It was therefore necessary to select embryos of each species for test-
ing at a slightly later stage of development than specified in the
contract. When embryos are selected at the late gastrula - neurula
stage after melanophores become visible, nearly 100 percent of the
selected embryos hatch when maintained under suitable conditions. This
stage can be readily distinguished from unfertilized eggs, arrested em-
bryos, and other developmental stages. Therefore embryos were selected
at this stage for all tests.

Acquisition of larvae from field or hatchery operations does not present
the same problems as are inherent with the eggs, at least with regard to
M. salmoides. Our experience has been excellent in obtaining healthy
fishes from both sources. Only two minor problems were encountered and
only one of these could probably be reduced through laboratory culture
of this species. The first problem is that certain larvae from a given
spawning could be referred to as the "runts of the litter"; i.e., they
do not grow as rapidly as the majority, and therefore are subject to
heavier pressures of competition by their siblings leading to death by
starvation or cannibalism. The second problem is a correlary of the
first. Whenever possible, hatchery personnel attempt to select larvae
of the same size. In doing this larvae of different ages may be selected,
and occasionally "runts" from an older group of larvae may be selected
along with the average of a younger group because of their similar size.
In some cases it appears that these "runts", in spite of their equality
in size, are still unable to compete with the younger individuals and
may starve to death or become victims of cannibalism.

Juveniles are subject to injury during collection and transport from
field to laboratory. In addition they may be diseased when collected.
Therefore all juvenile fishes were quarantined for 24 to 48 hours prior
to experimentation. Only juveniles from collections exhibiting low mor-
tality during this period were used in experiments.

With one exception, all acute and interaction determinations were con-
ducted utilizing styrofoam fish shipping boxes manufactured by Plasti-
Kraft of Ozona, Florida and lined with a 15 x 15 x 22 inch 0.004 mil
polyethylene bag. This apparatus was selected after considerable test-
ing based on the following criteria: (1) containers should provide
adequate and uniform life support for experimental fishes without filtra—
tion for a period of 96 hours and with filtration (i.e., corner filter)
for long term exposures; (2) containers should be of convenient size for
handling, maintenance, and storage; (3) containers should have better
insulation qualities than glass aquaria; (4) containers should be rea-
dily sealed for reduced oxygen experiments; (5) containers should be
inexpensive and disposable. Few of these criteria are met by the glass
containers (glass pickle jars) routinely used in bioassay experiments
or glass aquaria. It is widely recognized that many, if not all, of the
chlorinated hydrocarbon pesticides will adhere to glass and, in some
cases, will chemically bind to the glass. The use of a polyethylene
liner in a styrofoam "frame" does not reduce chlorinated hydrocarbon
adsorption to the container surface but it does provide for a low cost,

12.


Toxaphene (Cont'd)

Penaeus duorarum Nauplius, Mysis 0, 5, 10
Protozoea 0, 0.5, 1.0, 5.0
Rhithropanopeus harrisii st 0, 10, 20, 30,
40, 50

Reduced Dissolved Oxygen Experiments

Four levels of dissolved oxygen were tested; 100% sat., 75% sat., 30%
sat., and 10% sat. These oxygen levels were produced by passing regu-
lated amounts of an air-nitrogen mixture over the culture dishes placed
in large plastic bags. A beaker of water placed in each bag served as

a measurement vessel. Oxygen levels in the measurement vessel were de-
termined daily with an oxygen electrode (Galvanic Cell Oxygen Analyser,
Precision Scientific Co.) which was calibrated by Winkler titration. Sa-
linity and temperature were kept optimal for each test species.

Toxaphene Experiments

A stock solution with a concentration of 10 ppm in distilled water was
prepared using an emulsified concentrated preparation (available from
Southern Mill Creek Products, South Miami, Florida). Specifications for
the concentrate are given in Materials and Methods, Fish. Test solutions
were prepared by adding the requisite amount of the stock solution to
medium of the optimal salinity for each test species to produce the de-
sired concentration. Concentration levels to be tested were determined
in screening tests. All tests were run at the optimal temperature for the
test species. Animals were introduced immediately after solutions were
prepared. The levels tested for each species are given in Table 10.

Glassware used for these tests was segregated from that used in all other
parts of the study. These tests were conducted in a separate independent
laboratory module to avoid any possibility of contamination.

Upon completion of the bioassay studies reported herein, a contract modi-
fication was negotiated to test the concentration of Toxaphene in simu-
lated bioassay tests without animals. Three concentrations were prepared
in the same manner as for bioassay tests in 1 liter Pyrex® culture bowls.
The larger bowl size was necessitated by the sample volume required for
analysis. A separate bowl was prepared for each sampling time. Analyses
were made 3 hr., 48 hr. and 96 hr. after preparation of the solutions.
The samples were extracted with hexane and the extract analyzed by gas
chromatographic methods.

Since the concentration of Toxaphene recovered was less than expected, a

subsequent test was conducted to develop a Toxaphene budget to account
for all Toxaphene as one concentration level. The solution was prepared

33.


