Title: Ionic and osmotic regulation, metabolic response to salinity, and physiological response to pesticides of juvenile Callinectes Sapidus Rathbun
Full Citation
Permanent Link: http://ufdc.ufl.edu/UF00098681/00001
 Material Information
Title: Ionic and osmotic regulation, metabolic response to salinity, and physiological response to pesticides of juvenile Callinectes Sapidus Rathbun
Physical Description: v, 58 leaves. : illus. ; 28 cm.
Language: English
Creator: Leffler, Charles William, 1947-
Publication Date: 1974
Copyright Date: 1974
Subject: blue crab   ( lcsh )
Crabs   ( lcsh )
Zoology thesis Ph. D
Dissertations, Academic -- Zoology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
Thesis: Thesis--University of Florida.
Bibliography: Bibliography: leaves 54-57.
General Note: Typescript.
General Note: Vita.
 Record Information
Bibliographic ID: UF00098681
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000869360
notis - AEG6385
oclc - 014267353


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I would like to thank Drs. F. G. Nordlie, B. K. McNab, J. F.

Anderson, and F. J. S. Maturo, Jr. for their help during the course

of my research and the preparation of this dissertation and Dr. D. W.

Johnston for the use of his equipment (funded through NSF Grant GB-

25872) and expertise in pesticide analyses.

The help of George Zeigler, Lee Belcher, John Paige, and Buck

Parnell in maintaining a constant supply of juvenile blue crabs is

greatly appreciated.

I would also like to thank Donna Gillis for typing the manuscript.

Special thanks go to my wife, Robin.


ACKNOWLEDGMENTS . . . . . . . . .. ... ii

ABSTRACT. . . . . . . . . . . .... iv

INTRODUCTION . . . . . . . . ... . . ... 1

MATERIALS AND METHODS . . . . . . . . . .. 3

RESULTS . . . . . . . . . . . . . 8

Osmotic and Ionic Regulation . . . . . . . . 8
Salinity Effect on Metabolic Rate. . . . . . ... 16
Oxygen Concentration Effects on Metabolic Rate ...... 16
Pesticide Concentration in Juvenile Blue Crabs
from the Cedar Key Area. . . . . . . . . 23
Acute Levels of DDT and Mirex. . . . . . . ... 23
Pesticide Effects on Metabolic Rate. . . . . . ... 24
Oxygen Concentration Effects on the Metabolic
Rates of Pesticide Treated Juvenile
Blue Crabs . . . . . . . .... . . .25
Pesticide Effects on Ionic and Osmotic Regulation. .... . 32
Pesticide Effects on Autotomization of Limbs . . ... 32
Pesticide Effects on Carapace Thickness .. . . . ... 37

DISCUSSION. . . . . . . . . ... . . .... 40

SUMMARY .. . . . . . . . . . . .... 51

LITERATURE CITED. .... . . . . . . .... 54

BIOGRAPHICAL SKETCH . . . . . . . ..... 58

Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy



Charles William Leffler, II

Chairman: Frank G. Nordlie
Major Department: Zoology

The osmotic and ionic regulation and the metabolic response to

salinity of juvenile CalZinectes sapidus Rathbun were investigated.

Juvenile blue crabs at 250C hyper-osmoregulate in medium of less than

700 mOs/l and osmoconform at higher salinities. Hemolymph Na+ and C1

concentrations increase gradually with increasing external concentrations

below 700 mOs/l and increase more rapidly at higher concentrations: only

C1- is hypo-regulated. The internal K+ concentration is higher than the

external concentration at all external concentrations tested (as high as

15 meq/1). At 16C and concentrations less than 700 mOs/l total hemo-

lymph concentrations are higher than at 250C. The metabolic rates of

juvenile blue crabs acclimated to test concentrations between 50 and

1410 mOs/l are not significantly different. Abrupt transfer from a

higher acclimation concentration to a lower concentration results in a

large increase in the metabolic rate. At 02 concentrations greater than

2.0 ,.1 02/1 juvenile blue crabs are metabolic regulators.

DDT and Mirex concentrations in juvenile blue crabs from the Cedar

Key, Florida, U.S.A. estuarine zone were low. The effects of ingested

DDT and Mirex on the physiology of juvenile blue crabs were examined.

The crabs are sensitive to these pesticides when the compounds are

ingested--to Mirex more than to DDT. High, subacute internal levels

of DDT and Mirex result in pronounced metabolic rate elevations, re-

duction in critical oxygen concentration, inhibition of the autotomy

reflex (Mirex), and reduced carapace thicknesses. DDT and Mirex below

acute levels do not affect patterns of osmotic and ionic regulation.

I conclude that DDT and, to a far greater extent, Mirex are potentially

disastrous agents with respect to blue crab populations.



2. metabolic costs of osmotic regulation at different salinities,


3. effects of DDT and Mirex on the metabolic rate, ionic and

osmotic regulation, ability to autotomize limbs, and carapace thick-



The experimental animals were juvenile blue crabs, CaZZinectes

sapidus Rathbun, (0.75-2.6 g [dry], 35-50 mm [width]) of both sexes

from the estuarine zone near Cedar Key, Florida, U.S.A. A commercial

shrimp fisherman captured the crabs at night on sea grass flats during

all seasons (1971-1974).

The crabs were maintained in the laboratory for 35 days before

physiological measurements were made. The tanks were 103 liter aquaria

divided into 6 chambers. Water was circulated and filtered through

activated charcoal by a power filter maintaining an 02 concentration

above 4.0 ml/1 in the acclimation water. The full sea water (approxi-

mately 1000 mOs/l [34 O/oo]) was filtered ocean water from Marineland

(Marineland, Florida, U.S.A.). It was made more saline by the addition

of synthetic marine salt mix (Instant Ocean) or diluted to the desired

concentrations. The concentrations were measured with an Osmette

precision osmometer. Coquina (Donax variabilis) shell fragments were

placed on the floors of the tanks. The water was replaced and the

substrate rinsed after the experiments on each group of crabs were


Two acclimation temperatures 160C (+ 1) and 250C (+ 1) were

maintained. The photoperiod was 14h light and 1Oh dark.

