‘ FIELD AND LABORATORY STUDIES
. ON THE EFFECTS OF DDT ON ”EDS
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MECHIGAN STATE UNIVERSETY
Richard F. Bernard
1962
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' rthesis entitled
FIELD AND LABORATORY STUDIES ON THE

EFFECTS OF DDi_o§ BIRDS”

presented by

Richard F. Bernard

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Major inofessor

Date May 2].- 1962

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Walla-9.1

FIELD AND LABORATORY STUDIES ON THE EFFECTS
OF DDT ON BIRDS
by

Richard F. Bernard

AN ABSTRACT

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

DOCTOR OF PHILOSOPHY
Department of Zoology

1962

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FIELD AND LABORATORY STUDIES ON THE EFFECTS
OF DDT ON BIRDS

by Richard F. Bernard

Following the use of DDT (1,1,1-trichloro-2,2-bis (p-
chloropheny1)-ethane) sprays for the purpose of controlling
Dutch elm disease, many communities, especially in the
Middle West, have reported the finding of large numbers of
birds, particularly Robins (Turdus migratorius), either dead
or dying of symptoms commonly associated with chlorinated
hydrocarbon poisoning. This study was undertaken in an
attempt to gain additional information concerning the ef-
fects on birds of DDT used in areas treated for Dutch elm
disease control or for other related programs.

For this study 137 dead or dying birds (mostly Robins)
were obtained from known spray areas and analyzed for DDT.
Usually several tissues from each were tested. The
Schechter-Haller method of analysis (Schechter, gt,al., 1945)
was used for the quantitative determination of DDT. Levels
of DDT in the body tissues of these birds were compared
with quantities found in corresponding tissues of experi-
mentally poisoned birds of the same species. Experiments
with captive birds were also conducted to determine the
quantity of DDT stored in various body tissues following
varying degrees of exposure to the toxicant and to determine
the value of chemical analysis as a diagnostic tool in

attributing bird mortality to DDT poisoning.

 

 

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Richard F. Bernard

The results indicate that the analysis of entire
carcasses of birds is not an accurate means of attributing
bird mortality to DDT poisoning. However, comparison of DDT
levels in brains of experimentally poisoned House Sparrows
(Passer domesticus) and Robins with those in brains of birds
found in the field support the conclusion that analysis of
brain tissues is a valid criterion for determining Whether
or not a bird died from ingestion of DDT. The evidence ob-
tained also supports the conclusion that House Sparrows
having 65 or more, and Robins with 50 or more, pg. of DDT/g.
of brain tissue died directly from the effects of the
chemical.

The analysis of brain tissue from 54 of 70 adult Robins,
found dead or dying in sprayed areas, showed that 90.7 per
cent had sufficient quantities of DDT in the brain to
Justify the conclusion that it was the cause of death. All
birds found tremoring had DDT residues in their tissues,
while only one of the birds found dead was completely free
of DDT in all of the tissues tested.

DDT was also found in the female reproductive organs,
in develOping eggs, and in unhatched eggs, as well as in the
body tissues of young birds. Similarly, 19 of 20 male Robins
found dead or dying had concentrations of DDT in the testes
ranging from 6 to 109 pg. of DDT/g. of tissue.

A total of 52 birds of 27 species other than Robins

were also analyzed. Of these, 35 birds representing 21

 

 

 

 

 

 

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Richard F. Bernard
species had at least some DDT in their tissues. These do
not include some 60 or more birds of 34 species analyzed in
another project carried out for the Cranbrook Institute of
Science (Wallace, Nickell, and Bernard, 1961).

Analyses of plant and animal material in areas
treated with DDT indicate that the toxicant is both per-
sistent and widely available to ground-feeding, bark-
foraging, and seed-eating birds.

Experimental birds exposed to low DDT diets for
short periods of time were able to eliminate the toxicant
from vital organs. However, DDT stored in fat is cumulative
and less subject to elimination. When fat reserves are
utilized (as in starvation), stored DDT may be released to
more sensitive sites (such as the brain), resulting in
tremors followed by death.

Results using the "Student's t" distribution indi-
cate that the observed differences in the concentrations of
DDT between male and female Robins and between male and
female House Sparrows are not significant at the 1 per
cent level. Additional computations using the Pearson
Product Moment Correlation Coefficient suggest that concen-
trations of DDT in the brain of poisoned birds are largely
independent of the concentrations found in other tissues
except between brain and heart in male Robins, and between

brain and liver in female House Sparrows.

 

 

 

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Richard F. Bernard
It is concluded that bird mortality in areas sprayed
‘with DDT for the control of Dutch elm disease has been
'high, and that the birds died from continued ingestion of

this poison.

FIELD AND LABORATORY STUDIES ON THE EFFECTS

OF DDT ON BIRDS

by

Richard F? Bernard

A THESIS

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

DOCTOR OF PHILOSOPHY
DEPARTMENT OF ZOOLOGY

1962

ACKNOWLEDGMENTS

I wish to express my deep appreciation to Dr. George J.
Wallace, Professor of Zoology, for his interest, direction,
and encouragement during the course of this investigation.
Grateful thanks are also due to Dr. Erwin J. Benne, Professor
of Biochemistry, for his helpful advice on analytical methods
and for the use of facilities in the Department of B10-
chemistry, where a major portion of this study was conducted.
I am also indebted to Dr. Rollin H. Baker, Director of the
Museum and Professor of Zoology, and to Dr. Roger HOOpin-
garner, Assistant Professor of Entomology, for their able
assistance while serving on the author's guidance committee.

Special thanks are due to Dr. Philip J. Clark, Professor
of Zoology, for his help with the statistical analysis of the
data, and to Mr. Earle D. Harrison for his able assistance in
the care of the captive birds used in the study.

Acknowledgment is also made to the many individuals who
submitted dead and dying birds for analysis, and to Miss
Wanda Buckovac for typing the original draft of the manu-
script.

The author takes this opportunity to express his appreci-
ation to the United States Bureau of Sport Fisheries and Wild-
life for financial assistance in initiating the study and

securing essential facilities.

TABLE OF CONTENTS

INTRODUCTION . . . . . . .

ANALYSIS FOR DDT . . . . . . .
General Considerations . . .
Limitations . . . . . . . . .
Extraction . . . . . . . . .
Evaluation of DDT . . . . . .
Preparation of Standard Curves

Calculations . . . . . . . .

Special Methods for Fatty Materials

Separation of DDT from DDE .
ANALYSIS OF FIELD SPECIMENS . .
Samples . . . . . . . . . . .
Results . . . . . . . . . . .
Discussion . . . . . . . . .
Other Species . . . . . . . .
Availability of DDT to Birds
FEEDING EXPERIMENTS . . . . .'
Methods . . . . . . . . . . .
Analytical Procedures . .
Initial Feeding Tests . . . .
Effects of Starvation . . . .

Elimination of DDT . . . . .

PAGE

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11
12
13
14
16
16
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29
39
44
44
45
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Relationship of Starvation to Exposure Time . . . . . 58
Release of DDT from Fat Depots . . . . . . . . . . . 6O
Levels of DDT Stored in Fat Tissues . . . . . . . . . 62
Effects of Feeding Habits . . . . . . . . . . . . . . 63
Levels of DDT in Non-Captive House Sparrows . . . . . 66
ANALYSIS OF RESULTS . . . . . . . . . . . . . . . . . . 69
GENERAL DISCUSSION AND CONCLUSIONS . . . . . . . . . . 77
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . 82
LITERATURE CITED . . . . . . . . . . . . . . . . . . . 87

10.

_11.

12.

13.

LIST OF TABLES

DDT Found in Adult Male Robins . . . . . . . .
DDT Found in Adult Female Robins . . . . . . .
DDT Found in Adult Robins (Not-Sexed) . . . . .
DDT Found in Young Robins . . . . . . . . . . .
DDT Found in Adult Robins Fed DDT-Injected
Earthworms . . . . . . . . . . . . . . .'. .
DDT Determined in the Tissues of Miscellaneous
Birds . . . . . . . . . . . . . . . . . . . .
DDT Determined in Sparrows Fed a Diet Contain-
ing 300 ppm of the Toxicant . . . . . . . . .
DDT Determined in Sparrows Fed a Diet Contain-
ing 200 ppm of the Toxicant . . . . . . . . .
DDT Determined in Sparrows Fed a Diet Contain-
ing 100 ppm of the Toxicant . . . . . . . . .
DDT Determined in Sparrows Fed DDT and Then
Starved . . . . . . . . . . . . . . . . . .
DDT Determined in Sparrows Fed Partial DDT
Diets . . . . . . . . . . . . . . . . . .'. .
DDT Determined in Sparrows Alternately Fed on
Diets Containing DDT and Starved . . . . . .
DDT Determined in Sparrows Fed Initial DDT

Diets O O O O O O O O O O O 0 O O O I O O O 0

PAGE
18
2O
22
24

25

37

47

49

52

54

55

59

61

vi
TABLE PAGE
14. DDT Determined in Non-Captive House Sparrows . . 67
15. Average Concentration and Range of DDT (in
pg./g.) in Tremoring Robins . . . . . . . . . . 72
16. Average Concentration and Range of DDT (in
pg./g.) in Tremoring House Sparrows . . . . . 73
17. Average Concentration and Range of DDT (in
pg./g.) in Sacrificed Robins . . . . . . . . . 74
18. Average Concentration and Range of DDT (in
‘pg./g.) in Sacrificed House Sparrows . . . . . 75
19. Correlation Coefficient and Sample Size for

Robins and House Sparrows . . . . . . . . . . . 75

INTRODUCTION

Numerous birds have been reported dead or dying
following the application of DDT(1,1,1-trichloro-2,2-bis
(p-chlorOphenyl)ethane) to elms for the purpose of control-
ling the bark beetles, Scolytus multistriatus and Hylgg-
gapinus rufipes, believed to be the vectors of the fungus
Ceratocystis glmi, which produces Dutch elm disease, particu-
larly in American elms (glmgg americana). The many reports
of bird mortality raises a number of questions as to the
effects of DDT on non-target organisms. This study was
undertaken in an attempt to gain additional information
concerning the effects of DDT on birds in areas treated for
Dutch elm disease control or for related purposes.

The fungus which produces Dutch elm disease was first
discovered near Cleveland, Ohio, in 1930 (Whitten and Swingle,
1958). Since then the disease has spread into more than 20
states.

In Michigan, Dutch elm disease first appeared in
Detroit in the summer of 1950 and has since been reported in
many communities throughout the southern part of the Lower
Peninsula.

Attempts to control Dutch elm disease involve both
sanitation and the suppression of the beetle vectors which

introduce the fungus (particularly to one and two-year-old

twigs) during the time of feeding. Spraying elms with DDT
is carried out in order to kill the beetles before feeding
takes place. Since adult bark beetles emerge and fly to new
trees during the late spring, a dormant spray of DDT is
usually used any time between leaf-drop in the fall and
bud-swelling in the spring. However, when bark beetle
numbers are high, a July (foliar spray) treatment is also
recommended in order to reduce the adult beetle pOpulation
which lowers the number of overwintering grubs (Janes and
Strong, 1961).

Early studies on the toxicity of DDT to wildlife by
biologists of the U. S. Fish and Wildlife Service (Hotchkiss
and Pough, 1946) indicated that the dosages recommended for
use on elms would be lethal to birds. Field tests on the use
of DDT in Dutch elm disease control at Princeton, New Jersey,
beginning in 1947 (Benton, 1951; Blagbrough, 1952), and re-
search in Robin-earthworm relationships in Illinois (Barker,
1958) also indicated that mortality to birds was to be ex-
pected in Dutch elm disease control programs.

In spite of this, the use of DDT for possible control
of Dutch elm disease has been greatly expanded, especially
in the Middle West. As a result, about 60 different com-
munities have reported the loss of Robins (Iurdus
migratgrius) and some 80 other species of birds following
the application of DDT to the elms (Wallace, Nickell, and

Bernard, 1961).

