THE EFFECT OF MIREX ON REPRODUCTION OF

JAPANESE QUAIL AND ON CHARACTERISTICS

OF EGGS FROM JAPANESE QUAIL
AND CHICKENS
K. L. DAVISON, J. H. COX, and C. K. GRAHAM 1
United States Department of Agriculture
Agricultural Research Service
Metabolism and Radiation Research' Laboratory
Fargo, North Dakota 58102

Laying White Leghorn chickens were fed mirex at 0, 5, 10, 20, 40, 80, and
160 ppm for 12 weeks, and laying Japanese quail were fed mirex at 0, 5, 40, and
80 ppm for 12 weeks. The data suggest that dietary mirex at these levels did not
affect egg production, egg weight, shell thickness, shell calcium, the proportion of
broken eggs, or the proportion of soft-shelled eggs of either chickens or quail.
Statistical significance (P < 0.05) associated with dietary mirex was detected in the
analysis of eggshell weight for chickens; however, because a dose-response relation-
ship of shell weight vs. level of mirex fed was not evident, this observation was
attributed to chance. The data suggest that dietary mirex did not affect eggshell
weight, fertility, or hatchability of quail. Mirex accumulation in eggs and carcasses
of both species was proportional to dose and was slightly higher in quail than in
chickens.

Mirex [dodecachlorooctahydro-l,3,4-metheno-iH-cyclobuta[cd] pentalene] is applied

at 1.7 g/A in 1.25 Ib of corncob grits-soybean oil bait to control the imported fire
ant (Solenopsis richteri) in the southeastern United States (Baetche et al. 1972). In an
experiment involving very heavy application of mirex bait (up to 1000 lb/A), Baker
(1967) reported reduced fertility and hatchability of bobwhite quail in pens receiving the
highest rate of application. However, because of variability in reproductive success among
replicate pens of quail, Baker (1967) cautiously interpreted his data and suggested that
his observations be investigated further.

Nabor and Ware (1965) studied effects of mirex on egg production and hatchability of
chickens and on survival of hatchlings. Mirex fed at 300 or 600 ppm for 16 weeks did not
affect egg production. Mirex fed at 300 ppm for 12 weeks did not affect hatchability
but did reduce survival of hatchlings. Mirex fed at 600 ppm for six weeks reduced
hatchability and hatchling survival.

Two chlorinated hydrocarbon insecticides, dieldrin [1,2,3,4,10,10-hexachloro-6,7-

epoxy-l,4,4a,5,6,7,8,8a-octahydro-l,4-endo-exo-5,8-dimethanonaphthalene] and DDT

IStatistician, North Central Region, Ames, Iowa 50010.

Archives of Environmental Contamination 84

and Toxicology, Voi. 3, No. 1,1975, 9 1975
by Springer-Verlag New York Inc.
 Effect of mirex on Japanese Quail and Chickens 85

[1,1,1-trichloro-2,2-bis(p-cbAorophenyl)-ethane], cause eggshell thinning in ducks (Heath

et al. 1969, Davison and Sell 1974) but not in chickens (Davison and Sell 1972, Cecil et al.
1972). DDT caused eggshell thinning in Japanese quail (Coturnix coturnix japonica) in
some experiments (Bitman et al. t969, Stickel and Rhodes 1970) but not in another
experiment (Cecil et al. 1971). Since mirex is also a chlorinated hydrocarbon insecticide,
we thought that it should be investigated for eggshell thinning in different species of
birds.

We investigated effects of mirex on eggs and eggshells of chickens and Japanese quail
and on fertility and hatchability of Japanese quail. While our investigations were in
progress, we learned that Heath and Spann (1973) had conducted similar investigations
with mallard ducks (Anas platyrhynchos) and bobwhite quail (Colinus virginianus).

Methods
Chickens. Seventy White Leghorn hens, 19 weeks old, were housed in individual wire
cages. They were restricted to 90 g daily of a nutritionally balanced, commercially pre-
pared, laying diet containing 2.8% calcium. Individual hens served as the experimental
units. Artificial light was provided 14 hr per day.

