TOXICITY-METABOLISM RELATIONSHIP OF THE

PHOTOISOMERS OF CERTAIN CHLORINATED

CYCLODIENE INSECTICIDE CHEMICALS
M. A. Q. KHAN, R. It. STANTON, D. J. SUTHERLAND1,
J. D. ROSEN1,and N. MAITRA
Department o f Biological Sciences
University o f Illinois at Chicago Circle
Chicago, Ill. 60680

Photoaldrin, photodieldrin, and photoheptachlor are more toxic than their cor-
responding parent compounds (aldrin, dieldrin, and heptachlor) to freshwater in-
vertebrates and vertebrates, and to adult houseflies. The increase in toxicity is very
significant in the case of the amphipod, G a m m a r u s ( 1 . 5 - 1 2 times), bluegill fry
(3.6 5.7 times), mosquito larvae, A e d a s ( 2 . 3 - 6 times), minnow fry (2.5 times), and
the isopid, A s e l l u s (2 times). The greatest increases occur with photodieldrin which
is 12 and 5 times more toxic than dieldrin, respectively, to G a m m a r u s , and to blue-
gill fry, and with photoatdrin which is 6 and 4 times more toxic than aldrin, respec-
tively, to mosquito larvae and bluegill fry. The toxicities of the photoisomers of
isodrin and chlordene are generally less than those of their parent compounds to all
the organisms tested. The basis of the differences in toxicities of the chlorinated
cyclodiene photoisomers appears to be related to their chemical structure which
possibly affects their action at the site(s) of toxic action and/or detoxication. The
acidic proton present at the secondary chloride in photoatdrin, photodieldrin, and
photoheptachlor possibly is responsible for the formation of charge-transfer com-
plexes between components of the nerve and the m i x e d - f u n c t i o n oxidase; the lat-
ter enzyme apparently dehydrochlorinates these photo products to their corres-
ponding, more toxic ketones. The absence of such protons in photoisodrin and
photochlordene renders them incapable of forming such ketones. The inhibition
of these reactions by sesamex in house flies increases the stability of the chlorinated
cyclodiene insecticides and, thus, significantly affects their toxicity. The con-
version of photoaldrin, photodieldrin, and photoheptachlor to m o r e - t o x i c and
lipophilic ketones warrants additional studies of their accumulation and subsequent
concentration by the food chain.

The photolysis of chlorinated cyclodiene insecticide chemicals has been reported by

workers in several laboratories (Rosen and Sutherland 1966, Rosen e t al. 1969, Henderson
and Crosby 1967, Khan e t al., Benson e t al. 1971). Studies, in the laboratories of the pres-
ent authors, have shown that photoaldrin (PA), photodieldrin (PD), and photoheptachlor
(PH) are several times more toxic to houseflies, aquatic insects, crustacea, other aquatic

tPresent address: College of Environmental Sciences, Rutgers The State University of New Jersey,
New Brunswick, New Jersey

159

Archives of Environmental Contamination and Toxicology,

Vol. l, No. 2, 1973 ~) 1973 by
Springer-Vertag New York Inc.
160 M.A.Q. Khan et al.

invertebrates, fish, and amphibians than the corresponding cyclodienes, namely aldrin (A),
dieldrin (D), and heptachlor (H) (Fig. 1). However, photoisodrin (PI) is less toxic than iso-
drin (I) to all these organisms (Georgacakis and Khan 1971, Khan et al. 1969, Rosen et al.
1969). The toxicity of these photoisomers is apparently associated with their metabolism,
e.g., PA, PD, and PH are capable of the undergoing oxidative dehydrochlorination at the
secondary chlorine resulting in the formation of more toxic and more lipophitic ketones
(Khan e t al. 1969 and 1970a). On the other hand, PI (which lacks the acidic proton as-
sociated with the secondary chlorine) undergoes rapid oxidative hydroxylation.