The diet consisted oF three feedings weekly (on alternate days)

of one 3 mm3 piece of beef liver or shrimp per crab. Feeding was dis-

continued 5 days prior to the beginning of the actual experimentation.

Mirex (dodecachlorooctahydro-1, 3, 4-metheno-2H-cyclobuta [cd]

pentalene) and p,p'-DDT (1, 1, l-trichloro-2, 2-bis [p-chlorophenyl]

ethane) (chromatographically pure) were administered orally once per

week for a total of 2, 3, or 4 feedings. Food items were soaked in

acetone solutions of pesticides for measured lengths of time at 2C.

The treated food was frozen until used. Food type, particle sizes,

solution concentrations, soaking times, and the resultant pesticide

concentration of the food are given in Table 1. The treated food

particles were placed directly against the crab's mouth parts and were

devoured within a couple of minutes. Control groups were run simul-

taneously with experimental. One control group received food soaked

in acetone at one feeding each week while a second received only un-

treated food. As no physiological differences were observed between

these two groups, they are combined and are considered untreated crabs

in all further discussion. Metabolic rate determinations were made 7

days after the last treatment--one untreated feeding being given after

the final treatment.

Metabolic rates were measured by determining 02 uptake in closed

chambers. The chambers were opaque Erlenmeyer flasks fitted with

rubber stoppers pierced by three hypodermic needles. One needle was

fitted with catheter tubing that reached to the bottom. When the flask

was filled with water this arrangement allowed water samples to be taken

from the top and the bottom. A single, post-absorptive, intermolt (stage

C) crab was placed in each aerated chamber that was, in turn, placed in

a water bath maintained at the desired temperature. After the crab

became quiet, the aeration was terminated, the chamber sealed, and water


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samples were taken at hourly intervals for 3 h. Oxygen concentrations

of the samples were determined using a Radiometer p02 electrode. At the

conclusion of the test the crabs were removed from the chambers, weighed,

and returned to their acclimation treatment tanks. All metabolic

determinations were made between 10 A.M. and 4 P.M.

Two days after the metabolic rate measurements were made concentrations

of Na+, K+, C1-, and total osmotic concentrations of the hemolymph of the

same individuals used for metabolic experimentation were measured. Samples

were obtained by piercing the arthrodial membrane at the base of the

fifth walking leg with a glass capillary pipette. Hemolymph concentrations

were determined with a Clifton nanoliter osmometer or on pooled samples

with an Osmette precision osmometer. The hemolymph ionic composition

was analyzed using a Radiometer Model FLM 2 flame photometer (Na+, K+)

and a Radiometer Model CMT 10 chloride titrator.

The capacity of each crab to autotomize a damaged limb was

determined by crushing the merus of the forth walking leg. The crab

was then placed in hot water (600C). The extent of autotomization

resulting from an extreme generalized stimulus was recorded. All crabs

died within seconds and were removed. Crabs were dried at 1050C for

24 h. Twenty minutes after removal from the oven they were weighed.

Carapace widths and thicknesses (mean of three mid-dorsal measurements)

were measured with calipers.

Chlorinated hydrocarbon levels were determined using a Varian

Aerograph, Model 600-D, gas chromatograph containing a 6' X 1/4" (OD)

glass column of 1:1 6.4% OV-210:1.6% OV-17 on chromosorb W and equipped

with an H electron capture detector. The column and detector tempera-

tures were 2150 and 2138C respectively. Carrier gas (N2) flow rate

was 45 ml/min. Crabs were analyzed in groups of four that had under-

gone similar treatment. Chlorinated hydrocarbons were removed from

tissues by the following method: the crabs were ground along with

anhydrous Na2S04 in a Virtis "45" blender; the lipids and chlorinated

hydrocarbons were extracted from the powder into 1 acetone: 2 hexane

(Soxlet apparatus, 16 h); the sample was evaporated nearly to dryness;

cleanup was made by placing the sample (dissolved in hexane) on a

deactivated Florisil (10% water) column and removing the chlorinated

hydrocarbons with a 3 hexane: 1 benzene emulsion. The sample was

evaporated to about 5 ml and the volume increased, if necessary, with

benzene for injection into the gas chromatograph. Samples of DDT, DDD,

DDE, and Mirex of known concentrations were processed (Soxlet, Florisil

column) with each three samples analyzed to ascertain the percentage


mean % recovery DDT family (DDT, DDD, DDE) 85.1 + 9.6*

Mirex 88.0 + 5.5.

Pesticide concentrations reported have been corrected for the deviation

from 100% recovery.

*Numerals following + designate standard error of means.


Osmotic and Ionic Regulation

Juvenile blue crabs maintain hemolymph osmotic concentrations higher

than all external concentrations less than 700 mOs/l (25C) but crabs

acclimated to lower salinities have lower hemolymph concentrations than

those acclimated to higher salinities (Figure 1). For example, at an

external concentration of 700 m0sm/l the hemolymph concentration is about

700 mOs/l but this drops to 575 mOs/l at 50 mOs/l.* Above 700 mOs/l, juve-

nile blue crabs at 250C are approximately isosmotic with the environment

(Figure 1).