CRUSTACEA

INTRODUCTION

The purpose of this research was (1) to determine the median tolerance
limit of larvae of selected decapod crustaceans for extremes of tem-
perature, salinity and reduced dissolved oxygen concentration and for
the pesticide Toxaphene, (2) to determine the interaction effect of
pairs of the above parameters at sublethal levels (hereinafter called
interaction tests), and (3) to detect histopathological effects at
acute and sublethal concentration levels, especially for Toxaphene-
exposed animals.

The species selected for study were the blue crab, Callinectes sapidus;
the mud crab, Rhithropanopeus harrisii; the drift-line crab, Sesarma
cinereum; and the pink shrimp, Penaeus duorarum. Callinectes sapidus
and P, duorarum are commercially important species with major fisheries
throughout most of their ranges along the Atlantic coast of North
America. Rhithropanopeus harrisii and S. cinereum were selected for
study for two reasons; they are both ecologically important in their
respective habitats, and their larvae are better known physiologically
than perhaps any other species.

All larval stages for each of these species have been described in the
literature. Callinectes sapidus has 7 (occasionally 8) zoeal stages and
a megalopa (Costlow and Bookhout, 1959). The four zoeal and one mega-
lopal instars of R. harrisii were described from plankton specimens by
Connolly (1925) and verified in culture by Chamberlain (1962) and Costlow,
Bookhout and Monroe (1966). Sesarma cinereum has 4 zoeal and 1 megalopal
instar in culture (Costlow and Bookhout, 1960). The naupliar, protozoeal,
and mysis stages of Penaeus duorarum were described by Dobkin (1961) from
planktonic specimens and verified by Ewald (1965) from laboratory-reared
specimens.

The development of culture techniques has made available sufficient num-
bers of larvae for studies of larval ecology and physiology. Costlow
(1967) examined the effects of temperature and salinity on development
of the megalopa of C. sapidus but was unable to perform similar studies
with the zoeal instars. Results of a complete study of the effects of
temperature and salinity on all larval stages are available for S.
cinereum (Costlow, Bookhout, and Monroe, 1960) and R. harrisii (Chamber-
Tain, 1962; Costlow, Bookhout, and Monroe, 1966). In addition, studies
have been made concerning the effect of eyestalk extirpation on these
and other species for the larval stages (Costlow, 1966, 1968). Some data
is available on free amino acids in C. sapidus and R. harrisii larvae
(Costlow and Sastry, 1966), respiration for Uca, (Vernberg and Vernberg,
1964), and osmoregulation (for Cardisoma, Rhithropanopeus, and Libinia,
(Kalber and Costlow, 1966, Costlow and Kalber, 1968) and Sesarma (Kalber
and Costlow, unpublished). No data is available in the literature on the
internal morphology-histology of larvae of any species except the hermit
crab, Pagurus annulipes (Thompson, 1903).

29.


The temperature-reduced dissolved oxygen interaction test showed no sig-
nificant interaction effect for stage I zoeae or megalopae. For stage
II zoea, percent mortality increased markedly at the highest temperature
(34 C), but there was no effect of reducing the oxygen concentration.
The difference in mortality at 34 C in the acute temperature test and
the interaction test is attributed to the fact that the larvae were de-
rived from different females in the two tests. For stages III and IV,
there was no significant synergistic effect although the mortality when
both parameters were extreme was higher than expected. In salinity-re-
duced dissolved oxygen tests there were no significant differences at
any condition for any larval stage. In stage III, IV and megalopa there
was a higher mortality when both parameters were extreme than was ex-
pected. In the megalopa there was an effect of salinity but not reduced

dissolved oxygen. The data for interaction tests are summarized in Table
12.

TABLE 11.

TLyg VALUES AT 96 HR FOR EACH STAGE OF Sesarma cinereum

Reduced _
Lower Higher Dissolved
Temperature Salinity Salinity Oxygen Toxaphene
Stage (°C) (2/00) (2/00) (% sat.) (ppb)

I 37.0 12.8 36.5 55.0 0.054
II 37.0 7.9 42.0 50.5 0.760
Ill 36.8 7.3 41.8 52.3 0.740
Iv 36.3 8.7 46.0 49.0 6.8

Meg. 37.0 $2.0 $7.5 8.4

The temperature-Toxaphene tests show a significant synergistic effect

for zoeal stages I and IV. The results for megalopae are anomalous, show-
ing better survival when Toxaphene is present than in the controls. The
difference is statistically significant at the 95% confidence level. This
test should be repeated. The salinity-Toxaphene interaction tests also
show a striking synergistic effect of high salinity and Toxaphene for
stage I, with trends suggesting synergism in stages III, IV, and megalopa.
For stage II there was no significant difference in mortality between any
conditions in the test. In reduced dissolved oxygen-Toxaphene tests there
was a significant synergistic effect in stages II and IV. The results for

38.


caused fish kills, no one has investigated the less apparent but per-
haps more significant effects of sublethal levels of pesticides on the
important early life history stages of these organisms. Similarly,
there have been few studies on interaction effects of these pesticides
and fluctuations in environmental factors for these same life history
stages. These problems form the basis of the study reported herein.