This paper summarizes the results obtained, in these laboratories, concerning the pos-
sibility of ecological hazards of PA, PD, and PH, the parent compounds of which have been
and are still being extensively used in insect control. Also, it gives some toxicity data for
photochlordene (PC), [an isomer of chlordene (C)] considers the metabolism-toxicity
relationship of the photoconversion products studied, and explores their ecological
significance.
Materials and m e t h o d s
Toxicity assays were performed to determine the LCso value (lethal concentration,
ppb, to kill 50 percent of test animals exposed for 24 hours to graded suspensions of the
insecticide chemical under test), LDso value (lethal dose, gg/g, to kill 50 percent of test
animals treated by topical application or injection of the chemical, or exposed to films of
the chemical), and LT s o value (lethal time, hours, to achieve 50 percent mortality during
continuous exposure of test animal to a fixed concentration of the chemical) (Georgacakis
and Khan 1971, Khan et al. 1970a). Whenever used, sesamex and WARF antiresistant for
DDT (N, N - d i - n - b u t y l - - p - c h l o r o b e n z e n e sulfonamide) were applied topically at 20 #g/
fly about 30 minutes before the insecticide treatment was made.

The extraction of the insecticide chemicals and their metabolites involved the homog-
enization of the organisms in a Sorvall omnimixer in the least amount of water (10 ml/g).
The homogenate was shaken vigorously with three times of its volume with isopropanol
hexane mixture (1:4 by volume), followed by two extractions with hexane. The aqueous
layer was refluxed with three times its volume with hexane at 60°C for one hour and the
hexane extract was combined with the rest of the solvent extracts. The insecticide chemi-
cals and their lipophilic metabolites were determined by gas chromatography, using
authentic standards (Khan et al. 1970a).

Results and discussion

The data obtained in this study and in previous investigations (Rosen et at. 1969,
Khan et al. 1969 and 1970a, Georgacakis and Khan 1971) are given in Table I which
shows the effect of photoisomerization of cyclodiene insecticide chemicals on their
toxicity to insects, crustacea, and fish. PA, PD, and PH are more toxic, and PI and PC
less toxic than their parent compounds. Differences in species response to these toxicants
 CI CI CI CI

Ct C~

CI
0
CI CI " - ~ ~ ""4---O o
Cl Cl
Aldrin) Photoaldrin CI Dieldrin Cl Photodieldrin ~i
(A) (PA) (D) (PD) '<
o
CI CI CI "~
CI 0

Ci
Cl C ~
Cl
el o
C
CI CI CI
Isodrin Photoisodrin Chlordene Photochlordene O
(I) (PI) (C) (PC) =

el CI c~ ~-
Cl H

£D

Cl
C;
el 0 CI Cl
c,
CI
Photodieldrinketone Heptachlor F)hotoheptachlor
(PDK) (H) (PH)
Fig. t. Chemical structures of various chlorinated cyclodiene insecticide chemicals, their photoisomers, and p h o t o d i e l d r i n
ketone. The acidic p r o t o n ~r HC"I~~""~" - C ) is evident in photoaldrin, photodieldrin, and p h o t o h e p t a c h l o r but not in p h o t o -
O~
isodrin and p h o t o c h l o r d e n e .
 Table I. Toxicity of Certain Chlorinated Cyclodiene Insecticide Chemicals and Their Respective
Photoisomers to House Flies and Four Species o f Freshwater Animals

24--Hour mortality in terms of LC s 0, ppb, for all animals except Musca, or in terms of LD s o'/~g/fly, for
Animala Musca, for the indicated chemical b

A PA D PD H PH I PI C PC

Bluegill 260 90 170 30 12 25 218 346

Minnow 24 10 13 8 6 10
Asellus (isopod) 80 40 100 60
Aedes (mosquito) 3 0.5 6 3 5 2 19 19 130 150
Musca (house flYl 14 6.9 9.8 8.3 11 5.6 54 113 158 179

a T w o - m o n t h - o l d fish fry; late 3rd instar Aedes aegypti (susceptible) larvae; 3 - d a y - o l d female house flies.
bSee Fig. 1.
 Toxicity of Photoisomers of Chlorinated Cyclodiene Insecticides 163

are quite noticeable; for example, isodrin is the most toxic insecticide chemical to the fish
but it is intermediate in toxicity to other animals; photoaldrin is more toxic to Asellus
(isopod), and photoheptachlor is more toxic to mosquito larvae and house flies than the
other insecticide chemicNs.

