BLUE CRAB MORTALITY: INTERACTION OF

TEMPERATURE AND DDT RESIDUES

CHRISTOPHER C. KOENIG, ROBERT J. LIVINGSTON, and CLAUDE R. CRIPE

Department of Biological Science
Florida State University
Tallahassee. FL 32306

Serial observations of DDT-contaminated and uncontaminated salt marshes in the

northern Gulf of Mexico were made in November and December, 1973. Blue crab
(Callinectes sapidus) mortalities observed in the DDT-contaminated marsh during this
period were correlated with reduced daily temperature minima. Gas chromatographic
analysis of hepatopancreas and swimmeret muscle tissue of dead and dying crabs
revealed total DDT residue concentrations as high as 39.0 ppm and 1.43 ppm, respec-
tively. It is suggested that the DDT body burdens and reduced temperatures interact to
produce acutely toxic effects. Several physiological and behavioral mechanisms are
proposed.

Blue crab (Callinectes sapidus) sensitivity to DDT is well known from field
observations (Cottom and Higgins 1946, Commercial Fisheries Review 1946,
Springer and Webster 1951, Mills 1952) and from laboratory bioassays (Butler and
Springer 1963, Butler 1965, 1969, Lowe 1965). Recently Mahood et al. (1970)
reported massive blue crab mortalities and the concurrent decline of the blue crab
populations along the coast of the South Atlantic states which occurred in the late
1960's. A significant result of their investigations seeking the probable cause of the
mortalities was that all 1,950 blue crabs sampled contained p , p ' - D D T and the
metabolites p , p ' - D D D and p,p'-DDE.

Although the use of DDT has been greatly curtailed in the USA, it and its major
metabolites may persist in the biosphere for many years (Woodwell et al. 1971). In
estuarine environments certain physico-chemical factors which cause nutrients to be
concentrated may also concentrate DDT and other pollutants (Odum 1970). DDT
readily adsorbs to detritus and becomes part of estuarine sediments. As such it may
be assimilated directly by detritus-feeding organisms (Odum et al. 1969) or indi-
rectly by predation. Woodwell et al. (1967) showed that DDT residues can accumu-
late in estuarine sediments and can also concentrate systematically with an increase
in trophic level. Blue crabs are in an especially vulnerable position in the estuarine
community considering their predatory'habits (Carriker 1967) and their extreme
sensitivity to DDT. During the summer, fall and winter of 1973 serial observations
were made of the blue crab populations in a series of Northern Gulf salt marshes.
This paper presents the blue crab mortalities observed in a DDT-contaminated salt
marsh.

Archives of Environmental Contamination 119

and Toxicology Vol. 4, 119-128 (1976)
r by Springer-Verlag New York Inc.
 120 C.C. Koenig et al.

Materials and methods

Field observations were made biweekly from October to December, 1973; addi-
tional observations were made in the latter months during the movement of cold
fronts through the area. Both control and DDT-contaminated salt marshes were
observed on the same dates. When mortalities were observed in the DDT-
contaminated salt marsh, blue crab samples were taken from control and contami-
nated areas and frozen for not more than two months. Portions of the hepatopancreas
and swimmeret muscles (all of the muscle material of the left side of the eighth
thoracic somite) (Pyle and Cronin 1950) were then examined for p , p ' - D D T and its
metabolites according to a method modified from that of A. J. Wilson, Jr. (En-
vironmental Protection Agency, Gulf Breeze, Florida 32561). Samples o f 0.2 to 2.2
g were placed in a size 23 Kontes tissue grinder and ground for three min with five
ml of acetonitrile, stopping occasionally to loosen tissue which had become packed
in the bottom of the grinder. The liquid portion was decanted into a centrifuge tube
and the sample was extracted three more times in the same manner. The extracts
were centrifuged and the supernatant was poured into a glass vial along with 20 ml
of an aqueous 2% sodium sulfate solution and five ml of hexane. The vial was
sealed with a Teflon-lined cap and shaken for one min. The hexane layer was
removed with a pipet, placed in a concentrator tube, and the solution extracted three
more times. The combined hexane extracts were concentrated to about 1.0 ml and
added to a Florisil column. This consisted of a size 9 Chromoflex column (9 m m id)
plugged with glass wool and filled with 11.3 cm of Florisil (PR grade, 6 0 - 1 0 0
mesh, activated at 130 ~ C) which was tapped down to 10 cm. This was topped with
2.5 cm of granular sodium sulfate. Columns were prepared in advance and stored at
130 ~ C until needed. Prior to use, they were rinsed with ten ml of hexane. After the
hexane extract was added to the column and the concentrator tube rinsed twice with
about 1.0 ml of hexane, the sample was eluted with 20 ml of a 5% ethyl ether in
hexane solution.