Behind the urbanized coastal regions of Florida lies a large agricul-
tural complex. The climate of Florida, particularly that of central
and southern regions, provides a twelve month growing period for many
fruit and vegetable crops. Citrus fruit is the largest agricultural
crop. Around and south of Lake Okeechobee are thousands of square miles
which are utilized for vegetable and fruit production. Florida ranks
as the third largest cattle-producing state.

It has been estimated that 25 thousand tons of pesticides are used an-
nually in Florida, mostly by agriculture, with only a small amount used
for domestic pest control. Prior to 1969, DDT was the most commonly
used insecticide for agricultural crops in central and south Florida.
During 1969 the use of DDT was restricted and farmers began using Toxa-
phene instead (James E. Brogden, Department of Entomology, University of
Florida, personal communication). Exact figures on the amounts of spe-
cific pesticide useage in Florida are not available from any source.

Toxaphene is administered to crops in the same formulations as was DDT.
One of the most popular is a mixture of this chlorinated hydrocarbon
with an organophosphate insecticide, typically Parathion. It was used
in this manner on potatoes until the registry for use of Toxaphene on
this crop was cancelled (James E. Brogden, personal communication).

This formulation remains in use on corn, beans, cabbage, and for control
of caterpillars on domestic lawns. Toxaphene is also used by itself or
with Parathion on peanuts, soybeans, southern peas, cabbage, peppers,
eggplant, tomatoes (south Florida's major vegetable crop), and early sea-
son celery. Toxaphene is the active ingredient of many livestock sprays
for beef cattle and is also used for treatment of pastures (James E. Brog-
den, personal communication).

Toxaphene, therefore, has been one of the most likely chemical pollutants
to be found in south Florida waters since 1969, although this was not
tested in this study. The U. S. Geological Survey has found DDT in south
Florida waters and soils (Benjamin McPherson, personal communication)

and this indicates that its replacement, Toxaphene, is probably now pre-
sent in these same areas. Few data are available on the effects of Toxa-
phene on aquatic animals. It is for these reasons that Aquatic Sciences,
Inc. chose Toxaphene as the insecticide to be tested.

The aquatic animals utilized in this study were chosen because (1) they
are distributed in the waters described above, (2) they are important

as commercial, sport, or forage organisms, (3) knowledge of their physi-
ology and/or life history is available in the scientific literature, and




TABLE 16.

PERCENT MORTALITY AFTER 96 HOURS IN INTERACTION TESTS.
FOR THE FIRST THREE ZOEAL STAGES OF Callinectes sapidus

Stage I
Temperature (2c) Salinit (°/00)
25 35 25 35
Reduced 1100 7.5 35.0 5.0 12.5
Dissolved
Oxygen 75 25.0 100.0 27.5 67.5
(% sat.)
Stage II
Temperature (°c) Salinity (2/00)
25 35 30 35
Reduced 100 20.0 25.0 25 35
Dissolved
Oxygen 75 27.5 77.5 30 60
(% sat.)
Stage III
Temperature (°C) Salinity (°/o0)
25 35 30 40
Reduced 100 25.0 62.5 22.5 40.0
Dissolved
Oxygen 75 25.0 } 65.0 37.5 55.0
(% sat.)

In interstage comparisons for C. sapidus, the response to salinity
differed between all four stages tested. The response to temperature
was the same for stages I and III. The response of stage IV to re-
duced dissolved oxygen was significantly different from the other stages.

48.


TABLE 14 (continued)

Reduced
Temperature Salinity Dissolved
(°c (2/00) Oxygen (% sat.)
25 35 35 45 75 100
Toxaphene | 0.0 5.0 37.5 22.5 | 25.0]} 32.5 10.0
(ppb)
1.0 27.5 97.5 22.5 | 40.0}] 87.5 15.0
Mysis
Temperature (°C) Salinity (°/00)
25 35 35 50
Reduced 75 37.5 70.0 37.5 67.5
Dissolved
Oxygen 100 35.0 37.5 32.5 42.5
(% sat.)
Reduced
Temperature Salinity Dissolved
(°c (°/00) Oxygen (% sat.)
25 35 35 50 75 100
Toxaphene } 0 15 2s 17.5 | 32.5}) 12.5 | 15.0
(ppb)
1 30 90 15.0 | 67.5}) 12.5 15.0

In interstage comparisons for P. duorarum, the responses of all stages
to all parameters differed significantly even though 96 hr Tlsq values
were quite similar. This resulted from low survival under all conditions
including optimal for the mysis stages, relative to the other stages and
differences in the shape of the mortality curves for each stage.

Acute tests for C. sapidus were completed for stages I to IV for envi-

ronmental parameters only. This resulted from problems encountered in
obtaining ovigerous females and low survival rates of early stages. Low

46.


TABLE 19.

