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3 1293 01094 8127 flaggeesity

dissertation entitled

Photoperiodic Effects On The Avian Dietary LC50 With
Bobwhite (Colinus Virginianus) And Mallards (Anus

Platxrhxnchosl

presented by

William J. Breslin

has been accepted towards fulfillment
of the requirements for

Ph.D. degeem Animal Science

1 i
M o >
Major professo

Robert K. Rin
7-27—84 ger
Date

MSU i: an Affirmative Anion/Equal Opportunity Institution 0.12771

   

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Place in book drop to
remove this checkout from
your record. FINES will
be charged if book is
returned after the date
stamped below.

 

  
   

LIBRARIES
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William J. Breslin

 

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Animal Science
and

Center for Environmental Toxicology

 

 

 

TABLE OF CONTENTS

ABSTRACT ..............................................
INTRODUCTION . ....... ..... ............ . ..... . ..........
REVIEW OF LITERATURE ..................................
Regulation of Food Consumption ....................
Dose-Response Relationships ....... . ...............
Chemicals .........................................
ANTU ...................................... ....
Fenthion ......................................
Endrin ........... ' .............................
Sodium Secobarbital ...........................
Strychnine ....................................
PROCEDURE .............................................
Design and Chronology of Study ....................
Experimental Design ...............................
Range Finding - Preliminary Tests .................
Facilities ........................................
Animals ...........................................
Diets ....................................... t .....
Observations of Record ............................
Statistical Analysis ..............................
RESULTS ...............................................
Litchfield and Wilcoxon Analysis ..................
Direct Regression of Dose on Mortality ............
Inverse Prediction of the Regression of
Mortality on Dose ...............................
DISCUSSION ............................................
CONCLUSIONS ...........................................
REFERENCES ............................................
APPENDIX ..............................................

12
l7
l7
l8
19
20
21

22
22
24
24
26
27
30
31
33

35
35
48
48
74
79
8O

83

    

lung-Ioduh-IUIIII-nb -.- _ ..... ..'---" - I' ..

SI o--.--o.-n..- Illa... ' _ :H '
'1'

 

 

LIST OF TABLES

Table # Page
1 Chronology of LC50 trials ................. 23
2 Percent mortality for bobwhites and

mallards fed ANTU, secobarbital, and
strychnine during preliminary range

finding tests ............................. 25
3 Brooder and room light temperature ........ 28
4 Room light intensities for the

14D photOperiod during the

brightening and dimming sequence .......... 29
5 Dietary concentrations (ppm) used

during LC50 testing ....................... 32
6 LC50 values for ANTU, fenthion,

endrin, secobarbital, and
strychnine in bobwhites and
mallards calculated by the
probit analysis method of

Litchfield and Wilcoxon (1949) ............ 36
7 Percent mortality and LC50 values

for bobwhites fed dietary ANTU ............ 37
8 Percent mortality and LC50 values

for bobwhites fed dietary fenthion ........ 38
9 Percent mortality and LC50 values

for bobwhites fed dietary endrin .......... 39
10 Percent mortality and LC50 values

for bobwhites fed dietary seco—

barbital .................................. 40
11 Percent mortality and LC50 values

for bobwhites fed dietary

strychnine ................................ 41
12 Percent mortality and LC50 values

for mallards fed dietary ANTU ............. 43
13 Percent mortality and LC50 values

for mallards fed dietary fenthion ......... 44

II

 

 

 

Table #

14

15

l6

17

18

19

20

21

22

23

24

Percent mortality and LC50 values

for mallards fed dietary endrin ........

Percent mortality and LC50 values
for mallards fed dietary seco—

barbital ...............................

Percent mortality and LC50 values
for mallards fed dietary

strychnine .............................

LC50 values for ANTU, fenthion,
endrin, secobarbital, and
strychnine in bobwhites and
mallards calculated by log-
probit analysis using a direct

regression of dose on mortality ........

LC 0 values for ANTU, fenthion,
en rin, secobarbital, and
strychnine in bobwhites and
mallards calculated by a log—
profit analysis using an inverse
prediction of a regression

analysis of mortality on dose ..........

Mortality patterns of bobwhite
and mallards during LC50 testing

with secobarbital ......................

Mortality patterns of bobwhite
and mallards during LC50 testing

with strychnine ........................

Mortality patterns of bobwhite and
mallards during LC50 testing with

fenthion ...............................

Mortality patterns of bobwhite and
mallards during LC50 testing with

endrin .................................

Mortality patterns of bobwhite and
mallards during LC50 testing with

ANTU ...................................

Observed symptoms of toxicosis for

bobwhite and mallard LC50 trials .......

III

.....

.....

.....

Page

46

47

49

50

52

53

55

56

57

58

 

 

Table #

25

26

27

28

29

3O

31

32

Mean feed consumption (grams/bird/day)
by photoperiod of bobwhites and
mallards fed dietary ANTU, fenthion,
endrin, secobarbital, and strychnine
at day five of study

Mean feed consumption (grams/bird/day)
by photoperiod of bobwhites and
mallards fed dietary ANTU, fenthion,
endrin, secobarbital, and strychnine
(three day recovery period)

Initial mean body weights (grams)

by photoperiod of bobwhites and
mallards fed dietary ANTU, fenthion,
endrin, secobarbital, and

strychnine

Mean body weights (grams) by
photoperiod of bobwhites and
mallards fed dietary ANTU,
fenthion, endrin, secobarbital,

and strychnine at day five of
study

Mean body weights (grams) by
photoperiod of bobwhites and
mallards fed dietary ANTU,
fenthion, endrin, secobarbital,

and strychnine at day eight of
study

Mean feed consumption (grams/
bird/day) by treatment of bobwhites
and mallards fed dietary ANTU,
fenthion, endrin, secobarbital,

and strychnine for the five-day
treatment period

Mean feed consumption (grams/
bird/day) by treatment of bobwhites
and mallards fed dietary ANTU,
fenthion, endrin, secobarbital,

and strychnine for the three—day
recovery period

Initial mean body weights (grams)

by treatment of bobwhites and
mallards fed dietary ANTU, fenthion,
endrin, secobarbital, and strychnine

IV

ooooooooooooooooooooo

oooooooooooooo

...............................

oooooooooooooooooooooooooooooooooooo

....................................

ooooooooooooooooooooooooo

--------------------------

Page

60

61

63

65

66

68

69

71

 

 

 

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éfl' “ ' Mean body weights (grams) by
treatment of bobwhites and
mallards fed dietary ANTU, -
fenthion, endrin, secobarbital, :-
and strychnine (day eight of -- -
study) 73

 

LIST OF APPENDIX

Page
Appendix A. Composition of quail starter ...... ....... 83
Appendix B. Composition of duck starter . ............. 84
Appendix C. Room temperature, brooder
temperature, and room
relative humidity during
LC50 trating ............................. 85

VI

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912.115 ?-: ---.'.-.- '.' comm: . *-

'i

F.

 

 

ABSTRACT

PHOTOPERIODIC EFFECTS ON THE AVIAN DIETARY
LC50 WITH BOBWHITE (COLINUS VIRGINIANUS)
AND MALLARDS (ANAS PLATYRHYNCHOS)

 

by

William J. Breslin

Eight-day (five-day treatment and three-day recovery
period) dietary LC50 trials were conducted with bobwhite

(Colinus virginianus) and mallards (Anas platyrhynchos) using

 

a-napthylthiourea(ANTU), fenthion, endrin, sodium secobarbital,
and strychnine under 24 hours lightzo hours dark (24), 14 hours
lightle hours dark (14), and 14 hours lightle hours dark

with dimming and brightening of lights between light and dark
periods to simulate dusk and dawn (14D), to determine the
effect of photoperiod on the avian dietary LC50. No signifi-
cant differences in LC50 values between the three photoperiods
could be established for a particular chemical in bobwhites

or mallards. Mortality patterns and symptoms of toxicosis

were generally similar between the 24, 14, and 14D photoperiods
for each chemical. Photoperiod significantly affected feed
consumption during the five-day treatment and three—day reco-
very periods. The 24—hour photoperiod resulted in significantly
greater feed consumption than either of the two 14-hour photo—
periods. Significant differences in feed consumption between
the birds on the 14 and 14D photoperiods were less frequent

ed consumption c: birds

(D

ifferences between

(1:

than significant

 

 

 

 

 

 

 

 

INTRODUCTION

The Environmental Protection Agency has established testing
guidelines for the registration of pesticides in the United
States under the Federal Insecticide Rodenticide and Fungicide
Act (FIFRA) (U.S. EPA, 1982) and for the determination of
safety of industrial chemicals under the Toxic Substance
Control Act (TSCA) (U.S. EPA, 1983). Portions of both acts
deal with wildlife toxicity testing. At present, wildlife
toxicity testing guidelines are not final and information
required for a particular pesticide is determined on a case-
by-case basis (Federal Register, 1978a). The oral LD50 and
_ dietary LC50 are two tests frequently required early in the
registration process, supplying basic lethality and species
susceptibility data for a particular chemical. The avian
dietary LC50 protocol, having no similar mammalian counterpart
as does the LDSOI allows considerable variation in the methodo—
logy of experimentation, resulting in the potential for
significant variations in test results and thus making it
difficult for interpreting and categorizing a chemical’s
toxicity. One section of the proposed avian LC50 protocol
allowing considerable experimental variation deals with photo-
period (Federal Register, 1978b).

Photoperiod, the relative length of light and dark periods,
is one of many important factors which control the behavior
and physiological state of most domestic and wild bird species

(Morris, 1967; Van Tyne and Berger, 1976; Tucker and Ringer,

on: so! aeoliab:up

 

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1982). Of particular significance to toxicity testing is the
effect that light duration has on the bird's activity, feeding
behavior, reproductive success, and the biochemical inter—
actions which occur during these events.

Since in LC50 testing the quantity of toxic compound
ingested is dependent on food consumption, the effects of
photoperiod on feeding behavior is of critical concern. In
studies with domestic chickens, increasing photoperiod lengths
from 12 hours light:12 hours dark to 24 hours 1ight:0 hours
dark significantly altered the number of feedings, feeding
duration, and feed consumption (Squibb and Collier, 1979).
Overall, chickens reared under 24 hours light consumed signi—
ficantly greater amounts of feed than chickens reared under
diurnal lighting schemes. The birds reared under 24 hours
light consistently (at short intervals) consumed small amounts
of feed throughout most of the 24 hour period (Squibb and
Collier, 1979), while birds raised on diurnal lighting schemes
consumed large quantities of feed just prior to darkness and
shortly after the onset of lighting (Dingle, 1971; Squibb and
Collier, 1979). These differences in feeding patterns could
possibly result in significant variations in toxicity. Birds
which consume large quantities of treated feed at the initia—
tion or cessation of the lighting periods would be exposed to
greater doses of toxicants over a shorter period of time
compared to birds consuming smaller quantities of feed over an

extended period. Thus, chemicals which are more acutely toxic

 

 

and less persistent may be more toxic when administered to
birds on diurnal lighting schemes. Conversely, less acutely
toxic or more persistent chemicals may be more toxic when
administered to birds under continuous light due to the over—
all increased feed consumption and increased chemical intake.