All sediment samples (top 2 cm) were collected with a 6.5 cm id aluminum
coring device from submerged sediments in October and November, 1973. Sediment
samples were air dried at room temperature and 10- to 20-g samples were extracted
in soxhlets with a 10% acetone in petroleum ether solution for seven hr. The
solution was then concentrated, exchanged with hexane, and reconcentrated to about
one ml. The columns described above were used for the cleanup. Prior to injection,
the concentrated samples were placed in glass stoppered centrifuge tubes and shaken
vigorously with 0.1 ml of metallic mercury to minimize interference from sulfur
compounds.

Samples were analyzed on a Varian "2100 Gas Chromatograph with a tritium

electron capture detector. Temperatures of the injector, column, and detector (base,
not foil) were 220 ~ 192 ~ and 282 ~ C, respectively. The analytical column consisted
of a 180 cm glass column by 0.63 cm od and filled with 2% OV-101. The absence
of dieldrin was determined by eluting some samples with 20% ethyl ether in hexane
solution and injecting in a column which contained 1% OV-17 and 3% OV-210.
 Interaction of Temperature and DDT 121

Quantification was performed with a programmable calculator which was used to

compute a regression line from three standards.

Daily water temperature minima were estimated from Gulf water run through an
artificial tidal marsh (1000 gal.) located on the grounds of the Florida State Univer-
sity Marine Laboratory (Sopchoppy, Florida). The artificial marsh was designed
with an automatic 12-hr tide cycle and the early morning temperatures were taken as
an estimate of the daily temperature minima. Temperatures taken in the artificial,
contaminated, and control tidal marshes during observation periods confirmed the
close thermal similarity between these areas. Daily air temperature minima for the
Franklin County, Florida area were obtained from the weather bureau office in
Appalachicola, Florida; these data closely correlated with our water temperature
estimates. All o f the mortalities except those observed on 1 November 1973 and 3
December 1973 are known to have taken place not more than 24 hr prior to their
observation. The mortalities observed on l November and 3 December were proba-
bly not more than 48 hr old considering their degree of freshness.

The possible involvement of other contaminants in the observed mortalities is

considered unlikely. The contaminated marsh is at least 50 miles from any heavy
industry and the surrounding areas are sparsely populated. Pleasure boating is
minimal and the site borders the St. Mark's National Wildlife Refuge. The absence
of the pesticides dieldrin, mirex, and methoxychlor in blue crab tissues was con-
firmed using the techniques described above. The heavy metals copper and lead
were analyzed in whole fish (Adinia xenica) collected simultaneously from control
and contaminated marshes. The wet ash technique (conc HNO3) was used and
analyses were performed with a Perkin-Elmer model 303 Atomic Absorption
Spectrophotometer. The concentrations of copper (1.8 ppm) and lead (5.6 ppm) for
the DDT-contaminated fish correlated with those values for control fish, 2.0 ppm
and 6.8 p p m , respectively. Low PCB contamination (.089 ppm) of DDT-
contaminated whole fish (A. xenica) was confirmed in the pesticide research
laboratory of Dr. N. P. Thompson (University of Florida, Gainesville, Florida)
using the method of Thompson et al. 1974.

To test the null hypothesis that there is no association between reduced tempera-
ture and mortality in the DDT-contaminated population a 2 x 2 contingency table
was constructed. The criteria of positivity-negativity was used for mortality against
the possibility that the water temperature minimum on the day of the observed
mortality had been below the mean (16.6~ of the daily water temperature minima
for N o v e m b e r and December, 1973.

Results and discussion

The source of DDT contamination in the tidal marsh (Figure 1, Station 8a) was
from a dogfly (Stomoxys calcitrans) control program. Under this program beach
areas of Franklin County, Florida were sprayed once or twice a year. DDT spray
was last used in 1968 when 2500 gal (35% emulsion) were applied to 48 miles of
122 C. C. Koenig et al.

Marks

0 4
Nautical miles

APALACHEEBAY

96c "7c .as

"4a 9o.~f "6b .7b .8d 9b"
i~:^ .1~ "7a.,.:8c 9a-

Alligator

Fig. 1. Tidal marsh sampling stations in the northern Gulf of Mexico bordering Franklin and
Wakulla Counties, Florida.

beach (letter dated 3 January 1974 from Clayton E. Van Tassel, Mosquito Control
Director, Franklin County, Florida). The highest DDT concentrations appeared in
the sediments of the lower portion of the marsh and diminished in the upper portions
and seaward (Table I).