When the hens were 24 weeks old, they were randomly allotted to seven treatment
groups of ten hens each. Treatments consisted of mirex added to the diet as follows:
(1) 0, (2) 5, (3) 10, (4) 20, (5) 40, (6) 80, and (7) 160 ppm ~ g of mirex/g of diet). The
mirex was dissolved in 400 to 500 ml of acetone and premixed into six kg of feed - one
premix was made for each diet. The acetone evaporated during mixing. Diets were pre-
pared twice during the experiments from these premixes. Mirex levels in the diets were
verified by electron-capture gas-liquid chromatography (glc).

Eggs were collected, dated, and weighed to the nearest 0.1 g. At biweekly intervals,
the eggs from individual hens were arranged on numbered fiats according to date laid and,
with the aid of a table of random digits, one egg was selected for measurement of shell
thickness (without membranes), dried shell weight, and shell calcium (Davison and Sell
1972).

Japanese quail. Forty-eight female Japanese quail, six weeks old, were housed singly in
sheet metal and wire cages measuring 17.5 • 24 • 18 cm; and 48 pairs of quail (male and
female), also six weeks old, were housed in pairs in sheet metal and wire cages measuring
22.5 X 38 X 17 cm. They were fed ad libitum a nutritionally balanced, laboratory-
prepared, laying diet containing 2.9% calcium. Each female quail served as an experi-
mental unit.

When the quail were eight weeks old, they were randomly allotted to four treatment
groups of 12 single quail and 12 pairs of quail each. Treatments consisted of mirex added
to the diet as follows: (1) 0, (2) 5, (3) 40, and (4) 80 ppm. Mirex feeding began when the
quail were ten weeks old. Light was provided and the diets were prepared as described for
chickens.
 86 K.L. Davison e t al.

Eggs were collected daily and stored in a refrigerator in numbered canisters (one
canister for each female). The eggs were weighed at biweekly intervals and arbitrarily
arranged on a numbered flat. One egg was selected at random for measurement of shell
thickness, dried shell weight, and shell calcium (Davison and Sell 1972). Remaining eggs
from single birds were discarded. Remaining eggs from paired birds were incubated to
determine fertility and hatchability.

Residues. Mirex residues were determined in samples of freeze-dried eggs or carcasses

by electron-capture glc according to our modification (Davison 1970) of Johns on's (1965)
method for measuring chlorinated hydrocarbons in fatty materials. About 0.05 ppm of
mirex could be detected in the egg and carcass samples by this method. Because residue
analysis is tedious, the number of samples to be analyzed was reduced by pooling.

The contents of eggs broken for shell measurements were pooled so that two pools of
eggs represented each dietary level of mirex at each biweekly interval for the chickens,
and four pools of eggs (two pools for single birds and two pools for paired birds) repre-
sented each dietary level of mirex at each biweekly interval for the quail. Identity of the
birds whose eggs contributed to each pool was maintained so that eggs from the same
birds were pooled together throughout the experiment. The egg contents were blended
and freeze-dried.

All birds were killed at the end of the experiments. Carcasses of six chickens from each
treatment group and of all female quail were plucked and ground (including viscera).
The ground chickens were divided according to diet into 14 pools, each containing three
carcasses. The ground quail were divided, according to diet and housing, into 24 pools of
three carcasses each. After blending, a sample of the pooled carcasses was freeze-dried.

Statistical analysis. The data were analyzed statistically by analysis of variance, with
significance assessed at probabilities of 5% or less. Proportion data were transformed with
a method described by Gabriel (1963) for proportions based on small numbers of observa-
tions. The analyses of variance for data on egg weight, shell thickness, shell weight, and
broken eggs of quail are shown in Table I.

Results
Chickens. Data summarized for the 12-week period are shown in Table II. All hens
survived the experiment. Thirteen hens, on occasion and irrespective of diet, refused to
eat all of the allotted 90 g of feed per day. The average body weight for all 70 hens in-
creased 22 g during the experiment. Egg production for the experiment averaged 0.79
eggs/bird/day.

