An Investigation of DDT Degradation in the Starfish,
Sea Urchin, and Mussel
The me tabolism of DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane)
to less toxic derivatives has been shown to be the probable mechanism of
DDT resistance in a number of invertebrate species. Enzymatic degradation
of DDT to DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene) and small amounts
of DDA (bis-(p-chlorophenyl)acetic acid) has been demonstrated in DDT-resist¬
ant strains of the house fly Musca domestica (Sternberg et al. 1950). Enzy-
matic dehydrochlorination of DDT to DDE has also been demonstrated in DDT-
resistant strains of the pink bollworm Pectinophora gossypiella (Bull and
Adkisson 1963), the blood-sucking leech Hirudo nipponia (Kimura et al. 1967),
the mosquito Culex fatigans (Kimura et al. 1965), and by the Madeira roach
Leucophaea maderae and the European Corn Borer Pyrausta nubilalis (Lindquist
and Dahm 1956). Kallman and Andrews (1963) demonstrated the reductive de¬
chlorination of DDT to DDD (1,1-dichloro-2,2-bis(p-chlorophenyl)ethane) by
yeast, requiring no DDE intermediate.
A recent study by Risebrough et al. (1967) noted the presence of the
DDT residues DDE and DDD in a large number of marine invertebrates. Recent
investigations have shown that DDT, DDE, and DDD are also present in sea
water in low concentrations (Odeman et al. 1968). The following experiments
with the starfish Pisaster ochracesus, the purple sea urchin Strongylocen-
trotus purpuratus, and the mussel Mytilus californianus were designed to
determine whether the DDE and DDD found in these species by Risebrough are
products of an in vivo enzymatic degradation of DDT or result from direct
uptake of DDT residues from the environment.
5
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Materials and Methods
Specimens of Pisaster, Strongylocentrotus, and Mytilus were collected
at Point Cabrillo, Pacific Grove, California. Specimens were incubated for
twelve hours in closed one-gallon glass jars, in sea water containing 1 ppb.
5 ppb, or 10 ppb of C+ labeled p,p'-DDT (uniformly labeled in ring, obtained
from Amersham/Nuclear Chicago). After incubation, the specimens were trans¬
ferred to tanks containing fresh sea water, where they were allowed to equil-
ibrate for a period of four hours. They were then rinsed with 95% ethanol
and dissected. The viscera and body fluids were dissolved in equal parts
of perchloric acid and glacial acetic acid on a steam bath. After complete
dissolution, an equal volume of water was added (Stanley and LeFavoure 1965).
The lipid-DDT fraction was then extracted with petroleum ether. The extracts
were passed through a sulfuric acid-Celite column to remove lipid con¬
taminants and then concentrated to volumes of 3-5 ml. in a Kuderna Danish
concentrator. The nature and relative amounts of the radioactive products
were  determined by paper chromatography analysis.
For the paper chromatography, a cylindrical glass chamber, 8" in dia¬
meter and 17" in height, was used. The mobile solvent was 2,2,h-trimethyl-
pentane, and the immobile solvent was 8% (v/v) 2-phenoxyethanol in anhydrous
ethyl ether (Mitchell 1957). Strips of Whatman No. 1 chromatographic paper.
L" by 16", were spotted with the extract and subjected to ascending paper
chromatography for four hours. The strips were then air-dried and developed
by the method of Mitchell (1957). The radioactive portions of the strips
were cut into quarter-inch horizontal fractions and counted in a Nuclear
Chicago Unilux II two-channel scintillation counter. The identity of peaks
was confirmed by cochromatography with pure C+DDT. The activity of a.25
ml. aliquot of extract was determined and a quantity of C+-DDT approximately
equal in activity to this aliquot was then added. A knowm volume of this
86
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mixture was then spotted and chromatographed using the procedure outlined
above.
Results
Preliminary chromatograms run with pure standards indicated that good
separation of DDT, DDE, and DDD could be attained using the method outlined
above. However, consistent Re values for DDT, DDE, and DDD were not obtained.
Results are shown graphically in Figure 1, which consists of chromato¬
grams of extracts prepared as previously specified. As seen, only a single
peak appears in each graph. These peaks were tentatively identified as DDT
by comparison of Re values with pure standards of DDT, DDE, and DDD run on
the same strips with the extracts.
Confirmation of the identity of these peaks was obtained by cochrom-
atography of extracts combined with pure C++-DDT. The cochromatograms are
shown graphically in Figure 2. In each case the single peak was of sufficient
amplitude to include from 2/3 to 3/ of the total counts spotted from the
extract and C-DDT combination. This suggests that none of the C+-DDT
taken up by any of the three species studied was converted to DDE or DDD.
Discussion