ASSAYED TOXAPHENE CONCENTRATION IN SIMULATED BIOASSAY
TEST WITH GLASS CULTURE VESSELS

Nominal Calculated
Concentration Concentration Assayed Concentration (ppm) Days
(ppm) (ppm) 0 2 4
0.50 0.479 0.140 0.130 0.058
0.10 0.096 0.086 0.075 0.078
0.05 0.048 0.099 0.210 0.084

approximately equal to that at 3 hr, most probably by adsorption to the
glass. At 24 hr, 13 yg were present in particulate form, and 10 yg were
adsorbed to the glass. No absorbed Toxaphene was observed. The total
Toxaphene recovered was 892.1 yg or 93.1% of the amount introduced. Con-
sidering the number of assays ‘involved with their attendant errors, and
the crude nature of the procedure to estimate the particulate fraction,
this percent recover is very complete.

TABLE 20.

TOXAPHENE BUDGET AT 24 HR IN SIMULATED BIOASSAY
TEST WITH GLASS CULTURE VESSELS

Fraction PE

soluble 869.1
particulate 13.0
adsorbed 10.0
absorbed 0.0

TOTAL 892.1

ug Toxaphene introduced 958 pg
% recovered 93.1%

The lack of conformity between the measured concentration at 3 hr in the
concentration-time study and the Toxaphene budget test at a nominal con-
centration of 0.5 ppm is very puzzling. The anomalous results in the con-
centration-time study may provide an explanation since the percentage in
solution at this nominal concentration was surprisingly low. We place grea-
ter confidence in the second test because the data was repeatable in rep-
licate tests. The lack of conformity may also derive from the use of one
liter culture bowls in the concentration time study versus larger volume
glass containers with a different configuration and hence surface to volume
ratio in the budget test.

52.


tribution of these larvae. The upper salinity limit was very high at
ca 50 °/oo, far in excess of concentrations apt to be encountered in
nature.

C. sapidus migrates upstream during the juvenile period and grows to
adult size in brackish or fresh waters. When sexual maturity is reached,
it migrates in the reverse direction into regions where salinities ex-
ceed 20 °/oo where eggs hatch (Van Engel, 1958). It has previously been
demonstrated that eggs of C. sapidus do not hatch at salinities lower
than about 15 °/oo and complete development only occurs at salinities
above 20 °/oo (Sandoz and Rogers, 1944; Costlow and Bookhout, 1959).
This salinity is somewhat higher than the lower lethal limit based on

a TLso at 96 hr of 17.5 to 19.0 °/oo (and only 14.0 °/oo for stage III)
reported here. However, it was not determined as a property of the con-
tract research whether larvae which survived after 96 hours could com-
plete development to the juvenile at these low salinities. The upper
salinity limits of 37 to 42 °/oo are slightly higher than normal sea-
water and probably exceed levels ever encountered by larvae in nature.

Costlow (1967) investigated the effects of temperature and salinity on

the development of the megalopa of C. sapidus. The megalopa developed

to the juvenile over the range of 15 to 30°C at suitable salinities and
over a range of 5 to 40 °/oo at suitable temperatures. Development at

5 °/oo occurred only at 25 and 30°C and at 40 °/oo at all temperatures

tested. No data is available from this study for comparison.

R. harrisii is a brackish water crab which as an adult ranges from vir-
tually fresh water to mid salinity regions, although it is capable of
surviving in normal seawater. For stages I to IV zoea, the lower salini-
ty limit either does not exist or is well below 5 °/oo. The upper sali-
nity limit was 37 °foo or above for all zoeal instars. Costlow, Bookhout
and Monroe (1966) observed complete development for 8% of the larvae
cultured at 25°C, 40 °/oo, and 22% of the larvae cultured at 25°C,

35 Joo.

It has been reported in numerous studies that death under adverse condi-
tions is frequently associated closely with molting (see for example
Roberts, 1971a). It would seem that when the larva molts, its defenses
are lowered making it more susceptible to stress conditions. In these
tests, the same effect was noted in cases where molting occurred during
the course of the test. However, in the majority of the tests, molting
did not occur during the course of the test.

Because of the above observation, one must consider whether as deter-
minations after 96 hr are meaningful for these organisms. eh

value at 96 hr would presumably be higher than is actually the be
value, that is the animals appear more tolerant than they really are.
Analysis of this point was not required by contract. The data of Cost-
low, et al. (1960, 1962, 1966) is based on a different approach and en-
compasses this consideration; however, direct statistical comparisons of
their data with that presented here is not possible. The permissible

56.


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44,


TABLE 2.

RANGES OF TEMPERATURE, SALINITY, REDUCED DISSOLVED
OXYGEN AND CONCENTRATION LEVELS OF TOXAPHENE TESTED
FOR EACH SPECIES AND STAGE

Temperature (°c)
Micropterus salmoides embryo 25 - 39

sac-fry, juvenile 25 - 40

Mugil cephalus embryo 22 - 38
juvenile 25 - 40
Trachinotus carolinus juvenile 26 - 39
Salinity (°/00)
Micropterus salmoides all stages 0 - 20
Mugil cephalus embryo 0 - 65
juvenile 0 - 68
Trachinotus carolinus juvenile 0 - 60
Reduced Dissolved Oxygen (%)
Micropterus salmoides embryo 10 - 100
sac-fry 30 - 100
Mugil cephalus embryo 30 - 100
Trachinotus carolinus juvenile 30 - 100
Toxaphene (ppm)
Micropterus salmoides embryo 0.005 - 5.00
sac-fry 0.010 - 0.10
juvenile 0.010 - 0.25
Mugil cephalus embryo 0.005 - 0.50
juvenile 0.005 - 0.50
Trachinotus carolinus juvenile 0.010 - 0.25

16.


in the usual manner. The container was a larger volume glass vessel to
preclude possible errors in preparation of replicate solutions. Sam-
ples were taken in the same manner as described in the previous section
concerning tests on fish species.