Dietary mirex did not significantly affect body weight or egg production of the hens,
number of cracked eggs, egg weight, eggshell thickness, eggshell calcium, or number of
soft-shelled and membranous eggs. Statistical significance (P < 0.05) associated with
dietary mirex was observed in the analysis of the weight of the eggshells. However, a dose-
response relationship of shell weight vs. level of dietary mirex was not evident, so this
observation was attributed to chance.
 Table I. Statistical analysis of egg weight, shell thickness, shell weight, and
proportion of broken eggs of quail fed mirex

Degrees of Egg Shell Shell Broken

Source of variation freedom weight thickness weight eggs

Mean squares
Total 634
o
Diet (Mirex levels) 3 2.926 0.00091 0.0347 0.1020
t~
Caging (Single or paired) 1 3.832 0.00208 0.0015 0.4151
O

Diet X Caging 3 5.609 0.00006 0.0137 0.1276

Birds within Diet X Caging (Error A) a 88 3.314 0.00118 0.0333 0.1233

Time (Biweekly periods) 6 21.639 b 0.00152b 0.0692 b 0.1326 b ,O

g~

Diet X Time 18 0.247 c 0.00018 c 0.0054 0.0199

Caging X Time 6 0.336 c 0.00003 0.0019 0.0385 o

Diet X Caging X Time 18 -0.014 0.00008 0.0033 0.0289

Residual (Error B) a 491 0.132 0.00010 0.0042 0.0360

aTerm for testing effects of diet, caging, and diet X caging.

b Statistically significant, P < 0.01.
cStatistically significant, P < 0.05.
dTerm for testing effects of time and all interactions containing time as a factor.
OO
 O0
O0

Table II. Body weight, egg production, and some characteristics of eggs of chickens fed
diets containing various levels o f mirex

Mirex level, ppm

Parameter 0 5 10 20 40 80 160

Avg. hen wt. at beginning of expt., g 1556 1432 1438 1465 1415 1500 1471
Avg. hen wt. at end of expt., g 1553 1441 1489 1500 1456 1497 1506
Avg. no, of eggs collected per bird 69 68 68 62 70 62 68
Total no. of cracked eggs 3 3 0
2 2 1 1
Avg. w t. of eggs, g 53.9 53.2 54.7 53.0 52.9 56.4 55.4 O

Avg. wt. of dried shell, ga 4.9 4.9 5.1 4.9 4.9 5.3 5.2 ga

Avg. thickness of shell, mm X 102 34.5 34.5 35.2 34.4 34.5 36.1 35.6
Avg. calcium in shell, % 36.7 36.9 36.8 36.8 36.8 36.8 36.7
Soft-shelled and membranous eggs, no. b 12 4 1 4 6 40 c 3

aStatistical significance was detected among these means, P < 0.05,

bThese eggs were not collected. They are not included in the average number of eggs collected per bird.
CThirty-eight of these eggs were laid by one bird.
 Effect of mirex on Japanese Quail and Chickens 89

Seventy very thin-shelled and membranous eggs fell through the cage floors into the
droppings. Thirty-eight of these eggs were from a single bird fed 80 ppm of mirex. No
attempt was made to collect these eggs, and they were not included in the average
number of eggs collected per bird. The incidence of thin-shelled and membranous eggs
did not appear to be related to dietary mirex.

Residues in the eggs and carcasses are shown in Figure 1. Higher residues relative to
other time periods were detected during the first two weeks of the experiment in eggs
collected from birds fed no mirex (control). Accidental contamination of these samples
was suspected. Control eggs averaged < 0.5 ppm of mirex for the second through the
twelfth week, and control carcasses averaged 0.1 ppm of mirex.

Mirex accumulation in dry matter of eggs appeared to be directly related to the amount
of mirex fed. Thus, we wanted to see if a mathematical model could be developed to
predict mirex residue in eggs for the various dosage levels and the time during which the
chickens were fed. Equations containing reciprocal, logarithmic, and quadratic terms were
examined. The model selected was:

X1.
Y = 5.06 X 1 - 3.57 X2 Equation 1

where Y = estimated mirex residual in eggs,

X1 = dose in ppm, and

X 2 = time in weeks.

This model fitted the data well (R 2 = 0.99) and was relatively simple. (Some caution
should be used in interpreting the R2 value as the observations consisted of data obtained
from pooled samples rather than from individual observations.) The two parameters were
estimated by least squares techniques. Equations for the various dosage levels are plotted
in Figure 1, along with the mean values for the time periods. Examination of this graph
and equation 1 shows that initially the mirex accumulated rapidly. However, the rate of
mirex accumulation diminished with time so that eventually accumulation ran asymptoti-
cally to a level slightly over five times the dosage levels. It seems reasonable to assume
that this pattern would exist for time periods longer than 12 weeks and possibly for
doses larger than 160 ppm; however, caution should always be exercised in extrapolating
beyond the limits of the data.

