EFFECTS OF POLYCHLORINATED BIPHENYLS AND

TERPHENYLS AND POLYBROMINATED BIPHENYLS

ON PENTOBARBITAL SLEEPING TIMES OF
JAPANESE QUAIL
HELENE C. CECIL, SUSAN J. HARRIS and JOEL BITMAN
USDA, Agricultural Research Service
Animal Physiology and Gene~cs Institute
Bldg. 161 ARC-East
Belt~ville, Maryland 20703

Polychlorinated biphenyls (Aroclor 1221, 1232, 1242, 1248, 1254, 1260, 1262
and 1268), polychlofinated terphenyls (Aroclor 5460), a mixture of polychlofinated
biphenyls and terphenyls (Aroclor 4465) and polybrominated biphenyls (BP-6)
were administered in a single dose, I00 mg/kg body weight injected orally, to
mature male and female Japanese quail. Two hr after Aroclor treatment, pento-
barbital sleeping times were prolonged (2 to 3 X control) in both male and female
quail. Twenty-four hr after treatment, sleeping times were similar to those of con-
trois. However, 48 hr after treatment with Aroclor 1248 through Aroclor 5460 and
with BP-6, sleeping times of males were only 1/2 those of controls, whereas after
treatment with Aroclor 1221, 1232 and 1242, sleeping times were similar to those
of controls. The reduction in sleeping times of male quail 48 hr after administration
of PCBs was correlated with the percentage of chlorine in the PCB with greater re-
ductions in male than female quail. The response after three days ad l i b i t u m feeding
of 300 ppm of each substance was the same as the response 48 hr after a single oral
injection. Although very few birds died from the toxicity of the Aroclors and BP-6,
mortality was greatly increased during anesthesia when the pentobarbital was
administered two hr after single oral dosing.

Polychlofinated biphenyls (PCBs) are industrial chemicals that have been widely dis.
tributed in our environment. Recently, a mixture of polybrominated biphenyls was intro-
duced for use as a flame retardant in automobile seat covers and could be a potential
environmental pollutant. Like DDT, PCBs degrade very slowly and accumulate in the lipid
of animal bodies. Much of the research on PCBs indicates similarities in the effects of
PCBs and DDT.

Both DDT and PCBs have been shown to induce microsomal enzyme activity in birds
(Lincer and Peakall 1970, Nowicki and Norman 1972), rabbit (Villeneuve e t al. 1971), or
rat (Hart and Fouts 1963, Street e t al. 1969, Bickers e t al. 1972, Litterst and Van Loon
1972, Litterst e t al. 1972, and Norback and.Allen 1972). Rats fed DDT or PCBs also had
reduced pentobarbital sleeping times (Hart and Fouts 1965, Street e t al. 1969, Bitrnan
e t al. 1971, Bickers e t al. 1972, Villeneuve e t al. 1972) indicating increased metabolism of
pentobarbital by the induced liver microsomal enzymes. In quail, we found the opposite
effect, DDT and methoxychlor lengthened pentobarbital sleeping times (Bitman e t aL
1971, Cecil e t al. 1973).

Archives of Environmental Contamination 183

and Toxicology, Vol. 3, No. 2, 1975
1975 by Springer-Verlag New York Inc.
 184 H.C. Cecil et al.

In a preliminary study (Bitman e t aL 1972), we found that the sleeping time pattern
elicited by PCBs (Aroclor 1268) or by polychlorinated terpenyls (Aroclor 5460) was in
marked contrast to the pattern elicited by the organochlorine pesticide analogs of DDT in
quail (Bitman e t al. 1971, Cecil et al. 1973). The present study investigates several PCBs,
a mixture of polychlorinated terphenyls (PCT), a mixture of PCBs and PCTs, a mixture of
polybrominated biphenyls (PBB) and correlates their effects on liver metabolism of
Japanese quail as measured indirectly by pentobarbital anesthesia.