A summary of the mortality data in Table I and the data reported by Georgacakis
and Khan (1971) is given in Table !I. Species differences in the increased toxicity of
photoaldrin, photodieldrin, and photoheptachlor, compared to the respective parent com-
pound, are quite apparent; for example, photodieldrin exerts its toxic effects 12-times
faster than dieldrin on Gammarus, and 5 times faster on bluegill, and approximately 2 -
times faster on Asellus, Aedes, and guppies. Similarily photoaldrin is 6-times more toxic
to Aedes and 2 - and 3-times more toxic to Gammarus, Asellus, houseflies, guppies, and
bluegills, lit is of interest that photoaldrin is also more toxic than aldrin to mice (Rosen and
Sutherland 1966).] The toxicity of photoheptachlor is not importantly increased for
these test animals; insects are more seriously affected than other organisms as they
~are 2 - to 3-times more sensitive to photoheptachlor than to heptachlor. Chlordene
and isodrin photoisomers generally are less toxic than the respective parent compound to
all of these test animals.

The increased toxicity of the photoisomers of aldrin, dieldrin, and heptachlor to in-
sects is reported to be related to their rapid metabolism by oxidative dehydrochlorina-
tion to more toxic ketones (Khan et al. 1970a and 1969). The relationship of the
toxicity of PA and PD with the rate of their in vivo metabolism to the photodieldrin
ketone in mosquito larvae is demonstrated in Fig. 2. The toxicity of PA and PD increases
with the increase in the concentration of PDK in their tissues.

A similar relationship between the toxicity of PD and its conversion to photodieldrin

ketone exists for crayfish, tadpoles, guppies, and goldfish (Georgacakis et al. 1971). In the
case cited, the insecticide was directly injected into the body cavity to eliminate dif-
ferences caused by absorption and penetration. Crayfish and tadpoles appear to be more
sensitive than guppies and goldfish because the former convert photodieldrin faster to
photodieldrin ketone than the latter.

Detailed information about the affect of inhibition of the photodieldrin ketone forma-
tion by the antioxidant synergist, sesamex, on the toxicity of photodieldrin and other
photoisomers is presented in Table III. Sesamex lowers the toxicity of photodieldrin,
photoaldrin, and photoheptachlor while it synergizes the toxicity of photodieldrin ketone,
photoisodrin, and photochlordene. The synergism effect on some of these compounds is
probably related to the inhibition of their hydroxylation by sesamex (Khan et al. 1970).
These data also show that the photodieldrin ketone is in fact more toxic than photoaldrin
and photodieldrin.
 4a

Table II. Toxicity Ratios o f Certain Chlorinated Cyclodiene Insecticide Chemicals and Their
Photoisomers to Various Species o f Freshwater Animals and a Species o f House Flies,
Calculated From Respective 24-Hour L C5 o and L T 5 o Mortality Values

Animal Toxicity ratios a for indicated chemicals b

A/PA D/PD H/PH I/PI c/Pc

Crustacea
Daphnia pulex (water flea) 1.43 1.27 1.24 0.45 0.91
Gammarus spp. (amphipod) 1.83 12.22 1.45 0.43
Asellus spp. (isopod) 2.13 (2.0) 1.60(1.7) 0.64
Insects
g
Aedes aegypti larvae (mosquito) 5.68 (6.0) 2.30(2.0) 2.80(2.5) 0.30(.33)
Musca domestica (house fly) 2.13 (2.0) 1.18 (1.2) 1.90 (1.96) 0.41 (.48) 0.78 (.70)
Fish
m m
Lebistes reticulatus (guppy) 2.41 2.57 0.45
Pimephalus promelas (bass) 1.42 1.1 1 (2.4) 1.23 (1.63) 0.3 (.60)
Lepomis macrochirus (blue gill) 3.60 (2.9) 5.14(5.7) 0.75 (.48) 0.72 (.63)

a Ratios calculated from LT 5 o values (Georgacakis and Khan 1971 and Maitra, et al. 1973) and from LC s o values given in Table I;
the ratios in parenthesis are derived from the latter.
t'See Fig. 1.
 Toxicity of Photoisomers of Chlorinated Cyclodiene Insecticides 165

Table III. Effect o f Sesamex on the Toxicity o f the Photoisomers of Chlorinated

Cyclodiene Insecticide Chemicals and o f PhotodieMrin Ketone to
Susceptible House Flies
Degree of
L T s o , hours synergism b
Compound plus
Compound Compound alone sesamex a

Dieldrin 6.3 6.0 1.1

Photodieldrin 5.4 5.9 0.9
Photodieldrin Ketone 3.4 2.9 1.2
Photoaldrin 4.8 5.6 0.9
Photoheptachlor 7.8 9.0 0.9
Photoisodrin 20.8 12.4 1.7
Photochlordene 22.5 t0.7 2.1

a Dose: 20/ag/sesamex/fly applied topically 30 minutes before applying 14 ng compound/fly.

bRatio of LT 5 o for compound to LT 50 for compound -plus-sesamex.