At the end of the experiment, mirex residues in the carcasses were four times the
mirex concentration in the diets. The carcasses contained 36% dry matter, which con-
rained 39% lipid. The eggs contained 24% dry matter, which contained 35% lipid.

Japanese quail. Data for this experiment are summarized in Table III. Egg production
for the experiment averaged 0.86 eggs/bird/day. There were no significant differences
among any of the means listed in Table III although the higher percentages of broken
90 K.L. Davison e t al.

eggs for paired quail compared with those of single quail approached significance
(P = 0.066). For paired quail, the percentage of broken eggs was highest (about 6%)
during the two-week preliminary period, and this figure decreased as the experiment
progressed. For single quail, the percentage of broken eggs remained relatively constant
throughout the experiment.

As expected, time was a significant factor in the outcome of most parameters measured.
The average weight of the eggs increased throughout the experiment (Figure 2). The
average eggshell thickness (Figure 2) was maximum during the first to second week, and
then it decreased continuously to the ninth to tenth week. Other factors significantly
affected by time alone were the average weight of eggshells, the percentage of soft-
shelled eggs, fertility, and hatchability. The weight of eggshells increased from 0.72 g
initially to 0.80 g by the seventh to eighth week and remained near 0.80 g. The per-
centage of soft-shelled eggs decreased from 1.35% during the two-week preliminary
period to less than 0.70% during the 12--'week mirex feeding period. Fertility followed a
sigmoid pattern with time. Hatchability increased from 56% initially to 71% by three to
four weeks; it then decreased to 53% for the ninth through twelfth weeks.

Significant interactions (P < 0.05; see Figure 2) of diet X time and caging X time were
detected for egg weight, and of diet X time for eggshell thickness. Initially, eggs laid by pair-
ed quail were heavier than eggs laid by single quail;however, as the experiment progressed,

H
Dietary M i r e x
160 ppm =
80~ 80 p p m 9 =
40 p p m v =
20 p p m ,, = M i r e x in carcases
700 10 p p m o
5 ppm 9 o
Control ~ =
600
r / Mirexaccumulatelineggs 9
E 500

"o
c
300
x

.~ 200

1001 j 9 m im

2 o o o
C' | -- I -" i -" ! m
' "',"."

2 4 6 8 10 12 -" 12
Weeks
Fig. 1. Average mirex residues in dry matter of eggs and carcasses of chickens fed various
levels of technical mirex (points), and curve predicted by the equation: Y = 5.06 dose -
(3.57 dose + time).
 Table III. Mortality, egg production, and some characteristics of eggs of Japanese quail housed singly
or in pairs {male and female) and fed various levels of mirex

Mirex fed to single quail, ppm Mirex fed to paired quail, ppm
rt~
Parameter 0 5 40 80 0 5 40 80

Mortality, no. 0 1 0 0 3 0 1 0

Avg. no. of eggs/bird/day 0.86 0.85 0.92 0.84 0.82 0.89 0.87 0.80
2
O
Avg. weight of eggs, g 10.4 9.8 10.1 10.3 10.4 10.5 10.1 10.3

Avg. weight of dried shell, g 0.78 0.75 0.76 0.80 0.77 0.77 0.76 0.78

Avg. thickness of shell, mm 0.189 0.183 0.184 0.187 0.186 0.181 0.181 0.182 ,O
Avg. calcium in shell, % 33.9 33.9 33.8 33.8 34.0 33.5 33.8 33.7

Avg. percentage of broken eggs 1.7 3.4 2.1 0.9 4.8 3.8 2.3 3.4 ('3
~r
Avg. percentage of soft-shelled eggs 0.1 1.4 0.9 0.3 0.5 0.7 0.2 0.7

Avg. percentage of fertile eggs - - - 54.5 56.2 56.5 64.3

Avg. percentage hatchability of

fertile eggs 61.4 58.7 61.6 63.8

',D
 92 K.L. Davison e t al.

the eggs became similar in weight. The biological implication of the interactions of diet X
time for egg weight and eggshell thickness was not clear, and the interactions were quite
small. The large mean squares for time (Table I) relative to the mean squares for diet X
time interactions for these variables suggest that the interaction might be discounted.