Materials and m e t h o d s
Mature male and female quaff (40-80 days old, 100-125g)were housed individually
on a schedule of 14 hr of light and 10 hr of dark. Each test substance was administered as
a single feeding in olive oil by stomach tube or by ad libitum feeding as a 300 ppm diet
for three days. Eight PCBs, a mixture of PCBs and PCTs, a mixture of PCTs, and a mixture
o f PBBs were used. The PCBs were Aroclor 1221, 1232, 1242, 1248, I254, 1260, 1262
and 1268; the mixture of PCTs was Aroclor 5460; and the mixture of PCBs and PCTs
was Aroclor 4465. The last two digits of the Arodors (Monsanto Company) represent the
percentage of chlorine in the formulation. The PBB mixture used was BP-6 (Michigan
Chemical Company) containing 75% bromine.t

At appropriate times after administration of the test substance, sodium pentobarbital

was injected intramuscularly at a dosage rate of 50 mg/kg of the male body weight or
60 mg/kg of the corrected female body weight. Body weights of the females were correct-
ed for the weight of the reproductive tract by subtracting ten g and, if an egg were also
present, subtracting an additional ten g. Birds were considered asleep as long as they could
not right themselves when placed on their back. Control birds slept for 40 to 60 rain.
Duration of sleeping time for treated birds is expressed as percentage of control. Statistical
comparisons were made with the Student's "t" test, with correction for unequal group
size (Snedecor 1956).

Results
Single oral injection of PCBs. Two hr after PCB administration, pentobarbital sleeping
times of quail were two to three times longer than sleeping times of control birds
(Figure 1, p < 0.001), and five hr after PCB administration sleeping times were 1.5 times
longer than those of controls (p < 0.001). However, the effects of a single dose of PCB
were short.lived and when pentobarbital was given 24 hr after PCB, the PCB-treated birds
had sleeping times similar to those of controls. PCB had no further effect in female quail;
but in males the trend toward shorter sleeping times continued, and at 48 hr some male
groups had siguifieanfly shorter sleeping times than those of controls. Closer examination
of the 48-hr data revealed that the shorter sleeping times of male quail were related to
the percentage chlorine in PCBs (Figure "2). The PCBs containing only 21% and 32%

tTrade names wore used solely for the purpose of providing specific information. Mention of a trade
name does not constitute a guarantee or warranty of the product by the Federal Government or an
endorsement by the Government over other products not mentioned.
 Effect of PCBs, PTPs, and PBBs on Sleeping Times of Quail 185

==
lOOI !
200

:
Female

:
-G

C
8 200 ~ Male

,oo !_/ "

0 = = I ~ = I
0 2 5 24 48
Hours post PCB

Fig. 1. Pentobarbital sleeping times of Japanese quaff after a single oral injection of poly-
chlorinated biphenyls (Aroclor 1221, 1232, 1242, 1248, 1254, 1260, 1262, or 1268). The
administered dosage was 100 mg test substance per kg body weight. Each point represents
the mean for 4 to 6 animals for each point.

Sleeping time
Male

48 hours after PCB

o
u 100

0
50 9 9

0 I = I = I , I , I i
20 30 " 40 50 60
% chlorine in PCB

Fig. 2. Pentobarbital sleeping times of male Japanese quaff 48 hr after a single oral injec-
tion of polychlorinated biphenyls containing 21% to 68% chlorine. The administered
dosage was 100 mg test substance per kg body weight. Each point represents the mean
for 5 or 6 animals.
 186 H.C. Cecil et al.

chlorine did not reduce pentobarbital sleeping time; however, as the chlorination in-
creased, sleeping times decreased. With 54% chlorine, the reduction in sleeping time
reached a plateau, and male quail treated with the PCBs containing 54, 60, 62 and 68%
chlorine had sleeping times that were only half as long as those of controls (p < 0.001).