Certain field studies reveal that Robin mortality in
areas of high elm density may be 86-88 per cent in the first
year of DDT spraying (Hickey and Hunt, 1960) and at least 85
per cent in the second year (Hunt, unpublished). Following
three years of DDT spraying for Dutch elm disease, Robin
populations in three sprayed communities were 69, 70, and
98 per cent below the average for three unsprayed communities
(Hunt, 1960). Similar studies on the campus of Michigan
State University at East Lansing have shown that an esti-
mated pre-spraying (1954) population of at least one pair
of Robins per acre over a 184-acre study area dwindled to a
few scattered adults and one young by the end of June, 1957
(Mehner and Wallace, 1959). In 1958, small waves of Robins
which invaded the campus in April and May, died or dis-
appeared by the end of June (Wallace, 1959). In 1959, about
ten pairs of Robins were recorded on the campus in mid-April,
but by the end of June, 45 dead Robins had been reported or
collected from the same area (Wallace, 1960). In 1960, a
mid-April population of about 18 pairs of Robins declined
rapidly in late April and May, until only three were known
to be left by May 22 (Wallace, Nickell, and Bernard, 1961).

However, observed mortality of birds does not neces-
sarily constitute proof that the birds died as a direct or

indirect result of exposure to DDT, and many other explanations

have been offered.

Chemical analysis of tissues of birds from spray areas
to determine residues of DDT have been made (Mitchell,
Blagbrough, and Van Etten, 1953; Barker, 1958; Wallace,
Nickell, and Bernard, 1961), but data comparing the concen-
trations of DDT found in field specimens to those present
in laboratory animals fed lethal and sublethal levels of DDT
are not available except for Ring-necked Pheasant (ghasianus
colchicus) and Bobwhite (Colinus virginianus) (Dewitt, Derby,
and Mangan, 1955).

On the campus of Michigan State University a limited
prOgram for the prevention of Dutch elm disease was introduced
in 1954. Spraying was expanded in 1955, and from 1956 to
1958 a full scale program involving both foliar and dormant
sprays was conducted on campus. In 1959 and 1960, only the
dormant applications of DDT were used. Rotomist sprayers
were employed, using a 12-1/2 per cent solution of DDT. In
addition to this, aerial spraying for mosquito control and
other programs also took place in 1958 and 1959.

Dying birds were first noticed in 1955, and since
then a considerable number of birds (particularly Robins)
have been observed or reported dying on campus each year.
Usually (at least in the last three years) the number of
Robins recovered equals or exceeds the maximum April counts.

This investigation, rather than describing observed

mortality to birds, attempts to relate this mortality to
Dutch elm disease control and related prOgrams by comparing
quantities of DDT in tissues of dead and dying birds to
quantities found in corresponding tissues of experimentally
poisoned birds. Experiments on captive House Sparrows
(Passer domesticus) fed DDT to determine the quantity stored
in various tissues under varying degrees of exposure and

the value of chemical analysis as a diagnostic tool in
determining the cause of death in field specimenaare also

considered.

Ah ALYSIS FOR DDT

General Considerations

 

The Schechter —Ha ller method of analysis (Schechter,

et 1., 1945) was used for the quantitative determination of

 

DDT. Generally speaking, this consists of removing the DDT

ubstance being analyzed,

U)

with an organic solvent from the
evaporating the solvent, nitrating the residue and then sepa-
rating the nitrated DDT from the acidified aqueous solution
1th ether. Finally alcoholic sodium methylate is added to
a benzene solution of the nitrated product which produces a
characteristic blue-colored complex, the concentration of
which is evaluated lpectrophoto. tr ically. DDT values are

acrruted from a standard curve relating phOtORleteP readings

and known quantities of DDT.

Limitations

 

This method (Schesnte; 2711 r) is generally specific

(D

and accurate. However, other insecticides may interfere
with the results. The principal insecticides known to
interfere are TDE (dichloro-2 ,2— bis (chlorophenyl) ethane),
DDE (l, l~dichloro—2,2-bis (p—chlorophenyl) ethylene),
thoxychlor (l,l,l-trichloro-2,2—bis (p-methoxyphenyl)
ethane), DNB and DNP (the nitro 1,1 bis (p-chlorophenyl)

butane and propane analoques of DDT). Other insecticidal

7
compounds, are oxidized or degraded in the nitration process
and are then removed from ether or petroleum-ether solution
by washing with alkali, or they do not form compounds that
interfere with the colorimetric evaluation of DDT. The
presence of fats and waxes in sufficient amounts produces
yellow off-colors which are difficult to remove and special
separations must be made. Lastly, as with all determinations,
it is necessary to carry a blank (uncontaminated sample)
along with the tissues being analyzed, in order to correct
for deviations in technique and the effects of the tissue in
the analysis. '

Robins known to be free from DDT could not be procured
in sufficient numbers; therefore, substitute blanks were
used to make up, in part, for this limitation. These con—
sisted of benzene solutions, migratory thrushes, and various
other birds considered to be free from DDT. Most of the DDT-
free birds were procured at the base of television towers,
where they were presumably killed by collision with the
structure.

Since it was not possible to analyze the specimens
immediately, they were stored in a deep freeze until the
analyses could be carried out. Exceptions were two birds
preserved in formalin and the House Sparrows used in the
feeding experiments. The latter were analyzed within two or

three days after death. Usually, birds were kept individually

in plastic bags during storage in order to reduce loss of
moisture. Some birds stored earlier were not individually
wrapped and were found to have lost considerable moisture.
The moisture content of random samples of breast
muscle and liver tissues was calculated to determine the
amount of moisture lost during storage. Values for Robin
breast muscle averaged 66.5 per cent in stored tissues as
compared to 70.3 per cent in fresh muscle slices. Stored
Robin liver tissue averaged 60 per cent moisture as com-
pared to 66 per cent in fresh liver. Loss of moisture
invariably concentrates the solid constituents of the
tissue, and results in higher concentrations of DDT when

results are expressed on a wet-weight basis.

Extraction

Prior to analysis the frozen carcasses were allowed
to thaw, and tissues or sections of tissues were remoVed
and analyzed separately. The tissues were weighed and then
macerated with anhydrous sodium sulfate in a mortar contain-
ing some quartz sand (the amount of sodium sulfate used
varied with the size and consistency of the sample). The
pulverized tissue was then stored in a small flask until
the analysis was resumed. I i

The next step consisted of extracting the DDT from
the pulverized tissue by adding a mixture of diethyl ether

and petroleum ether (1:4 ratio by volume of ether-petroleum-

ether) and grinding it with the ether mixture in a mortar
several times (again depending on the quantity and con-
sistency of the material) and pouring the ether layer each
time through filter paper in a funnel leading into an
Erlenmeyer flask. This method of extraction is relatively
simple and requires a minimum of time.

In order to determine the efficiency of the extraction,

1 to 1 part 0,p1 -

a known quantity of pure DDT (3 parts p,p
DDT) was macerated with the tissues and extracted with
ether and the quantity of DDT present was determined. The

percentage of recovery ranged from 91 to 112.

valuation DT

After the extraction from the sample tissue, the
general procedure followed for determining DDT was that of
Schechter, 91. gl., as prescribed by the Association of
Official Agricultural Chemists (1956): First, evaporate the
solvent on a steam bath with the aid of a gentle current of
air. When the flask is dry, remove it from the steam bath
and chill it thoroughly in an ice bath. While the flask is
immersed in ice water, add 5.0 ml of the chilled nitrating
mixture (mixture of fuming HNO3 with equal volume of con-
centrated H2804) from a burst or pipet, taking care to wet
all portions of the residue. Then nitrate by placing the

flask in a water bath and heat so that solutions reach 85°C.

10
in 20-30 minutes (lead rings may be used to weight flask).
Next, place the flasks on a steam bath for one hour. Remove
the flasks and cool under the tap or leave overnight. 0001
the flask and pour the cooled acid mixture slowly from each
flask into a 500 ml separator funnel containing 25 ml of
ice-cold water; rinse the flasks with several portions of
ice water, then pour the rinsings into the separator. Rinse
again with 25 ml of the ether solution (1:4 ether - petroleum-
ether) and finally with a second 15 ml portion of ether,
pouring last ether rinse into a second separator. Extract
by shaking the first separator vigorously for one minute;
then drain the aqueous (lower) layer into the second sepa-
rator and repeat the extraction. Discard the aqueous layer
and drain the second separator into the first, rinsing with
small portions of ether. Draw off any residual aqueous layer
as completely as possible, add 10 ml of 10 per cent KOH
solution (a second or even third wash may be necessary to
remove all yellow color from the ether layer), and shake
vigorously for 30 seconds. Allow time for solutions to
separate, then drain off the KOH layer and wash with two
successive 15 ml portions of saturated NaCl solution. Drain
well and filter extract through a plug of Pyrex glass wool
held in a funnel into a 125 ml. Erlenmeyer flask containing a
glass bead (the glass wool should be saturated with ether
before filtration). Rinse the separator and filter with a

11
few small portions of ether and evaporate off the ether on a
steam bath. Cool the flask, making sure that the interior
is thoroughly dry, and take up the residue with exactly 10
or 25 ml of benzene, the amount depending on the quantity of
DDT expected (for small quantities use 10 ml). Stopper the
flask and swirl for one minute to ensure complete solution
of the residue (procedure may be interrupted overnight at
this stage). Transfer a 5.00 ml aliquot of the benzene
solution to a small flask and develop characteristic DDT
color by adding exactly 10.0 ml of the sodium methylate
solution (10 per cent sodium methylate in dry C° P. methanol).
Mix well, allow solution to stand for 15 minutes, and then
determine the transmittancy (absorbance may also be used)
at 600 my in a cell of appropriate length by means of a
Beckman B spectrophotometer (other photometric instruments
may be used). Calculate DDT content from a standard curve

prepared with the 3 to 1 p,p1 - o,p1-DDT mixture.

Preparation of Standard Curves

In routine work, resolution of separate standard

1

curves of the p,p1 and o,p isomers of DDT is not necessary.

Instead, a single standard curve determined at 600 mu, using

1 1 - DDT may be used.

a mixture of 3 parts p,p and 1 part o,p
The color of the p,p1 - DDT will, therefore, predominate in
the color mixture.

To prepare the standard curve, dissolve 40.0 mg of the

12
DDT mixture in benzene and dilute to 200 ml (1 ml : 0.20 mg).
Measure 1.00, 2.00, 3.00, 4.00, and 5.00 ml into 50 ml Erlen-
meyers and evaporate the solvent on a steam bath. Nitrate
the residue in the same manner as used for unknown samples
except that the period of time on the steam bath is reduced
to 30 minutes. Extract as stated above for unknown samples,
using 10 ml of 5 per cent NaOH solution instead of 10 ml of
10 per cent KOH solution. Carry the samples through the re-
mainder of the procedure as described above and plot trans-
mittancy readings against corresponding concentrations of
DDT. The entire procedure should be repeated a number of
times in order to obtain values with which to establish a

standard curve that can be closely duplicated.

Calculations

The quantity of DDT nitrated ranges up to 1.0 mg in
steps of 0.2 mg. The actual transmittancies found will vary
with the wavelength setting and slit width for spectrophotoe
meters, but curves obtained with a particular instrument
should be straight lines and be reproducible within small
error.

For actual determinations,measure the transmittancy
of the unknown sample with the same cells used in making the
standard curve, and read the quantity of DDT from the
standard curve plotted on semi—log paper and calculate back
to the weight of sample represented in the aliquot taken to

obtain the ppm of DDT residue.

13

Special Methods for Fatty Materials
Since fat may interfere with the determination,

special methods are used for its removal. That is, after
evaporation of the solvent on a steam bath, quantitatively
wash the residue from evaporation into a 500 ml separator
with 150 ml of CHClB. Place 100 ml of CHCl3 in a second 500
ml separator, and extract the CHCl3 solution successively
with two 50 ml portions of sodium sulfate - sulfuric acid
solution (100 g. anhydrous Na2804 in 1 liter H2504), 50 ml
of fuming sulfuric acid - concentrated sulfuric acid mix-
ture (equal volumes of fuming H2504 and H2804), and 50 ml of
the Na2SO4 - H2804 solution. If the last wash is not light
in color, use still another Na2304 - H2804 wash. Drain each
acid wash (lower layer) from first separator into second
separator and finally into a 250 ml cylinder. (Extraction in
the second separator is used to minimize the loss of DDT by

slight emulsification of CHCl in acid washings.) Shake

3
separators vigorously after each extraction and then allow
solutions to stand 10-15 minutes before draining off the
acid layer.