Table IV. Effect of Pretreatment with Sesamex and WARF Antiresistant on the
Metabolism o f Photodie!drin and Photodieldrin Ketone in DDT-Resistant House Flies

Amount of com round recovered from flies, ng/fly

Treatment of flies a
Photodieldrin Photodieldrin
Total
(PD) Ketone (PDK)

PD only 100 34 134

PD + sesamex 138 5 143
PD + sesame× + WARF antiresistant 152 NIL 152
PDK only 160 160
PDK + sesamex 182 t82

aFlies of a D D T - r e s i s t a n t strain were topically treated with either 20/ag sesamex/fly or

20 /ag sesamex plus 20/~g WARF antiresistant/fly 30 minutes before topical treatment
with 285 ng/fly of either PD or PDK. The flies were homogenized and extracted 2 hours
after the treatment with PD or PDK.
166 M.A.Q. Khan et al.

The effect of sesamex on the inhibition of in vivo formation of the photodietdrin

ketone from photoaldrin or photodieldrin in house flies is seen in Table III. Sesamex does
inhibit the oxidative dehydrochlorination. In fact, sesamex with WARF antiresistant for
DDT, both of which are competitive inhibitors of the mixed-function oxidase which
catalyzes oxidative dehydrochlorination (Khan, et al. 1970b), completely inhibits ketone
formation. Also, it is possible that they inhibit hydroxylation as evidenced by the in-
creased recovery of the applied photodieldrin. In addition, sesamex increases the recovery
of the photodieldrin ketone, probably by blocking its conversion to hydrophilic metabo-
lite(s), ana this action explains the synergism of this ketone by sesamex (Table III).

The toxicity-metabolism studies indicate that photodieldrin ketone is responsible for

the increased toxicity of photoaldrin and photodieldrin. The alarmingly high toxicity of
some of the photoisomers, if related with the formation of lipophilic ketone, can be
ecologically hazardous if these photoisomers and their lipophilic ketone(s) become con-
centrated through food-chain organisms. This aspect needs thorough investigation.
Studies are underway, in these laboratories, to determine the biological concentration of
the photodieldrin ketone and the various photoisomers of chlorinated cydodiene in-
secticide chemicals, and their metabolites, by food-chain organisms (Maitra et al. 1973).

The effect of sesamex, which inhibits mixed-function oxidase, on the toxicity of all the
photoisomers under study suggests the involvement of this enzyme system in the metabo-
lism of these compounds. Preliminary investigations, in these laboratories, indicate that these
compounds are in fact metabolized by the mixed-function oxidase in rat, mouse, certain
fish (bluegill and bass) crayfish, and house flies (Khan, et al. 1972, 1970a, and 1970b,
Stanton and Khan 1972).

The chemical basis of the differences in the toxicity of these photoisomers deserves
some consideration. Photoaldrin, photodieldrin, and photoheptachlor all have the secon-
dary chlorine with an acidic proton. This is the site at which oxidative dehydrochlorina-
tion occurs to yield corresponding ketones (Fig. 1). It is possible that the acidic proton
makes these molecules more reactive at the site of toxic action and of oxidative dehy-
drochlorination. However, it must be mentioned that the photodieldrin ketone, which
lacks this proton, is more toxic than PA or PD (Table III), indicating that other factor(s)
possibly are responsible for the toxicity of the photodieldrin ketone. One apparent reason
is the increased liposolubility of photodieldrin ketone. [Detailed investigations on the
differences in liposolubility of these compounds and photodieldrin ketone will be re-
ported elsewhere (Khan et al. 1973)]. The absence of the acidic proton in the bird cage
of photoisodrin and photochlordene makes them incapable of undergoing oxidative de-
hydrochlorination. However the mixed-function oxidase attacks other sites on the
molecules of the photoisodrin and photochlordene, resulting in their hydroxylation
which leads to their detoxication. The parent compounds, isodrin and chlordene, are ac-
tivated in vivo by the mixed-function oxidase and this makes them become more toxic
 ,~
I 100 100
PHOTOALDRIN PHOTODIELDRIN