In addition to feeding behavior, significant increases in
feeding efficiencies have been reported in chickens exposed to
continuous light, resulting in increased early growth rates
and heavier body weights (Squibb and Collier, 1979). This
increase in feeding efficiency has been attributed to a
continuous period of food processing, which is interrupted in
birds exposed to alternate lightzdark periods. Test birds
housed under continuous light for the standard five-day pre-
test acclimation period may show significantly greater body
weights than diurnally acclimated birds at the onset of
testing, making test results incompatable between studies
using different lighting schemes.

Another potentially important effect of photoperiod on
the avian LC50 is diurnal fluctuations in the level and
activity of microsomal mixed function oxidase (MFO) enzymes.
Fluctuating MFO enzyme activities may result in significant
diurnal changes in the toxification—detoxification reactions
producing oscilations in the levels of toxic compounds within
the animal.

The purpose of this study was to assess the potential
effects various lighting schemes have on dietary LC50 deter-

minations in bobwhite (Colinus virginianus) and mallards (Anas

 

 

u-i mart-m Milli-isle .':-:..z-.—.= -

 

Elatyrhynchos). The study was funded by the Environmental
Protection Agency on a contract basis in order to help develop
a more uniform testing protocol for subacute dietary toxicity

testing with avian wildlife species.

REVIEW OF LITERATURE

Regulation of Food Consumption

Sturkie (1976) states "Animals eat to satisfy energy
requirements and volume receptors or to attain a state of
fullness or satiety". The amount of feed consumed is depen-
dent on many factors, some of which include photoperiod,
activity, age, size, environmental temperature, reproductive
stage, appearance and taste of food, and availability of water.

In mammals, it is generally recognized that the satiety
and appetite centers are located in the ventromedial and late-
ral nucleus of the hypothalamus, respectively. Studies in
various species of birds utilizing hypothalamic lesion
techniques also indicate that the ventromedial nucleus and the
lateral hypothalamus play an important role in the regulation
of feed intake (Sturkie, 1976). Lesions in the ventromedial
nucleus at the base of the third ventricle above the optic
chiasma caused hyperphagia in chickens (Gallus domesticus) and
white—throated sparrows (Zonotrichia albicollis) while lesions
in the lateral hypothalamus just caudal to the ventromedial
nucleus produced aphagia (Smith, 1969; Kuenzel, 1972).

The exact mechanism by which the hypothalamus controls

feed intake has not been determined. Certain investigators

 

 

theorize that adipose tissue stores and energy requirement
regulate appetite and feed intake. Mu gt 31. (1968) proposed
that there is a "set point" for tissue fat concentration. If
tissue lipid concentrations rise above this "set point", feed
intake decreases and lipolysis is accelerated. Conversely,
when the tissue lipid concentrations drop below the "set
point" feed intake increases and lipogenesis and fat deposition
are stimulated. When fat deposits approach or equal the "set
point" an equilibrium forms stabilizing feed intake and body
weight (Lepkovsky, 1973). Feed intake and indirectly the size
of the fat droplets are regulated by the hypothalamus. Long
term changes in eating behavior and fat deposits caused by
hyperphagia or hypophagia are thought to be due to the develop-
ment of new set points.

Lepkovsky (1973) supported the "set point" theory of
feeding regulation by reporting that continued force feeding
of chickens at twice their normal ad libitum intake and nutri—
ent requirements made the birds obese. After the force feeding
was terminated and the birds were allowed to feed ad libitum,
they refused to eat for 7 to 10 days, lost weight, and depleted
their abnormally high fat stores, theoretically to a level
low enough (set point) to trigger the feeding center. Hill
and Dansky (1954) also provided data which support energy
requirements as a controlling factor in feed intake. When
these authors varied the caloric content and nutrient density
of feed, chickens altered their food consumption. Hill and

Dansky also noted that the differences in energy intake

 

 

 

between birds of the same weight and egg laying performance
could be attributed to other factors such as the volume of
feed eaten or the stimulation of hypothetical volume receptors
in the crop and esophagus.

Polin and Wolford (1973) conducted studies in chickens,
the results of which contradict the "set point" hypothesis
while supporting the theory that volume receptors are impor—
tant in controlling the feeding behavior of birds. The authors
fed groups of chickens dd libitum, dd libitum and force fed,
and force fed 150 percent of the ddlibitum group. The data
showed a direct relationship between increasing blood lipids
and adipose tissue with increasing feed intake, but failed
to show any difference in feed consumption 2 to 3 days after
the termination of force feeding even though there was
considerable differences in fat deposits among the groups.
Polin and Wolford concluded that their study provided evidence
that volume receptors, which are influenced by rate of filling,
capacity, and discharge of feed, control the feeding process
and suggested that hormones or other factors such as energy
requirements regulate the "set point" at which these receptors
operate. They also pointed out that it is well known that the
emptiness, fullness, or distention of the digestive tract in
mammals influence food intake.

Squibb and Collier (1979) studied the individual eating
patterns of broiler chicks from day one to 20 days of age
under three different lighting schemes (12 hours light:12

hours dark (LD); continuous light (LL); and continuous dark

 

 

(DD)). The LL and LD lighting regimes were initiated on the
first day after hatch while the DD regime started with a light
dark period on day one after hatch, followed by a gradual
decrease in light intensity until total darkness was reached
on day 8. Previous studies showed that when chicks were
immediately placed in total darkness upon hatching 75 percent
of the birds died due to their inability to find food and
water. As early as day 2 of the trial, the chicks on conti—
nuous lighting were eating roughly twice as many meals per day
and spending approximately 40 percent more total time eating
per 24 hour period than the LD chicks. Although the LL chicks
consumed more meals and spent a greater amount of total time
eating per 24 hour period early in the study, the LD chicks
spent more time eating per meal. At day 3 the LD chicks

ate for approximately 13 minutes per hour; the LL chicks ate

6 minutes per hour; and the DD chicks 11 minutes per hour.

At day 11 the LD chicks had increased their hourly eating time
to 25 minutes while the LL and DD chicks ate an average of

12 and 15 minutes per hour, respectively. The differences
between the LL and LD lighting schemes in the number of

meals and the time spent eating per meal continued and inten-
sified throughout the study. During days 8 through 20, the
birds on the continuous lighting schedule ate an average 1.6
times the number of meals than the birds on the diurnal photo-
period but spent approximately 50 percent less time eating per

meal. The total time spent eating per 24 hour period was

 

 

 

 

 

10

greatest in the chicks under the diurnal lighting scheme at
the completion of the study while the chicks reared in conti—
nuous light or dark spent roughly an equal amount of time
eating per 24 hour period. Throughout the study, the LL and
DD chicks ate each hour while the chicks on the diurnal photo—
period ate every hour during the 12 hour light period. The
LD chicks did not attempt to feed at any hour during the 12
hours of darkness.

In this same study by Squibb and Collier (1979), at day
3, hourly feeding duration within a lighting regime remained
relatively constant under all lighting schemes, except for
a sharp surge during the hour the caretaker entered the room.
Individual variability in minutes eating per hour at day 3
was greatest in the LD chicks. These birds were unable to
anticipate "lights out" at this time, as the time spent
feeding in the 2 hours prior to the beginning of the dark
period was decreasing. By day 11, the LD chicks still showed
a high degree of variability in hourly feeding time, but were
able to anticipate lights out, as indicated by increasing
time spent feeding during the four hours prior to darkness.
At day 20, the chicks had developed a strong sense of timing
in anticipation of lights out as shown by a very low indivi-
dual variability in hourly feeding time and by a steep rise in
the hourly feeding time just prior to the 12 hour dark period.

There was no significant difference in the total 20 day
feed consumption between the LL and DD chicks but the LD

chicks ate significantly less feed than either the LL or DD

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11

chicks. Twenty day body weights of chicks on continuous light
were significantly heavier than body weights of the LD chicks,
while the DD Chick's 20 day body weights were intermediate to
the LL and LD birds. Efficiency of feed utilization was high—
est in the LL chicks, intermediate in the LD chicks, and lowest
in the DD birds. Water to feed ratios were significantly lower
in the DD group, but similar in the LL and LD birds.

An earlier study by Weaver and Siegel (1968), in which
commercial broiler crosses were reared under continuous
diurnal photoperiods, supports the feeding behavior conclu-
sions of the Squibb and Collier study. Weaver and Siegel
reported that the average percentage of birds feeding per hour
during the light period was consistently lower under conti-
nuous light than that for the flock under the diurnal lighting
regime. These researchers identified a highly significant
time-light regime interaction in feed consumption in all
photoperiods studied. Although the birds on the continuous
light schedule showed feeding rhythms, the rhythms were less
uniform and dramatic than those under the light-dark photo—
period, and produced no evidence of a day—night pattern. The
birds on the diurnal lighting regime showed 2 large peaks in
the percent of birds feeding; one just after the onset of
light and the other just prior to darkness. These increases
in percent feeding were due to the birds consuming large
quantities of feed after the onset of lighting and prior to
darkness, and showed that the chickens were able to condition

themselves to the anticipated dark period.

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12

Dose—Response Relationships

One of the major fundamental principles of pharmacology
and toxicology is that of the relationship between the inten-
sity of a response caused by a drug or xenobiotic and the dose
administered. This dose—response relationship can be best
summarized with three basic principles. (1) There are mole-
cular or receptor sites in which the chemical reacts to produce
a response. (2) The production and intensity of the response
are related to the concentration of the chemical or toxic
agent at the reactive site or receptor. (3) The concentration
of the agent at the receptor site is related to the dose
administered or exposure level. In addition, the intensity
of the response elicited by the agent is thought to be propor—
tional to the number of receptors occupied by the agent, with
maximal response occurring when all receptors are occupied.
This receptor occupancy is a function of drug concentration
and its ability to react or combine with the receptor. The
ability of a compound to react with a receptor or the drugs
affinity for the receptor is generally considered to be
constant (Klaassen and Doull, 1980).

In general, the toxic effects of a chemical in an organism
are not produced unless the agent, its metabolites, or conver-
sion products reach and react with receptor sites or macro-
molecules in concentrations and for a length of time to cause
an alteration in normal function. Thus, the chemical's
inherent toxicity, or its inherent ability to produce harm in

an organism, and the degree of exposure of an organism to the

 

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13

toxin are two important factors which determine toxicity. In
addition, many factors contribute to the degree of inherent
toxicity of a chemical and the exposure situation. Two
important aspects of exposure which play a role in the develop-
ment of toxicosis are the duration and frequency of exposure.

Many agents show quite different effects between acute and
chronic exposure. Acute exposure to agents that are rapidly
absorbed tends to produce immediate toxic effects, while
chronic exposure to a toxin may produce both an acute effect,
after each successive dose, and a long term effect due to the
chronic presence of low levels of the compound. Fractionization
of the dose can also reduce the intensity of the chemical's
effect. Administering a specific quantity of chemical in two
doses over a period of several hours may result in less than
one half of the effect that would have been obtained had the
same amount of chemical been given in a single dose. This
"fractionization effect" occurs due to the compound being
metabolized or excreted between successive doses, thus
reducing the peak tissue or blood concentrations or by the
ability of the organism to partially or fully reduce the
injury caused by the first dose prior to the second. In
certain incidences, the production of a toxic response can be
totally dependent on the frequency rather than the duration
of exposure (Klaassen and Doull, 1980).