After completing the acid extractions, filter the
CHClB from the first funnel and then the second funnel
through a 5 cm tightly packed plug of cotton in a glass
Gooch holder into a third 500 ml separator funnel. Pipet

off any CHCl3 that has risen to the surface from the

14

combined acid washings in the cylinder and run it through the
plug of cotton. Rinse the two separators and cotton with
50-100 ml of CHClj. Add a sufficient amount of 5 per cent
NaHCO3 solution (about 40 ml) to keep the combined CHCl3
filtrates alkaline to litmus paper. Allow 10 minutes for

clear separation, then filter the CHCl layer through a 5

3
cm plug of tightly packed cotton in a glass Gooch holder

into a 500 ml Erlenmeyer. Wash the NaHCO3 solution remain-
ing in the separator with two 30 ml portions of CHClB, and
filter these washings through the cotton into the Erlenmeyer.
If filtrate is not clear, refilter. Add a glass bead to the
Erlenmeyer and evaporate or distill the CHCl3 on a steam

bath, using an all-glass system, until a 10 ml residue re-
mains. Wash the residue quantitatively into a large test
tube, or a second Erlenmeyer, with acetone, add a glass bead,
and cautiously evaporate the solvent on a steam bath. Nitrate

the residue with 5 ml of the nitrating mixture and complete

the analysis as described in the foregoing section.

Separation of DDT from DDE

In most cases no attempt was made to distinguish DDT
from its known degradation products. However, when sepa-
ration of DDT from DDE was carried out, the method of Stern-
burg and Kearns (1952) was employed. This method consists
of extracting the residue from animal tissues with alkali

to remove DDA and puchlorobenzoic acid if present, then

15
evaporating the solvent on a steam bath. The residue is
taken up in 5 ml of petroleum ether and passed through a
chromatographic tube with a 12 mm. inside diameter plugged
with glass wool, and packed lightly with activated alumina.
This is repeated twice more with 5 ml portions of petroleum
ether, followed by 60 ml of petroleum ether. The entire
eluate is collected in one flask. If DDE is present in the
extract, it will appear in the eluate. When the petroleum
ether has sunk into the alumina, a clean flask is placed
under the column and 25 ml of C014 is passed through the
alumina. The C014 eluate will contain DDT if present. The
solvents are evaporated from the flasks and analyses

continued as previously described.

ANALYSIS OF FIELD SPECIMENS

Samples

Birds to be analyzed were obtained from known spray
areas, including in particular the campus of Michigan State
University at East Lansing. 0f the 137 birds analyzed in
thhsstudy (85 Robins and 52 other birds representing 27
species), 44 (32 per cent) were observed or reported dying
with the tremors commonly associated with chlorinated
hydrocarbon poisoning. The other birds were found dead.
The total includes three Robins trapped for determination
of possible sublethal quantities of DDT, three other Robins
shot in a non-spray area as checks, and seven birds other
than Robins collected at the base of television towers as
controls in the analyses. However, eight Robins which were
fed DDT-injected earthworms at the University of Wisconsin
and sent to me for analysis are not included. Excluded also
are some 60 birds of 34 species analyzed in another project
carried out for the Cranbrook Institute of Science (Wallace,
Nickell, and Bernard, 1961), as well as 15 House Sparrows
listed in Table 14.

In most cases, the brain, liver, heart, kidney, and
breast muscle of each bird were analyzed separately. The
testes of 23 male Robins were also checked for DDT, as were

the eggs within the uteri of female Robins and eggs from

 

 

Dirt,“ ... $1.1

 

17
deserted nests.
The determination of the sex of the individual birds
was not always possible since a number of birds (particularly
Robins) were not sexually mature, or at least the gonads were

not apparent.

Results

The following tables summarize the results obtained
from the chemical analysis of various tissues. The figures
in all tables are expressed in terms of micrograms of DDT/
gram of moist tissue (pg. of DDT/g. of tissue).

Table 1 presents the results obtained from the analysis
of 38 adult male Robins, including two trapped at the Kellogg
Biological Station in Kalamazoo County, Michigan, and two
others collected from Rose Lake, Clinton County, Michigan,
to serve as controls in the analysis. Table 2 summarizes
the results obtained from the analysis of 19 adult female
Robins, including one trapped in a mist net at the Biological

Station and another shot at Rose Lake.

 

18

 

 

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19

 

 

 

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mos mm mm mm 1 mnosuca mendedq mm\m \e me1m
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21
The following table (Table 3) gives the results ob-
tained for 19 additional Robins whose sex was not or could

not be determined.

 

22

 

 

 

 

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23

Young Robins at various stages of deveIOpment were
occasionally collected or turned in from the campus area or
from other communities. This group included a newly hatched
nestling which was found dead under one of the few campus
nests. Due to its small size, the entire carcass of this
bird was ground up and analyzed as a whole. The other birds
in the table were all fully feathered and spotted. All of
these birds are listed under the collective heading of

"young robins."

 

24

 

 

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1 1 o o o s_ .eeH seem managed mm\om\s _m1m
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25

Table 5 lists the results obtained from the analysis
of eight Robins which were experimentally poisoned with DDT
at the University of Wisconsin, and submitted for analysis
through the courtesy of Dr. J. J. Hickey. All of the birds
were captured at least 14 days prior to the start of the
feeding experiments on 13 October 1959. From 13 October to
26 October 1959, the birds were offered earthworms injected
with a DDT-peanut oil solution. The daily dosage was 110 mg
of DDT/Kg. of body weight based on an average initial weight
of 82.94 grams per Robin. All birds exhibited tremors prior
to death.

TABLE 5
DDT FOUND IN ADULT ROBINS FED DDT-INJECTED EARTHWORMS

 

 

 

 

 

Specimen Data ng. of DDT/g. of_Tissue
Breast
No. Date Condition Brain Muscle Heart Liver
R-86 10/16/59 Tremors 71 78 67 170
R-87 10/16/59 Tremors 84 62 60 207
R-88 10/16/59 Tremors 86 171 141 313
R-89 1o/18/59 Tremors 86 50 78 126
R-9O 10/19/59 Tremors 61 53 26 104
Rr91 10/24/59 Tremors 86 89 72 194
R-92 10/25/59 Tremors 64 7o 95 109
R-93 10/27/59 Tremors 116 207 141 263

W

Discussion
As can be seen from the preceding tables, all of the

tremoring Robins analyzed had DDT present in at least some

 

26
of their tissues. The average dose of DDT in tremoring
adult females appears to be higher than that found in
tremoring adult males, in all tissues listed except for
breast muscle. Adults of both sexes had much greater
quantities of DDT in their tissues than the young.

The testes of 23 male Robins were also analyzed.
Except for two males shot in a non-spray area as controls,
one trapped in a mist net, andenother which was free of DDT
in all of its tissues, all of the male Robins checked had
concentrations of DDT in the testes ranging from 6 to 109
pg. of DDT/g. of tissue. Albert (1962) has shown that DDT
at high levels can reduce sperm production in white leghorn
cockerels, but this phenomenon has not been investigated in
Robins.

In two cases, the ovaries of females with large
numbers of developing eggs in the ovaries were analyzed and
found to contain 171 (R-43) and 211 (R-48) pg. of DDT/g.,
respectively. In addition, fully developed eggs in the
uteri of two other females contained 56 (27 April) and 102
(15 May) ug. of DDT/g. of tissue, respectively. These re-
sults are not included in Table 2.

Similarly, eggs from abandoned Robin nests on the MSU
campus were analyzed for DDT. In each case, the egg shell
was removed prior to analysis, to eliminate the possibility

that DDT was present only on the egg surface. One egg,

 

27
collected on 24 May 1960, contained 15 pg. of DDT/g., while
four eggs (two of which had fairly well develOped embryos,
approximately 9 days old), taken from another nest on 15 May
1960, contained 0, 16, 8, and 25 pg. of DDT/g., respectively.
One nestling (R-84), found dead under a nest, contained 18
pg. of DDT/g. in its carcass, while eight young Robins at
different stages of deveIOpment had concentrations ranging
from 0 to 282 pg. of DDT/g. in their tissues, as indicated
in Table 4 above.

It is of interest to note that some and possibly all
of the DDT found in these young birds could conceivably have
been passed on to them by the female through the egg. The
possibility exists that the eggs failed to develOp and that
some of the young may have died from the effects of DDT with-
out ever having been exposed to it directly. Unfortunately,
insufficient information is available on concentrations of
DDT that are lethal to young, as well as on the effects it
may have on various stages of develOpment and reproduction.

The results obtained from the analysis of breast
muscle, heart, liver, and kidney pose a number of problems
in interpreting the data. As may be noted from the above
tables, the quantities of DDT found in these tissues were
variable and in a few cases tremoring birds had low
quantities or no DDT at all in breast muscle, heart, liver,

and kidney. Most young Robins had no DDT in the breast

 

28
muscle, even when it was present in other tissues. This may
be related to the limited exercise of the breast musculature
in young birds.

The liver contained higher concentrations of DDT than
found in any of the other tissues analyzed. This may be
partly due to the storage function and high lipid content of
the liver. The highest concentration of DDT found in the
liver was 794 mg. of DDT/g. of tissue listed for No. R-62 in
Table 3.

The tremors associated with DDT poisoning (as well as
other nerve poisons) points to the nervous system as the
likely site of lethal action. This suggests that the brain
furnishes the best criterion in relating DDT in tissues to
mortality. The lowest amount of DDT present in the brain of
a tremoring Robin was 53 pg. of DDT/g. of tissue found in
No. R-50 in Table 2. The three seemingly normal birds live-
trapped for analysis had significantly lower amounts in
their brain tissue, while the three birds shot in an un-
sprayed area as controls had no DDT present in the brain.

All of the Wisconsin Robina fed on the DDT-injected
earthworms, contained amounts of DDT in the brain which fit
well within the range of the birds found dying with tremors.

Quantities of DDT determined were variable in all
Species, but levels for Robins, based on tremoring birds of

bOth sexes, averaged 93.29 pg. of DDT/g. in the brain, 115.73

 

29
in breast muscle, 143.82 in heart tissues, and 187.08 in
liver. Barker (1958) suggested that 60 pg. of DDT is close
to the median lethal dose for adult Robins when brain tissue
is used as a criterion. Based on the quantities of DDT
determined in the brain of tremoring adult Robins, results
from this study indicate that the highest number of Robins
found dying of tremors had concentrations of DDT ranging
from 50 to 120 ug. of DDT/g. in the brain. Since the lowest
concentration of DDT found in a tremoring Robin was 53 us. of
DDT/g. in the brain, it appears that adult Robins found dead
or dying with levels of DDT greater than 50 ug./g. in the
brain died as a direct result of exposure to this compound.

Based on this conclusion, the data indicate that from
a total of 70 adult Robins found dead or tremoring in sprayed
areas, 49 of the 54 whose brains were analyzed had 50 or more
pg. of DDT/g. in their brain tissue. This figure indicates
that 90.7 per cent of the analyzed Robins found dead or
tremoring had sufficient quantities of DDT in the brain to

justify the conclusion that they died from its toxic action.

Other Specigg

An additional 52 birds, representing 27 species, were
also analyzed for DDT (see Table 6). Of these, seven birds
were recovered at the base of a television tower in Onondaga,
Michigan, for possible use as uncontaminated controls. 0f

the remaining 45 birds listed, 35 birds of 21 species had at

 

30
least some DDT in their tissues. Most of the specimens were
turned in by staff and students to G. J. Wallace, who sup-
plied the field data included below. The following list
describes the species examined and Table 6 shows the analyti-
cal results obtained.

Mallard (Anas platyrhynchos).~-In May of 1960 a considerable
die-off was observed among the 100 or more semi- 5m
domesticated Mallards resident along the campus portion of

the Red Cedar River. Six specimens, three observed with 4*

 

tremors, were turned in and nine others were reported but
not retrieved. The brain of the only Mallard analyzed had
37 ug. of DDT/g. of tissue.

Ring—necked Pheasant (Phasianus colchicus).——A pheasant, pre-
sumed to have died from crashing into an obstacle, was
picked up at the Frandor ShOpping Center and checked for
DDT. Both breast muscle and brain samples were negative.

Migratory Quail (Coturnix coturnix).--The only quail
analyzed was one of a group kept on campus for experi-
mental use. The bird was found on campus in an eXhausted
condition by the campus police, but was already dead when
turned in. DDT was recovered in the brain and liver, but
not in breast muscle and heart samples.

Mourning Dove (Zenaidura macroura).--Several "sick" or dead
Mourning Doves were found on campus in the spring of 1959,

but none were found in 1960. The "sick" doves did not

31
appear to have typical poisoning symptoms. Of the two
tested, one was negative and the other had 8 pg. of DDT/g.
in the heart, but none in the breast muscle, liver, or
kidney.