,/
t
3 N 60 ~ 60
co < 3

7--"
"-4

20 20

I I I " ~'-" I ! I ........... I

0 1 2 3 4 0 1 2 3 4

HOURS OF EXPOSURE

Fig. 2. Relationship between the toxicity of photoaldrin or photodieldrin (% m o r t a t i t y , © ~ ©), the degree of absorption of
these photoproducts (ng/larva, A -A ), and their metabolism to photodieldrin ketone (ng/larva, X . X ), and hours of
exposure of mosquito larvae to 70 ppb of photoaldrin or photodieldrin.
168 M.A.Q. Khan et al.

(Khan et aL 1970a and 1969). In the case of DDT, a similar acidic proton has been shown
to be responsible for the formation of a charge-transfer complex with lecithin (Tinsley
et aL 1971). Studies are in progress, in these laboratories, to investigate the possibility of
the formation of a similar charge-transfer complex between photodieldrin and phos-
phatidylcholine resulting in proton shifts which should be absent with photoisodrin and
photochlordene.

Acknowledgments

Thanks are given to Professor E. B. Hadley, Head of the Department of Biological

Sciences for the use of facilities in his laboratory. This research was supported by a grant
from the National Institutes of Environmental Health Sciences (ES-00808). Standard
chlordene and photochlordene were kindly provided by Velsicol Corporation, Chicago.

References

Benson, W. R., P. Lombardo, I. J. Egry, R. D. Ross, R. J. Barron, D. W. Mastbrook, and

E. A. Hansen: Chlordane photoalteration products: their preparation and identifi-
cation. J. Agr. Food Chem. 19, 857 (197t).
Georgacakis, E. and M. A. Q. Khan: Toxicity of the photoisomers of cyclodiene in-
secticides to freshwater animals. Nature 233, 120 (1971).
Georgacakis, E., S. R.Chandran, and M. A. Q. Khan: Metabolism-toxicity relationship of
the photoisomers of cyclodiene insecticides to aquatic animals. Bull. Environ.
Contain. Toxicol. 6, 535 (1971).
Henderson, G. L. and D. G. Crosby: Photodecomposition of dieldrin and aldrin. J. Agr.
Food Chem. 15,888 (1967).
Khan, M. A. Q., W. Coello, A. A. Khan, and H. Pinto: Some characteristics of the mixed-
function oxidase in the freshwater crayfish, Cambarus. Life Sci. 11,405 (1972).
Khan, M. A. Q., D. J. Suthertand, J. D. Rosen, and W. F. Carey: Effect of sesamex on the
toxicity and metabolism of photoisomers of cyclodienes. J. Econ. Entomol. 16,
470 (1970a).
Khan, M. A. Q., J. L. Chang, D. J. Sutherland, J. D. Rosen, and A. Kamal: Housefly
microsomal oxidation of some foreign compounds. J. Econ. Entomol. 63, 1807
(1970b).
Khan, M. A. Q., J. D. Rosen, and D. J. Sutherland: Insect metabolism of photoaldrin and
photodieldrin. Science 168, 318 (1969).
 Toxicity of Photoisomers of Chlorinated Cyclodiene Insecticides 169

Maitra, N., H. M. Khan, and M. A. Q. Khan: Biological concentration of the photoisomers

of cyclodiene insecticides and their metabolites. Bull. Environ. Contam. Toxicol.,
submitted for publication.
Rosen, J. D. and D. J. Sutherland: The photochemical isomerization of dieldrin and
endrin and effects of toxicity. Bull. Environ. Contain. Toxicol. I, 133 (1966).
Rosen, J. D., D. J. Sutherland, and M. A. Q. Khan: Properties of photoisomers of hepta-
chlor and isodrin. J. Agr. Food Chem. 17,404 (t969).
Stanton, R. H. and M. A. Q. Khan: Oxidation of cyclodiene insecticides by sunfish, mouse
and rat liver mixed-function oxidase. Amer. Zoot. 12 (3), XXXVIII (1972).
Tinstey, L. J., R. Haque,and D. Schmedding: Binding of DDT to lecithin. Science 174,
145 (1971).

Manuscript received September 2, 1972; accepted October 20, 1972