerating

tissues of the circular muscle layer of the body wall. He

also noted lesions and swellings of the body wall.

In a similar paper, Cooper (1968) reported tumors
and growths in earthworm body wall, which were the results

of foreign materials being released into the coelomic

cavity. 'Thus, it appears possible that the histopathological

(flanges I have seen in the earthworm in response to carbaryl

treatments are generalized responses and not specific

responses to carbaryl as such.

O'Brien (1967) suggested that carbaryl, like many
ckhernmthylcarbamates, is a fairly potent inhibitor of

cholinesterase in mammals and suggested that many symptoms

whhflxmay accompany their action, such as lachrymation,

60

salivation, excretion,

convulsions and even death are
caused by the anticholinesterase activity.

He noted that
only a few studies have been reported in insects and

mammals showing the mode of action of the carbamate carbaryl.

Casida (1963) suggested that the carbamates act in

insects and mammals as autonomic drugs, stimulating
stxnnrtures innervated by cholinergic nerves. This
parasympathetic action results from inhibition of

acetylcholinesterase to allow local accumulation of
acetylcholine.

Even though no studies have been found on the mode

of action of carbaryl in earthworms, it is possible that

earthworms respond in a very similar manner to the mode of
action reported previously for insects and mammals.
Since the importance of acetylcholine and

cholinesterase in earthworms has not been clearly estab-
lished, it will only be through further detailed investiga-

tions that it will be possible to determine precisely the

sitecflfaction of carbaryl in the earthworm.

 

\I
1‘ i.-.

"h.

61

SUMMARY

1. An LD50 of 20 pgs of carbaryl per gm of wet

bedding has been established for earthworms. The. method

ofzumflication was topical, the temperature during the

tzeatment period was 15.5°C, the solvent used was.distilled

water and the period of treatment was 21 days.

2. Using non-lethal doses of carbaryl, several

behavioral and morphological effects were seen: .lack of
"withdrawal responses" to chemical and light stimuli,
coiling, twisting and constricting of the body and dis-

coloration of the epidermis of the treated animals.

3. There were increased rates of oxygen consump-

tion for Lumbricus terrestris, after three weeks of treat-

ment, with non—lethal doses of carbaryl, even in animals

which did not show any external symptoms of treatment.

4. The histological studies show that treatment

of worms with non-lethal concentrations produce a number of

structural changes in the body wall. Some of these effects

*were: proliferation of epithelial cells of the epidermis

into tine circular muscle layer and lesions throughout the

surface of the body wall.

5. These lethal and sub-lethal effects were

observed at concentrations of carbaryl as low as 0.3 pgs
per gm of bedding and the effects of carbaryl treatment at
these low concentrations seems not to be specific for the

earthworm, since other compounds may cause the same types

of effects in the earthworm.

 

‘1

 

LIST OF REFERENCES

 

LIST OF REFERENCES

An Der Lan, Von H. 1962. Histopathologische Auswirkungen
von Insektiziden (DDT and Sevin) bei Wirbellosen
und ihre cancerogene Beurteilung. Mikorskopie Bd.
51: 85-112.

An Der Lan, Von H. and H. Aspock, 1963. Zur Wirkung von
Sevin auf Regenwurmer. Anzeiger fur Schadlingskunde
55(12): 180-82.

Baker, W. L., 1946. DDT and Earthworm Populations. J. Econ.
Entomol. 55(3): 404-405.

Casida, J. E., 1963. Mode of Action of Carbamates. Ann.
Rev. of Entomol. 5: 39-58.

Cooper, E. L., 1968. Multinucleated giant cells, granulomata
and Myoblastomas in annelid worms. J. Invert. Pathol.
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Doane, C. C., 1962. Effects of certain insecticides on
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Edwards, C. A., 1965. Effects of pesticide residues on soil
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Fleming, W. E. and C. H. Hadley, 1945. DDT, ineffective
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Gersch, Von M., 1951. Untersuchungen uber Zellentartung
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63

Gersch, Von M., 1957. Zellentartung und Zellwucherung bei
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Kohlenwasserstoffen. Archiv. fur Geschwulstforschung
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HOpkins, A. R., 1957. Effects of several insecticides on
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Hoy, J. M., 1955. Toxicity of some hydrocarbon insecticides
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Humanson, G. L., 1972. Animal Tissue Techniques. W. H.
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Johnson, M. L., 1942. The respiratory function of the
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Lavarack, M. S., 1963. Physiology of the Earthworm.
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Louloudes, S. J.; T. J. Shortino and N. L. Brown, 1962.
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Martin, L. W. and S. C. Wiggins, 1959. The tolerance of
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Metcalf, R. L., 1961. Carbamate insecticides. J. Agr.
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Moriyama, H.; H. Sugiyama and H. Shigematsu, 1972. A
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silkworm, Bombyx mori. Pest. Biochem, and Physiol.
2: 1-70

 

O'Brien, R. D., 1967. Insecticides: Action and Metabolism.
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Reeves, A. J. 1962. The insecticidal action and toxicology
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288-293.

1 jar

 

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i
q

64

Ruppel, R. F.; M. J. Zabik; C. W. Laughlin and R. Fogg,
1973. Effects of some pesticides used in field
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Sugiyama, H., T. Kanzi and H. Shigematsu, 1971. Difference
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and the bIaCk-tipped leafhopper, Bothrogonia

japonica, Ishihara (Hemiptera: Tilligellidae).
App . Ent. Zool. 5(2): 57-62.

 

Sugiyama, H., H. Shigematsu and T. Kanki, 1972. Conversion
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Thompson, A. R., 1971. Effects of nine insecticides on the
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