Screech Owl (Otus asio).~-0ne reported dying of convulsions
on June 16, 1959, in a heavily sprayed area of East
Lansing had 28 pg. of DDT/g. in the breast muscle and 33
in the heart, but none in the brain or liver. Additional
specimens from the Detroit area have been checked, but
nearly all tests on predatory birds have failed (Wallace,
gt gl., 1961).

Chimney Swift (Chaetura pelagica).--Four emaciated swifts,
found dead on campus in 1960 after a cold rainy period,
were presumed to have starved, but two were tested to see
if they had possibly accumulated DDT. A sample of the
combined brains was negative.

Belted Kingfisher jgggaceryle alcyon).--A "ripe" kingfisher,
found dead by the MSU Library on June 4, 1959, was checked
to see if it had accumulated DDT from fish or other food
in the Red Cedar River. It had low, presumably sublethal,
amounts of DDT in all of the tissues tested.

Horned Lark (Eremophila alpestris).--A fledgling lark,
picked up by Ornithology class students in the vicinity of
recently sprayed elms on April 28, 1960, exhibited typical

symptoms and died within an hour. A brain sample had 46

 

32
pg. of DDT/g. of tissue.

Blue Jay (Cyanocitta cristata).--A dozen or so Blue Jays were
found dead or dying following various local spraying pro-
grams, but only four were tested. Two specimens were
negative, including a young Blue Jay reportedly dying with
tremors (some other poison is suspected). One Blue Jay,
picked up in a campus parking lot (had apparently been
run over), had 87 pg. of DDT/g. in the breast muscle and
127 in the brain. Another specimen from Battle Creek,
Michigan, had DDT in all of the tissues tested.

Black-capped Chickadee (Parus atricapillgg).--Two chickadees
were available for analysis in the fall of 1959. The test
on one failed; the other had 56,pg. of DDT/g. in the
breast muscle, 122 in the heart, and 68 in the liver.

White—breasted Nuthatch (Sitta carglinensis).--Two East
Lansing nuthatches were examined (three others reported
but not retrieved). Both birds tested had DDT in their
tissues.

House Wren (Tgoglodytes ggdgn).--The specimens recovered at
East Lansing included one adult and six young. The car-
cass of the adult wren, picked up on June 18, 1959, had 92
pg.of DDT/g. of tissue. This was believed to be due to
the aerial spraying for mosquitoes in early June, as
foliar sprays were not used on the elms in 1959.

Brown Thrasher (Toxostoma rufum).~-A Brown Thrasher

 

npparvntly took up residence on csmnus in mid-April of
1950 (the first one recorded here in several years). On
April 27 it was observed in labored flight and the follow-
ing day two students reported it ”affected" but still able
to fly. Later in the day it was brought in dead. The re-
sults of the analysis are shown in Table 6.

Swainson's Thrush (Hylocichla ustulata).--Three Swainson's
Thrushes were picked up at the base of a television tower
in Onondaga, Michigan, in early October. These were used
as checks. All three were negative for DDT.

Gray-cheeked Thrush (Hylocichla minima).--Two Gray-checked
Thrushes were also picked up at the base of the Onondaga
television tower for use as checks. Both were negative
for DDT.

Cedar Waxwing (Bombycilla cedrorum).~~An East Lansing spring
specimen (May 1, 1959) was negative and a campus bird
(November 16, 1959) had 8 pg. of DDT/g. in the brain, but
none in the breast muscle, heart, or liver. Waxwings are
believed to accumulate DDT by budding in sprayed elms in
the spring. This conclusion is supported, in part, by a
previous study (Wallace, gt.gl., 1961) in which concen-
trations of DDT were high in two spring specimens, while
two fall specimens (when waxwings are feeding on fruit)
were negative.

Starling (Sturngg vulcaris).-~Several Starlings have been

 

1
A

 

34
available for examination, but not in proportion to the
number exposed to DDT. Starlings appear to be resistant
to DDT, either by ability to avoid it, or the capacity to
withstand high dosages. Of the six examined in this
study, only two had fairly high levels of DDT in their
tissues. The four others were negative or had low quanti-
ties, although they were recovered in heavily sprayed .-

areas.

Black-throated Green Warbler (Dendroica virens).--A specimen

 

(one of six or seven reported dying following the spraying
of elms in Berkley, Michigan, in the spring of 1956) was
sent to MSU in 1956. Several years later the bird was
found in an icebox (kept at about 40 degrees F.). The
dried carcass was analyzed (four years later) and con-
tained 122 pg. of DDT/g. of tissue.

Ovenbird (Seiurus aurocapillus).--Both Ovenbirds analyzed
in this study were negative. One was used as a control.
Eastern Meadowlark (Sturnella magna).--A specimen found dead
on the Red Cedar School lawn in East Lansing on June 12,

1959, several days following aerial spraying for mos-
quitoes, had 40 pg. of DDT/g. in its breast muscle, 28 in
the heart, and 128 in the liver.

Red-winged Blackbird (Agelaius phoeniceus).--A tremoring
red-wing from Oakland County, Michigan, had 157 MS. of
DDT/g. in breast muscle and 98 in the heart.

Baltimore Oriole (Icterus galbula).--Three specimens examined.

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eatemner

The carcass was t-
DDT/g. of tissm
.

13'

41 a
' cl I

' I- a .
~*-h—.ww‘.-‘J.C->- u...-

’ A - . V u u‘ ’.‘e
1 I .1 ,1.. g 1 -‘ .
‘11 \l . ‘x‘ 4 \JLA

osted for DDT, but it had only

 

35

leged fly on Haslett Road, East Lansing,
f 1959, was negative. An East Lansing

l foand deal following aerial treatment

r mosquitoes, was much emaciated (may

1 p3. of DDT/g. the era in aid 41
The third specimen,from Sylvan Glen,
dying on a golf course near sprayed

102 pg. of DDT/g. in the breast muscle

 

 

).
of DDT in

e
,"11 9“ n 1 r
.‘I. l. :1 .1 .51 1. :1.
w _l‘--“.‘A‘_

”“Of the fOLlI‘ 81.5.10 Lles

i;ifi1.letfls their brains

negative. Like the Starling, these

in heavily sprayrnl areas.

ter).w~A specimen, unable

—--

/.,.__ fl ,
(nuluiiru s

tbs r.a:>le remqrs, was picked up on

21, 1959, and died overnight in a cage.

1 pg. of

).m~Five of the six Cardinals

analysed had high Te els of DDT in their tissues. Other
Cardina-s Hare nee« lwcp )rte ed dying in DDTwsprayed areas,

they may be exposed to the toxicant

1taminated prey, or even grit

 

. .. . ‘ ,. r ..
UHOG“ t-c elms (Wallace, et al., 190-).
Grasshogpcr Sparrow (A; 1od1c1us savannargg).~uA tremoring

 

1
i

 

36
(exhausted ?) bird, picked up on Point Pelee, Ontario,
Canada, on May 7, 1960, presumably after a long flight
across Lake Erie, had 17 pg. of DDT/g. in its carcass.
Field Sparrow (Spizella pusilla).--One picked up at the base
of a television tower in Onondaga, Michigan, for use as a

control, was negative.

 

 

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AemscfipsOOV c 22229

39
Availabilit1_of DDT to Birds
From the wide variety of feeding habits represented
by the different species of birds found to contain some DDT
(at least in a few tissues examined), it becomes apparent

that a number of possible sources of contamination in the

diet must exist. Barker (1958) has already pointed out that

earthworms build up and store DDT residues by feeding on
leaf litter and then serve as a source of contamination to
birds (particularly Robins) which consume earthworms in the
spring. Unfortunately, stomach analyses to determine the
source of contamination yield inadequate results in most
cases. Most birds found dead or dying in sprayed areas had
little or no food in their stomachs. In some instances (as
With Robins) the birds feed on material which is readily
digested or quickly eliminated. It is quite probable also
that after the onset of tremors, the affected birds stOp
feeding and may not ingest any food for the duration of the
tremoring period, which may extend over a period of several
hours, or even days.

Included among the few items found in the stomachs
of dying birds were ants, a number of maple pods, cherry
seeds, parts of a dragon fly, a few unidentified crickets,
some plant materials, corn, and numerous stones of various
sizes.

During application, quantities of DDT are broadcast

over a substantial area surrounding the elms being sprayed.

 

4O
Spraying of areas of high elm density may lead to complete
coverage not only of the elms, but also most of the surround-
ing ground area and shrubbery. Therefore, many foods of birds
having territories or feeding in heavily sprayed areas would
be contaminated with DDT. Since so few reports of bird
mortality have come from the eastern part of the United
States, where elms occur at a much lower density than in the
Middle West (Wallace, et al,, 1961), it appears that smaller
numbers of birds are fatally affected by the spray programs
in areas with few elms.

The following samples were randomly collected in
known spray areas and examined for DDT.

A sample of grass collected under a sprayed elm imme-
diately after spraying contained 5.3 ppm of DDT.

A soil sample, collected at a .25" depth in December
of 1959 in an area sprayed earlier in the fall, contained
48 ppm of DDT.

Samples of myrtle leaves,also collected in December
of 1959 in the same area, contained 14 and 18 ppm of DDT,
respectively.

Similarly, a sample of leaf litter, collected at the
same time and place, contained 31.7 ppm of DDT.

Elm bark, collected in December of 1959 from an elm
sprayed earlier in the fall, contained 28 ppm of DDT nearly

three months after the application of the toxicant.

 

41

In the spring of 1960 samples of earthworms (mostly
Lgmbricus terrestris) were collected from various parts of

the campus. The first sample consisted of ten medium-sized

earthworms collected on April 15, 1960. The area had been
sprayed two days earlier. The worms were macerated and ex-
tracted. An aliquot portion of the extract was removed and
analyzed for DDT. Results disclosed that no DDT was present
in the sample. A second sample of earthworms was collected
on the same date on a different area of the campus which had
been sprayed in the fall of 1959 and on which no further
Spraying had been done at the time the sample was taken.
Analysis of an aliquot, taken from the combined extracts of
10 medium-sized worms, yielded no DDT. A third sample of
earthworms, collected in an area intermediate between the
previous two, was collected on April 15, 1960, two days after
spraying had started in the area. An aliquot taken for
analysis from a sample of ten worms ground together contained
25 H8. of DDT/g. of sample. Ten worms, collected on April
30, 1960, in an area sprayed two weeks earlier, contained 86
pg. of DDT/g. of sample. The last sample consisted of a
number of earthworms which were collected on the ground
surface following a heavy rain. Three of the larger speci-
mens from the sample were selected and analyzed separately.

The three worms contained 88, 57, and 831pg. of DDT/g. of

 

42

tissue, respectively. No attempt was made to analyze
tissues separately.

The results from the analysis of the few samples
listed above are not intended to illustrate the build—up of
residues in plant or animal tissues, nor to indicate any
direct source of contamination for any particular species
of bird. Instead these tests were carried out to gain

1mg:

information regarding the persistence and distribution of

A...

the toxicant and its availability to birds.

As is suggested by these results, DDT may be retained
in the soil, leaf litter, bark, and other sites for periods
of time following application in quantities sufficiently
high to allow rapid build-up of the material in the tissues
of birds. The finding of earthworms with no DDT in an area
recently sprayed demonstrates clearly that not all worms are
immediately reached by the compound, but several days to
several weeks may lapse before the material is present in
any quantity in these organisms.

It is not surprising that a number of different
species of birds were found to contain variable quantities
of DDT. The availability of the toxicant makes it diffi-
cult to avoid. Ground-feeding, bark-foraging, and seed-
eating birds are all susceptible to contamination. Aerial
feeders, due to their method of acquiring food, are rela-

tively immune to exposure from ground programs. However,

43
these birds may be indirectly affected by a drop in insects

following applications of DDT.

 

FEEDING EXPERIMENTS

To supplement the information obtained from the
analysis of field specimens, captive House Sparrows were
maintained on diets containing different levels of DDT in
order to measure the lethal and sublethal quantities of
the poison in their tissues.

Unfortunately, earlier attempts to keep adequate
numbers of Robins in captivity were unsuccessful, and House
Sparrows were subsequently selected due to their availability

and to the small amount of care required to maintain them in

captivity.

Methods

Adult House Sparrows were captured around the campus
dairy barns (where DDT is not used) by means of mist nets
and then kept in captivity for at least 10 days before any
tests were conducted. The birds were divided into a number
of groups and were allowed ad libitum access to diets of
chick starter mash containing known amounts of DDT. The DDT
was weighed and added to a previously weighed quantity of
chick starter mash in the desired ratio. During preparation

of the feed DDT was added in dry powder form and blended
with the mash for at least 10 minutes in a Twin Shell dry

blender.