The absorption or elimination of most drugs and xenobio-
tics follow expotential (first order) kinetics. A constant

proportion of the compound administered is absorbed or

1h
manure ‘o usages erasing-l

   
   

- ME. 35135114' .--="' 11".:- . ';‘.-'.'- H“ .‘-'.I'.-r.:

 

has a '1. 2: .".-‘.'!E*\-'1'..":§ 3?"; i"--

 

 

 

 

l4

excreted per unit of time, since the chemical concentration
within the organism usually does not saturate the mechanism
responsible for absorption and excretion. In certain cases
where the elimination process is restricted due to the limited
quantity of carrier molecules, drug metabolizing enzymes, or
cofactors in the metabolism pathway or the concentration of
the toxic compound saturates the excretion mechanism, zero
order elimination (constant quantity per unit time) kinetics
results.

In first order kinetics an absorption or elimination rate
constant (K) which expresses the fractional change per unit
of time can be calculated. From this constant, a half—time,
(.693/K) or the time required for 50 percent of the admini—
stered dose to be absorbed or excreted, can be determined.
Both the rate constant (K) and the half—time are independent
of drug concentration and dose. From these formulas, it can
be determined that the effect of a single dose can be charac-
terized by its latency, time to peak effect, time to peak
concentration, magnitude of peak concentration, and the dura-
tion of effect. As the dose increases, the latency is reduced
and the peak effect is increased without altering the time of
peak effect. The duration of effect increases with increasing
dosage but proportionally less than peak effect.

In first order kinetic models, 4 half-times are required
for near complete elimination (94%) of a compound from the

organism, and any dosing interval shorter than this results

 

 

15

in the accumulation of the compound. The accumulation of the
compound continues during successive doses until the rate of
elimination equals the rate of absorption. Once this equili-
brium has been reached the body concentration becomes a func—
tion of the maintenance dose (dose/dose interval) or exposure
rate and the half-time for elimination. Thus, if two dosing
intervals are used for a compound but the total drug admini-
stered within a 24 hour period is equal between the two dosing
schedules, the plateau levels of tissue stores would be equal,
but the peak tissue concentrations would be different.

Light has been known to alter the metabolism and response
to drugs in many animal models. In general, light can alter
the organism's response to drugs or xenobiotics by two
mechanisms; photochemical reactions or changes in the levels
and activity of drug metabolizing enzymes. Photochemical
reactions can be either deleterious or beneficial. Chemicals,
such as selected sulfonamides, tetracyclines, nalidixic acid,
sulfonylureas, thiazides, phenothiazines, and coal tars, when
chronically ingested, result in photosensitivity after expo—
sure to sunlight or artificial fluorescent light (Klaassen,
1980). Two types of photosensitive reactions have been obser—
ved; a phototoxic reaction which is characterized by the
fundamental dose-response relationship and results from the
production of a reactive chemical metabolite, and the photo—
allergic reaction which is an idiosyncratic response charac-
terized by the promotion of a chemical reaction between the

drug and subcutaneous proteins resulting in the formation of

 

16

a photoantigen (Klaassen, 1980). Conversely, photochemical
reactions such as that observed in the phototherapy of pre-
mature infants with neonatal hyperbilirubinemia result in the
formation of less toxic photometabolites. When infants with
hyperbilirubinemia are exposed to blue or visible light, the
radiation penetrates the skin photooxidizing bilirubin to a
more hydrophilic product which can be easily eliminated by
both biliary and urinary excretory pathways (Sanvordeker and
Lambert, 1974). Similarly, alterations in drug metabolizing
enzymes can be deleterious or beneficial depending on the
nature of the chemical and its metabolites. Light affects
the drug metabolizing enzyme system by promoting rhythmic
changes in the activity and production of enzymes. In rat
studies conducted by Joir SE Ei' (1971) using female Long-Evan
rats kept under 12 hours light:12 hours dark, liver microsomal
drug metabolizing enzyme levels peaked during the dark phase
of the diurnal photoperiod, then declined to minimum levels
during the 12 hour light period. Nair and Casper (1969)
reported similar diurnal fluctuations in hepatic drug metabo—
lizing enzyme actitivy in male Sprague—Dawley rats raised
under a 12 hour light:12 hour dark schedule. In addition to
the diurnal photoperiod, Nair and Casper also studied the
effects of continuous light and continuous darkness on the
hepatic drug metabolizing enzymes of rats. Both of the
continuous lighting schedules abolished the daily rhythms in
enzyme activities. The rats raised in total darkness main-

tained significantly higher enzyme levels than the rats raised

 

' - . ' 5..1
J
u. d 1 “.|- f
I. I '(_ u
‘1-
u
{.4
.

 

17

under continuous light. When phenobarbital sleeping times were
measured in these two groups of rats, the animals exposed to
continuous light slept significantly longer than the rats on
the dark schedule indicating light can indirectly alter the

response to drugs by affecting drug metabolizing enzyme levels.

Chemicals

The chemicals used in this study were selected on the
basis of their ability to affect the nervous system. The five
chemicals represent agents that stimulate, inhibit, or have
no effect on the nervous system.

ANTU

 

Synomyms: a-napthylthiourea; a-naphylthiocarbamide;
Krysid

Description: A colorless to gray, odorless, bitter
tasting, crystalline compound with a melting point of 198°C
and solubilities of 0.06 g/100 ml water and 2.43 g/100 ml
acetone.

Molecular weight: 202.27

Molecular formula: CllHlONZS

Use, mode of action, and clinical signs: ANTU is a
rodenticide that is highly specific for the adult Norway or
brown rat (Rattus norvegicus) while being less toxic to other
Rattus species and safe to most domestic animals. Of the
domesticated animals, dogs, cats, and swine are the most sus—
ceptable.

ANTU is a fast acting toxicant which causes an increase

in pulmonary capillary permeability leading to plural

 

   

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entreoibnl slnheflss swab an!

    
   

 
  
  

llodsaam put! n ' w'. "a : . . 'H“th7

 

 

18

effusions, pulmonary edema, and anoxia. Clinical signs include
salivation, vomitation, diarrhea (general gastric irritation),
dyspnea, coughing, tachycardia, and pulmonary rales. Post-
mortem examinations reveal cyanosis, hydrothorax, inflammation
of the trachea, bronchi, and gastrointestinal tract, and
hyperemia of the kidneys and liver.

Chemical purity: 95%

Source: K and K Laboratories - ICN, 121 Express St.,
Plainview, NY.

Lot number: 45898A

Fenthion

Synonyms: Baytex, Baycid, ENTEX, Lebaycid, Mercap-
tophos

Description: A yellow, oily liquid with a slight odor
of garlic, a boiling point of 87°C (commercial product boiling
point 105°C) and a vapor pressure of 3.0 x 10"5 mmHg at 20°C.
It is readily soluble in methanol, ethanol, ether, and other
organic solvents. Its solubility in water is 5.5 mg/100 ml
water.

Molecular weight: 278.34

Molecular formula: C10H15O3P82

Use, mode of action, and clinical signs: Fenthion is
an organophosphate insecticide which can be readily absorbed
through the skin, mucous membranes, lungs, or digestive tract.
Its toxicity results from irreversible binding to acetyl-

cholinesterase causing acetylcholinesterase inhibition which

 

 

 

 

 

19

leads to overstimulation of the parasymphathetic nervous
system. Clinical signs include lacrimation, salivation,
tracheal congestion, gastrointestinal disturbances (hyper-
motility), muscle stimulation, convulsions, ataxia, immobility,
and dyspnea. Post—mortem findings after acute exposure may be
minimal and nonspecific while subacute or chronic exposures
result in excessive secretions within the respiratory tract.

Chemical purity used: 94%

Source: Mobay Chemical Corp.; Agricultural Chemicals
Division; P.O. Box 4913; 8400 Hawthorn Road; Kansas City, MO.

Batch number: 8030130

Endrin

Synonyms: Mendrin, Nendrin, Hexadrin, Compound 269

Description: A white crystalline solid or powder
(technical powder tan) with a melting point of 226-230°C, a
vapor pressure of 2.0 x 10'7 mmHg at 25°C and solubilities
of 17 g/100 ml acetone, 13.8 g/100 ml benzene, and 7.1 g/100
ml hexane.

Molecular weight: 380.93

Molecular formula: C12H8Cl6O

Use, mode of action, and clinical signs: Endrin is an
organochlorine insecticide which was used mainly on field
crops, particularly cotton. Its specific mechanism of action
has yet to be established, but toxicity is thought to result
primarily from the effects endrin and endrin metabolites have
on the central nervous system. Chlorinated hydrocarbon insec—

ticides in general act as diffuse stimulants or depressants

20

of the central nervous system. Clinical signs due to endrin

toxicity are: convulsions, tremors, vomiting, abdominal
distress, drowsiness, lethargy, and ataxia. Respiratory
failure is considered to most common cause of death. The

liver, being a secondary site of action, shows signs of fatty
infiltration, hypertrophy, and proliferation of smooth endo-
plasmic reticulum.

Chemical purity used: 99%

Source: The Anspec Company, Inc.; P.O. Box 7730; 122
Enterprise Drive; Ann Arbor, MI.

Lot number: 5—171

Sodium secobarbital

Synonyms: Sodium Meballymal, Sodium Seconal, Sodium
Bipinal, Immenoctal, Pramil, Quinalspan, Sebar, Sedutain

Description: A white, bitter tasting, hygroscopic
powder with a melting point of 100°C. It is very soluble in
water, soluble in alcohol, and practically insoluble in ether.

Molecular weight: 260.27

Molecular formula: C12H17N2Na03

Use, mode of action, and clinical signs: Sodium seco—
barbital is a short acting barbituate. Barbituates in general
act to reversibly depress the activity of all excitable tissue.
Depression at sensitive CNS synapses may result from a pre—
synaptic decrease in transmitter release or by enhancing the
postsynaptic GABA responsive membranes. In the peripheral

nervous system, barbituates depress the threshold of spinal

 

 

21

reflexes by diminishing the response of sympathetic ganglia
to preganglionic stimulation. Respiratory complications
result from the depression or elimination of the hypoxic and
chromoreceptor drives controlling respiration. Barbituates
also have various effects on the liver, kidneys, gastrointes—
tinal tract, and skeletal muscles. Symptoms of poisoning
include drowsiness, confusion, loss of coordination, lethargy,
and ataxia. Death may result from cardiovascular collapse or
respiratory arrest.

Chemical purity used: 99%

Source: Sigma Chemical Co.; P.O. Box 14508; St. Louis,
MO .

Lot number: 059C0207

Strychnine

Synonyms: Mole death, Mouse—nots

Description: A colorless to white crystalline powder
with a melting point of 270~280°C and solubilities of 1.56 x
10'6 g/100 ml water, 6.67 x 10‘1 g/100 ml alcohol, and 5.56
x 10"1 g/ml benzene.

Molecular weight: 334.4

Molecular formula: C21H22N202

Use, mode of action, and clinical signs: Strychnine
is an indole alkaloid used as a pesticide, ruminatoric, and
stimulant. Its primary use as a pesticide is in bird and
mammal control. Strychnine causes neural stimulation by

antagonizing spinal and medullary post—synaptic inhibition,

 

 

 

 

22

preventing the moderating and controlling effects in normal
reflexes. Early poisoning signs of nervousness, tenseness,
and stiffness are followed by tetanic seizures, convulsions,
muscle tremors, ataxia, and rigor. Convulsive seizures can
be initiated by external stimuli such as light, sound, and
touch (hypersensitivity). Clinical signs may appear within
ten minutes of exposure and remain for up to two hours if
untreated. Death usually results from anoxia brought about
by convulsions.