Regulation of Na+ and C1- follows similar patterns to that of total

osmotic concentration (Figures 1, 2, and 3). Sodium ions are hyper-

regulated up to an external concentration of about 325 meq/1 (equivalent

to a total osmotic concentration of 760 mOs/1). Within this hyper-

regulatory range hemolymph Na+ concentration increases in response to

increases in external concentration. For example, at an external Na

concentration of 25 meq/l the hemolymph Na+ concentration is approxi-

mately 280 meq/1 but the hemolymph concentration increases to about

320 meq Na+/l at an external concentration of 300 meq Na /1. Sodium

ions are slightly hyper-regulated and conforming to external concentrations

between 325 and 500 meq/1 (about 1200 mOs/l). Hemolymph and external Na

concentrations are equal at 625 meq/1 (1410 mOs/l). Hemolymph C1 con-

*All differences cited (hemolymph concentration, metabolic rate, etc.) are
significant at 95% confidence (Student-t).

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meq/1 (660 mOs/1). Within this hyper-regulatory range hemolymph C1 is

higher at high external concentrations than at low. For example, the

hemolymph Cl concentration is 325 meq/1 when the crab is acclimated to

water containing 30 meq C1-/l increasing to about 390 meq/1 at 350 meq

C1-/1. Hemolymph Cf-, unlike Na+ and total osmotic particles, is hypo-

regulated with respect to external concentrations between 400 and 650

meq Cl-/l (700 and 1200 mOs/l). At an external concentration of 650

meq C1-/1 the hemolymph C1 is isoionic with respect to the medium but

at 800 meq C1-/l (1410 mOs/l) the hemolymph C1- concentration is far

below that of the environment.

The hemolymph K+ concentration is strongly hyper-regulated at all

external K+ concentrations tested (maximum external K+ concentration =

15 meq/1 at 1410 mOs/l).

Temperature also influences the hemolymph concentration of juvenile

blue crabs. Below an external concentration 650 mOs/1 the hemolymph

concentrations of crabs acclimated to 16C are higher than those of

crabs acclimated to 25C (Figure 1). For example, the mean hemolymph

concentration of a crab acclimated to 250C and 200 m0s/l is about

600 mOs/l but at 16C and the same external concentration the hemolymph

concentration is about 8% higher (647 mOs/l). Similarly, at 250C and

600 mOs/l, the mean hemolymph concentration is about 700 mOs/l but is

about 770 mOs/l at 160C and the same external concentration. However,

at 1000 mOs/l the hemolymph concentrations at 250C and 160C were roughly


Figure 4. The relationship between the hemolymph K+ concentration
and the external K+ concentration of juvenile blue crabs
not treated with pesticides (o), DDT1 treated (*), DDT2
treated (*), and Mirex3 treated (X) at 250C and untreated
juvenile blue crabs at 160C (+).
(Diagonal is equality.)

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At 16C, the hemolymph Na+ concentration is, like total concentration,
higher than at 25C at external Na concentrations less than 400 meq/1

(950 mOs/l) (Figure 2). For example, at an external Na+ concentration

of 300 meq/1 the hemolymph Na concentration is 375 meq/1 when the crab

is acclimated to 160C but only about 325 meq/1 at 25C. The hemolymph

K and C1- concentrations appear to be unaffected by temperature (160

and 250C) (Figures 3 and 4).

At 16C juvenile blue crabs cannot survive exposure to 75 mOs/l

if abruptly transferred from 220C, 800 mOs/l. Of six crabs transferred,

none survived more than 12 h.

Salinity Effect on Metabolic Rate

No significant differences in metabolic rate are observed in crabs

acclimated to and tested at different salinities between 50 and 1410 mOs/l

(25C) (Figure 5, untreated). However, the metabolic rates of crabs

acclimated to one salinity and tested at a lower salinity are much

higher at the lower salinity. For example, one juvenile blue crab which

had a metabolic rate of 0.221 ml 02/g-h when acclimated to 456 mOs/l

showed an approximately 65% increase in metabolic rate (to 0.36 ml

02/g-h) when transferred to63 mOs/l. Another crab abruptly transferred

from its 1235 mOs/l acclimation medium to 387 mOs/l showed an even

greater metabolic rate increase (87%; 0.21 to 0.38 ml 02/g-h) (Figure 5).

Oxygen Concentration Effects on Metabolic Rate

In oxygen rich water, juvenile blue crabs appear to be metabolic

regulators. Above 2.0 ml 02/1, the ambient 02 concentration did not

aFFect the metabolic rates of juvenile blue crabs at any salinities

tested (between 50 and 1410 mOs/l) (Figure 6).

Figure 5. The effect of the environmental concentration on the
weight specific 02 consumption of juvenile blue crabs
(25"C, 02 concentration: 4.5 ml 02/1) not treated
with pesticides (o), DDT1 treated (e), DDT2 treated
(*), Mirex1 treated (+) and Mirex2 3 treated (X).
T indicates crabs treated with DDT'which were exhibit-
ing convulsions. (Ranges indicated are standard
errors of means.) The arrows indicate metabolic
responses of juvenile blue crabs to sudden changes
from higher (origin of arrow) to lower (point of
arrow) concentrations.




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Pesticide Concentration in Juvenile Blue Crabs
from the Cedar Key Area

The mean DDT family concentration in juvenile blue crabs collected

from the estuarine zone around Cedar Key was 0.031 + 0.003 ppm. No

Mirex was detected in any sample from this area.

Acute Levels of DDT and Mirex

Ingested Mirex is highly toxic to juvenile blue crabs. At intermediate

salinities (200-600 mOs/l) four feedings of 0.14 ig Mirex each over a five-

week period resulted in convulsions in most of the 0.75-1.25 g crabs.

Convulsions were induced in 1.25-2.5 g crabs by five such feedings.