 

 

45

To estimate the quantity of food consumed per day for
each bird, the food wasted, as well as that remaining after a
period of six days, was weighed and subtracted from the
initial amount of food fed to a group of 15 caged birds.
The same procedure was repeated twice more, and the resulting
figures were taken to approximate the amount of food con-
sumed by the 15 birds during that 6-day period (it was not
always possible to recover all of the food wasted and some
of it contained feces Which were difficult to remove
completely). From these figures the amount of food taken in
by each bird per day was roughly calculated to be between
5 and 7 grams.

During the course of the experiment, a few birds from
each group were randomly selected and sacrificed. These

birds were then stored in a deep freeze for future analysis.

Analytical Procedures

Specimens found dead or dying of tremors, as well as
those sacrificed during the experiment, were analyzed by the
methods previously described for field specimens. The brain
of each bird was analyzed separately, in most cases, while
the analysis of the liver, breast muscle, and heart was

carried out for only a few birds of each group.

Initial Feeding Tests

The first feeding tests in the series were started on

 

46
December 7, 1960. Four groups of 15 sparrows each (except
for 16 sparrows in Group I) were placed in four separate
cages and fed on diets of chick starter mash containing 0,
100, 200, and 500 mg. of DDT/Kg., respectively. Group I was
fed a diet containing 300 mg. of DDT/Kg.,Group II on a diet
having 200 mg. of DDT/Kg.,and Group III a diet containing
100 mg. of DDT/Kg. The fourth group received a diet of F.

chick starter mash with no DDT added and served as a control.

 

Group I. The following table (Table 7) summarizes
the results obtained from the analysis of birds fed on a

diet containing 300 mg. of DDT/Kg.

47

TABLE 7

DDT DETERMINED IN SPARROWS FED A DIET CONTAINING
300 ppm OF THE TOXICANT

W

 

 

Specimen Data ing. of DDT/g. of Tissue
* Breast
No. Date Sex Condition Brain Muscle Heart Liver

 

 

s-1 12/14/60 M Sacrificed 43 - - 63
s-2 12/14/60 F Tremoring 108 238 74 -
s-3 12/14/60 M Dead 56 - - -
3-4 12/14/60 M Dead 83 - - -
3-5 12/14/60 F Dead 60 - - -
S-6 12/14/60 F Dead 78 - - -
s-7 12/16/60 M Sacrificed 41 61 87 -
S-8 12/16/60 M Tremoring 70 175 60 -
s-9 12/19/60 M Tremoring 134 205 74 -
s-10 12/19/60 M Dead 139 - - -
S-11 12/19/60 M Tremoring 149 - - -
s-12 12/20/60 F Tremoring 125 - - 367
s-13 12/30/60 M Tremoring 99 - 88 -
s-14 1/ 4/61 F Sacrificed 62 - - -
s-15 1/ 4/61 M Tremoring 181 - 103 460
s—16 1/ 5/61 F Dead 184 - - -

 

*Starting date, December 7, 1960.

The dates listed in the table above refer to the day
on which the birds died. As can be seen, all of the birds
(except for three which were sacrificed) died after a period
of exposure of from 7 to 29 days. The two male House
Sparrows sacrificed after 7 and 9 days of exposure,
respectively, had less DDT in the brain than any of the males
found dead or tremoring. The female House Sparrow (S-14),
sacrificed 28 days after the start of the experiment, con-

tained less DDT in the brain than any of the other females

48
found dead or dying (except for the female House Sparrow,
S-5 Which had 60 pg. of DDT/g. in the brain).

Generally the amount of DDT in the brain increases
with the time of exposure to it. The variation in the
level of DDT in the brain suggests a wide range of
susceptibility. However, none of the birds sacrificed had
more than 62 pg. of DDT/g. in the brain, while few of those
found dead or dying had less than this amount. The few
analyses carried out for liver tissue indicate a similar
relationship between the amount of DDT present in that
tissue and the length of exposure. Analysis of heart
tissue indicates that it is not a good criterion in relating
the dosage in tissues to the cause of death, since one of the
sacrificed birds contained more DDT in the heart than that
found in three of the birds with tremors, and only 1 pg. of

DDT/g. less than a fourth tremoring House Sparrow.

Group II. Table 8 lists the results obtained from the
analysis of birds maintained on a diet containing 200 mg. of
DDT/Kg. of food. These birds started dying after 22 days of
exposure, compared to 7 days for birds fed on a diet having
300 mg. of DDT/Kg. Again, brain analyses of birds found dead
or dying in this group yielded higher results than in those
sacrificed. The quantities of DDT found in the brain of
these birds were generally greater than the quantities

determined in House Sparrows of Group I. It appears that

 

49
the quantity of DDT present in the brain increases with the
time of exposure.

Male and female House Sparrows sacrificed during the
experiment had 58‘pg. or less of DDT/g. in the brain; whereas,
the brains of all the birds found dead or dying contained 85
or more pg. of DDT/g.

At the 200 mg./Kg. level, a total of 49 days of ex-

posure was required to kill all of the birds.

 

 

TABLE 8

DDT DETERMINED IN SPARROWS FED A DIET CONTAINING
200 pan OF THE TOXICANT

 

Specimen Data

 

Breast

 

No. Date* Sex Condition Brain Muscle Heart Liver
s-17 12/16/60 M Sacrificed 37 0 0 -
s-18 12/29/60 M Dead - - - -
s-19 12/30/60 M Dead 134 - - 139
s-20 12/30/60 M Dead 132 - - -
S-21 12/30/60 M Dead 154 - - -
s-22 12/31/60 M Dead 119 - - -
s-23 12/31/60 M Tremoring 127 - - -
S-24 1/ 4/61 F Sacrificed 58 - - 49
s-25 1/ 5/61 F Dead 200 - 60 -
s-26 1/ 6/61 M Tremoring 136 - - -
s-27 1/ 6/61 F Tremoring 120 - - -
S-28 1/ 6/61 F Tremoring 150 - - -
S-29 1/ 6/61 F Tremoring 158 - - -
S-BO 1/ 6/61 M Tremoring 85 243 103 -
3-31 1/25/61 F Dead 124 - - -

 

*Starting date, December 7, 1960.

ing 100 mg. of DDT/Kg.

Group III.

Theoretically,

Birds in Group III were fed a diet contain-

this amount represents

 

.l...‘ 14le

 

50
the chronic level of DDT intake for the species under con-
sideration. As can be seen from the following table (Table
9), 41 days of exposure were required before the first bird
died. Two of the birds (both females) were still alive
after more than 90 days of exposure, which was the cut-off
date predetermined for the experiment.

Theoretically, it is assumed that these birds would
not have died from poisoning. As with the two previous
groups fed DDT, none of the birds sacrificed during the
course of the experiment contained more than 62 (maximum 48)
pg. of DDT/g. in the brain, while those found dead or dying
had more than this amount. Again, analysis of heart tissue
revealed that some tremoring birds had less DDT present in
heart tissue than that found in some of the House Sparrows
which were sacrificed.

It is interesting to note that the amounts of DDT
present in the brains of House Sparrows fed 100 mg. of
DDT/Kg. were comparable to those found in the brains of
birds fed BOO mg. of DDT/Kg. This strongly suggests that at
chronic levels, the amounts of DDT found in the tissues are
not necessarily related to the time of exposure as seems to
be the case at higher exposure levels.

Brain analyses of the last three birds sacrificed in
this group, especially S-45 and S-46, yielded levels of DDT

comparable to the quantities found in the brain of birds

 

 

IEAIIMAIIMA‘

11 T. lrll‘lll ‘11.:

 

 

51
sacrificed much earlier in the test. These results demon-
strate that some birds are able to eliminate DDT from their

tissues at the lower exposure levels, or else they fail to
absorb as much of the toxicant. However, although it is
conceivable that birds would show wide variation in the
amount of DDT which is absorbed from the intestine, it seems
logical to assume that some of the birds fed at higher
levels should also absorb it slowly and that these would
have been able to survive beyond the 90 day cut-off period.
Unfortunately, incomplete data on the rate of absorption of
DDT make it impossible to elucidate this point further. On
the other hand, if birds are able to metabolize the compound
to some other form, this derivative should be present, at
least in some cases, in their tissues. In order to check
this latter view, separation of DDT from DDE (which is

known to be one of the major breakdown products of DDT) was
attempted by the method outlined in the laboratory methods
for field specimens. The brain of House Sparrow S-36 had 67
pg. of DDE/g., while s-37 contained 13. House Sparrow s-38
had 22 pg. of DDE/g. of tissue, while s-39 had DDT but no
DDE present. Similar analysis of the male bird, numbered
S-44, yielded no DDE in the brain tissue. Since the number
of birds analyzed was so small, and the results so variable,
no definite conclusions may be drawn except that in some

cases, at least, DDT may be broken down to DDE in variable

ilflJlnflthxn.‘

 

52

quantities and might be a factor in longer survival.

TABLE 9

DDT DETERMINED IN SPARROWS FED A DIET CONTAINING
100 ppm OF THE TOXICANT

 

 

13":

 

 

ugi_or DDT/g. of Tissue
Breast

Specimen Data

 

No. Date* Sex Condition Brain Muscle Heart Liver
3-32 12/15/60 F Dead 25 - - -
s-33 12/16/60 F Sacrificed 59 - - -
s-34 1/ 4/61 M Sacrificed 48 - - 27
s-35 1/15/61 M . Sacrificed 47 51 87 76
S-36 1/17/61 F Tremoring 105 - 64 -
8-57 1/17/61 M Dead 67 - - -
5-38 1/18/61 M Dead 81 - - -
S-39 1/24/61 M Tremoring 72 233 64 -
S-4O 1/24/61 M Tremoring 64 - - -
s-41 1/25/61 M Dead 98 - - 157
s-42 1/25/61 F Dead 87 - - -
s-43 1/31/61 F Dead 91 - - 415
s-44 2/14/61 M Sacrificed 50 - - 57
s-45 3/ 9/61 F Sacrificed 15 95 211 149
s-46 3/15/61 F Sacrificed 17 95 200 43

 

W
*Starting date, December 7, 1960.

Group IV. The last group of 15 birds served as a con-

trol. Birds in this group were fed the same diet as those
in the other three groups except that no DDT was added to
the mash. None of the controls died during the course of

the experiment.

Effects of Starvation*

In conjunction with the experiments outlined above, 20

*Starvation, as used in these experiments, means de-
prived of food for short periods of time.

53
House Sparrows were divided into two groups of 10 birds each.
The birds in Group I were maintained on a diet containing
300 mg. of DDT/Kg. for 6 days and then starved. The birds
in Group II served as controls and were kept on a DDT-free
diet for the same length of time and then starved. As with
all of the birds used in these experiments, the birds found
dead or dying were analyzed in the same manner as outlined

previously.

Group I - FedeDT and then starved. All of the birds
exposed to DDT in this group died when they were subjected
to starvation. Eight of the birds were dead following 16
hours of starvation, but two lived for an additional hour.

The following table (Table 10) summarizes the re-
sults obtained from the analysis of tissues of the birds

from Group I.

 

:1
3‘1
' F

 

54
TABLE 10

DDT DETERMINED IN SPARROWS FED DDT AND THEN STARVED

 

 

 

 

Specimen Data ug. of DDT/g. of Tissue#_
Breast

No. Date Sex Condition Brain Muscle Heart Liver
S-47 2/7/61 F Tremors 112 238 - -
S-48 2/7/61 F Tremors 139 - - 302
s-49 2/7/61 F Tremors 103 - - 265
s-50 2/7/61 F Dead 11o - - 454
s-51 2/7/61 F Dead 122 165 75' -
s-52 2/7/61 M Dead 114 191 - 526
s-53 2/7/61 M Dead 12o - - 465
s-54 2/7/61 M Dead 89 104 - 210
8-55 2/7/61 M Dead 104 148 75 248
S-56 2/7/61 M Dead 99 -' - 255

W

As may be seen from the results, the quantities of DDT
found in the brains of these birds compare favorably with
those found in birds in the other experiments. In all cases,
levels greater than 88 pg. of DDT/g. of tissue (minimum 89)
were found in the brain. The quantity of DDT found in the
heart was less than that found in some birds sacrificed in
the previous experiments.