Chemical purity used: 96%

Source: Pfaltz and Bauer, Inc.; Division of Aceto
Chemical Co., Inc.; 375 Fairfield Ave.; Stamford, CT.

Lot number: 24763

PROCEDURE

Design and Chronology of Study

The study consisted of 30 individual LC50 tests conducted
over an 11 month period from March 1982 through January 1983.
The 30 tests were run in ten separate trials each consisting
of three tests per trial (see Table l for chronology of
trials). During each trial one of five chemicals (ANTU,
fenthion, endrin, secobarbital, or strychnine) was tested in
bobwhite or mallards. Individual tests within a trial were
identical with the exception of photoperiod. The three photo—
periods used were: 24 hours light:0 hours dark (24); 14 hours
light:10 hours dark (14); and 14 hours light:10 hours dark

with dimming and brightening of lights between light and dark

 

 

 

23

 

 

 

Nm\m \N Nm\a \k -Nm\s \N me\s NM -Nm\_ \k Nm\_ NM Ne\_ \N Mmm\nwws w:_:;uxtnm
Nm\kw\__ Nm\aw\__-wm\ew\__ Nm\em\__-we\a_\__ Ne\a_\__ Nw\a_\l_-we\e_\__ .es_srmsouam
~m\_m\m Nm\_w\m -Nm\m_\m Nm\e_\m -Ne\m_\m Ne\m_\m Nm\m_\m -~e\m \@ =_rs:L
Nm\mw\a Nm\mm\a -Na\om\k Nm\om\k -Nm\m_\k ~e\m_\k Nm\m_\k -Nm\o_\a :o_;3:wl
Nm\mm\o_ ~e\am\o_-mm\o~\o_ Ne\em\o_-Ne\_N\o_ Nm\_m\o. Nm\_m\o_-ww\e_\o_ :_z<
Seawmm:
~w\e_\m Nm\e_\m -~e\__\m Nm\__\m -Nn\e \m Ne\e \m Ne\o \m -Nm\_ \m w:_:;ustsm
mm\e_\_ mm\e_\_ -mm\__\_ mm\__\_ -ms\o \_ me\e \_ mm\e \_ -mm\_ \_ _ao_2tesouam
Ne\mw\e ~w\mw\e -Nm\ow\e Ne\oN\e -Ne\m_\e Na\m_\e Nm\mi\e -Ne\o_\e :_rs:u
Nw\m \e ~m\m \e -~w\e \e Nm\e \e -Nm\_ \4 Nm\_ \4 ~w\_ \e -Na\am\m =o_;s:at
mm\m \_ mw\m \_ -Nm\_M\N_ Nm\_M\N_-Ns\oN\N. Nm\o~\m_ Nw\em\~_-~w\_~\w_ =_z<
amazemmm
:owum:_snoh uo_noa :o_umtom_mM5cm broom : uo_gwa - . .mwmmmem
ato>ooom _ao_eon do eo_noa amok :owume__oo<

 

 

 

24

periods to simulate dusk and dawn (14D). Eight treatments
(two controls and six chemically-treated groups) of ten birds

per treatment were used within each LC50 test.

Experimental Design
Photoperiods = 3 (24L:0D; 14L:10D; l4L:lOD with dimming

and brightening)

Chemicals/photo. = 5 (ANTU, fenthion, endrin, secobarbital,
strychnine)
Species/chemical = 2 (bobwhite, mallard)

Treatments/chem. = 8 (2 controls, 6 treated diets)
Birds/treatment = 10
Range Finding — Preliminary Tests

A literature review of the chemicals used in this study
indicated that dietary LC50 values for fenthion and endrin had
been previously determined in bobwhite and mallards. The
reported LC50 estimates for these two compounds (Heath 3E dl.,
1972) were used as the approximate median concentrations in
the LC50 trials.

ANTU, secobarbital, and strychnine trial concentrations
were determined, in part, through preliminary testing using
three widely spaced dietary concentrations per chemical with
ten birds per dietary concentration. The mortality results
of these preliminary tests were used to set the median dietary
concentrations and the concentration spacing for the LC50
trials (see Table 2 for preliminary test dietary concentra-

tions and mortality).

 

 

 

25

Table 2. Percent mortality for bobwhitesand mallards fed ANTU, seco—
barbital, and strychnine during preliminary range finding

tests.
Dietary Mortality

Chemical concentration (%)

Bobwhite
ANTU 2 ,0001 10
5,000 20
l0,000 30
Secobarbital 2.000 0
5,000 O
l0,000 3O
Strychnine lOO 0
375 20
l,000 3O

Mallard
ANTU 2,000 30
5,000 60
l0,000 50
Secobarbital l,000 0
3,000 l0
5,000 20
Strychnine lOO 0
375 30
l,000 80

1 Parts per million.

 

 

 

26

Facilities

All birds were housed in the Michigan State University
Poultry Research and Teaching Center during the acclimation
and testing period. The three rooms used, one for each photo-
period, were entirely enclosed and measured 2.95 m X 4.50 m X
2.54 m (W X L X H). Each room contained one Petersime 250—24
battery brooder in which the birds were maintained. The
brooders were divided into six levels, each of which was
separated into four equally sized sections measuring 34.3 cm
X 99.4 cm X 24.1 cm (W X L X H). Only two levels (eight sec-
tions) with ten birds per section, allowing a minimum of 134.2
cm2 floor space per bird, were used during each test. The
galvanized steel wire mesh of the sides and floors measured
1.3 cm X 1.3 cm and 0.8 cm, respectively, for bobwhite and
1.3 cm X 2.4 cm and 1.7 cm X 1.7 cm, respectively, for
mallards. Each section was provided with an individual feeder,
measuring 5.7 cm X 61.0 cm X 1.9 cm (W X L X H) for bobwhite
and 8.8 cm X 63.5 cm X 6.1 cm (W X L X H) for mallards, and
waterer, one quart mason jar for bobwhite and 8.8 cm X 33.0 cm
X 5.1 cm (W X L X H) trough for mallards. Mallard feeders and
waterers were attached to the outside of each section while
bobwhite feeders and waterers were placed within each section.

Photoperiod was controlled by Paragon model 41005—D auto-
matic timers that were set for 24 hours light:0 hours dark or
14 hours light:10 hours dark. In addition, a voltage regula—
tor constructed by the Department of Agricultural Engineering,

Michigan State University, was used in one of the 14 hours

 

27

light rooms to create gradual dimming and brightening sequences
of 17 min duration before dark and light periods. Light was
provided by a 150 watt incandescent light bulb centered in the
ceiling of each room. Light intensities for each room and
battery level are given in Table 3 and 4.

A thermometer and humidity gauge were located on the wall
inside each testing room. One 1,500 watt convection floor
heater with variable temperature controls was used to heat

each room when temperatures dropped below 21°C (70°F).

Animals

Bobwhite chicks were reared at Michigan State University
from eggs collected from an established breeding population
of second generation wild bobwhite. The original stock of
birds was obtained from the Illinois State Game Farm, Mt.
Vernon, IL. Upon hatching, all bobwhite were wing banded and
placed in quarantine until five days of age.

All mallard ducklings were purchased from Whistling Wings
Duck Farm, Hanover, IL. Prior to each trial, approximately
300 one-day old ducklings were shipped (shortly after hatch-
ing) and received at Michigan State University, Department of
Animal Science within 36 hours, whereupon they were wing
banded and placed in quarantine until five days of age.

During the quarantine periods, all birds (bobwhite and
mallards) were observed and all deformed, sick, injured, or
abnormal birds were removed from the flock. At five days of

age all birds in apparent good health were transported to the

 

28

Table 3. Brooder and room light intensities].

 

Photoperiod 242 143 1404
Room 69.9 69.9 69.9
Brooder upper level 32.3 32.3 32.2
Brooder lower level 30.l 30.l 30.l

1 Light intensity in luxes.

2 24 hours lightzo hours dark.

3 l4 hours light l0 hours dark.

4 l4 hours lightle hours dark with dimming and brightening
between light and dark periods.

 

 

29

Table 4. Room light intensities1 for the MD2 photoperiod during the
brightening and dimming sequence.

Bri htenin se uence Dimmin se uence
Time min ntensity ime (min) Intensity

0 0 0 69.9 (maximum)
1 0 l 64.6

2 2.2 2 59.2

3 5.4 3 53.8

4 8.6 4 48.4

5 l0.8 5 43.8

6 l6.l 6 37.7

7 l9.4 7 34.4

8 2l.5 8 30.l

9 26.9 9 26.9

l0 32.3 l0 2l.5

ll 37.7 ll l6.l

l2 43.8 l2 l2.9

l3 48.4 l3 l0.8

l4 56.0 l4 5.4

l5 64.6 l5 0 (minimum)
l6 66.7 l6 0

l7 69.9 (maximum) l7 0

Light intensity in luxes.

2 l4 hours lightle hours dark with brightening and dimming between

dark and light periods.

 

 

30

testing facility and placed in their testing batteries for a
five day acclimation period preceding each trial. At ten days
of age the birds were randomly assigned to their testing
sections. Prior to the assignment of birds to sections, any
abnormally under-weight or over—weight birds were removed from
the flock to increase flock uniformity and prevent bias caused
by unequal body weights. All birds used for testing were ten
days of age at the onset of testing. At trial terminations,
all birds were killed by either cervical dislocation or

chloroform asphyxiation.

Diets

 

All test diets were prepared by the Department of Animal
Science, Michigan State University using a mash as the basal
diet (Appendix A and B). Control diets consisted of basal
mash plus any carriers used in the preparation of treated
diets. Premixes treated with ANTU, endrin, secobarbital, or
strychnine were prepared by adding the powdered form of these
compounds to 1,500 grams of fine mash which had been filtered
through a number 20 sieve. The entire 1,500 grams of mash
and chemical were mixed and filtered through a number 20
sieve to prevent chemical clumping and obtain an equal distri—
bution of the compound throughout the feed. The unfiltered
feed which failed to pass through the sieve in obtaining 1,500
grams of fine mash, and any additional feed required to bring
the premix to the desired concentration, were added to the
chemical—fine mash mixture and tumbled for ten minutes on a

mechanical tumbler. Treated diets used during the test were

 

 

 

 

 

 

31

prepared by dilutions of the premix diet. Specific quantities
of premix and untreated feed were combined to reach the
desired dietary concentration and tumbled for ten minutes to
assure mixing. The fenthion premix was prepared by dissolving
liquid Baytex in corn oil and adding this oil—chemical mixture
to a specific quantity of mash. The corn oil content of the
premix did not exceed one percent of the total feed—oil—chemi—
cal mixture. The premix was tumbled for ten minutes on a
mechanical tumbler after the oil-chemical mixture had been
thoroughly mixed manually into the mash. Fenthion test diets
were prepared by the dilution method described for ANTU,
endrin, secobarbital, and strychnine. Nominal dietary con-
centrations of all chemicals tested are listed in Table 5.
Eight treatments per test were used in each trial; two
controls plus six concentrations of the test chemical.
Chemical concentrations within the six treated diets were
geometrically spaced at intervals of 1.3X to 1.6x, depending
on the chemical used. Feed and water were provided dd

libitum throughout the quarantine, acclimation, and testing

periods.