Previous studies of enzymatic degradation of DDT generally have been
limited to species showing evidence of the development of resistance to pro-
longed exposure to high levels of DDT. In this study we attempted to deter-
mine whether similar mechanisms of enzymatic degradation of DDT also exist
in three marine invertebrates. Under the conditions of these experiments,
Pisaster ochraceeus, Strongylocentrotus purpuratus, and Mytilus californianus
show no evidence of enzymatic degradation of DDT to DDE or DDD. This observa¬
tion implies that the DDE and DDD residues found in these species by Risebrough
are the result of direct uptake of these DDT derivatives from the marine
environment.
page 1
However, our conclusions are subject to certain limitations. Different
periods of incubation or of equilibration in fresh sea water might yield
different results. The time periods in these experiments may have been too
short to allow for the induction of a DDT-degrading enzyme system; or, if
suchsn a system were present in the organism, degradation of DDT to DDE
or DDD may be so slow as to produce negligible end products in the period
of time studied. Incubations at higher or lower concentrations of C+-DDT
might also alter our results.
59
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Acknowledgments
We would like to thank all of those people at the Hopkins Marine
Station in Pacific Grove, California, who were so helpful throughout this
study. Special thanks are extended to Dr. David Epel and to John Miller
of the Hopkins Marine Station staff. This work was supported in part by
the Undergraduate Research Participation Program of the National Science
Foundation, Grant No. 6Y-5878.
Bibliography
Bull, D. L., and P.L. Adkisson. 1963. Absorption and Metabolism of C+-Labeled
DDT by DDT-Susceptible and DDT-Resistant Pink Bollworm Adults. J. Econ.
Entomol. 56: 641-643.
Kallman, Burton J., and Austin K. Andrews. 1963. Reductive Dechlorination
of DDT to DDD by Yeast. Science 1h1: 1050-51.
Kimura, Takuji, J.R. Duffy, and A.W.A. Brown. 1965. Dehydrochlorination and
DDT-Resistance in Culex Mosquitos. Bull. Wld. Health Organ. 32: 557-561.
Kimura, Takuji, Hugh L. Keegan, and Thomas Haberkorn. 1967. Dehydrochlorination
of DDT by Asian Blood-sucking Leeches. Am. J. Trop. Med. and Hyg. 16:
688-690.
Lindquist, Donald A., and Paul A. Dahm. 1956. Metabolism of Radioactive DDT
by the Madeira Roach and European Corn Borer. J. Econ. Entomol. 19;
579-584
Mitchell, Lloyd C. 1957. Separation and Identification of Chlorinated Pes¬
ticides by Paper Chromatography. J. Assoc. Off. Agr. Chem. 40: 291-302,
Odeman, Melvyn W., Paul W. Wild, and Kenneth C. Wilson. 1968. A Survey of
the Marine Environment from Fort Ross, Sonoma County, to Point Lobos.
Monterey County. State of California Department of Fish and Game,
MRO Ref. No. 68-12.
Risebrough, Robert W., Daniel B. Menzel, D. James Martin, Jr., and Harold
S. Olcott. 1967. DDT Residues in Pacific Sea Birds: A Persistent
Insecticide in Marine Food Chains. Nature 216: 589-590.
Stanley, Ronald L., and Herbert T. LeFavoure. 1965. Rapid Digestion and
Clean-up of Animal Tissues for Pesticide Residue Analysis. J. Assoc.
Off. Agr..Chem. 18: 666-667.
Sternberg, James, C.W. Kearns, and W.N. Bruce. 1950. Absorption and Metabolism
of DDT by Resistant and Susceptible House Flies. J. Econ. Entomol. 13:
211-219.
60
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Footnotes
1. Permanent address: Jeffrey D. Davis
42h Newcastle Way
Madison, Wisconsin 53704
Figure Legends
Figure 1. Chromatograms of extracts of Strongylocentrotus purpuratus, Pisaster
ochraceus, and Mytilus californianus. The origin corresponds to fraction
No. 2 and the solvent front to fraction No. 40. DPM: Disintegrations
per minute. Background counts of 25-35 DPM were not subtracted.
Figure 2. Cochromatograms of extract-C--DDT combinations for Strongylocentro-
tus purpuratus, Pisaster ochraceus, and Mytilus californianus. The
origin corresponds to fraction No. 2 and the solvent front to fraction
No. 40. DPM: Disintegrations per minute. Background counts of 25-35
DPM were not subtracted.
6e
500
a 250.
600-
300
1200-
600
70
Strongylocentrotus
purpuratus
30
Fraction Number
Pisaster
ochraceus
30
Fraction Number
Mytilus
californianus
30
Fraction Number
40
40
20
6
500-
a 250-
250:
125-
800-
400
Strongylocentrotus
purpuratus

20
10
Fraction Number
Pisaster
ochraceus

70
Fraction Number
Mytilus
californianus
70
Fraction Number

30
C
30
30
40
40
C
Addendum
Experiments performed after the conclusion of the writing of the above
report indicate that there is a possibility that a mechanism of enzymatic
degradation of DDT is present in the starfish Pisaster ochraceus. The
procedure used in this experiment was identical to that of the experiments
above except that the period of equilibration in fresh sea water after expo¬
sure to C++-DDT was extended from four hours to five days. Two peaks appeared
in the chromatogram. The identity of the two peaks has not been determined.
68