The distribution of DDT residues throughout the marsh was probably the result of
tidal exchange since only beach areas were treated and the marsh was not sprayed
directly (Van Tassel, personal communication). The extremely high proportion of
p,p'-DDT in the upper two cm of submerged sediment attests to its extreme stability
and persistence.

It was observed that blue crab mortalities occurred in the DDT-contaminated

population simultaneously with a decreased water temperature whereas no mor-
talities were observed in uncontaminated blue crab populations. Although total DDT
residues in blue crabs from the contaminated "marsh (Table II) were quite variable,
they were, in all cases, higher than those from uncontaminated marshes. A total of
36 blue crab mortalities were recorded for the ten-A DDT-contaminated salt marsh.
The mortalities began on 1 November 1973 and continued through 26 December
1973 (Figure 2). No mortalities were observed in the contaminated marsh prior to 1
November 1973. The water temperatures remained above 18~ throughout the
 Interaction of Temperature and DDT 123

s u m m e r and fall until 31 O c t o b e r 1973. The null hypothesis o f no association

b e t w e e n low water temperatures and mortality was rejected for the Yates-corrected
Chi square value of 7.900 (P < .01). That is, the blue crab mortalities occurred in
the D D T - c o n t a m i n a t e d salt marsh when the temperature on the day o f the observed
mortalities fell below the mean o f the daily water temperature m i n i m a for N o v e m b e r
and D e c e m b e r 1973.

In salt marshes, blue crabs are major predators as well as scavengers (Carriker
1967). Fishes from the contaminated marsh (Table III) may therefore add signific-

Table I. DDT residues (ppm) in the top 2 cm of submerged sediments from DDT
contaminated (4-8) and uncontaminated (10) salt marshes

Station DDE DDD DDT DDT (total)

4a Ta T 0.010 0.010
4b T T 0.016 0.016
5a 0.017 0.040 0.041 0.098
5b 0.012 0.016 0.082 0.110
5c 0.005 0.006 0.011 0.022
5d T 0.006 0.015 0.021
5e _ _ b - T T
5f T T 0.085 0.085
5g T T 0.056 0.056
6b T T 0.009 0.009
7a T T 0.022 0.022
7b T T 0.013 0.013
7c T T 0.109 0.109
8a I 0.128 0.848 0.559 1.535
8a 2 0.048 0.271 0.180 0.499
8a 3 0.111 0.402 0.337 0.850
8a 4 0.025 0.163 0.300 0.488
8a s 0.094 0.483 0,624 1.201
8a 6 0.053 0.044 2.66 2.77
8a7 0.238 1.64 17.2 19.1
8a 8 0.292 2.12 10.8 13.2
8a 9 0.373 1.90 23.8 26.1
8b 0.277 0.495 7.32 8.09
8c T T .079 0.079
8d T T .043 0.043
8e .025 0.025
9a T T T
9b T T .035 0.035
10a T T T T
10b T T T T

aT = trace = < 0 . 0 0 2 ppm; b--indicates none detected; all samples collected Fall, 1973.
 124 C . C . Koenig et al.

antly to the D D T b o d y burdens o f the crabs. The high aquatic trophic level main-
tained by the crabs in addition to their direct contact with the substrate apparently
exposes them to quite high D D T concentrations.

It was observed that the blue crabs remaining in the marsh d u r i n g the winter
would burrow into the mud, c o m p l e t e l y coverning themselves with as much as ten
c m o f sediment. This behavior could buffer them from low temperature extremes
since temperatures recorded 10 to 20 cm beneath the surface o f the mud were as
much as 8 ~ C higher than the overlying water. T h e y were o c c a s i o n a l l y o b s e r v e d to
come out o f the mud in the late afternoon and evening, p r e s u m a b l y to feed. This
could be a response to w a r m e r water temperatures since d a i l y fluctuations as much
as 12 ~ C have been o b s e r v e d in the area.