Observations of Record

Feed consumption, body weights, mortality, signs of intoxi-
cation, room and brooder temperatures, and relative humidity
were recorded throughout the testing period. Average feed
consumption and feed wastage per battery section (dietary

concentration) were measured on days 2, 4, 5, 7, and 8 during

 

 

 

32

 

0px 0cm

 

00m._ 0N0 0mg mmm 0.0 0.0
0mm.0_ m00.m 00¢.m 0N0.m 000.N 000.N 0.0 0.0
v.0m 0.0N 0.NN m.m— m.m— v.0_ 0.0 0.0
00m mvp mop me mm NM 0.0 0.0
0m~.0_ 000.K 00v.m 0N0.m 000.N 000.N 0.0 0.0
vw_.0 0¢0.m 00v.N 00m._ 0M0 00m 0.0 0.0
0m~.0_ mmo.~ 00v.m 0N0.m 000.N 000.N 0.0 0.0
«.mm m.0~ 0.0— 0.N_ w.0 _.N 0.0 0.0
0.0m 0.Nv 0.0m e.FN m.m~ 0.0_ 0.0 0.0
0mm.0_ mmw.m 00v.m 0N0.m 00m.m 000.N 0.0 —0.0
a r e .;m m. .. ..ll-.....fl.w-fl..w N T

.:o____e to; worse

 

udoEHmotL xcmuo_m

 

_

w:_:;thom
_mo_ntmcooom
:_to:L
:o_:o:od
zez<

era__ms

m:_czoxtom
Fauwncmaooom
:_tucw
:o_zo:ol
sez<

amlezsom

.m:_ommo 0m0L m:_t=u com: Asaev meowoacocoocoo zimoo_0 .m m_ame

 

 

 

33

all bobwhite trials. Any significant amount of feed spilled
into the litter pans beneath bobwhite sections was separated
from feces and weighed. Mallard feed consumption was measured
at identical times during the testing periods but feed

wastage could not be accurately determined due to the diffi-
culty in separating feed from feces and water which had
collected in the litter pans. All birds were weighed at days
0, 5, and 8 or on their day of death. All weighings (feed
consumption and body weights) within each trial were conducted
at similar times of day in order to maintain consistency in
weighing intervals.

Observations were made three times during the first day
of exposure and twice daily thereafter until study termination.
All signs of intoxication and the number of birds showing
specific signs were recorded for each dietary concentration
within a test. Band numbers and body weights of birds which
died during the trial were also recorded at these times.
Brooder temperature, room temperature, and room relative
humidity were measured daily during the first observation

period of each day.

Statistical Analysis

LC50 values with 95% confidence limits and the slope of
the concentration response curves were determined for each
test run using three different established methods; the probit
analysis method described by Litchfield and Wilcoxon (1949),

a log—probit analysis using a direct regression of dose on

 

 

 

 

 

34

mortality (Montgomery and Peck, 1982), and a log-probit analy—
sis using an inverse prediction of the regression of mortality
on dose (Gill, 1978). Statistical contrasts of LC50 values
between photoperiods for a specific compound were accomplished
by confidence limit comparisons. If the LC50 : confidence
limits of two photoperiods were separated (nonoverlapping) by
> 10% of the smallest confidence limit, the LC50 values of the
two photoperiods were considered significantly different at

a < 0.05 (Browne, 1979).

Initially, a two—way analysis of variance (photoperiod by
dietary concentration), which compensates for unequal replica-
tion and missing blocks when appropriate, was run on all body
weight and feed consumption data on a trial basis. The two—
way analysis of variance on body weight was run at day 0, 5,
and 8 of every trial, while the feed consumption analysis was
run on the five-day treatment and three—day recovery periods.
Feed consumption was measured three times during the five—day
treatment period and twice during the three-day recovery
period. Sampling units for body weights and feed consumption
were individual bird weights and mean feed consumption per
dietary concentration (expressed as grams/bird/day), respec—
tively. Means of body weights and feed consumption for each
of the photoperiods were compared by use of Tukey's all pair-
wise comparisons test, while means of body weights and feed
consumption for each treatment were compared using Dunnett's
two-tail t—test (Gill, 1978). The analysis of variance pro-

cedures used were run on the Michigan State University Cyber

 

 

35

750 Computer using the SPSS (Nie SE d£., 1975) and JENSTAT

(Alvey SE d£., 1977) statistical packages.

RESULTS
Three trials resulted in insufficient mortality to pro-

perly determine LC50 values. The bobwhite—ANTU, bobwhite—
secobarbital, and mallard—secobarbital trials failed to pro—
duce greater than 50% mortality at a dietary concentration
exceeding 10,000 ppm, in at least two of the three photo—
periods. Although insufficient data were obtained from these
trials, LC50 values were calculated with the available infor—

mation and are presented along with the other trials.

Litchfield and Wilcoxon Analysis

No significant differences in LC50 values between the
three photoperiods could be established in bobwhite or mallards
using the probit analysis method of Litchfield and Wilcoxon
(Table 6). The photoperiod effects on LC50 determinations
varied considerably among the two species and five chemicals
tested. Bobwhite mortality on the 24 photoperiod produced
lower LC50 values than either of the two 14—hour light photo—
periods when chicks were exposed to fenthion, endrin, and
strychnine, but produced higher and intermediate LC50 values
compared to the LC50 values for the 14-hour photoperiods when
chicks were exposed to ANTU and secobarbital, respectively
(Tables 6 through 11). Mallard mortality under the 24 photo-
period resulted in higher LC50 values than the 14—hour photo-

periods when ducklings were exposed to fenthion and endrin,

 

36

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37

Table 7. Percent mortality and LC50 values for bobwhites fed dietary ANTU].

Percent mortalit
Dose 24 hrs. )4 hrs. 14 hrs.

(ppm) n light light light w/d2

0.0A l0 0 0 o

0.05 lo 0 o 0
2.000 l0 l0 0 30
2,800 l0 lo 20 0
3,920 10 l0 20 40
5,488 lo 20 20 lo
7,683 10 l0 so 50
10,755 l0 40 50 20
LC503 28.000 3,200 l5,000
95:. C.L. (ll,755-55,540) (5,563-l2,087) (5,865-38.365)
Slope 2,205 244 3,072
0504 l3,836 5,745 5,470
95% C.L. ( 3,020-53,24l) (4,853- 9,375) (Z,805-lO,7l5)
Slope 0.004 0.0l0 0.02l
1.0505 4l .495 7 ,534 2l ,749
95:; c.L.5 --- (2,05l-m)
Slope 47l l2l 333

l
2

Chemical purity = 95%

Fourteen hours light:10 hours dark with dimming and brightening of lights
between light and dark periods.

LC50 values, 95% C.L., and Slopes were detennined by the method described by
Litchfield and Wilcoxon (l948).

LC50 values, 95% C.L., and Slopes were detennined by a log-probit analysis
using a direct regression of dose on mortality (Montgomery and Peck, l982).

UI

LC50 values, 95% C.L., and Slopes were determined by a log-probit analysis
using an inverse prediction of mortality on dose (Gill, l978).

Lack of confidence limits resulted from the inability to reject the hypothe—
sis of slope equaling zero.

 

38

l

 

Table 8 . Percent mortality and LC50 values for bobwhites fed dietary fenthion .
Percent mortality

Dose 4 hrs. )4 hrs. l4 hrs.“

(ppm) h light light light w/de

0.0A l0 0 0 0

0.08 lo 0 O O

10.9 l0 0 0 0

lS.3 lo 20 10 l0

2l.4 lo 60 30 60

30.0 10 80 80 90

42.0 l0 l00 l00 lOO

58.8 lo 100 lOO 9O
LC503 20.5 23.7 2l.0
95% C.L. (l6.8-25.l) (19.5-28.8) (l7.9-24.6)
Slope 0.25 0.22 0.l2
L0504 2l .2a 22.2b 24.0C
95% C.L. (2l.l-2l.3) (2l.9-22.5) (23.0-25.l)
Slope 3.04 2.93 2.35
L0505 2l.l 22.2 23.5
95% C.L. (l2.9-34.5) (l5.2-32.9) ( 3.4-l22.7)
Slope 0.3l 0.3l 0.48

l
2

Chemical purity = 94%

Fourteen hours light l0 hours dark with dimming and brightening of lights
between light and dark periods.

LC50 values, 95% C.L., and Slopes were detenhined by the method described by
Litchfield and Wilcoxon (l948).

LC50 values, 95% C.L., and Slopes were determined by a log-probit analysis
using a direct regression of dose on mortality (Montgomery and Peck, l982).

LC50 values, 95% C.L., and Slopes were determined by a log-probit analysis
u5lng an inverse prediction of mortality on dose (Gill, l978).

 

39

Table 9 . Percent mortality and L050 values For bobwhites fed dietary endrinl.

Percent mortalit
Dose :3 hrs. 4 hrs. l4 hrs.

 

(ppm) n light light light w/dz

0A l0 0 0 0

08 lo 0 0 0

7.l lo 30 30 0

9.2 lo 40 4O 30

l2.0 l0 l00 40 40

l5.6 lo 70 80 50

20.3 lo 80 l00 90

26.4 l0 lOO l00 90
LC503 l0.8 l0.8 13.5
95% C.L. (8.2-l4.2) (8.7-l3.3) . (ll.3-l6.4)
Slope 0.23 0.l6 0.l7
1.0504 11.131D 10.4a 14.3b
95% C.L. (7.6-16.4) (8.9-l2.2) (l4.0-l5.6)
Slope 7.4l 5.50 6.42
LC505 7.7 9.9 15.1
95% C.L.6 ( 6.l-44.9)
Slope 0.34 0.l5 0.20
1 Chemical purity = 99%
2 Fourteen hours ligntle nours dark with dimming and brightening of lights

between light and dark periods.
3 L050 values, 95% C.L., and Slopes were detennined by the method described by

Litchfield and Wilcoxon (l948).
4 LC50 values, 95% C.L., and Slopes were determined by a log-probit analysis

using a direct regression of dose on mortality (Montgomery and Peck, l982).
5 L050 values, 9 % C.L., and Slopes were determined by a log-probit analysis
6 using an inverse prediction of mortality on dose (Gill, l978).

Lack of confidence limits resulted from the inability to rejeCt the hypothe—
sis of slope equaling zero.

 

40

Table l0. Percent mortality and L050 values for bobwhites fed dietary seco—

barbital).
Percent mortalit
Dose 24 hrs. l4 hrs. l4 hrs.2
(ppm) n light light light w/d

0.0A 10 0 0 103

0.08 10 0 103 0
2,000 10 0 10 0
2,800 10 0 0 0
3.920 10 20 20 0
5,488 10 0 100 10
7.683 10 0 50 10
lO,756 10 40 30 30
L050“1 16,300 12,300 23,000
957. C.L. (7,739-34,329) (7,153-22,904) (l0.892—48.567)
Slope 1,279 529 550
L0505 7,211 4,943 11,614
951; C.L. (2,018-25,763) (4,140- 5,902) ( 5,420-24,831)
Slope 0.0l8 0.023 0.005
LC505 41,933 6,366 l2,892
957; c.1..7 ---
Slope 1,560 110 495

Chemical purity = 99%

2 Fourteen hours lightle hours dark with dimming and brightening of lights

between light and dark periods.