The reduction in exposure time required to kill birds
brought about by starvation indicates that DDT is evidently
released from less sensitive areas to more vital sites
during starvation. Birds fed the same level of DDT, but not
starved, were able to survive for as long as 29 days in some

cases.

 

55
Group II - Controls. None of the birds in the control
group died during the 17-hour period of starvation to which

they were also subjected.

Elimination of DDT

To obtain additional information regarding the elimi-
nation of DDT in birds, 20 House Sparrows were divided into
two groups of 10 birds each and kept in separate cages.
Birds in Group I were fed a diet containing 500 mg. of DDT/
Kg. for 6 days and 18 hours, then put on a DDT-free diet.
Following this, one bird from the group was sacrificed each
week for the next 4 weeks, a fifth 2 days later, and another
4 days after the fifth bird. The table below (Table 11)

summarizes the results of the analysis of these birds.

TABLE 11
DDT DETERMINED IN SPARROWS FED PARTIAL DDT DIETS

 

 

 

 

Specimen Data __ngi of DDT/gL of Tissue
Breast

No. Date Sex Condition Brain Muscle Heart Liver
S-57 2/7/61 F Tremoring, 93 303 158 -
S-58 2/7/61 F Tremoring 99 188 166 189
S-59 3/7/61 M Tremoring 79 - 150 528
s-6o 2 14/61 M Sacrificed 9 14 25 27
s-61 2/24/61 F Sacrificed 15 6 23 55
S-62 2/28/61 M Sacrificed 9 6 18 2o
s-65 5/7/61 F Sacrificed 4* 1o 0 o
S-64 3/8/61 M Tremoring 32 76 O 52
5-65 3/9/61 F Sacrificed o o o o
S-66 3 13/61 F Sacrificed O 10 O O

 

 

 

—-

*Starved for 22 hours following 28 days on a DDT-free
diet. It appears that some factor other than direct DDT-
poisoning may have been involved.

 

56

Group I - Fed_partial DDT diets. From the table, it
may be seen that three of the birds died with tremors after
the initial 6 days and 18 hours on the DDT diet, which is
quite comparable to the results obtained earlier for birds
exposed for 7 days on the same diet.

Subsequent tests on birds, which were maintained on a
DDT-free diet following an initial 6-day exposure to it, show

a marked decline in the amount of DDT in their tissues.

 

House Sparrow S-66 contained no DDT in its tissues except
for 10 pg/g. in the breast muscle. After 28 days on a DDT-
free diet, birds numbered S-64 and S-65 were assumed to con-
tain little or no DDT in their brain tissues and were
starved. The male bird, S-64, died with tremors after 22
hours of starvation, while the female, S-65, did not die
after 30 hours of starvation. Following the 30-hour period
of starvation, the remaining female was placed on a DDT-free
diet for 18 hours and then sacrificed. As indicated by the
table, the male bird, S-64, contained more DDT in its
tissues than any of the other sacrificed birds analyzed.
Analysis of tissues from the female House Sparrow, S-65,
shows that no DDT was present in the tissues tested.

A number of interesting considerations are suggested
from the results of this experiment. In the first place, it
seems reasonable to state that, after exposure to DDT, birds

are able to eliminate the toxicant from their tissues or to

57
redistribute it to some other part of the body where it may
be stored in a passive manner. The first idea was tested,
in part, by separating the DDT from DDE in the brain tissues
of the first three birds sacrificed. No DDE could be de-
tected in the brain of any of the three birds checked in
this manner. This does not eliminate the possibility that
DDT was converted to some other analogue which was not de-
tectible by the analytical methods employed, or that the
converted DDT had been eliminated before the analyses were
conducted. Another possibility is that tissues other than
the brain are able to break down the DDT to DDE. The fact
that one bird died of tremors following a period of starv-
ation indicates that, at least in some cases, the DDT is
still present in the body and is released to more sensitive
tissues during starvation. This latter idea requires the
assumption that the brain contained little or no DDT prior
to the starvation period. Results from the analysis of the
tissues of all other sacrificed birds tend to support this

assumption.

Group II - Controls. The birds in Group II in this
experiment were used as controls and fed on a DDT-free diet
throughout the experiment except for a period of 30 hours
when they were starved to correspond with the starvation

periods described above. None of the controls died.

 

58

Relationship of Starvatipp to Exposure Time

As outlined earlier, subjecting birds to a period of
starvation following six days of feeding on a diet contain-
ing 300 mg. of DDT/Kg. resulted in the death of all the
birds. To determine the effect of starvation on birds fed
at the same level for shorter periods of time, 12 sparrows
were separated into two groups and subjected to the follow-

ing conditions.

Group If: Starved between feeding_period§. Group I
consists of five birds which were fed a diet containing 300
mg. of DDT/Kg. for three days and then starved for 19 hours.
At the end of the 19-hour period of starvation, one bird
died. Following this, the four remaining birds were again
fed on a diet containing 300 mg. of DDT/Kg. for 24 hours.

A second bird died in tremors during this period. After ex-
posure to DDT for four days, the three remaining birds were

again starved for 21 hours, during which time another died.

The second starvation period was followed by 24 hours on the
DDT diet. The two remaining birds were starved for a third

time and died during the following 16 hours.

The following table (Table 12) gives the results of
the analysis of the five birds kept on a DDT-diet alter-
nating with periods of starvation. The birds are listed
in the order in which they died.

 

59
TABLE 12

DDT DETERMINED IN SPARROWS ALTERNATELY FED ON DIETS
CONTAINING DDT AND STARVED

 

 

man— M
specimen Data pg! of DDT/g. of Tissue
Breast
No. Date Sex Condition Brain muscle Heart Liver

 

Tremoring 89 120 58 158

s-67 2/14/61 M

S—68 2/14/61 F Tremoring 93 - - -
S-69 2/16/61 M Tremoring 1OO - - 242
s-7o 2/17/61 F Tremoring 115 - - -
s-71 2/18/61 M Tremoring 155 557 208 576

 

 

_—
—-—:

The results indicate that the birds contained quanti-
ties of DDT in the brain comparable to those found in
tremoring birds in the other experiments. The amount of
DDT present increases with the increase in exposure time.

It is interesting to note that periods of starvation showed
a marked effect in the reduction of time required for
tremors to appear in poisoned birds.

Since it is already well known that DDT is stored in
fat tissue, it is logical to assume that DDT is released to
more sensitive areas during periods of starvation. Starving
birds for short periods of time following periods of expo-
sure to DDT prevent build-up of fat reserves and simul-
taneously reduce the amount of DDT stored passively. It
seems logical to assume, therefore, that DDT is retained for

fairly long periods of time in the fat depots, and that the

toxicant may be released to more sensitive areas during

6O

periods of starvation.

Group_II - Controls. This group contained seven
House Sparrows which served as controls. All sparrows in
this group were subjected to the same conditions outlined
for the first group described above, except that they re-
ceived no DDT in their diet. None of the birds in this

group died during the experiment.

Release of DDT from Fat Depots
The importance of fat depots as a source of stored
DDT suggested the following experiment in which 10 sparrows

were separated into two groups of five birds each.

Group I - Fed initia;_DDT;diet. Birds in Group I were
fed a diet containing 500 mg. of DDT/Kg. of food for six days
followed by a DDwaree diet for 10 days. Following this 10-
day period, two of the birds were sacrificed and the remain-
ing three were starved for 29 hours.

As may be seen from the results expressed in Table 13,
the two birds sacrificed before starvation had only small
amounts of DDT in their tissues as was expected from the re-
sults found in a similar experiment previously described.
Following starvation there was a marked increase in the
quantity of DDT found in the tissues of the birds. All three
House Sparrows were observed tremoring before they died.

The levels found in these birds are fairly comparable to the

 

61

quantities determined previously in other tremoring birds.

In this case, the submcutaneous fat of the two birds sacri-
ficed before the period of starvation contained 780 and 950
ug. of DDT/g. of fat. Of course, the birds which were starved
had no sub-cutaneous fat present and, therefore, no fat
analysis could be made. These results strongly support the
view that DDT stored in fat tissue is released during
starvation and is transferred to vital organs.

It was also noted, during the course of these experi-

 

ments, that not only the starved birds, but also tremoring
House Sparrows had little or no submcutaneous fat present.
It appears that the violent tremors observed prior to death
in cases of poisoning increases the energy expenditure of
the bird and leads to a rapid utilization of stored fat
tissue.
TABLE 13
DDT DETERMINED IN SPARROWS FED INITIAL DDT DIETS

 

 

 

 

 

Specimen Data _4ug. of DDT/g. of Tissue__
. Breast

No. Date Sex Condition Brain Muscle Heart Liver
s-72 5/6/61 M Sacrificed o 15 - 11
s-75 5/6/61 F Sacrificed 4 15 - o
S-74 3/7/61 F Tremoring 65 97 - -
S-75 3/7/61 M Tremoring 73 - - 177
S-76 3/7/61 F Tremoring 60 104 O 96

 

—_ w."— 1—

62

The fact that the last three sparrows died in tremors
following starvation after 10 days on a DDT-free diet also

indicates that the poison is not readily eliminated from fat

depots.

Group II - Controls. The five sparrows in this group
served as a control, and were fed a DDT-free diet for 16
days followed by 29 hours of starvation. None of the con-
trols died during the experiment.

At the termination of this experiment, the five check
birds were sacrificed. Analyses of brain, liver, heart,
breast muscle, and adipose tissue in these birds were all

negative.

Levels of DDT Stored in Fat Tissues

In order to acquire some estimate of storage levels in
fat depots of House Sparrows fed sublethal quantities of DDT,
the subcutaneous fat from sacrificed birds fed in previous
experiments on diets containing 100, 200, and 300 mg. of
DDT/Kg., respectively, was analyzed. It was found that some
of these birds, at all levels, had quantities of DDT ranging
as high as 800 pg./g. in the fat tissue. Since these birds
were apparently normal (as far as their activity was con-
cerned), it seems evident that even at high levels of storage,
DDT in adipose tissue apparently is passively stored and

causes no obvious symptoms in birds. From these results,

 

63
it is also concluded that analyzing whole carcasses of birds
for insecticidal content would take into account residues in
adipose tissue, if present, as well as unabsorbed material
in the digestive tract. Therefore, it would seem that accu-
rate appraisal of DDT poisoning by means of chemical analysis
to determine quantities of DDT present in tissues should be
based on individual tissue analysis rather than entire car-

08.8888.

Effects of Feedinngabits

 

Considering that it required at least seven days on a
constant DDT diet at the 500 mg. of DDT/kg. level before
tremors appeared, and that starvation can reduce the period
of exposure by releasing DDT stored in fat tissues to more
sensitive areas, a rather limited attempt was made to deter-
mine what would occur in cases where birds were fed on
alternating diets of DDT-contaminated food and DDT-free food.
Unfortunately, spraying of DDT for Dutch elm disease was al-
ready in progress in the area, and in order to avoid the
possibility of using recently contaminated birds, this experi-
ment was limited to six House Sparrows still available from
the stock used throughout the winter. Consequently, the six
sparrows were separated into two groups of three birds each

and subjected to the following conditions.

 

Group I - Fed alternating diets. The first three birds

 

64

were fed on a diet containing 300 mg. of DDT/Kg. for one day,
followed by a DDT-free diet on the second day, and this
alternation of diets was continued throughout the experiment.
On the second day of the test, one of the birds died from
causes (undetermined) other than DDT poisoning. Unfortu-
nately, this reduced the group to only two birds. With
alternating diets, tremors first appeared in a male bird on
March 13, 1961, 31 days following the start of the experi-
ment. The second bird (a female) showed signs of excita-
bility for two days prior to the appearance of tremors and
died after being exposed for 35 days.

Both birds were sacrificed as soon as possible after
the appearance of vigorous tremors, and it was noted that a
small amount of subcutaneous fat tissue was present in each
case. Subsequent analysis showed that the male bird con-
tained 82 pg. of DDT/g. of tissue in the brain, 252 in
breast muscle, 157 in the heart, and 234 in the liver. The
female House Sparrow had 163 pg. of DDT/g. in the brain, 353
in breast muscle, and 648 in the liver. The results ob-
tained from the analysis of these two birds are not in- I
cluded in Tables 16 and 19.