M o r i b u n d crabs from the D D T - c o n t a m i n a t e d marsh a p p a r e n t l y lacked e q u i l i b r i u m

Table II. DDT Residues (ppm) in Hepatopancreas (HP) and Swimmeret Muscle (SM}
Tissue of Callinectes sapidus from DDT-Contaminated (Sa)
and Control (3, l OJ Salt Marshes

Station DDE DDD DDT D D T (total)

3 SM Ta T T Tb
(6 individuals)
10 SM 0.013 0.004 T 0.017 b
HP 0.243 0.037 0.010 0.290
SM 0.009 0.005 T 0.014 b
HP 0.159 0.077 T 0.236
SM 0.004 0.005 T 0.009 b
HP 0.461 0.218 0.029 0.708
SM 0.008 0.004 T 0.012 b
HP 0.374 0.133 0.021 0.528
8a SM 0.575 0.374 0.109 1.058 a
(I-9) IIP 23.0 12.0 3.97 39.0
SM 0.151 0.244 0.010 0.405 d
HP 6.56 8.89 0.885 16.33
SM 0.190 0.117 0.023 0.330 d
HP 0.302 0.158 0.950 1.410
SM 1.02 0.350 0.030 1.400 e
HP 2.06 0.772 0.071 2.90
SM 0.568 0.102 0.041 0.711 c
SM 1.13 0.26'4 0.031 1.43 c
SM 0.241 0.159 0.012 0.412 d

aT = trace = < 0.00 z ppm; all results on fresh-weight basis; all samples collected N o v . - -
Dec. 1973.
blive crabs
Cdying crabs
ddead crabs
 Interaction of Temperature and DDT 125

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-= H I iJ= I E
i 9 e~| |174 | i| | iJ | ~boee | 1 7 4 | |174 i|
24 1 8 15 23 30 8 15 23 30
November December

Fig. 2. Blue crab mortalities observed in a DDT-contaminated salt marsh (8a) during
November and December, 1973. Corresponding daily air and water temperature minima are
shown with arrows indicating drops in water temperature minima below the mean (16.6 ~ C)
for the two months.

Table III. DDT Residues (ppm) in Fish Muscle Tissue from DDT-Contaminated (8a) and
Control (1, 2, 3) Salt Marshes

Station DDE DDD DDT DDT (total)

1 FundulusgrandiU .086 .064 Tc .150
2 .163 .153 .041 .367
3 .011 .010 T .021
8a .463 .682 .253 1.398
8a Poecil~ ~tipinna ~ .733 1.047 .141 1.921
8a .800 .600 .222 1.622
8a .530 .475 .110 1.115
3 Fundu~ssimilis .026 .022 T .048
3 T T T T
3 T T T T
3 .011 .015 T .026
3 .008 .009 T .017
3 .006 .006 T .012

aF. grand& samples collected simultaneously.

bfrom DeGrove (1973).
CT = trace = < 0.002 ppm; all samples taken in 1973.
126 C.C. Koenig et al.

and were either tremoring or in an extremely torpid state. Tremors are symptomatic
of DDT poisoning as the primary effect is throught to be on the sensory nerves
(O'Brien 1967). It is possible that the crabs may have come out of the mud in
response to the warmer afternoon temperatures and were unable to burrow again
when the cooler evening and early morning temperatures caused the sympomatic
tremoring and loss of equilibrium.

At this time it is not certain how reduced temperatures interact with DDT body
burdens to produce toxic symptoms. Cooler winter conditions may cause a higher
dependency of the crabs upon their fat reserves and thereby mobilize stored DDT
residues into the crab's system. It is also possible that extreme low temperatures
may favor the toxic mechanism of DDT on nerve membranes. Darnell (1959)
suggested that some blue crabs may "hibernate" during the winter months and
demonstrated that there is virtually no growth during this time. Our observations in
the tidal marshes support this view. Burrowing behavior would seem to be a
significant aspect of winter dormancy in blue crabs. If the crabs remain in an
inactive state for considerable periods and utilize a stored lipid supply, then the
availability of DDT residues to the sites of action would be increased.

In addition to the release of DDT residues during lipid metabolism the toxic
effect of DDT on the crabs may haste been intensified at low temperatures as Figure
2 suggests. Anderson (1971) demonstrated that p,p'-DDT in salmonid fishes caused
their lower lethal temperature to be significantly raised at two acclimation tempera-
tures. Peterson (1973) found that temperature selection in salmonid fishes could be
raised by p , p ' - D D T and the related compounds o,p'-DDT, p , p ' - D D D , p,p'-DDE,
and methoxychlor. If temperature selection is related to the raising of the lower
lethal temperature then the above compounds could be additive in their action at low
temperatures. Therefore, the metabolites p,p'-DDD and p,p'-DDE may have contri-
buted significantly to the low temperature toxic effects on the blue crabs.

The mechanism for the toxic action of DDT residues on blue crabs at low
temperatures could be similar to that suggested for insects. Holan (1969) presented
evidence of the formation of molecular complexes of DDT with protein components
of the nerve membrane based upon steric factors and van der Waals forces. As
O'Brien (1967) points out low temperatures should favor such complexes.