Percent mortality values used to compute LC50 values were adjusted to com-
pensate for control mortality.

L050 values, 95% C.L., and Slopes were detennined by the method described by
Litchfield and wilcoxon (l948).

LC50 values, 95% C.L., and Slopes were determined by a log-probit analysis
using a direct regression of dose on mortality (Montgomery and Peck, l982).

U1

LC50 values, 95% C.L., and Slopes were determined by a log-probit analysis
using an inverse prediction of mortality on dose (Gill, l978).

Lack of confidence limits resulted From the inability to reject the hypothe-
sis of slope equaling zero.

 

 

41

Tablell . Percent mortality and L050 values for bobwhites fed dietary

strychninel.

Percent mortalit
Dose 24 hrs. l4 hrs. l4 hrs.

(ppm) n light light light w/d2
0.0A 10 0 0 0
0.08 10 0 o 0

586 10 0 0 10

938 10 10 20 10
1,500 10 20 40 50-
2.400 10 100 70 60
3.840 10 100 100 100
6,144 10 100 100 100
LC503 1,650 1,700 1,700
9 % C.L. (l,284-2,l2l) (1,197-2,414) (l,l8l-2,448)
Slope 2l.O 37.l 40.3
L0504 1,309a 1,549b 1,371a
95% C.L. (l,l83-l,449) (1,521-1,574) (1,259-1,493)
Slope 0.051 0.028 0.025
LC505 3.122 1,552 -1,337
95% C.L.5 ----- ( 674-3,573) ( l4-23,659)
Slope 18.2 32.5 33.3
1 Chemical purity = 96%.
2

UI

Fourteen nours ligntle hours dark with dimming and brightening 3f lights
between light and dark periods.

L050 values, 9 % C.L., and Slopes were determined by the method described
by Litchfield and Nilcoxon (l948).

LC50 values, 95% C.L., and Slopes were detenmined by a log-probit analy-
sis using a direct regression of dose on mortality (Montgomery and Peck,
l982).

LC50 values. 95% C.L., and Slopes were determined by a log-probit analy-
sis using an inverse prediction of mortality on dose (Gill, l978).

Lack of confidence limits resulted from the inability to reject the
hypothesis of slope equaling zero.

42

however, mortality on the 24 photoperiod resulted in inter—
mediate LC50 values to the 14—hour photoperiods when exposed
to strychnine, ANTU, and secobarbital (Tables 6 and 12 through
16).

Similarly, mortality between the two 14—hour light photo-
periods varied with species and chemicals (Table 6). Bobwhite
LC50 values in the 14 photoperiod were lower than LC50 values
of the 14D photoperiod when chicks were exposed to ANTU,
endrin, or secobarbital (Tables 6, 7, 9, and 10). The LC50
values for bobwhite fed strychnine were equal for the two
14-hour photoperiods, while the 14 photoperiod produced a
slightly higher LC50 value than the 14D photoperiod when the
chicks were fed fenthion (Tables 6, 8, and 11). Mallard
mortality resulted in higher LC50 values under the 14 photo-
period compared to the 14D photoperiod when ducklings were
exposed to fenthion and secobarbital, but produced lower LC50
values under the 14D photoperiod when ducklings were exposed
to ANTU and strychnine (Tables 6, 12, 13, 15, and 16). LC50
values for mallards between the two 14-hour photoperiods were
equal for endrin (Tables 6 and 14).

In summary, the 24 photoperiod produced the highest LC50
values in three of the 10 trials while the 14 and 14D photo—
periods produced the highest LD50 values in three and four of
the trials, respectively. The lowest LC50 values between the

three photoperiods showed a similar pattern of being equally

distributed.

 

43

Table 12. Percent mortality and LC50 values for mallards fed dietary ANTU].

Percent mortalit
Dose 24 hrs. l4 hrs. l4 hrs.

(ppm) h light light light w/d2
0.0A 10 0 0 0
0.08 10 0 0 0

2,000 10 273 30 10
2,800 10 70 50 40
3,920 10 564 40 40
5,488 10 70 60 70
7,683 10 50 90 60
10,756 10 80 90 80
1.0505 4,000 3,400 4,900
95% C.L. (l,786-8,960) (2,378-4,862) (3,490—6,880)
Slope l06 l04 l24
1.0505 3,855 3,589 4,742
953201. (2,8l8-5,284) (3,105-4,159) (4,677-4,797)
Slope 0.0106 0.0086 0.0096
LC507 2,931 3,436 4,753
951401.8 --- (l,l6l-7,586) (1 ,879-l2,388)
Slope 268 l38 136

l
2

Chemical purity = 95%

Fourteen hours lightle hours dark with dimming and brightening of lignts
between light and dark periods.

Mortality percentage based on ll animals (n = ll).
Mortality percentage based on nine animals (n = 9).

01 pm

L050 values, 95% C.L., and Slopes were determined by a log-probit analysis
described by Litchfield and Wilcoxon (l948).

LC50 values, 9 % C.L., and Slopes were determined by a log-probit analysis
using a direct regression of dose on mortality (Montgomery and Peck, l982).

L050 values, 95% C.L., and Slopes were determined by a log-probit analysis
using an inverse prediction of mortality on dose (Gill, l978).

Lack of confidence limits resulted from the inability to reject the hypothe-
sis of slope equaling zero.

 

 

44

Table l3. Percent mortality and LC50 values for mallards fed dietary
fenthion .
Percent mortaliv

Dose 24 hrs. l4 hrs. l4 hrs.

(ppm) h light light light we?
0.0A l0 0 O 0
0.08 10 0 0 0

37 10 lo 30 lo

52 lo 40 4O 60

73 lo 30 40 60

l02 lO 60 70 70

l43 10 80 80 l00

200 l0 l00 9O l00
LC503 82 69 50
95% C.L. (6l.7-l09.l) (48.5- 98.2) (45.l- 79.3)
Slope l.57 2.09 l.l2
LC504 74.5 68.5 61.0
95% C.L. (66.8- 82.8) (63.4- 74.l) (5l.3- 72.4)
Slope 0.46 0.38 0.78
LC505 71.4 67.6 57.9
95% C.L.6 (l4.6-23l.7) (40.6-l05.4) ( O -3l0.0)
Slope l.7 2.6 l.2

l
2

pl

Chemical Purity = 9 m.

Fourteen hours light:l0 hours dark with dimming and brightening of lights
between light and dark periods.

LC50 values, 95% C.L., and Slopes were detennined by the method described
by Litchfield and Wilcoxon (l948).

LC50 values, 95% C.L., and Slopes were detennined by a log-probit analy-
sis using a direct regression of dose on mortality (Montgomery and Deck,
l982).

LC50 values, 95% C.L., and Slopes were determined by a log-probit analy—
sis using an inverse prediction of mortality on dose (Gill, l978).
Lack of confidence limits resulted from the inability to reject the
hypothesis of slope equaling zero.

45

Tablel4 . Percent mortality and LC50 values for mallards fed dietary

endrin].
Percent mortalit
Dose 24 hrs. l4 hrs. l4 hrs.
(ppm) h light light light w/dZ
0.0A 10 0 0 0
0.08 10 0 0 0
10.4 10 0 0 10
13.5 10 10 20 o
17.5 10 30 10 10
22.8 10 10 40 30
29.6 10 40 50 50
38.4 10 60 60 7
L0503 34.0 29.6 29.6
95% C.L. (24.6—46.9) (22.4-39.1) (22.4-39.1)
Slope 0.68 0.60 0.59
Lcso4 27.9 26.5 25.9
95% C.L. (20.8-37.5) (20.9-33.4) (l8.9-35.5)
Slope 2.72 3.09 3.66
L0505 31.9 29.4 31.5
95% C.L.6 (l2.0-l.786) (ll.2-0.48) ---—
Slope 0.4 0.3 0.4

l
2

Chemical purity = 99%.

Fourteen hours light:l0 hours dark with dimming and brightening of lights
between light and dark periods.

LC50 values, 95% C.L., and Slopes were determined by the method described
by Litchfield and Wilcoxon (l948).

LC50 values, 95% C.L., and Slopes were determined by a log-probit analy-
sis using a direct regression of dose on mortality (Montgomery and Peck,
l982).

LC50 values, 95% C.L., and Slopes were detennined by a log-probit analy-
sis using an inverse prediction of mortality on dose (Gill, l978).

UI

Lack of confidence limits resulted from the inability to reject the
hypothesis of slope equaling zero.

 

 

46

Table l5. Percent mortality and LC50 values for mallards fed dietary seco-
barbital).
Percent mortalit
Dose 24 hrs. l4 hrs. )4 hrs.
(ppm) h light light light w/d2
0.0A l0 0 0 O
0.08 l0 0 0 0
2,000 l0 0 0 l0
2,800 l0 0 0 l0
3,920 l0 20 20 l0
5,488 l0 20 0 l0
7,683 lo 50 lo 50
l0,756 l0 40 20 70
LC503 10,600 98,000 7,683
95% C.L. (6,463—l7,384) (l9,l03-502,740) (5,583-l0,573)
Slope 3S4 l04,309 l53
L0504 7,656 8,250 7,656
95% C.L. (S,023-ll,64l) ( 2,698- 25,35l) (4, 32-ll,885)
Slope 0.0098 0.0066 0.0080
LCSOS 8,959 20,502 9,354
95% C.L.6 --- ---
Slope lZl 53l 209

l

2

Chemical purity = 99%

Fourteen hours lignt2l0 nours darK with dimming and brightening of
between light and dark periods.

lights

U]

LC50 values, 9 % C.L., and Slopes
Litchfield and Wilcoxon (l948).

LC50 values, 9 % C.L., and Slopes
using a direct regression of dose

LC50 values, 95% C.L., and Slopes

were determined by the method described by

were determined by a log-probit analysis
on mortality (Montgomery and Peck, l982).

were detennined by a log-probit analysis

using an inverse prediction of mortality on dose (Gill, l978).

Lack of confidence limits resulted from the inability to reject the hypothe—
sis of slope equaling zero.

47

Table l6. Percent mortality and LC50 values for mallards fed dietary stry-
chninel.
Percent mortalit
Dose 24 hrs. l4 hrs. l4 hrs.
(ppm) h light light light w/d2
0.0A l0 0 0 0
0.08 lo 0 0 0
323 lo 50 40 30
420 lo 50 80 60
546 10 90 90 80
710 lo 80 9O 70
923 lo 90 80 90
l.200 l0 lOO l00 l00
LC503 370 270 410
95% C.L. (284-482) (l69-430) (320-524)
Slope 5.97 8.43 7.l3
LC504 432 424 460
95% C.L. ‘ (3l9-585) (285-628) (367—577)
Slope 0.054 0.067 0.070
LC505 ~ 379 343 421
951:. 01.5 ( l8-927) -—- ( 69-955)
Slope , l3.6 l5.9 ll.6

l
2

03

Chemical purity = 9 %

Fourteen hours lightle hours dark with dimming and brightening of lights
between light and dark periods.

LC50 values, 95% C.L., and Slopes were determined by the method described by
Litchfield and Wilcoxon (l948).