Comparison of polychlorinated biphenyls and terphenyls and a polybrominated bi-

phenyl mixture (Figures 3 and 4). A mixture of PCBs and PCTs containing 65% chlorine
had the same effect as PCBs of comparable chlorination, with sleeping times twice that of
controls two hr after treatment (i0 < 0.001), while the PCT mixture (60% chlonne) and
the PBB mixture (75% bromine) only slightly lengthened sleeping times two hr after
treatment (males, PCT, p < 0.001 ;PBB, p < 0.005; females, PCT, p = n.s.; PBB, p < 0.1301).
However, at later times the PCT and PBB had more effect than the PCBs. Twenty-four
hr after PCT or PBB treatment, sleeping times of treated male quail were half as long as
those of control males (Figure 3, p < 0.001) and at 48 hr after treatment, all the higher
halogenated compounds reduced sleeping times in male quail (p < 0.001). In female
quail, the polybrominated compound (PBB) was the only compound which reduced
sleeping times (Figure 4, p < 0.001).

Ad libitum feeding. The effects 48 hr after a single oral injection were compared with
those of ad libitum feeding for three days (Figure 5)~ The single oral injection was
100 mg/kg of body weight, which is comparable to the daily intake of birds eating feed

Sleeping t i m e
Male

PCB - - 60, 62, 68% CI

3O0
PCB + PCT -- 65% CI
m-.a PCT -- 60% CI
~.Q~,,~ +,,..oPBB - - 7 5 % Br
200

~o+I ~ " + . . . oo
10o

= I I .,~ I |
0 2 5 24 48
Hours post injection

Fig. 3. Pentobarbital sleeping times of male Japanese quail after a single oral injection of
higher chlorinated polychlorinated biphenyls (Aroclor 1260, 1262 or 1268), a mixture of
polychlorinated biphenyls and terphenyls (Aroclor 4465), a polychlorinated terphenyl
(Aroclor 5460) and a polybrominated biphenyl (BP-6). The administered dosage was
I00 mg test substance per kg body weight. Each point represents the mean for 4 to 6
animals.
 Effect of PCBs, PTPs, and PBBs on Sleeping Times of Quail 187

t Sleepingti me
Female

300 0-0 PCB - 60, 62, 68% CI

PCB + PCT -- 65% CI
r PCT -- 60% CI
o...oPBB -- 75% Br

100

0
0 2 5 24 48
Hours post injection
Fig. 4. Pentobarbital sleeping times of female Japanese quail after a single oral injection of
higher chlorinated polychlorinated biphenyls (Arodor 1260, 1262 or 1268), a mixture of
polychlofinated biphenyls and terphenyls (Arodor 4465), a polychlorinated terphenyl
(Aroclor 5460) and a polybrominated biphenyl (BP-6). The administered dosage was
100 mg test substance per kg body weight. Each point represents the mean for 4 to 6
animals.

100

50

100

50

0
PCB PCB PCB PCB-T PCT PBB
%CI 21 48 68 9 68 60 75% Br

Fig. 5. Pentobarbital sleeping times of Japanese quail 48 hr after a single oral injection or
3 days ad libitum feeding of polychlorinated biphenyls, a mixture of polychlorinated bi-
phenyls and terphenyls, polychlorinated terphenyls and a polybrominated biphenyl. The
administered dosage for the oral injection was 100 mg test substance per kg body weight;
and the dosage for feeding ad libitum was 300 ppm of the test substance in the diet. Each
bar represents the mean of 4 to 6 animals.
188 H.C. Cecil et al.

containing 300 ppm. The response after a single oral injection was similar to the response
after three-day ad libitum feeding for each substance. The PCB containing 21% chlorine
did not affect sleeping times, whereas higher chlorinated or brominated compounds re-
duced sleeping times of male quail. In female quail, the mode of administration of the
test substance made very little difference in the response to pentobarbital. The only
exception was the PCB containing 68% chlorine, which reduced sleeping times in females
more after ad libitum feeding than after a single oral dose.