Interaction Tests

Five combinations of factors were tested: temperature-reduced dissolved
oxygen, salinity-reduced dissolved oxygen, temperature-Toxaphene,
salinity-Toxaphene, and reduced dissolved oxygen-Toxaphene. Conditions
were established and maintained in the same manner as described above
for the acute tolerance tests.

Levels for each parameter were selected to be sublethal on the basis of
the acute tolerance tests, ranging from the optimal level to just below
the 96 hr TLsg level. Each experiment was conducted at two (in a few
cases three) levels of each parameter with all possible combinations.

In most tests there were two replicates of 20 animals each or a total of
40 animals at each test level. In a few tests, four replicates of 10
animals each were used. In tests with Penaeus duorarum, some experi-
ments involved four replicates of 20 animals each for a total of 80
animals.

Specimens for histopathological study were preserved in 7% formalin in
tap water with calcium carbonate buffer. The sections were stained with
eosin-hematoxylin, a general stain. Selected slides were then examined
for histopathological abnormalities. Control animals served as a basis
for morphology.

Data Analysis

TLs5g values after 96 hr. were determined in the acute tolerance tests by
the graphical method of Doudoroff, et al. (1951, Anon., 1960) with the
data for replicates pooled.

The data for rgplicates within each experiment were tested for homogene-
ity with the 2? test. The data for replicates was then pooled and the
differences between test conditions evaluated by x2 tests. The data

for control conditions is the best available data concerning the survi-
val rates one can expect under optimal conditions for these animals

which have a natural mortality greater than zero under natural conditions.

The mortality rates of all stages of each species were compared with a

2 test. Interspecific comparisons were not attempted because the num-
bers of stages of the several species varied. If this had been attempted
the number of comparisons would have been astronomical.

The results of each interaction test was subjected to a 2-way classifi-

cation analysis of variance test to define significant interaction ef-
fects. This test determines whether there is a statistically signifi-

34.


ACKNOWLEDGEMENTS

Aquatic Sciences, Inc. gratefully acknowledges the following for valu-
able assistance in obtaining experimental animals for use in this pro-
ject: John Anderson, U. S. Bureau of Sport Fisheries and Wildlife

fish hatchery, Welaka, Florida; William Ashe, U. S. Bureau of Sport
Fisheries and Wildlife fish hatchery, Frankfort, Kentucky; C. Yancey
Byrd, III, Delray Beach, Florida; Roger M. Cooper, U. S. Bureau of

Sport Fisheries and Wildlife fish hatchery, Yankton, South Dakota;
Sheldon Deal, Riviera Beach, Florida; J. Walter Dineen, Central and Sou-
thern Florida Flood Control District, West Palm Beach, Florida; Peter
Dingler, Miami, Florida; Fisher Wholesale Seafoods, Port Canaveral, Flo-
rida; 0. Earl Frye, Jr., Director, Florida Game and Fresh Water Fish
Commission, Tallahassee, Florida; Maj. Brantley Goodson, Chief of Law
Enforcement, Florida Game and Fresh Water Fish Commission, Tallahassee,
Florida; Robert M. Ingle, Florida Department of Natural Resources, Tal-
lahassee, Florida; Marilyn Metzger, Port Salerno, Florida; Lt. Morgan,
Titusville, Florida; Lt. Purdon, West Palm Beach, Florida; Vernon C.
Ogilvie, Florida Game and Fresh Water Fish Commission, West Palm Beach,
Florida; Larry Schafer, St. Georges Packing House, Ft. Myers Beach, Flo-
rida; Herman Summerlin, Ft. Pierce, Florida; David Sweat, Sea Farms Inc.,
Key West, Florida; John W. Woods, Chief of Fisheries, Florida Game and
Fresh Water Fish Commission, Tallahassee, Florida. Robert Zensen of the
Central and Southern Florida Flood Control District, West Palm Beach,
Florida provided a permit and levee key allowing us to have access to
water conservation areas for the purpose of collecting animals. The fol-
lowing people are acknowledged for their assistance in conducting ex-
periments with fish: Karen Ellerie, Martin Hyatt, Edward Mistarka, Jackie
Schroeder, Brad Stirn, Ron Weiss. The studies with decapod larvae were
carried out through the assistance of: Margaret Cutts, Maura Haran, Gail
Holland, Linda Kapsch, Mary Maynard, Dianne Rocheleau, Peggy Van Arman,
Margaret van Montfrans, Mark Widmer, Cathy Wood. The collection of many
specimens and responsibility for life support by Kent Norris, Buddy Payne,
and Robert Rutter are gratefully acknowledged. Phyllis Graglia and Joel
Van Arman are acknowledged for preparation of animals for histological
examination. Teen Kline and Jeff Peterson assisted with photography.
Mike Vaillant prepared the final drawings for this report and was respon-
sible for routine chemical analysis. Dr. Al Smith kindly examined slides
of decapod larvae for histopathological changes. Dr. Richard G. Domey
gave advice on statistical questions and performed the statistical tests.