The internal concentrations of the poisoned crabs were 0.48 + 0.05 ppm

Mirex. In dilute (50 mOs/1) and concentrated (1000 mOs/l) media the

crabs were slightly more sensitive to Mirex: three weekly feedings of

0.14 pg Mirex each produced acute effects in 0.75-1.25 g crabs and four

feedings resulted in convulsions and death in the 1.25-2.5 g crabs:

internal concentrations averaged 0.42 + 0.05 ppm.

DDT is a far less toxic stomach poison to juvenile blue crabs than

is Mirex. No acute effects were elicited by DDT at four times the acute

Mirex intake levels (four weekly feedings of 0.53 ig DDT). Such treat-

ment resulted in DDT family tissue concentrations approximately equal to

Mirex tissue concentrations resulting from 1/4 the dietary intake: 0.13

+ 0.01 ppm DDT family, 0.19 + 0.03 ppm Mirex. Three weekly feedings of

3.2 pg DDT resulted in typical symptoms of chlorinated hydrocarbon poison-

ing at 50 mOs/l and 430 mOs/l in 1.5-2.5 g crabs (internal concentrations:

1.0 + 0.1 ppm). Crabs killed by DDT (same intake levels as above) at 1000

mOs/l showed none of the characteristic symptoms of chlorinated hydrocarbon

poisoning. The DDT levels in these crabs were 1.1 + 0.15 ppm. Crabs

were treated with high levels of DDT in Mg++ enriched (Mg++ concentration

approximately 1.2 times that of full sea water) brackish water (400 mOs/l)

to test the hypothesis that the higher Mg+ levels in the sea water might,

by relaxing muscles, inhibit the typical chlorinated hydrocarbon syndrome

in sea water. These crabs showed typical signs of DDT poisoning at the

same input levels as those in non-Mg enriched brackish water. High

Ca++ levels were tried in an attempt to raise the threshold of nerves

and thus suppress the symptoms. The results were as with Mg Mirex

treated crabs at high salinities (1000 and 1450 mOs/l) showed typical

symptoms of chlorinated hydrocarbon poisoning.

Pesticide Effects on Metabolic Rate

High subacute internal levels of Mirex and DDT (0.19 + 0.03 ppm

Mirex = Mirex3, 0.82 + 0.05 ppm DDT = DDT2*) cause pronounced elevations

of juvenile blue crab metabolic rates (Figure 5). The metabolic rates of

these pesticide treated crabs are more than twice those of untreated crabs.

Some subtle behavioral abnormalities such as greater than normal reactions

to food, extreme excitability when pursued, and a tendency to move in a

"tip-toe" manner were observed at these subacute pesticide levels. Tissue

Mirex levels causing metabolic rate increases are much lower than the

internal DDT family concentrations that elevate the metabolic rate: DDT

family concentrations (0.15 ppm = DDT1) approximately equal to the Mirex

*See Table 1.

levels (0.19 ppm) that result in metabolic rates double those of un-

treated crabs have no effect on the metabolic rates of the crabs (200

and 600 mOs/l) (Figure 5). Mirex concentrations (Mirex1) as low as

0.02 parts per million parts of body tissue caused significant metabolic

rate elevations (Figure 5). Although the differences are not significant

at the 95% confidence level (Student-t), the pesticide caused metabolic

rate elevation is more pronounced at higher salinities (Figure 5). For

example, the mean metabolic rate of Mirex3 treated crabs at 1000 mOs/l

is 1.25 times those at 50 or 200 mOs/l. Mirex3 crabs at 1450 mOs/l

had metabolic rates averaging 1.8 higher than at 50 mOs/l. Similar

increases were observed with DDT treatments. DDT1 crabs had the same

rates of metabolism as controls at 200 and 600 mOs/l but the same treat-

ment caused significant metabolic rate increases at 1000 m0s/l (Figure


Oxygen Concentration Effects on the Metabolic Rates of
Pesticide Treated Juvenile Blue Crabs

Metabolic rates of crabs fed pesticides are independent of environ-

mental 02 concentration at high 02 levels (Figures 7, 8, and 9). Crabs

that were exposed to 02 concentrations of less than 2.0-2.5 ml 02/1 consumed

02 more slowly (Figures 7, 8, and 9). Although the sample size is too

small to draw a definite conclusion, Mirex treated crabs at higher

salinities (1000 and 1450 mOs/l) appear more sensitive to falling 02

concentrations than crabs in other groups (i.e. the critical oxygen

concentration may be as high as 2.5 ml 02/1) (Figure 9).



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Pesticide Effects on Ionic and Osmotic Regulation

DDT and Mirex do not significantly affect osmotic and ionic

regulatory patterns of juvenile blue crabs at subacute concentrations

(Figures 1, 2, 3, and 4).

Pesticide Effects on Autotomization of Limbs

High levels of Mirex inhibit the ability of juvenile blue crabs to

autotomize limbs. The inhibition applies to both the autotomy of a

single damaged limb (Figure 10) and to the number of limbs autotomized

in response to extreme generalized stimuli (Figure 11). Internal Mirex

concentrations as low as 0.02 ppm (Mirex,) inhibited autotomy of limbs

by crabs placed in hot water (one limb autotomized per crab compared to

an average of more than 3 limbs per untreated crabs). The same Mirex

concentration blocks autotomy of a severely damaged limb by most crabs.

Ninety-five percent of the untreated crabs autotomized a walking leg with

a crushed merus while only 29% of Mirex treated crabs autotomized a limb

with the same damage (Figure 10, results from all crabs with Mirex levels

between 0.02 and 0.2 ppm are combined as no concentration-dependent

response was observed). Extremely high internal DDT concentrations

(0.82 ppm [DDT2]), although causing some inhibition (less than 2 limbs

per crab compared to an average of more than 3 limbs per untreated crab)

of limb autotomy in response to hot water (Figure 11), did not inhibit

autotomy of a damaged limb (Figure 10). Juvenile blue crabs exhibiting

convulsions resulting from either DDT or Mirex poisoning were unable to

autotomize limbs. Seven convulsive animals (4 Mirex induced, 3 DDT

induced) were tested for ability to autotomize d damaged walking leg

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or to autotomize limbs when placed in hot water. One chela was autoto-

mized by a Mirex treated crab in hot water--no other autotomy occurred.