Mortality. Although very few birds died from the toxicity of the PCBs alone, a signifi-
cant number of birds died when pentobaxbital was administered (Table I). Mortality
during anesthesia was greatest two hr after a single oral injection of PCB. This is also the
time when sleeping times were increased two to three times those of controls. The greatest
mortality during anesthesia was in birds who were fed the PCBs containing 32% to 48%
chlorine by stomach tube. PCBs containing 21% chlorine and 58% chlorine had little
effect on mortality of male quail during anesthesia. Female quail were more susceptible
to these compounds, and 20% to 36% of the treated females died during anesthesia.

Table I. Mortality o f male and female Japanese quail during pentobarbital

anesthesia after a single oral feeding o f polychlorinated biphenyls,
polychlorinated terphenyls or polybrominated biphenyl
(100 mg per kg body weight)a

Mortality (%) after feeding test substance for

Test 2 hr 5 hr 24 hr 48 hr
substanceb M F M F M F M F

PCB 1221 0 29 0 8 0 20 0 0
PCB 1232 45 55 17 0 20 0 0 17
PCB 1242 25 58 0 0 0 20 0 0
PCB 1248 83 41 20 8 0 0 0 17
PCB 1254 0 33 60 33 0 0 0 0
PCB 1260 17 33 17 0 0 0 0 0
PCB 1262 0 36 0 0 0 0 0 0
PCB 1268 17 20 0 33 0 33 0 0

PCB-PCT4465 33 0 10 0 0 0 0 0
PCT 5460 0 18 17 17 8 9 10 17
PBB BP-6 0 0 "50 17 0 18 0 9

aControl mortality = 4% for males, 8% for females.

b2 and 5 hr PCB treatment: Males, n=6-14; females, n=10-17.
24 and 48 hr PCB treatment: Males n=5-6; females, n=5-6.
2, 5, 24 and 48 hr PCT and PCB treatment: Males, n=11-12; females, n=l 1-12.
 Effect of PCBs, PTPs, and PBBs on Sleeping Times of Quail 189

Because administration of the PCBs containing 32% to 48% chlorine at a dosage of

i00 mg/kg of body weight greatly increased mortality during anesthesia, these com-
pounds were administered at lower levels (Table II). When the dose was reduced to
50 mg/kg of body weight, the number of deaths during anesthesia was greatly reduced;
however, sleeping times of the treated quail were still two to three times those of con-
trois. At 25 mg/kg of body weight, mortality was reduced, and sleeping times approached
those of controls.

Neither ad libitum feeding of 300 ppm of PCBs for three days increased mortality;
nor did ad libitum feeding of PCT or PBB.

Table II. Duration o f pentobarbital anesthesia and mortality during

anesthesia o f male and female Japanese quail 2 hours after a single
oral feeding of polychlorinated biphenyls containing
32 to 54% chlorine

Sleeping time Mortality

Test % control %
substance M F M F

Arodor 1232
100mg/kg BW 256 213 45 55
50 337 183 0 33
25 153 114 20 0

A.rodor1242
100m~kg BW 330 340 36 58
50 320 107 27 0
25 184 169 27 20

Aroclor1248
100 mg/kg BW 218 222 83 41
50 205 217 33 0
25 159 123 20 0

Arodor1254
100 mg/kg BW 330 224 0 33
50 295 214 33 40
25 147 104 25 0

Discussion
The biphasic sleeping times induced by chlorinated hydrocarbon industrial pollutants
in quail are extremely interesting. Initially, they appear to exert a depressing effect upon
liver metabolizing enzymes and respiratory systems. At later times after administration
 190 H.C. Cecil et al.

they induce the well-known stimulatory effects upon liver enzymes which have been
characterized as microsomal enzyme induction. The initial increases in sleeping times
two hr after PCB treatment indicate suppression of pentobarbital metabolism by the
liver. Respiratory and central nervous system depression undoubtedly occurred as
evidenced by the increased mortality during anesthesia. Unfortunately, we could not
establish a dose-response relationship. Two hr after a dosage of 100 mg/kg of body
weight of Aroclors containing 32% to 48% C1, sleeping times lengthened and mortality
was greatly increased during anesthesia, whereas 25 and 50 mg/kg had much less effect. A
graded response may exist between 50 and 100 mg.