Considering that only two birds were used in this
test, any suggestions indicated by the results must be viewed
with reservation. However, the presence of at least some fat

in the two tremoring birds helps to support the View that

 

65
subcutaneous fat depots are depleted during tremors. The
results also suggest that birds feeding consistendy on
contaminated food are unable to metabolize the toxicant
fast enough and are less likely to resist poisoning, while
birds feeding on both contaminated and uncontaminated food
would be less affected by poisons.

Under natural conditions, a bird utilizing a wide
feeding range, as well as one feeding on a wide variety of
food items, would be much less affected by a spray program
than a bird limited to some particular food item in a single
area for some period of the year. The fact that Robins, for
example, feed almost exclusively on earthworms (which have
been shown to accumulate DDT - Barker, 1958) during the
spring months, and that they utilize a small feeding area
(a single lawn in some cases) would make them especially
vulnerable in areas which have been sprayed. On the other
hand, House Sparrows, Starlings, and Common Grackles, which
are not so limited in their food selection or feeding areas,
may not be as adversely affected, even in highly sprayed
areas. If an avoiding mechanism is included among
resistance factors, then these particular birds would appear

much less susceptible to DDT poisoning than Robins.

Group II - Controls. The three birds in Group II
served as a control, and were fed on a DDT-free diet during

the time of the experiment. None of the control birds died

66
during the test.

Levels_p§_QDT in Non-captive House Sparrows

To compare the results from the analysis of captive
House Sparrows to quantities of DDT in House Sparrows found
in the field, 14 sparrows found dead or dying with tremors
were analyzed for DDT. The following table summarizes the
results obtained from the analysis of these birds, as well

as of one which was trapped.

 

67

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Qmwmhm COfipHUQOU mwwg

 

 

 

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68

The results indicate that all of the tremoring House
Sparrows found on the MSU campus had DDT concentrations in
their brain comparable to those found in experimentally
poisoned birds. The House Sparrow (S-80) which was trapped
off campus, had much lower quantities of DDT in its tissues
than any tremoring bird examined. Concentrations of DDT in
the brain of most specimens found dead also compare
favorably to those found in experimentally poisoned birds.
The data indicate that chemical analysis of brain tissues
constitutes a valid criterion in attributing bird mortality

to DDT poisoning.

ANALYSIS OF RESULTS

The data accumulated from the analysis of Robins and
House Sparrows demonstrate a wide variability in the concen-
trations of DDT in different tissues in the same bird as
well as in different species of birds.

Amounts of DDT in the heart of tremoring male Robins
. ranged from 10 to 257 pg. of DDT/g. of tissues; whereas, in
tremoring female Robins values for the same tissue ranged
from 25 to 263 pg. of DDT/g. One apparently normal Robin,
trapped in August of 1960, contained 11 pg. of DDT/g. 1n the
heart. Similar comparisons in the heart tissues of House
Sparrows, based on tremoring birds, show a range of from 10
to 208 pg. of DDT/g. of tissue in males (with the exception
noted below) and from O to 166 in females. One male House
Sparrow (S-64) had 32 pg. of DDT/g. in the brain, 76 in
breast muscle, 0 in the heart, and 52 in the liver. However,
it is not included in this discussion, due to the experimental
conditions to which it was subjected. Concentrations of DDT
found in heart tissues of male and female House Sparrows
sacrificed during these experiments ranged from O to 87 pg.
of DDT/g. in males and from O to 211 in females. DDT levels
in the heart of two sacrificed male House Sparrows were
greater than those found in 6 of 11 tremoring male birds.

Further, the level of DDT found in the heart of two sacrifiCed

 

 

70
female House Sparrows was greater than that found in any
tremoring female House Sparrows. These data clearly indicate
that concentrations of DDT found in the heart of dead or
dying birds do not constitute valid criteria in relating
bird mortality to DDT poisoning.

Similar comparisons for concentrations of DDT in the
breast muscle of tremoring male and female Robins show a

range of 11 to 264 pg. of DDT/g. in males and 29 to 205 in

 

females. Birds of both sexes trapped as controls had lower
concentrations of DDT present in the breast muscle. For
House Sparrows, quantities of DDT in the breast muscle of
tremoring males ranged from 120 to 357 pg. of DDT/g. of
tissue and in females from 97 to 303. Analysis of breast
muscle samples of sacrificed male and female House Sparrows
gave results ranging from 0 to 61 pg. of DDT/g. of tissue
in males and from O to 95 in females. These results indi-
cate that sacrificed sparrows generally had smaller amounts
of DDT in breast muscle than tremoring birds.

Liver concentrations in tremoring male Robins ranged
from 0 to 335 pg. of DDT/g. of tissue as compared to a
range of 77 to 486 in tremoring females. One male trapped
in the field had 12 pg. of DDT/g. in the liver. Quantities
of DDT recovered in the liver of tremoring House Sparrows
ranged from 63to 576 pg. of DDT/g. in males and from 96 to
367 in females. Levels of DDT in the liver of sacrificed

71
male House Sparrows ranged from 11 to 76 pg. of DDT/g. of
tissue while females had from O to 149. It appears that the
analyses of liver samples yield results which are not con-
sistent enough to ascribe bird mortality to DDT poisoning.

Levels of DDT in the brain of tremoring male Robins
ranged from 62 to 175 pg. of DDT/g. and from 55 to 115 in
tremoring females. Concentrations of DDT in the brain P‘
tissue of two trapped male Robins were 16 and 29 pg. of DDT/g.,

respectively, while one trapped female had 13. Quantities .5!

 

in the brain of tremoring male House Sparrows ranged from 60
to 286 pg. of DDT/g.; whereas, females had from 95 to 150.
Concentrations of DDT in the brain of sacrificed male House
Sparrows ranged from 0 to 50 pg. of DDT/g. and in females
ranged from O to 62.

In general, these comparisons support the conclusion
that levels of DDT in the breast muscle and brain tissues
can be used in relating bird mortality to DDT poisoning.
However, the association of tremors with poisoning suggests
that of the two,amounts in the brain may be the more dependable
criterion.

The wide variation in levels of DDT determined in
various tissues of male and female birds of both species
indicates wide variability in lethal levels between
individuals, regardless of sex.

Generally, however, the collective levels of DDT,

72
expressed in terms of average concentrations found in the
tissues of tremoring female Robins, were greater than those
found in males for all tissues tested except breast muscle.

Similar comparisons between tremoring male and fe-
male House Sparrows indicate that males are able to tolerate
higher levels of DDT than females in all tissues tested ex-
cept for heart tissue.

The following tables list the mean concentrations,
sample size (N), and range of DDT computed from the tremor-

ing Robins and House Sparrows for the various tissues tested.

TABLE 15

AVERAGE CONCENTRATION AND RANGE OF DDT (in pg./g.)
IN TREMORING ROBINS

 

Tissue N Male N Female Average Range
Brain 11 92.36 6 95.00 93.29 53-173
Breast muscle 15 118.06 8 111.37 115.73 11-264
Heart 15 140.26 8 150.50 143.82 25-263
Liver 15 174.86 8 215.00 187.08 0-486

-_—* _:—. ————_*—~_—.——
-—- -— m

 

73
TABLE 16

AVERAGE CONCENTRATION AND RANGE OF DDT (in pg./g.)
IN TREMORING HOUSE SPARRows

W

Tissue N Male N Female Average Range

 

Brain 17 117.64 18 107.16 112.25 60*-286
Breast muscle 8 208.12 7 188.42 198.93 97-357
Heart 11 88.27 6 89.00 88.52 0-208
Liver 9 270.33 6 234.83 256.13 63-576

W
1eHouse Sparrow, S-64, found tremoring, had 32 pg. of

DDT/g. in the brain, but it is not included in the table due
to the experimental conditions to which it was subjected.

The apparent differences suggested by these results
were clarified, to some extent, by statistical analyses using
the "Student's t" distribution. The differences in the
concentrations of DDT between males and females were not
significant at the 1 per cent level.

Comparison of means between brain levels of DDT in
experimentally poisoned Robins and tremoring field-
recovered Robins of both sexes was also computed using the
"Student's t" distribution. Results indicate that no
significant difference exists at the 1 per cent level.

Similar comparisons between concentrations of DDT
in tremoring male and female Robins and House Sparrows with

the concentrations determined in the tissues of sacrificed

birds of the same Species was also computed by means of the

74

"Student's t" distribution. The differences in the concen-
trations of DDT determined were significant at the 1 per
cent level for all tissues tested except for heart levels
between tremoring and sacrificed House Sparrows of both
sexes.

The following tables list the mean concentrations,
sample size (N), and range of DDT computed from the six

sacrificed Robins and 20 sacrificed House Sparrows.

TABLE 17

AVERAGE CONCENTRATION AND RANGE OF DDT (in.pg./g.)
IN SACRIFICED ROBINS“

W

 

 

 

Tissue N Male N Female Average Range
Brain 4 11.25 2 6.50 9.66 0-29
Breast muscle 4 3.00 2 4.50 3.50 0-9
Heart 4 2.75 2 0.00 1.83 0-11
Liver 4 3.00 2 0.50 2.16 0-42
M M

 

*Includes one male trapped in June of 1960, a male and
female trapped in August of 1960, and one female and two
males shot in April of 1961.

 

75
TABLE 18

AVERAGE CONCENTRATION AND RANGE OF DDT (in pg./g.)
IN SACRIFICED HOUSE SPARRows*

WII—II—Iw

 

Tissue N Male N Female Average Range
Brain 10 28.40 10 21.20 24.80 0-62
Breast muscle 6 20.83 7 32.71 27.23 0-95
Heart 6 38.83 6 72.33 55.58 0-211
Liver 8 35.37 8 34.50 34.93 0-149

 

*Sacrificed during the feeding experiments.

Levels of DDT determined in the brain of sacrificed
House Sparrows fed diets containing 100, 200, and 300 mg. of
DDT/Kg., respectively, and not subjected to starvation or
DDT-free diets during the experiment were also significantly
different at the 1 per cent level from levels determined in
the brain of tremoring House Sparrows.

Additional comparisons between quantities of DDT
found in the brain of tremoring male and female Robins with
levels determined in the brain of dead Robins of both sexes
Show that no significant difference exists at the 1 per cent
level. Similar comparisons between quantities of DDT in the
brain of tremoring House Sparrows of both sexes with levels
in the brain of male and female House Sparrows found dead
also Show that no significant difference exists at the 1 per

cent level. These computations show that a large number of

76
Robins and House Sparrows found dead had levels of DDT in
the brain comparable to those found in tremoring birds and
strongly suggest that they also died as a direct result of
the toxic action of the chemical.

The importance of the brain in attributing bird
mortality to DDT exposure makes it advantageous to demon-
strate what relationship exists, if any, between levels of
the toxicant in the brain and quantities in other tissues of
the same bird. This relationship was investigated by the
Pearson Product Moment Correlation Coefficient. These
correlations were significant at the 5 per cent level only
between brain and heart in male Robins, and between brain
and liver in female House Sparrows. The actual correlation

coefficients (r) and sample sizes (N) are shown in Table 19

 

 

 

 

 

 

 

below.
TABLE 19
CORRELATION COEFFICIENT AND SAMPLE SIZE FOR
ROBINS AND HOUSE SPARROWS
Male Female
N_ r N_ r

Robins

Brain-Breast muscle 11 -.11 6 -.O6

Brain-Heart 11 .77* 6 .15

Brain-Liver 11 .04 6 .37
House Sparrows

Brain-Breast muscle 9 .23 7 .73

Brain-Heart 12 .00 6 .50*

Brain-Liver 9 .48 6 .90

W
*Significant at the 5 per cent level.

GENERAL DISCUSSION AND CONCLUSIONS

The evidence presented in this study indicates that
there is a considerable specific as well as individual vari-
ation among birds in their susceptibility to DDT. Part of
this apparent variation, especially at the specific level,
may be due to rate of accumulation rather than to suscepti-
bility.

Results from the analysis of Robins found dead or
dying in areas sprayed with DDT showed that 90.7 per cent
had sufficient quantities of DDT in the brain to justify the
conclusion that they died from exposure to it. In all cases,
Robins found tremoring in sprayed areas had DDT residues in
their tissues, while only one of the birds found dead was
completely free of it in all of the tissues tested. It
appears, at least in Robins, that it is very unlikely that
a bird could live for long in an area sprayed with DDT with-
out accumulating at least some of the toxicant.

The finding of DDT in the female reproductive organs
and in unhatched eggs, as well as in young birds, points
to the need for additional research to determine the ef-
fects of this chemical on deveIOpment of eggs, hatchability,
and nestling survival. The limited data from this study
indicate that DDT may be passed on directly from the female

to the eggs and young.