Residues accumulated in the eggs and carcasses are shown in Figure 3. Residues in eggs
and carcasses of control birds probably represent accidental contamination because mirex
was not detected in the control feed or in the eggs collected before mirex was fed.

A mathematical model similar to the one for chicken eggs was developed for predicting
mirex residues in quail eggs. This model was:

Y = 6.42 X1 - 3.04 X1 Equation 2

X2 "

It fitted the data well (R2 = 0.97, where observations again were obtained from pooled
samples) although there was more variation with quail eggs than with chicken eggs.

Single or paired birds vs. t i m e Mirex level vs. time

Dietary Mirex
Single birds ---.o---
.195 Paired birds 9 80 ppm --~-
E 20ppm --=---
E
.190 -- " ,~,~, \ 5 ppm .......
,/ ~ "\ \ Control . 9
.18E

e-
.180

u~.175

.170

11.0

10.5

9~ 10.0

~ 9.5
/•e/"/
o" /

9.0

8.5 . . . . . , , ' , , , , . . . . . , ,
- 2 2 4 10 12 -- 2 0 6 8 1 12
Weeks

Fig. 2. Average eggshell thickness and egg weight of quail. Left half of figure shows
eggshell thickness and egg weight for quail housed singly or in pairs (male and female),
regardless of the amount of mirex fed. Right half of figure shows eggshell thickness and
egg weight according to the level of mirex fed, regardless of the kind of housing.
 Effect of mirex on Japanese Quail and Chickens 93

Possible reasons for this increased variation are that quail were fed ad libitum, whereas
the feeding of the chickens was restricted; the quail were housed both singly and in
pairs; and only three dosage levels were examined for quail but six were examined for
chickens.

Equations for the three dosage levels are plotted in Figure 3, along with the mean
values for both paired and single birds. Again there was a rapid initial accumulation and a
leveling off at about three weeks, with the prediction line running asymptotically to a
level slightly less than 6.5 times the dosage level. Caution should again be used when
extrapolating beyond the limits of the data.

Mirex accumulation in carcass dry matter was about five times the mirex concentration
in the diet. Carcasses of single quaff contained 31% dry matter, carcasses of paired quail
contained 29% dry matter, and eggs contained 28% dry matter. On a dry matter basis,

H
Paired birds Single birds
6OO 80 ppm 9 o
40 ppm = *
5 ppm 9 * 9
Control 9 9

500

o o 0
...=

E400 Mirex in carcases

C

ated in eggs

=>.
30(;
c

o .

o
20(;

100

Q
' B a 9 = B
i l l I i i ~m

2 4 6 8 10 12 "" 12
Weeks

Fig. 3. Average mirex residues in dry matter of eggs and carcasses of quail fed various
levels of technical mirex (points), and curve predicted by the equation: Y -- 6.42 dose -
(3.04 dose + time).
94 K.L. Davison et al.

carcasses of single quaff contained 37% lipid, carcasses of paired quaff contained 31%
lipid, and eggs contained 42% lipid.

Discussion
Experimentally, mirex has been shown to accumulate through two-level food chains
(Lowe et al. 1971). Residues of mirex were determined in beef, milk, deer, fish, birds,
earthworms, and insects (other than fire ants) in an area one year after mirex bait (two to
three applications of the corncob grits bait) had been spread (Baetcke et al. 1972). Mirex
residues were < 0.02 ppm in milk fat and up to 0.113 ppm in beef fat. The highest resi-
dues reported were 11.3 ppm in adipose tissue of fish and 104 ppm in adipose tissue of
birds.

Mirex fed at a very high level (600 ppm) caused reproductive difficulties in chickens
(Nabor and Ware 1965). Mirex fed at 300 ppm (still a very high level) did not affect egg
production or hatchabffity but did reduce survival of hatchlings in chickens (Nabor and
Ware 1965). Fertility, hatchabffity, and survival of hatchlings were normal when mallards
were fed one or ten ppm of mirex and when bobwhite quaff were fed 40 ppm of mirex
(Heath and Spann 1973) although 40 ppm ultimately was lethal to some of the adult
quaff. As found by us, fertility and hatchability were not affected when Japanese quail
were fed up to 80 ppm of mirex. Dietary mirex did not thin eggshells from the mallards
or bobwhite quail (Heath and Spann 1973) nor did it thin eggshells of the chickens or
Japanese quail. Thus, based on present knowledge, small amounts of mirex apparently do
not threaten reproduction in the species of birds examined to date.