Pesticide Effects on Carapace Thickness

When I measured the carapace thicknesses and widths of crabs molting

during captivity (during molt cycle stage C4), I discovered that the

thicknesses compared to the widths of Mirex3 treated crabs were considerably

less than those of the controls (Figure 12). For example, an average

42 mm wide untreated juvenile blue crab has a carapace that is 0.27 mm

thick while an average 42 mm wide Mirex3 juvenile blue crab has a

carapace that is only 0.22 mm thick mid-dorsally. Crabs treated with

lesser amounts of Mirex (Mirex2) and DDT1 did not have unusually thin

carapaces. The data on carapace thicknesses of DDT2 crabs are sparse.

However, these data are similar to those from Mirex3 crabs (Figure 12).




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Hemolymph osmotic, Na+, and Cl concentrations in adult blue

crabs have been extensively studied at external concentrations of up

to 1000 mOs/l. No significant differences in regulatory patterns of

juveniles and adults are observed (Figures 1, 2, and 3). Gifford

(1962) and Ballard and Abbott (1969) have measured adult hemolymph

concentrations at very high salinities. Their results differ (Figure 1).

Gifford (1962) reports hypo-osmoregulation at all salinities above

1100 mOs/l while Ballard and Abbott (1969) report slight hypo-

osmoregulation between 900 and 1100 mOs/l and osmoconformity above

these salinities. My results on juveniles are intermediate with

respect to the conclusions of these authors. I detected very slight

hypo-osmoregulation above 1100 mOs/l (250C) (Figure 1) due apparently

to C1 hypo-regulation. Chloride hypo-regulation in juveniles is

similar to that of total hemolymph concentration reported by Ballard

and Abbott (1969) between 900 and 1100 mOs/l. However, C1- is also

strongly hypo-regulated at 800 meq/1 (1410 mOs/l) (Figure 3). Lynch

et al. (1973) observed hypo-regulation of C- in adults between external

concentrations of 400 and 550 meq C1-/I. Chloride and Na+ regulatory

patterns are noticeably different which is what one would expect if these

ions are independently transported as suggested by Mantel (1967).

Temperature affects osmoregulation in juvenile blue crabs. Numerous

investigators have noted that adult blue crabs have higher hemolymph

salt concentrations in cold water than in warmer water of the same

salinity (for example, Lynch et al., 1973). This phenomenon was

also observed in juvenile blue crabs acclimated to 250 and 16C at

salinities less than 700 mOs/l (Figure 1). Part of the hemolymph

concentration increase in cool water is due to a hemolymph Na con-

centration increase (Figure 2). Chloride and K are not affected by

temperature (Figures 3 and 4). The inability of juvenile blue crabs

to tolerate low temperature (160C)/low salinity (76 mOs/l) combinations

is important. This limitation may explain the observation that in

temperate regions small crabs migrating into the estuarine zone in

the fall after completing their larval development do not migrate

into fresh water bays and rivers until the following spring (Van Engel,


There were no significant differences between metabolic rates of

crabs acclimated to different salinities between 50 and 1410 mOs/!

(Figure 5). Such absence of metabolic rate variation with salinity

is expected in an animal that does not strongly hypo-osmoregulate.

Hyper-osmoregulation does not appear to be an energetically expensive

function. Potts (1954) proposed a minimal theoretical energy expenditure

for osmotic regulation by Eriocheir in hard, fresh water as low as 0.5%

of the standard metabolic rate of a 60 g (wet) crab. This value is

based on the assumptions that all ion loss is via urine and that active

transport is 100% efficient. Seventy-five percent of the C1 loss by

Eriocheir is at the body surface Shaw (1961). Most transport processes

are between 20 and 80% efficient (Potts and Parry, 1964). Taking these

factors into account, the expected energetic cost of osmotic regulation

by a 60 g Eriocheir in hard, fresh water is between 2.5 and 10% of

its standard metabolic rate. My results, as well as earlier experiments

on an euryhaline fish (fugil cephalus; Nordlie and Leffler, 1974), support

the prediction that the steady-state energetic cost of hyper-osmoregulation

may be very low. Metabolic rates of crabs acclimated to the low test

salinity and those acclimated to salinities higher than the test

salinity may be greatly different. While juvenile blue crabs acclimated

to the test salinities have similar metabolic rates at 50, 200, 600,

1000, 1200, and 1410 mOs/l, crabs abruptly transferred from higher

salinities to lower ones have greatly elevated metabolic rates. The

fact that the steady-state and transitory relationships between metabolic

rate and salinity are so dissimilar indicates that increases in metabolic

rate following transfer from one medium to a more dilute one are not

totally the result of increases in the metabolic rates of the salt

absorbing tissues per se, but, also, the result of metabolic rate increases

in tissues not directly associated with osmoregulation. Further supporting

this contention, the magnitude of the difference between acclimation salinity

and test salinity is apparently more important than the level of the test

salinity in determining the extent of the increase. For example, a

crab acclimated to 1200 mOs/l had a metabolic rate 87% higher than expected

at 450 mOs/l but a crab acclimated to 450 mOs/l and tested at 160 mOs/l had

a metabolic rate only 65% higher than expected (Figure 5). No pronounced

increase in metabolic rate was expected at high salinities because

juvenile blue crabs do not strongly hypo-osmoregulate. The absence of

observable metabolic rate differences between crabs acclimated to

different salinities confirmed this expectation (Figure 5).