65.


ABSTRACT

Laboratory studies were conducted to determine lethal limits (96 hr TL.,)
for Toxaphene, salinity, temperature, and dissolved oxygen and their in-
teraction effects on developmental stages of selected warm-temperate and
subtropical fishes and crustaceans. Species tested were Micropterus
salmoides (largemouth bass), Mugil cephalus (striped mullet), Mugil cu-
rema (silver mullet), Trachinotus carolinus (pompano), Callinectes sapi-
dus (blue crab), Penaeus duorarum (pink shrimp), Sesarma cinereum (drift
line crab), and Rhithropanopeus harrisii (mud crab). Histopathological
and gross morphological studies were conducted on all early life history
stages of the species included.

Earliest developmental stages of the fish species treated are more resis-
tant to high levels of salinity, and to low levels of dissolved oxygen,
but more sensitive to high temperatures than are later stages. Decapod
larvae showed increasing tolerance to Toxaphene with increasing develop-
mental age. Synergistic effects between Toxaphene and the three environ-
mental factors were suggested in the species tested. Some histopathology
was noted in fry of bass and mullet, and in larvae of S. cinereum, C. sapi-
dus, and R. harrisii.

This report was submitted in fulfillment of Project Number 18080DLR, Con-
tract 14-12-532, under the sponsorship of the Office of Research and Moni-
toring (formerly Water Quality Office), Environmental Protection Agency.

In requesting information about this report from Aquatic Sciences, Inc.
refer to the identification number ASI 216-02.

iii


LITERATURE CITED (continued)

Costlow, J. D., Jr., and F. A. Kalber. 1968. Osmoregulation in lar-
vae of the land crab, Cardisoma guanhumi Latreille. Amer. Zool., 8:
411-416. ~

Costlow, J. D., Jr., and A. N. Sastry. 1966. Free amino acids in
developing stages of two crabs, Callinectes sapidus Rathbun and Rhith-
ropanopeus harrisii (Gould). Acta Embryo. Morph. Exp., 9:44-55.

Cowart, C. A. 1971. Comparative development of the feeding mechanism
in three species of western Atlantic mullet (Pisces: Mugilidae).
Master's thesis, Florida Atlantic University. 73 pp.

Dobkin, S. 1961. Early developmental stages of pink shrimp Penaeus
duorarum from Florida waters. Fish. Bull., 61:321-396.

Doudoroff, P., B. G. Anderson, G. E. Burbick, P. S. Galtsoff, W. B.
Hart, R. Patrick, E. R. Strong, E. W. Surber, and W. M. Van Horn.
1951. Bioassay methods for the evaluation of acute toxicity of indus-
trial wastes to fish. Sewage Industr. Wastes, 23:1380-1397.

Egler, F. E. 1964. Pesticides in our ecosystem: Communication II.
BioScience, 14:29-36.

Ewald, J. J. 1965. The laboratory rearing of pink shrimp, Penaeus
duorarum Burkenroad. Bull. Mar. Sci., 15:436-449.

Hansen, D. J. 1969. Avoidance of pesticides by untrained sheepshead
minnows. Trans. Amer. Fish. Soc., 98:426-429.

Harder, W. 1952a. Einrige Gerate zur Herstellung von Wasserschichten
verschiedener Dichte. Kurze Mitt. Fischbiol. Abt. Max-Planck-Inst.
Meeresbiol. Wilhelms, 1:21-27.

Harder, W. 1952b. Uber das Verhalten von Zooplankton in geschichtetem
Wasser. Kurze Mitt. Fischbiol. Abt. Max-Planck-Inst. Meeresbiol.
Wilhelms, 1:28-43.

Holden, A. V. 1966. Organochlorine insecticide residues in salmonid
fish. J. Appl. Ecol., 3(Suppl.):45-53.

Johnson, D. W. 1968. Pesticides and fishes--A review of selected litera-
ture. Trans. Amer. Fish. Soc., 97(4):398-424.

Kalber, F. A. and J. D. Costlow, Jr. 1966. The ontogeny of osmoregula-
tion and its neurosecretory control in the decapod crustacean, Rhithro-
panopeus harrisii (Gould). Amer. Zool., 6:221-229.

68.


LITERATURE CITED

Anonymous, 1960. Standard methods for the examination of water and
wastewater. 11th Edition. American Public Health Association, Inc.,
New York, 626 pp.

Anonymous, 1970. Summary of Florida commercial marine landings--1969.
Tallahassee, Florida Department of Natural Resources.