LC50 values, 95% C.L., and Slopes were determined by a log-probit analysis
using a direct regression of dose on mortality (Montgomery and Peck, l982).

LC50 values, 95% C.L., and Slopes were determined by a log-probit analysis
using an inverse prediction of mortality 0n dose (Gill, l978).

Lack of confidence limits resulted from the inability to reject the hypothe-
sis of slope equaling zero.

48

Direct Regression of Dose on Mortality

Predicting LC50 values using the log—probit analysis with
a direct regression of dose on mortality produced similar LC50
values to the Litchfield and Wilcoxon method in those trials
in which sufficient mortality was obtained (Tables 7 through
17). The two analytical methods also produced similar fre—
quencies in the relative toxicity (magnitude of LC50 values)
of the chemicals between the three photoperiods. Although the
LC50 values between the two methods were similar, the confi-
dence limits for the LC50 values were more narrow for the
direct regression of dose on mortality procedure (Tables 7
through 16), resulting in the determination of significant
photoperiodic effects in the bobwhite—fenthion, bobwhite—
endrin, and bobwhite-strychnine trials. In the bobwhite-
fenthion trial all three LC50 values were significantly differ—
ent (P < 0.05) between the photoperiods. The bobwhite—endrin
trial resulted in the 14D photoperiod producing a significantly
(P < 0.05) higher LC50 value than the 14 photoperiod while the
bobwhite—strychnine trial resulted in the 14 photoperiod
producing a significantly (P < 0.05) lower LC50 value than

the 24 and 14D photoperiods.

Inverse Prediction of the Regression of Mortality on Dose

As was seen with the probit analysis of Litchfield and
Wilcoxon, no significant differences in LC50 values between
the three photoperiods could be established (Table 18 and 7
through l6). The 24 photoperiod resulted in the lowest toxi—

city (highest LC50 values) in 50% of the trials. The 14 and

 

49

 

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51

14D photoperiods produced the lowest toxicity values in 10

and 40% of the trials, respectively. The confidence limits on
the LC50 values calculated by the inverse prediction of the
regression of mortality on dose were greatly widened in com—

parison to the other two analytical methods used.

(LC50 max

In comparing the variation LC50 min

) of LC50 values for
a particular chemical and species over the three photoperiods
tested, a range of 1.00:1.03 to 1.00:1.52, 1.00:1.08 to 1.00:
1.42, and 1.00:1.09 to 1.00:2.34 was obtained for the Litch—
field and Wilcoxon, direct regression, and inverse prediction
methods, respectively. If the variation in LC50 values for

a particular photoperiod, chemical, and species is contrasted
.by analytical method, a range of 1.00:1.03 to 1.00:2.39 is
obtained. The average variations in LC50 values between photo-
periods for a particular chemical and species were 27.6, 18.6,
and 64.3% for the Litchfield and Wilcoxon, direct regression,
and inverse prediction methods, respectively, while the
average variation for individual LC50 determinations between
analytical methods was 27.3%.

Mortality patterns of the three photoperiods for any
particular chemical were similar regardless of the chemical's
mechanism or speed of action. Secobarbital and strychnine
caused high mortality during the first 24 hours of exposure
followed by reduced rates of death in the remaining treatment
period in both species and throughout all photoperiods (Tables

19 and 20). Conversely, the fenthion and endrin treatments

resulted in minimal or no death during the first 24 hours of

 

 

 

52

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54

treatment but, mortality sharply increased after three or four
days of exposure (Tables 21 and 22). Also, as the dietary
concentration of fenthion increased, the time required for the
onset of mortality decreased. Thus, the higher dietary con-
centrations produced mortality during the second day of expo-
sure while the lower dietary concentrations did not produce
mortality until the third or fourth days of exposure. Morta-
lity patterns due to ANTU ingestion were similar between
photoperiods, but bobwhite and mallard reactions were somewhat
different. The majority of bobwhite deaths occurred during
day two or day four through six, while mallard mortality began
during the first 24 hours of exposure and continued through
day six at a relatively constant rate (Table 23).

In general, bobwhites were more sensitive than mallards
to the more toxic chemicals fenthion and endrin, but showed
less sensitivity than mallards to the less toxic chemicals
ANTU, secobarbital, and strychnine (Table 6).

Clinical observations of the symptoms of toxicosis for
each trial are reported in Table 24.

There were no significant differences in slopes between
photoperiod for any of the trials. The slopes of the dose—
mortality curves were relatively uniform between photoperiod
for a particular trial with the exception of the three trials
in which insufficient mortality was obtained for proper
analysis. The maximum difference between the dose-response
slopes for the three photoperiods of a partucilar trial was

2.17-fold.

 

 

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58

Table 24 . Observed symptoms of toxicosis for bobwhite and mallard LC50 trials.

Species

Chemcial Bobwhite Mallard
ANTU ataxia ataxic
lethargic lethargic
prostrate withdrawal
wing drop
withdrawal
Fenthion ataxic ataxic
frequent falling difficulty standing
hyperactivity lack of coordination
inability to stand lethargy
inability to walk withdrawal
lack of coordination
lethargic
wing drop
withdrawal
Endrin ataxic ataxic
lethargic , lack of coordination
tetanic seizures lethargic
wing drop tetanic seizures

Secobarbital

Strychnine

withdrawal

ataxic

lack of coordination
lethargic

prostrate

sleeping

wing drop

withdrawal

ataxic

convulsions
hyperacusis
hyperexcitability
lack of coordination
tetanic seizures
tremors

withdrawal

ataxic

lack of coordination
lethargic

prostrate

sleeping

withdrawal

ataxic

convulsions

lack of coordination
lethargic

prostrate

staggers

tetanic seizures
withdrawal

Severe ataxia;inability to carry out voluntary movements.

 

59

In four of the ten trials there was a significant photo—
period effects on feed consumption during the five-day treat—
ment period (Table 25). Bobwhite and mallard feed consumption
were significantly (P S 0.05) greater under the 24 photoperiod
than under the 14 photoperiod during exposure to endrin and
secobarbital (Table 25). In addition, bobwhite exposed to
secobarbital consumed significantly (P < 0.05) less feed during
the treatment period on the 14D photoperiod than on the 24
photoperiod.

A nonstatistical comparison of mean feed consumption
indicated that the birds under continuous light consumed a
greater amount of feed during the treatment period in seven
out of ten trials when contrasted to the feed consumption of
birds under the 14 photoperiod and nine out of ten trials
when compared to birds on the 14D photoperiod (Table 25).

Both bobwhite and mallard ANTU trials resulted in signi—
ficant photoperiod effects on feed consumption for the three—
day recovery period (Table 26). Bobwhite exposed to ANTU
consumed significantly greater (P S 0.05) amounts of feed
under the 24 photoperiod than under the 14D photoperiod during
the recovery period (Table 26). Similarly, mallard food con-
sumption during the three-day recovery period following expo-
sure to ANTU was significantly greater (P < 0.05) under the
24 and 14 photoperiods than under the 14D photoperiod (Table
26). The only other significant photoperiod effect on feed
consumption during the recovery period was in the mallard—

strychnine trial. During this trial ducklings on the 24 and

 

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62

14 photoperiods consumed significantly greater (P < 0.05)
quantities of feed than ducklings on the 14D photoperiod (Table
26). Generally, there was no trend toward increased feed con-
sumption in the 24 photoperiod during the recovery period.
Only three trials resulted in continuous lighting having the
highest feed consumption value of the three photoperiods during
recovery (Table 26).

Significant differences (P < 0.05) in initial mean body
weights between photoperiods occurred in three of the ten
trials (Table 27). For the trial in which ANTU was admini-
stered to bobwhite, the chicks on the 24 photoperiod were
significantly heavier at the initiation of the trial than
chicks on the 14 or 14D photoperiods (Table 27). Also during
the ANTU-bobwhite trial, chicks on the 14 photoperiod had
significantly greater initial mean body weights than chicks
on the 14D photoperiod. In the mallard trials, ducklings
exposed to ANTU and the 14 photoperiod were significantly
heavier at the study onset than ducklings exposed to ANTU and
the 24 or 14D photoperiods (Table 27). Ducklings fed the
fenthion treated diets and maintained on continuous lighting
had significantly lower initial body weights than birds
exposed to fenthion on the 14 photoperiod (Table 27).

The birds on the 24 photoperiod had the highest initial
mean body weights of the three photoperiods in 50% of the
trials run, while the 14 and 14D photoperiods produced the
heaviest weights in 30 and 10% of the trials, respectively

(Table 27).

 

 

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64

On day five of the LC50 studies, photoperiod caused a
significant (P < 0.05) alteration in body weight in seven of
the trials (Table 28). In all seven trials, the 24 photoperiod
produced significantly greater mean body weights than one or
both of the l4—hour lighting regimes. Bobwhite on the 24
photoperiod were significantly heavier at the end of the five-
day treatment period than birds under the 14 and 14D photo-
periods when exposed to ANTU, fenthion, endrin, or secobarbi—
tal (Table 28). In addition, bobwhite exposed to ANTU and the
14 photoperiod were significantly heavier than birds exposed
to ANTU on the 14D photoperiod (Table 28). Mallards maintained
on the 14D photoperiod had significantly lighter five-day body
weights than ducklings exposed to the 24 and 14 photoperiod
regimes during the ANTU and strychnine trials (Table 28).
During the mallard—endrin trial, ducklings under the 14 photo—
period had significantly lower body weights than ducklings
under the 24 and 14D photoperiods (Table 28).

Mean body weights at the study termination (day eight) of
birds exposed to the 24 photoperiod were significantly (P <
0.05) greater than at least one of the 14 hour photoperiods in
five of the ten trials (Table 29). Bobwhite fed ANTU, endrin,
and secobarbital treated diets produced greater body weights
at day eight under the 24 photoperiod than those under either
of the l4—hour lighting regimes (Table 29). The 14D photo-
period also produced significantly lower body weights than the
14 photoperiod at day eight of the fenthion and secobarbital

trial. Mallards fed ANTU showed significantly higher day

 

 

 

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67

eight body weights on the 24 photoperiod than on the 14D
photoperiod, while ducklings exposed to secobarbital weighed
significantly more on the 14D photoperiod than ducklings on
the 14 photoperiod (Table 29).

Overall, eight—day body weights were heavier on the 24
photoperiod than on either the 14 or the 14D photoperiods in
seven trials, while the 14 photoperiod produced heavier day
eight body weights than the 14D photoperiod in seven trials
(Table 29).

Treatment effects on feed consumption for bobwhite and
mallards during the five-day exposure period were highly
significant (P < 0.001) in all trials except the bobwhite—
secobarbital trial. The trial in which bobwhite were fed
secobarbital resulted in fairly uniform feed consumption
throughout all treatments, while all other trials produced a
general trend for decreasing feed consumption with increasing
dietary concentrations (Table 30).

The treatment effect on feed consumption for the three—
day recovery period did not result in the level of signifi-
cance produced during the treatment period. For the recovery
period, only four trials resulted in significant differences
in feed consumption between levels (Table 31) indicating feed
consumption of birds exposed to the various chemicals returned
to control values in most cases. Of the four trials producing
significant treatment effects, only seven diets resulted in
significantly (P < 0.05) lower feed consumption than the

control (Table 31).