Juvenile blue crabs are nonburrowing inhabitants of open portions of

the estuarine zone. They are not frequently exposed to low 02 concentra-

tions. These crabs are metabolic regulators in the high environmental 02

concentration range to which they are commonly exposed. The mean standard

metabolic rates of juvenile blue crabs are the same at 2.0 and 5.5* ml

02/1 (Figure 6). Earlier measurements of juvenile blue crabs' metabolic

rates at lower 02 concentrations indicated that these crabs were metabolic

conformers (Leffler, 1972). The critical oxygen pressure cannot be pin-

pointed because of differences between the methods used. It is below

2.0 ml 02/1. The hyperbolic curve of metabolic rate on 02 concentration

is much different from the linear relationship (with a very gradual slope)

observed in the burrowing Xanthid crabs, Menippe mercenaria and Panopeus

herbatii (Leffler, 1973), that naturally encounter low 02 concentrations.

Chlorinated hydrocarbons are ubiquitous in the environment. DDT

has been applied globally since 1946. The application of another

chlorinated hydrocarbon, Mirex, has, thus far, been more restricted.

Since 1963 it has been applied to vast areas of southeastern U.S.A. in

an attempt to control the imported fire ant, Solenopsis invicta (Bellinger

et at., 1964; Coon and Fleet, 1970; Collins et at., 1973).

Aquatic, estuarine, and marine carnivores, scavengers, and detritus

feeders take up chlorinated hydrocarbons mainly via their food. Chlorinated

hydrocarbons have low solubilities in water. They adhere to various

organic particles allowing input into detritus feeders to be high

(Odum et at., 1969). Biological magnification of persistent pesticides

*5.5 ml 0 /1 was thp highest 02 concentration at which experiments were
performed. Saturation at 250C, 50 mOs/1 is 5.7 ml 02/1.

in estuarine food chains has been demonstrated (Woodwell et aZ., 1967).

Therefore, the food of scavengers and predators would contain DDT

family and Mirex concentrations far above those in the water. Macek

and Korn (1970) support this contention. Their work shows that brook

trout accumulate DDT at a ratio of 10 parts from food to each part

taken up directly from the water. Catfish denied access to their

natural Food chain in Mirex treated ponds did not accumulate Mirex

while free-living catfish in the same ponds contained 0.65 ppm after

6 months (Collins et al., 1973). Pesticide input into a detritus

feeder, scavenger, and active predator such as CaZlinectes would also

be mainly via its food. In addition, juvenile blue crabs have been

shown to ingest Mirex bait with fatal results when such bait is

accessible (Mahood et al., 1970).

With food being the main route of chlorinated hydrocarbon input

into many aquatic, estuarine, and marine animals, it is surprising to

find that nearly all laboratory studies of the effects of persistent

pesticides on these animals have been conducted by pesticide application

to the aqueous medium. This approach can produce misleading results.

Stomach poisons (i.e. input through the digestive system) may be

relatively nontoxic contact poisons while potent contact poisons may be

relatively nontoxic stomach poisons. Butler (1963) states that Mirex

in solution is relatively nontoxic to juvenile blue crabs (48 h, EC20

2 ppm [compared to DDT with a 48 h EC50 = 0.01 ppm]). In contrast,

ingested Mirex has far greater effects on juvenile blue crabs than does

ingested DDT. Acute Mirex intake levels are about 0.06 those of DDT.

However, the internal DDT family concentrations oF juvenile blue crabs

fed amounts of DDT 17 times greater than the amounts of Mirex fed to

other crabs were only twice the Mirex levels in the latter group.

Either (1) the digestive system absorbs Mirex more readily than it

does DDT, or (2) DDT and its metabolites are excreted at greater rates

than Mirex. The second alternative must be at least in part responsible

for the lower retention of ingested DDT compared to Mirex because

several DDT poisoned animals exhibiting mild chlorinated hydrocarbon

syndrome recovered while no Mirex treated crab showing signs of poison-

ing ever recovered. Mirex is also more potent than is DDT in eliciting

subacute responses (i.e. increased metabolic rate, inhibition of

autotomy reflex). Mirex produces pronounced metabolic rate

increases at levels less than 0.25 internal DDT family concentrations

resulting in similar responses.

Acute and subacute DDT family and Mirex concentrations being

discussed are not above those encountered in many estuarine food

chains. For example, the maximum Mirex levels that Mahood et al.

(1970) detected in the tissues of adult blue crabs from Georgia coasts

was 0.389 ppm. A juvenile blue crab (1.25 g) ingesting 1.44 g of such a

carcass in 4 weeks would receive a lethal Mirex dose. Mahood et aZ.

(1970) measured levels of DDT and its metabolites as high as 0.231

ppm in adult blue crabs from Georgia. Forty grams of this tissue

eaten in 4 weeks would be acute to 1 g crabs. Other prospective

food items have pesticide concentrations far in excess of these:

(Long Island, New York, U.S.A.) white perch, 1.99 ppm DDT family;

menhaden, 1.53 ppm DDT family (Foehrenbach, 1972); sheepshead minnow,

0.94 ppm DDT family; chain pickerel, 1.33 ppm DDT family; Atlantic

needle fish, 2.07 ppm DDT family (Woodwell et aZ., 1967). Even the

vegetation may contain high pesticide levels. Vegetation in ponds

treated with Mirex bait at the manufacturer's recommended application

rate for field use contained Mirex residues as high as 4.0 ppm

(Van Valin et at., 1968). Juvenile blue crabs devour more than 55%

of their wet weights in food per day (Holland et at., 1971). I found

the wet weight/dry weight ratios were greater than 5. Therefore,

disregarding growth, a 1 g blue crab would consume at least 70 g of

food in 4 weeks.