Butler, P. A. 1966. The problem of pesticides in estuaries. In: A
symposium on estuarine fisheries, ed. by R. F. Smith, A. H. Swartz,
and W. H. Massmann. Amer. Fish. Soc. Spec. Publ.. 3:110-115.

Chamberlain, N. A. 1962. Ecological studies of the larval develop-
ment of Rhithropanopeus harrisii (Xanthidae, Brachyura). Chesapeake
Bay Institute, John Hopkins Univ., Tech. Rep., 28:1-47

Connolly, G. J. 1925. The larval stages and megalopa of Rhithropan-
opeus harrisii (Gould). Cont. Can. Biol., 2:327-334.

Costlow, J. D., Jr. 1966. The effect of eyestalk extirpation on lar-
val development of the mud crab, Rhithropanopeus harrisii (Gould).
Gen. Comp. Endocrin., 7:255-274.

Costlow, J. D., Jr. 1967. The effect of salinity and temperature on
survival and metamorphosis of megalops of the blue crab, Callinectes
sapidus Rathbun. Helgol. Wiss. Meeresuntersuch, 15:84-97.

Costlow, J. D., Jr. 1968. Metamorphosis in crustaceans. P. 3-41.
In: W. Etkin and L. I. Gilbert (ed.) Metamorphosis: A problem in
developmental biology. Appleton-Century-Crofts, New York.

Costlow, J. D., Jr. and C. G. Bookhout. 1959. The larval development
of Callinectes sapidus Rathbun reared in the laboratory. Biol. Bull.,
116:373-396.

Costlow, J. D., Jr. and C. G. Bookhout. 1960. The complete larval
development of Sesarma cinereum (Bosc) reared in the laboratory. Biol.
Bull., 118:203-214.

Costlow, J. D., Jr., C. G. Bookhout and R. Monroe. 1960. The effect
of salinity and temperature on larval development of Sesarma cinereum
(Bosc) reared in the laboratory. Biol. Bull., 118:183-202.

Costlow, J. D., Jr., C. G. Bookhout and R. Monroe. 1966. Studies on
the larval development of the crab, Rhithropanopeus harrisii (Gould)
I. The effect of salinity and temperature on larval development.
Physiol. Zool., 39:81-100.

67.


Whittmore, C. M., C. E. Warren and P. Douderoff. 1960. Avoidance
reactions of salmonid and centrarchid fishes to low oxygen concen-
trations. Trans. Amer. Fish. Soc., 89:17-26.

Williams, A. B. 1965. Marine decapod crustaceans of the Carolinas.
Fish. Bull., 65:1-298.

70.


DISCUSSION

Mugil cephalus juveniles were most tolerant of elevated salinity (TL., =

62 °/oo) followed by Trachinotus carolinus juveniles (Tls5g = 50.3 0788.
Juveniles of all those species tested tolerated fresh water (TL., = 0.0 °/oo).

Micropterus salmoides showed decreasing tolerance of elevated salinity with

increasing developmental age (TL,, = 20 °/oo for embryos, 15.8 °/oo for
larvae and 12.5 °/oo for juveniles).

The results of temperature tests are somewhat different. None of the ju-
veniles used in tests were acclimated to laboratory temperatures of 25°C
for more than 48 hours before these fishes entered temperature tests. The
results for juveniles are in rather close agreement (96 hr TLcg for M.
salmoides = 37.2°C; for M. cephalus = 36.5°C; and for T. carolinus = 36.2°C).
This coincides well with results for upper temperature limits of many other
warm acclimated organisms (Prosser, 1961). Larvae of M. salmoides are not
as resistant to higher temperatures (96 hr TL5y = 33.5°C) and bass embryos
are even less tolerant (96 hr TLso = 31.5°C). Therefore, temperature tol-
erance increases with development from embryos to juveniles. This is highly
significant with respect to injection of thermal effluents into areas in
which fishes spawn, i.e., estuaries and bays.

Juveniles of M. salmoides and M. cephalus and larval M. salmoides showed
close agreement in dissolved oxygen tests with 96 hr TL.,'s of 47.5%, 49%,
and 48% saturation respectively. Embryos of MicropteruS were most resis-
tant to low levels of oxygen with a 96 hr TL., of 15.3% saturation. Juven-
ile T. carolinus were quite resistant to reduced oxygen with a 96 hr They

of 18% saturation. It was noted that their activity during these tests
slowed as oxygen content of the water was lowered. The gills of juvenile
T. carolinus are probably more efficient in removing oxygen from water than
are those of the other species, perhaps representing an adaptation to pe-
lagic life.

The results of the chemical assay tests indicate that Toxaphene is rapidly
removed from the test solution by the container. After as little as 3
hours only 13% is in solution and after 48 hours only a trace. This means
that all 96 hr TLeg's were grossly overestimated because virtually no Toxa-
phene was present during the majority of the test. The rapidity of re-
moval precludes calculation of valid Tle estimates for any time interval.

Interaction between sublethal levels of paired test factors was suggested

in many if not all combinations tested. This conclusion is based on the

fact that mortality for the combined action of variables at levels less

than that required to produce 50% mortality (i.e. the 96 hr TL.,) was great-
° * 2 . 0

er than 50% and in several cases was 100%. However the incomplete factor-

ial design used precludes valid statistical treatment.