78

Although the Robin seems to be the most affected
species of bird in the study area, some 35 other birds, repre-
senting 21 species, were also found to contain at least some
DDT in their tissues. The lethal level of DDT for these
other species is not known, but the finding of residues in
their tissues Shows that they were exposed to it.

Analysis of plant and animal material in areas treated
with DDT indicates that the toxicant is both persistent and
widely available to ground-feeding, bark-foraging, and seed-
eating birds.

The results of the feeding tests showed that experi-
mentally poisoned birds developed tremors comparable to those
observed in birds dying in the field and that levels of DDT
stored in their tissues were generally proportional to the
severity of exposure. Concentrations of DDT found in House
Sparrows, which died of acute and Chronic poisoning, were
much greater than Concentrations found in birds which were
sacrificed before receiving lethal amounts. Birds fed high
levels of DDT in the diet died earlier than those fed on
lower levels.

One of the main objectives of this study was to
determine the value of chemical analyses as a means of re-
lating bird mortality to DDT poisoning in the field. Con-
centrations of the chemical in tissues of Robins and House

Sparrows found in DDT-treated areas were comparable to those

79
found in laboratory-poisoned birds. However, the presence
of high levels in the fat depots Of sacrificed birds suggests
that analysis of entire carcasses of birds does not consti-
tute a valid test for attributing bird mortality to DDT
toxicity. Moreover, analyses of individual tissues, such as
brain, heart, breast muscle, and liver, showed that wide vari-
ations exist in amounts found in all of these tissues.
Levels of DDT determined in the liver and heart of sacrificed
House Sparrows fed sublethal quantities frequently exceeded
the values determined for the same tissues in tremoring
birds. On the other hand, comparisons of DDT levels in the
brain and breast muscle of experimentally poisoned House
Sparrows and Robins with levels in the brain and breast
muscle of birds found in the field support the conclusion
that analysis Of brain and breast muscle is a good criterion
for determining whether or not a bird died from continued
ingestion of DDT. However, the association of tremors with
poisoning suggests that of the two, amounts of DDT in the
brain may represent the better criterion.

Of 62 dead and dying adult Robins (including eight which
were experimentally poisoned in Wisconsin) whose brains were
analyzed, 49 had levels of DDT ranging from 50 to 120 pg. of
DDT/g. of brain tissue, five had less than 50, and eight had
more than 120. Similarly, 54 Of 71 adult House Sparrows

found dead or dying during the feeding experiments or in the

80
field had levels of DDT ranging from 65 to 140 pg. of DDT/g.
in the brain, eight had less than 65, and nine had more than
140. All tremoring Robins had levels greater than 50 pg. of
DDT/g. in the brain (minimum 53% and none of those shot or
trapped as controls had levels exceeding 29. Similarly,
tremoring House Sparrows had more than 65 pg. of DDT/g. in
the brain (except for two which had 60 and 64, respectively),
while none of the House Sparrows sacrificed during this
study had more than 65 (maximum 62). Therefore, it is con-
Cluded that House Sparrows having 65 or more pg. of DDT/g.
in the brain and Robins with 50 or more died as a reSult of
DDT poisoning.

Analysis of results, using the Pearson Product Moment
Correlation Coefficient, suggests that levels of DDT in the
brain of tremoring Robins and House Sparrows are largely
independent of quantities found in other tissues except for
a correlation between brain and heart concentrations in male
Robins, and between brain and liver in female House Sparrows.
However, the finding of statistical significance does not ex-
clude the possibility that the results are due to Chance nor
does the absence of statistical significance prove that the
results are due to chance.

The results of the feeding tests also indicate that
birds maintained on low DDT diets or exposed to higher
dosages for short periods of time are either able to elimi-

nate the toxicant or redistribute it to more passive sites.

81
However, DDT stored in fat depots is cumulative and may be
less subject to breakdown or elimination. The storage of
DDT in fat tissues, even at high levels, apparently does not
poison birds directly, but serves as a storage depot. How-
ever, when the fat reserves are utilized (as in starvation),
the DDT may be released to more sensitive areas (such as the
brain) resulting in tremors followed by death. Theoretic-
ally, some birds might retain sublethal amounts of DDT in
fat all summer and perish in winter or during migration
when fats are utilized.

The evidence presented in this report demonstrates
Clearly that bird mortality in areas sprayed with DDT for
the control of Dutch elm disease is high and that the birds
died directly as a result of the toxic effects of the com-
pound.

In conclusion, therefore, it may be stated that any
sound program designed to control Dutch elm disease should
take into account the possible adverse effects on other than
the intended target organisms. Various suggestions have been
submitted by conservation-minded peOple for reducing wildlife
losses in control programs, but, unfortunately, none offers
an effective solution. Future research on better control
measures may offer a solution, but it appears questionable
that this much needed research will be accelerated until it
is realized that a real and not a fabricated problem does

exist.

SUMMARY

The use of DDT, particularly for the control of Dutch
elm disease in the Midwest, has resulted in numerous
reports of birds found dead or dying in DDT-sprayed
areas.

This study was undertaken in an attempt to gain addi-
tional information concerning the effects of DDT on
birds in areas treated for Dutch elm disease or for
other related programs.

A method for the determination of DDT in the tissues of
birds is described and a technique for the differenti-
ation of DDT from DDE is included.

A total of 137 birds was recovered from known spray
areas (mostly the MSU campus and vicinity) and samples
of various tissues from each bird were analyzed for DDT.
The results show that all tremoring Robins had high
levels (minimum was 55 pg. of DDT/g. of tissue) of DDT
in the brain, and at least some DDT in the other tissues
tested.

Robins fed DDT-injected earthworms (in Wisconsin) died
of tremors similar to those observed in field Specimens,
and the quantities of DDT in their tissues were comparable
to the levels found in tremoring field specimens.

Results support the conclusion that adult Robins found

10.

11.

12.

83
dead or dying with 50 or more pg. of DDT/g. in the brain
died from the effects of the toxicant.

Of 54 dead or dying adult Robins (whose brains were
analyzed), 49 (90.7 per cent) had sufficient quantities
of DDT in the brain to justify the conclusion that they
were killed by the poison.

Nineteen of 20 male Robins found dead or dying, had
concentrations of DDT in the testes ranging from 6 to
109 pg. of DDT/g. Of tissue.

DDT was also present in the developing egg follicles in
the ovaries of female Robins, in fully develOped eggs

in the uteri of two females, in unhatched eggs in de-
serted nests, in developing embryos, and in one nest-
ling.

Fifty-two birds of 27 species other than Robins were
also analyzed. Of these, 35 birds of 21 species had at
least some DDT in their tissues. The DDT-free birds in-
cluded 7 birds which were picked up at the base of a
television tower in the fall. These were used as con-
trols.

House Sparrows were kept in captivity and fed diets con-
taining 0, 100, 200, and 300 mg. of DDT/Kg” respectively.
Some of the birds were sacrificed during the experiment,
while others were analyzed after they died. All birds
fed at the 200 and 500 mg. of DDT/Kg. levels died;

 

 

13.

14.

15.

16.

17.

84
whereas,to of 15 kept at the 100 mg. of DDT/Kg. level
survived beyond the 90-day experimental period. None
of-the 15 birds used as Controls died.

Generally, the amount of DDT in the tissues of experi-
mentally poisoned birds increased with the time of ex-
posure.

Levels of DDT in the brain and breast muscle of experi-
mentally poisoned birds were greater than levels found
in birds sacrificed during the experiment. Most House
Sparrows dying with tremors had amounts greater than 65
pg. of DDT/g. in the brain.

Birds starved (deprived of food) after exposure to DDT
soon developed tremors and died. Levels of DDT deter-
mined in the brains of these birds were comparable to
those found in tremoring birds which were not starved.
Birds on a diet containing 500 mg. of DDT/Kg. of food,
followed by a diet free from DDT, were able to elimi-
nate the poison from most of the tissues tested except

fat. Following starvation, however, birds from the

same group died with tremors and again contained high
levels of DDT in most tissues tested.

DDT stored passively in fat depots may be released to
more sensitive sites following periods of starvation.
Amounts of DDT recovered in the subcutaneous fat of

apparently normal birds were as high as 780 and 950

18.

19.

20.

21.

22.

85
pg. of DDT/g. of fat.
Tremoring House Sparrows, collected on campus, had DDT
concentrations in the brain comparable to the levels
found in the brain of experimentally poisoned House
Sparrows. Hence, it is concluded that House Sparrows
with 65 or more pg. of DDT/g. in the brain were killed
by this toxicant. I
Comparisons between levels of DDT found in the tissues
of tremoring birds and those in birds exposed to sub-
lethal levels indicate that quantities in the brain and
breast muscle furnish a better criterion for attributing
mortality to DDT than do quantities in heart and liver
tissue.
Results based on the "Student's t" distribution indicate
that the observed differences in the concentrations of
DDT between male and female Robins and House Sparrows
are not significant at the 1 per cent level.
The relationship of the concentrations of DDT in the
brain with those in other tissues of tremoring birds
was investigated by means of the Pearson Product Moment
Correlation Coefficient. These correlations were
significant at the 5 per cent level only between brain
and heart in male Robins, and between brain and liver
in female House Sparrows.

It is concluded that bird mortality in areas sprayed

86
with DDT for the control of Dutch elm disease has been
high and that the birds died as a direct result of in-

gesting the compound.

LITERATURE CITED

Albert, T. F. 1962. The Effects of DDT on the Sperm Pro-
duction of the Domestic Fowl. Auk, 19:104-107

Association of Official ricultural Chemists. 1955. Off.
Meth. Of Anal. :406- 15.

Barker, R. J. 1958. Notes on Some Ecological Effects of
DDT Sprayed on Elms. Jour. Wildl. Mgmt. 22:269r274.

Benton, A. H. 1951. Effects on Wildlife of DDT used for
Control of Dutch Elm Disease. Jour. Wildl. Mgmt. 15:
20-27 0

Blagbrough, H. P. 1952. Reducing Wildlife Hazards in Dutch
Elm Disease Control. Jour. Forestry 5Q:468-469.

Dewitt, J. B., J. V. Derby, Jr., and G. F. Mangan, Jr.
1955. DDT vs. Wildlife. Relationship Between
Quantities Ingested, Toxic Effects, and Tissue Storage.
Jour. Ag. Pharp, Assoc. 44:22-24. ”

Hickey, J. J. and Hunt, L. B. 1960. Initial Songbird
Mortality Following a Dutch Elm Disease Control Program.

Hotchkiss, N. and Pough, R. H. 1946. Effects on Forest
Birds of DDT Used for Gypsy Moth Control in Pennsylvania.
Jour. Wildl. Mgmt. 19:202-207.

Hunt, L. B. 1960. Songbird Breeding Populations in DDT-
Sprayed Dutch Elm Disease Communities. Jour. Wildl.
Egg-t)... 12—1—4'1: 139-1460

Janes, R. L. and Strong, F. C. 1961. Dutch Elm Disease
Control. Extension Folder F-195 (revised). COOperative
Extension Service, M.S.U., East Lansing. 10 pp.

Mehner, J. F. and Wallace, G. J. 1959. Robin Populations
and Insecticides. 'Atlantic Naturalist. 14:4-9.

Mitchell, R. T., Blagbrough, H. P. and Van Etten, R. C.
1953. The Effects of DDT upon the Survival and Growth
of Nestling Songbirds. Jour. Wild;. Mgmt. llj45-54.

88

Schechter, M. S., Soloway, S. B., Hayes, R. A. and Haller,
H. L. 1945. Colorimetric Determination of DDT. I Q.
E S. Chem., Anal. 2;. 17:704-709.

Sternberg, J. and Kearns, C. W. 1952. Chromatographic
Separation of DDT and Some of Its Known and Possible
Degradation Products. Jour. Econ. Ent. 35:505-509.

Wallace, G. J. 1959. Insecticides and Birds. Aud. Mag.
61:10-12, 35.

. 1960. Another Year of Robin Losses on a University
Campus. Aud. Mag. 62:66-69.

 

, Nickell, W. P. and Bernard, R. F. 1961. Bird
Mortality in the Dutch Elm Disease Program in Michigan.
Bull. 41, Cranbrook Inst. Sci. 44 pp.

Whitten, R. R. and Swingle, R. U. 1958. The Dutch Elm
Disease and Its Control. Bull. 193, U. S. Dept. Agric.

12 pp.

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