High, subacute concentrations of DDT and Mirex result in pronounced

elevations of juvenile blue crab standard metabolic rates (Figure 5).

This elevation was observed at all concentrations above the minimal

level eliciting the response. Even crabs exhibiting pesticide induced

convulsions had as high metabolic rates as crabs with high subacute

concentrations. This is in contrast with Physa gyrina (pond snail),

Gambusia affinis (mosquitofish), and Lepomis macrochirus (sunfish) in

which low Mirex concentrations result in metabolic rate elevations while

higher concentrations depress rates of metabolism (De La Cruz and Naqvi,

1973). I believe the metabolic rate increases are the result of increased

muscle and nerve metabolism. DDT (and presumably other chlorinated hydro-

carbons) has an unstabilizing effect on nerves and muscles (Yeager and

Munson, 1945; Roeder and Weiant, 1945, 1948; Bodenstein, 1946; Welsh

and Gordon, 1947). Increased muscular and nervous activity must increase

the metabolic rates of these tissues substantially. In addition, DDT delays

the turning off of the Na+ influx associated with action potential

development (Harahashi and Haas, 1963). The resulting overlap night

increase the necessary energy expenditure associated with returning the

nerve to its "resting" state following a discharge.

Juvenile blue crabs with pesticide induced metabolic rate

elevations might face difficulties in obtaining sufficient food and

oxygen to accommodate both normal growth and increased metabolism.

Blue crabs with elevated metabolic rates as juveniles caused by

high ambient temperatures are smaller at maturity than are crabs

that grow up in cooler water (Leffler, 1972). Increased metabolic

rates of juvenile blue crabs containing substantial subacute chlorinated

hydrocarbon concentrations might similarly result in a reduced molt

increment and, thus, reduced size at the terminal molt. Reduced adult

size, if it did result from high internal pesticide concentrations, as

well as increased resting energy expenditure might adversely affect

reproductive success. In hypoxic waters crabs with high metabolic

rates face insufficient 02 at higher concentrations than those that

limit crabs with lower metabolic rates. Thus, chlorinated hydrocarbons

may act synergistically with other conditions in making particular waters

unsuitable for blue crab populations.

Mirex concentrations far below acute levels inhibit the autotomy

reflex of juvenile blue crabs (Figures 10 and 11). It may be that this

inhibition is associated with a reduction of internal acetylcholine.

High acdy!choline levels (via injection) facilitate autotomy in crustaceans

while compounds (e.g. atropine) that prevent normal acetylcholine action

result in partial or complete inhibition of the reflex (Welsh and Haskin,

1939). High DDT levels reduce acetylcholine concentrations in the

cerebral cortex and striatum oF rats (Hrdina et a7., 1973). Rosenblueth

and Morison (1937) suggest that high frequency stimulation of

vertebrate nerves reduces the quantal yield of acetylcholine per

impulse. Such a mechanism has been proposed for the inhibition of the

autotomy reflex as more legs are autotomized (Welsh and Haskin, 1939).

Such increased nervous activity is caused by chlorinated hydrocarbons.

Whatever the cause of the inhibition of autotomy may be, Mirex is more

effective than DDT in producing the altered internal conditions which

result in this inhibition. In fact, DDT does not elicit the response

until acute levels are reached.

Chlorinated hydrocarbons tend to reduce the thicknesses of completed

(Stage C4) juvenile blue crab carapaces (Figure 12). Persistent

pesticides have been implicated in thinning or reduction in total bulk

of other calcified tissues. Several chlorinated hydrocarbons when

ingested by female birds result in a reduction of the shell thicknesses

of their eggs (Peakall, 1970). Immature Roman snails fed small amounts of

DDT grow thinner shells than controls (Cooke and Pollard, 1973). Chlorinated

hydrocarbons can reduce oyster shell growth (Butler, 1966). Extensive

investigations have been and are being carried out into the mechanisms of

pesticide related bird egg shell thinning. Apparently, no single

mechanism is sufficient to explain this phenomenon. The thinning

appears partly associated with enzyme inhibition and affected enzyme

production in complex integrated enzyme and hormonal systems associated

with egg laying (Peakall, 1970; Cooke, 1973). Similarly, crustacean

molting and carapace formation is a complex process involving numerous

enzymatic reactions mediated by neurosecretions and hormones (Passano,

1960). Thus, probably no single mechanism is responsible for the

reduction in carapace thicknesses of juvenile blue crabs containing

appreciable Mirex and DDT concentrations from Stage E (exuviation)

until C4 ("intermolt").

Since the mid 1960's the blue crab industry along the coasts of

the southeastern U.S.A. has experienced a sharp decline in production.

Extensive adult mortality has been observed in some areas. However,

such crab kills are not sufficient to explain the nearly 50% drop in

production between 1964 and 1968 (Mahood et aZ., 1970). My results

raise the possibility that pesticide contamination of estuarine food

chains might result in a reduction in juvenile blue crab populations

and a subsequent reduction in adult populations. It is not surprising

that juvenile blue crab kills are not observed. The estuarine

environment abounds with scavengers and predators. Carcasses are only

recovered when large animals die and wash ashore or when massive

mortality of huge populations occurs--as, for example, red tide caused

fish kills. Further, many detrimental effects of chlorinated hydro-

carbons on juvenile blue crabs would reduce the likelihood of survival

to maturity without mortality directly attributable to acute toxicity.