Acknowledgments

We thank David Voeller, Carol Klein, Maurice Bush, Daniel CottreU, Kenneth Mize,
and Robert Johnson for assisting us with these experiments.

Technical mirex was donated by the Allied Chemical Co., Baltimore, Md.: purity,
98.9%; lot No. SN-6-6988-45.

Reference to a company or product name does not imply approval or recommendation

by the U. S. Department of Agriculture to the exclusion of others that may be suitable.

References

Baetcke, K. P., J. D. Cain, and W. E. Poe: Mirex and DDT residues in wildlife and
miscellaneous samples in Mississippi - 1970. Pest. Monit. J. 6, 14 (1972).
Baker, M. F.: Studies of possible effects of mirex bait on the bobwhite quail and other
birds. Proc. 18th Ann. Conf. S. E. Ass. Fish and Game Comm., 1964, pp. 153-160
(1967).
 Effect of mirex on Japanese Quail and Chickens 95

Bitman, J., H. C. Cecil, S. J. Harris, and G. F. Fries: DDT induces a decrease in eggshell
calcium. Nature 224, 44 (1969).
Cecil, H. C., J. Bitman, and S. J. Harris: Effects of dietary p,p'-DDT and p,p'-DDE on
egg production and eggshell characteristics of Japanese quaff receiving an adequate
calcium diet. Poul. Sci. 50, 657 (1971).
Cecil, H. C., G. F. Fries, J. Bitman, S. J. Harris, R. J. Lillie, and C. A. Denton: Dietary
p,p'-DDT, o,p'-DDT, or p,p'-DDE and changes in eggshell characteristics and pesti-
cide accumulation in egg content and body fat of caged White Leghorns. Poul. Sci.
51,130 (1972).
Davison, K. L.: Dieldrin accumulation in tissues of the sheep. J. Agr. Food Chem. 18,
1156 (1970).
Davison, K. L., and J. L. Sell: Dieldrin and p,p'-DDT effects on egg production and egg-
shell thickness of chickens. Bull. Environ. Contam. Toxicol. 7, 9 (1972).
Davison, K. L., and J. L. Sell: DDT thins shells of eggs from mallard ducks maintained on
ad libitum or controlled-feeding regimens. Arch. Environ. Contain. Toxicol. 2,
222 (1974).
Gabriel, K. R.: Analysis of variance of proportions with unequal frequencies. J. Am. Stat.
Ass. 58, 1133 (1963).
Heath, R. G., and J. W. Spann: Reproduction and related residues in birds fed mirex.
Pesticide Symposium, 8th Inter-American Conference on Toxicology and Occupa-
tional Medicine, North Miami, FI., pp. 421-435 (1973).
Heath, R. G., J. W. Spann, and J. F. Kreitzer: Marked DDE impairment of mallard
reproduction in controlled studies. Nature 224, 47 (1969).
Johnson, L. Y.: Collaborative study for a method for multiple chlorinated pesticide
residues in fatty foods. J. Ass. Offic. Agr. Chem. 48,668 (1965).
Lowe, J. I., P. 1L Parrish, A. J. Wilson, Jr., P. D. Wilson, and T. W. Duke: Effects of mirex
on selected estuarine organisms. Trans. 36th North Am. Wildlife and Nat. Resources
Conf., pp. 171-186 (1971).
Nabor, E. C., and G. W. Ware: Effect of kepone and mirex on reproductive performance
in the laying hen. Poul. Sci. 44, 875 (1965).
Stickel, L. F., and L. I. Rhodes: The thin eggshell problem. Proceedings of the Symposium,
The Biological Impact of Pesticides in the Environment, Environmental Health
Sciences Series No. 1, Oregon State Univ., Corvallis, pp. 31-35 (1970).

Manuscript received March 16, 1974; accepted May 26, 1974