The initial increases in sleeping times two hr after PCB treatment were similar to the
increases after a single oral injection of DDT (Cecil e t al. 1973). Three PlEBs, Aroclors,
1232, 1242, and 1248, increased mortality during anesthesia to a greater extent than DDT
or other Aroclors with lower or higher chlorination. We also found these three Aroclors
to be more embryotoxic when fed to White Leghorn chickens (Cecil e t al. 1974). Al-
though all of the PCBs studied increased sleeping times two hr after a single dose, PCT
and PBB did not. The lack of action by the PCT and PBB two hr after a single dose could
be due to their larger molecule size. As larger molecules there may be less absorption from
the gastrointestinal tract. Also, penetration into the liver or brain may not have occurred
as readily as penetration by the smaller PCBs.

Although the action of the PCBs resembled that of DDT two hr after a single dose,
DDT andPCB did not have comparable effects 48 hr after a single dose or afterad l i b i t u m
feeding. In this study, we found that the Aroclors had no effect on sleeping times of
females after 48 hr, whereas, males fed the higher chlorinated Aroclors had reduced
sleeping times. The PBB had an even more marked effect and reduced sleeping times in
both males and females. These reductions in sleeping times contrast with the lengthened
sleeping times elicited by DDT feeding (Bitman et al. 1971). Here, too, we f'md the action
of the PCBs, PCT and PBB appearing to be dependent on the degree of halogenation and
consequently, molecule size. It appears that the larger or heavier molecule, either the
brominated biphenyl, which is twice as heavy as a comparable chlorinated biphenyl, or a
terphenyl, which has three phenyl rings, exerts less immediate effect on sleeping time but
has a greater effect at later times (48-hr or three~day continuous feeding).

This observed reduction in sleeping time elicited by the polyhalogenated polyphenyls

is probably the result of induction of liver microsomal enzyme systems capable of
metabolizing pentobarbital. Street (1969) reported that the higher chlorinated PCBs fed
to rats for 15 days reduced sleeping times and increased liver microsomal activity.
ViUeneuve e t al. (1972) also found that only the higher chlorinated Aroclors reduced
sleeping times in rats. Litterst e t a t (1972) studied the effect of the chlorine content of
PCBs on the production of hydroxylated metabolites of pentobarbital by rat liver. He
found that hydroxylation was greater after Aroclor 1254 and 1260 treatment than after
Aroclor 1242 or 1248 treatment.

The commercial Aroclors and BP-6 used in this study are complex mixtures of many
chlorinated biphenyls. Chen e t al. (1973) studied the effects of two isomeric tetra-
 Effect of PCBs, PTPs, and PBBs on Sleeping Times of Quail 191

chlorobiphenyls (2, 3, 4, 5, and 3, 5, 3', 5'-tetrachlorobiphenyl) and compared their

results with those of Vos (1972) who used a hexachlorobiphenyl. As a result of their
study, Chen and coworkers concluded that "the effects of individual isomeric PCBs on
rats is quite different from commercial PCBs. Thus, the total effect of Aroclors is probably
the result of interactions between each component in them and is not merely the cumula-
tive effect of each individual component." Our report suggests that the action of PCB on
the liver metabolism of birds is similar to the effect of PCBs in rats. However, the re-
sponse of Japanese quail to a short exposure of PCBs is quite different from the response
after a short exposure to DDT.

The differential effects of the PCBs, which are probably related to the extent of
metabolism and permeability (relative ease or difficulty in crossing biological membranes),
appear to be correlated with their differing degrees of chlorination. In experiments in
which PCBs and PBB were administered to White Leghorn chickens, Fries e t al. (1973a, b)
found that the lower chlorinated isomers present in the PCB mixtures were readily
metabolized and the residues in fat contained only the chlorinated PCBs of longer GLC
retention times. These residue studies also indicated that the more highly chlorinated
PCBs or the heavier PBB were resistant to transfer across the biological membrane
(Fries e t al. 1973a, b).

References
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Manuscript received April 26, 1974; accepted August 24, 1974