For example, increased excitability and lack of maximal muscular

coordination, both components of chlorinated hydrocarbon syndrome,

undoubtedly increase susceptibility to predators. DDT induced hyper-

activity in frog tadpoles greatly increases their vulnerability to

predation by newts (Cooke, 1971). The thinner carapaces characteristic

of crabs depending on chlorinated hydrocarbon contaminated food chains

might result in greater probability of attacks upon such crabs being

successful. Ability to autocomize limbs is important to young crabs.

Inhibition of this ability must have profound effects upon the proba-

bility of successfully transversing the juvenile period. Often a

limb grasped by a predator will be autotomized and the crab will

escape. A damaged appendage is a handicap. Openings in the carapace

at the injury site as well as dying or dead tissue increase the likeli-

hood of infection. A damaged leg hampers effective locomotion and

hinders both predatory ability and ability to escape predation. A

damaged limb as well may impede successful exuviation as the damaged

tissue may not easily slip out of the old carapace. Finally, inability

to autotomize damaged appendages results in an inability to regenerate

new ones. Until the old limb is removed a limb bud cannot form and

the useless leg cannot be replaced by a functional one.

The possibilities of ingested chlorinated hydrocarbon pesticides

resulting in outright poisoning, metabolic rate elevations, decreased

muscular coordination, inhibition of the autotomy reflex, and reduced

carapace thickness/width ratio leave little doubt that chlorinated hydro-

carbon compounds (specifically DDT and Mirex) are potentially disastrous

agents with respect to blue crab populations. Additionally, Mirex, at

least with respect to crustaceans (see also Lowe et al., 1971), is far

more hazardous than is DDT.


Experiments were conducted to (1) observe the regulation of

hemolymph Na+, K+, Cl-, and total osmotic concentration by juvenile

blue crabs, (2) measure nietabolic costs of osmotic regulation by

juvenile blue crabs at different salinities, (3) discover DDT and

Mirex effects on metabolic rate, osmotic and ionic regulation, ability

to autotomize damaged limbs, and carapace thicknesses of juvenile blue


1) Juvenile blue crabs at 250C hyper-osmoregulate when in media

less than 700 mOs/l and osmoconform at higher concentrations.

2) Hemolymph Na+ and C1- concentrations gradually increase with

increasing external concentrations below 700 .Os/l and increase more

rapidly at higher concentrations. Chloride is hypo-regulated between

ambient concentrations of 400 and 600 meq C1 /1 and above 700 meq C1-/1.

3) The hemolymph K+ concentration is higher than the external K

concentration at all external concentrations tested (as high as 15 meq/1)

(1410 mOs/l).

4) At 160C and external concentrations less than 700 mOs/l the

total osmotic concentration and Na+ concentration of the hemolymph are

higher than at 250C. Hemolymph C1- and K concentrations are the same

at 160C as they are at 25C.

5) The metabolic rates of juvenile blue crabs acclimated to test

concentrations between 50 to 1410 mOs/1 are not different. Abrupt

transfer from a higher acclimation salinity to a lower test salinity

results in a large increase in the metabolic rate.

6) At 02 concentrations greater than 2.0 ml 02/1 juvenile blue

crabs are metabolic regulators. This finding in conjunction with my

earlier work suggests that the metabolic response of juvenile blue crabs

to 02 concentration results in a hyperbolic curve.

7) The mean DDT concentration in juvenile blue crabs from the

Cedar Key, Florida, U.S.A., estuarine zone was 0.031 ppm. No Mirex

was detected in any sample from this area.

8) Juvenile blue crabs are sensitive to ingested Mirex and DDT.

Mirex is a much more potent stomach poison than is DDT.

9) High, subacute internal levels of Mirex (0.02-0.2 ppm) and DDT

(0.8 ppm) cause pronounced metabolic rate elevations. Possible mechanisms

behind this phenomenon are discussed.

10) The critical oxygen concentration is higher in crabs with high

internal levels of DDT and Mirex than in untreated crabs.

11) Subacute levels of DDT and Mirex do not affect osmotic and ionic

regulation by juvenile blue crabs.

12) Mirex internal concentrations 0.02 ppm and above inhibit the

ability of juvenile blue crabs to autotomize limbs. Possible mechanisms

behind this phenomenon are discussed.

13) High internal levels of Mirex during carapace formation following

a molt seem to result in decreases in carapace thicknesses relative to

carapace widths.

14) The probable impact of Mirex and DDT contamination of estuarine

food chains is discussed. I conclude that DDT and, to a far greater


extent, Mirex are potentially disastrous agents with respect to blue

crab populations.


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Charles William Leffler, II was born May 21, 1947, at Cleveland,

Ohio. In June, 1965, he graduated from Marion Harding High School,

Marion, Ohio. From 1965 until 1968, he attended DePauw University,

Greencastle, Indiana. In June, 1969, he received the degree of Bachelor

of Science with a major in biology from the University of Miami, Coral

Gables, Florida. In 1969, he enrolled in the Graduate School of the

University of Florida where he received the degree of Master of Science

with a major in zoology in 1971. Since that time he has worked toward

the degree of Doctor of Philosophy with a major in zoology. He worked

as a graduate assistant in the Zoology Department from September, 1969,

until September, 1973. He was awarded a Graduate Council Fellowship

for the 1973-1974 academic year.

Charles William Leffler, II is married and the father of one


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

F. G. Nord 1 e, Chairman
Associate Professor of Zoology

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

F. Anderson
associate Professor of Zoology

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

B. K. McNab
Professor of Zoology

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

F. J/7S. Mat fo, Jr.
Pr ssor of Zoology

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

Ariel l.ugo
Assistant Professor of Botny

This dissertation was submitted to the Grad,.ate Faculty of the Department
of Zoology, in the College of Arts and Sci-erces and to the Graduate Council,
and was accepted as partial fLifillment r, the requirements for the degree
of Doctor of Philosophy.

August, 1974

0ean, Graduate School

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