We suspect, therefore, the simultaneous operation of several mechanisms. First,

the release of DDT residues by the mobilization of stored lipids during periods of
reduced temperature. This would make the DDT residues available to the postulated
site of action, the nerve membranes. Secondly, the sharp drops in temperature
during the winter may render circulating DDT residues acutely toxic because low
temperatures favor DDT-nerve membrane complexes. Thirdly, the acute effects may
be amplified through the impairment of the postulated behavioral low temperature
buffering mechanism.
 Interaction of Temperature and DDT 127

Acknowledgments

This study was supported in part by Florida Sea Grant Project R/EA-3 (NOAA,
U. S. Department of Commerce). We wish to give special thanks to Dr. Allen
Sampson for his suggestions on the statistical treatment of the data and to Janis S.
Koenig for aid in the preparation of the manuscript.

References

Anderson, J. M.: Assessment of the effects of pollutants on physiology and be-

havior. Proc. Roy. Soc. Lond. B. 177, 307 (1971).
Butler, P. A.: Commercial fishery investigations, p. 65-77. In Effects of pesticides
on fish and wildlife. U. S. Fish and Wildlife Serv., Circ. No. 226 (1965).
Butler, P. A.: The significance of DDT residues in estuarine fauna, p. 205-220. In
M. W. Miller and G. G. Berg (eds.) Chemical fallout: Current research on
persistent pesticides. Illinois: C. C. Thomas Company (1969).
Butler, P. A., and P. F. Springer: Pesticides - - a new factor in coastal environ-
ments. Trans. 28th N. Amer. Wildlife Nat. Res. Conf., p. 378-390 (1963).
Carriker, M. R.: Ecology of estuarine benthic invertebrates: A perspective, p.
442-487. In G. H. Lauff (ed.) Estuaries. Amer. Assoc. Adv. Sci., Publ. No.
83 (1967).
Commercial Fisheries Review DDT. Commercial Fisheries Review 8, 30 (1946).
Cottom, C., and E. Higgins: DDT: its effects on fish and wildlife. U. S. Fish and
Wildlf. Serv., Circular No. 11, 14 p. (1946).
Darnell, R. M.: Studies of the life history of the blue crab (Callinectes sapidus) in
Louisiana waters. Trans. Amer. Fish. Soc. 88, 294 (1959).
Holan, G.: New halocyclopropane insecticides and the mode of action of DDT.
Nature 221, 1025 (1969).
Lowe, J. I.: Chronic exposure of blue crabs, Callinectes sapidus, to sublethal
concentrations of DDT. Ecology 46, 900 (1965).
Manhood, R. K., M. D. McKenzie, D. P. Middaugh, S. J. Bollar, J. R. Davis, and
D. Spitzbergen: A report on the cooperative blue crab study - - South Atlantic
states. U. S. Dept. Interior, B.C.F. 32 p (1970).
Mills, H. R.: Deaths in Florida marshes. Audubon 54, 285 (1952).
O'Brien, R. D.: Insecticides action and metabolism. New York: Academic Press,
332 p (1967).
Odum, W. E.: Insidious alteration of the estuarine environment. Trans. Amer. Fish.
Soc. 99, 836 (1970).
Odum, W. E., G. M. Woodwell, and C. F. Wurster: DDT residue adsorbed from
organic detritus by fiddler crabs. Science 164, 576 (1969).
128 C.C. Koenig et al.

Peterson, R. H.: Temperature selection of Atlantic salmon (Salmo salar) and brook
trout (Salvelinus fontinalis) as influenced by various chlorinated hydrocar-
bons. J. Fish. Res. Bd. Can. 30, 1091 (1973).
Pyle, R. W., and L. F. Cronin: The general anatomy of the blue crab, Callinectes
sapidus. Chesapeake Biological Laboratory, Solomons, Md., Publ. No. 87, 40
p (1950).
Springer, P. F., and J. R. Webster: Biological effects of DDT applications on tidal
salt marshes. Mosquito News 11, 67 (1951).
Thompson, N. P., P. W. Rankin, and D. W. Johnston: Polychlorinated biphenyls
and p,p'-DDE in green turtle eggs from Ascension Island South Atlantic
Ocean. Bull. Environ. Contamin. Toxicol. 11, 399 (1974).
Woodwell, G. M., C. F. Wurster, and P. A. Isaacson: DDT residues in an east coast
estuary: a case of biological concentration of a persistent insecticide. Science
156, 821 (1967).
Woodwell, G. M., P. P. Craig, and H. A. Johnson: DDT in the biosphere: Where
does it go? Science 174, 1101 (1971).

Manuscript received August 7, 1974; accepted January 18, 1975