 

 

 

 

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70

Significant differences in initial mean body weights
between treatments occurred in the bobwhite—ANTU trial,
mallard—ANTU trial, and mallard-secobarbital trial (Table 32).
The LC50 tests in which bobwhite were exposed to ANTU
resulted in the number three and four (3,920 and 5,488 ppm,
respectively) treatment birds being significantly (P < 0.05)
lower in initial body weights than the control birds (Table
32).

Conversely, in the mallard trial using ANTU, the control
birds were significantly (P < 0.05) lower in initial body
weights than the number one, two, three, and six treatment
(2,000, 2,800, 3,920, and 10,756 ppm, respectively) birds
(Table 32). The mallard—secobarbital trial started with the
number four treatment (5,488 ppm) significantly (P < 0.05)
lower in initial mean body weight than the control (Table
32).

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effects on body weights at day five and day eight of the tests
(Table 33 and 34), with most chemically—treated diets produ-
cing significantly lower body weights than the control diets.

Significant photoperiod-dietary concentration interaction
occurred in initial, five—day, and eight—day body weights of
bobwhite fed endrin and in feed consumption during the three-
day recovery period of mallards fed strychnine. The inter-
actions were attributed to inconsistent effects of dietary

treatments across dietary concentrations and between photo—

periods. Body weights and feed consumption were highly

 

 

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74

irregular and did not follow the decreasing weights with
increasing dietary concentration noted in all other studies.
Mean room temperature, brooder temperature, and room

relative humidity are presented in Appendix C.

DISCUSSION

The avian subacute dietary LC50 is a fundamental para-
meter which may be required by the Federal Insecticide,
Fungicide, and Rodenticide Act (FIFRA) and the Toxic Substance
Control Act (TSCA) as a routine data point in evaluating the
hazards of pesticides and other toxic chemicals when dietary
exposure is the most probable route of intoxication. In
addition to the subacute lethality and species susceptibility
data, the LC50 provides a method for the comparisons of the
short-term dietary toxicity of chemicals to a given species
or between various species. Some authors have devised a
toxicity ranking system, based on the LC50, to categorize the
inherent toxicity of compounds in an attempt to establish and
predict the relative chemical toxicities by and within chemi—
cal classes (Heath et al., 1972). In addition, the slopes of
the dose—response curves can be used to establish the rate of
increase of mortality resulting from a proportional increase
in exposure. The greater the slope, the more rapid is the
increase in mortality and the lower the margin of safety.

When chemical contamination in the bird's natural diet
can be established by analytical techniques, the degree of

hazard may be predicted by comparing the concentration of the

 

 

75

chemicalin the natural diet to the lethality of dietary
concentrations in laboratory studies. When the contamination
of natural diets cannot be determined, the potential letha—
lity of the compound may be estimated by comparing the
chemical in question with a chemical in which the field
mortality is known for specific application rates. For
example, if the LC50 for compound A and B are 10 and 20 ppm,
respectively, and the field mortality of a species for com-
pound B is known at various application rates or general
residue levels, the potential mortality caused by compound

A should be predictable. Certain authors use a similar
analogy when they attempt to develop analytical methods of
predicting lethality values, for a particular species, on the
chemical and physical properties of toxic compounds (Rekker,
1980). Of key importance to the value of any classification
or prediction scheme is the reliability and repeatability of
the results.

There are several established and acceptable methods of
analysis of the dose—mortality response. Three accepted and
frequently used methods are the probit analysis of Litchfield
and Wilcoxon (1948), the analysis of the regression of percent
mortality on dose (Boyd, 1958; Gill, 1978), and the regression
of dose on percent mortality (Boyd, 1965; Gill, 1978;
Montgomery and Peck, l982). Boyd (1972) states that values
for the LDSO determined by one method have been found to be
repeatedly significantly different from values calculated by

another method with experiments run in his laboratory.

 

76

In looking at the analysis of data from this experiment
using the Litchfield and Wilcoxon procedure, the variation
between LC50 values of the three photoperiods in a particular
trial, in which sufficient mortality was obtained to accurately
calculate an LC50 value, was relatively low. The LC50 ratios
between photoperiods for these trials ranged from 1.00:1.15
to 1.00:1.52, which was somewhat smaller than the range in
LC50 values of intratrial replications (1.00:1.02 to 1.00:1.89)
reported by Hill and Camardese (1981) in studies exposing
Japanese quail (Coturnix coturnix japonica) to carbofuran,
dicrotophos, and thionazin. LC50 variations between photo-
periods were also considerably lower than the two-fold and
four-fold variations in LC50 values of intertrial LC50 repli-
cations exposing bobwhite and mallards, respectively, to diel—
drin (Heath gt $1., 1972). The variations between photoperiod
for a specific chemical and species using the direct regres-
sion method ranged from 1.00:1.08 to 1.00:1.42 which was also
considerably less than the intratrial replication of Hill and
Camardese (1981) and the intertrial replication of Heath et
31. (1972). The inverse prediction of the regression of
mortality on dose resulted in greater photoperiodic variation
1.00:1.09 to 1.00:2.34 in LC50 values within a trial than
the intratrial variation of Hill and Camardese but produced
intermediate variations to the intertrial replications of
Hill et 31. (1975). The variation in LC50 values between
analytical methods ranged from 1.00:1.03 to 1.00:2.39 and

was also greater than, and intermediate to the intratrial

 

77

and intertrial variance, respectively, of Hill and Camardese
(1981) and Hill et 31. (1975). Although there were considerable
variations in LC50 determinations between analytical methods,
the LC50 values calculated in this study for endrin and fen—
thion were in agreement with values published in the literature
with the exception of the LC50 of fenthion for mallards which
was somewhat lower in this study than that published in the
literature.

The slope of the dose—response curves for a particular
trial also showed less variation between photoperiod than the
variation of intra and intertrial replication reported by
Hill and Camardese (1981). The maximum variation in slopes of
the dose—response curves between photoperiods of a trial was
2.17-fold in comparison to the 2.64 and 4.30-fold variations
obtained in intra and intertrial replications, respectively,
of Hill and Camardese (1981).

Due to the considerable variation in photoperiod effects
on mortality between the two species and five chemicals tested
and the general inability to obtain significant differences in
LC50 values between photoperiods, it must be concluded that
photoperiod did not affect LC50 determinations in this study.
Although feed consumption and body weights, especially during
the five—day treatment period, proved on occasion to be signi-
ficantly greater on the 24 photoperiod than the 14-hour photo—
periods, the effects of increased chemical intake or altered
feeding patterns were not strong enough to significantly affect
mortality in these studies. Also, the significant differences

in feed consumption and body weight between the two 14—hour

 

78

photoperiods were less frequent than the significant differences
between the 24—hour and l4—hour photoperiods. Additional
studies utilizing intratrial replication of photoperiod or
alternate chemcials may prove otherwise, but based on this

study and the data supplied by Heath 33 31. (1972), Hill 33

31. (1975), and Hill and Camardese (1981), it appears that
intertrial variability of LC50 testing and analytical methods

of analysis causes greater problems in evaluating and classi—

fying chemical toxicities than does photoperiodic effects.

 

 

4.

79

CONCLUSIONS

Photoperiod significantly affected feed consumption during
the five—day treatment and three—day recovery periods. The
24-hour photoperiod tended to result in significantly
greater feed consumption than either of the two 14-hour
photoperiods. Significant differences in feed consumption
between the 14 and 14D photoperiods were less frequent
than significant differences between the 24 and 14 or 24
and 14D photoperiods.

Photoperiod significantly affected body weights at day 0,
5, and 8 of the trial. The 24-hour photoperiod tended to
produce significantly greater body weights than the 14—
hour photoperiods. Again, the frequency of significant
differences between the l4—hour photoperiods was less than
the frequency of significant differences between the 24-
hour and 14-hour photoperiods.

Mortality patterns and symptoms of toxicosis were gener—
ally similar between the 24, 14, and 14D photoperiods.

Photoperiod did not significantly affect LC5os.

 

80

REFERENCES

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Heath, R.G., J.W. Spann, E.F. Hill, and J.F. Kreitzer, 1972.
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Hill, E.F. and M.B. Camardese, 1981. Subacute toxicity testing
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Hill, E.F., R.G. Heath, J.W. Spann, and J.D. Williams, 1975.
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83

APPENDIX A

Composition of guail starter.

Ingredient Parts per kg
Corn, #2 yellow 375.2
Soybean meal, dehulled (49% protein) 420
Dist. dried grains solubles, corn 40
Fish meal 40
Alfalfa meal, dehy. (l7%protein) 50
Animal fat, stabl. 37.6
Dicalcium phosphate 20.0
Choline chloride (50%) 3.0
Methionine hydroxy analogue 0.7
Salt 3.5
Mineral mixa 5.0
Vitamin mixb 5.0

a Mineral mix: Supplies per kg diet: Cobalt, 50 mcg;
Manganese, 55 mg; Magnesium, 500 mg; Iron,
80 mg; Copper, 4 mg; Zinc, 80 mg; Selenium,
(from sodium selenite), O.l mg; Carrier
(dist. dried sol., corn with % tallow) to
5.0 g.

b Vitamin mix: Supplies per kg diet: Vitamin A, l5,000
I.U.; Vitamin D3, l,500 I C.U.; Vitamin E,
l5 I.U.; Vitamin K (menadione sodium bisul-
fite complex), 2.7 mg; Thiamine, 6.0 mg;
Riboflavin, l0.0 mg; Niacin, loo 0 mg;
Pyridoxine, l0.0 mg; Biotin, 220 mcg;
Folacin, 5.0 mg; Vitamin 812, ll.0 mcg;
Carrier (dist. dried sol., corn with %
tallow) to 5.0 g.

 

 

 

 

84

APPENDIX B

Composition of duck starter.

Ingredient Parts per kg
Corn, #2 yellow 503.l
Soybean meal (48% protein) 3l0
Alfalfa (l7% protein) 50
Wheat bran 6O

Corn oil, stabl.a 40
dl-methionine 0.9
Limestone

Dicalcium phosphate 22

Salt

Choline Clz, 50%
b

C

Vitamin mix

Mineral mix

Selenium mixd

a Ethoxyquin add

Vitamin mix:

C . .
Mineral mix:

From Calcium C

oowww
UTU‘IOOO

ed at l25 mg/kg diet.

Supplies per kg diet: Vitamin A, l5,000
I.U.; Vitamin D3, l,500 I.C.U.; Vitamin E,
l5 I.U.; Vitamin K (menadione sodium bisul-
fite complex), 2.7 mg; Thiamine, 6.0 mg;
Riboflavin, l0.0 mg; Niacin, lO0.0 mg;
Pyridoxine, l0.0 mg; Biotin, 220 mcg;
Folacin, 5.0 mg; Vitamin Biz, ll.O mcg;
Carrier (dist. dried sol., corn with %
tallow) to 3.0 g.

Supplies per kg diet: Cobalt, 50 mcg;
Manganese, 55 mg; Magnesium, 500 mg; Iron,
80 mg; Copper, 4 mg; Zinc, 80 mg; Selenium,
(from sodium selenite), O.l mg; Carrier
(dist. dried sol., corn with % tallow) to

0.50 g.

arbonate Co. at recommended levels.

 

 

 

 

 

 

85

 

 

 

 

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