27.


disposable liners which is destroyed after each test with Toxaphene.
Each apparatus contained twenty liters of water. Water circulation
and oxygen saturation were maintained with a single 1 inch air stone
in a corner of the unit.

The experimental apparatus utilized for juvenile T. carolinus was dif-
ferent than that described above. Two containers were tested with T.
carolinus: (1) styrofoam fish shipping boxes with plastic liners con-
taining 20 liters of water and (2) large cylindrical polyethylene ves-
sels with plastic liners containing 80 liters of water. Survival rates
were examined in these containers with and without filtration for 5, 10,
15, 20, and 25 fish per tank and several different feeding regimes in-
cluding live adult Artemia and Tetramin® flake food. Juvenile pompano
prefer live adult Artemia although they readily accept Tetramin®. They
feed selectively on the latter diet, rejecting the green flakes alto-
gether. Water samples were tested for pH and ammonia content at the end
of 96 hours (Table 1). Based on these tests, the large plastic lined
polyethylene container without filtration, but with an airlift device for
aeration and water circulation, was selected.

TABLE 1.

WATER QUALITY DATA FOR TWO EXPERIMENTS DESIGNED TO SELECT CULTURE
CONDITIONS FOR TESTS INVOLVING JUVENILE Trachinotus carolinus

A - 20 liters volume
July 2 July 3 July 6

Tank Food Filtration Animals/Tank pH NH, pH NHz pH  NHz
1 Tetramin® with 25 7.1 9.37 7.3 14.3 7.8 30.3
2 Artemia with 2s 7.3 7.40 7.5 10.6 7.8 20.5
3 Tetramin® without 25 7.3 9.37 7.4 12.1 --- ----
4 Artemia without 25 7.5 6.25 7.6 10.5 7.9 19.8
S Tetramin® without 0 7.1 0.75 7.8 1.94 7.9 8.1

B - 20 liter versus 80 liter
13 April 19 April

Tank Volume _Animals/Tank Filtration pH NH, pH NH,
1 80 25 without 7.6 1.62 7.4 2.42
2 80 20 without 8.0 1.21 7.4 1.54
3 80 15 without 7.9 0.90 7.5 0.82
4 80 10 without 7.8 0.59 7.4 0.57
5 80 5 without 7.9 0.45 7.3 0.53
6 80 25 with 7.9 0.57 7.5 0.38
7 80 20 with 7.9 0.49 7.4 0.45
8 20 15 without 7.6 6.25 7.2 9.37
9 20 10 without 7.7 2.21 7.6 6.25

10 20 15 with 7.7 6.70 7.3 7.85

ant 20 10 with 7.7 5.16 7.6 8.10

13.


RESEARCH RESULTS

All three life history stages of Micropterus salmoides were included
in acute and interaction exposures. The 96 hr temperature Ths for all
stages of M. salmoides ranged from 31.5°C for embryos to 37.2 iy for ju-
veniles. The 96 hr salinity TLo, for embryos was not established at
levels up to 20 °/oo but was 1528 °/oo for larvae and 12.5 °/oo for ju-
veniles. The majority of embryos tested at 20 °/oo salinity were de-
formed at hatching. The 96 hr reduced dissolved oxygen Ths ranged
from 15.3% for embryos to 47.5% saturation for juveniles. ese data
are provided in Table 3.

TABLE 3.
96 hr TL5gq VALUES FOR EACH STAGE OF EACH FISH SPECIES
Reduced

Lower Higher Dissolved
Temperature Salinity Salinity Oxygen

Species Stage (OC) (foo) _(°/oo) _(% sat.)
icropterus | embryos 31.5 Does not | 20.0 15.3
salmoides | exist

sac-fry 33.5 0.0 15.8 48.0
juveniles | 37.2 0.0 12.5 47.5
|

lugil embryos | 26.4 - - -
cephalus :

juveniles : 36.5 0.0 62.0 49.0
i :

[frachinotus | juveniles , 36.2 0.0 50.3 18.0

carolinus | i |
i i !

The temperature-reduced dissolved oxygen interaction test was conducted
on larval M. salmoides. Highest mortality occurred at a temperature of
33°C (Table 4). An interaction test between salinity and reduced dis-
solved oxygen was conducted for both larvae and juveniles. Larvae ex-
perienced total mortality at a salinity level of 12 °/oo when oxygen con-
centration was 75% saturation (Table 4).

Because of problems encountered in acquiring larval Mugil cephalus and in

obtaining sufficient numbers of embryos, only one test was conducted on
embryos. All tests required for juvenile M. cephalus were completed.

19.


exclude bias from this source because it did not insure complete randomi-
zation.

Temperature Experiments

In temperature tests, temperature was controlled by thermoregulated hea-
ters. Over the range of temperature to be tested, experimental units were
maintained at 5°C increments. Salinity was maintained constant and oxygen
at saturation. The range tested for each species is given in Table 2.

Salinity Experiments

In salinity experiments, tests were conducted at 5 700 increments over
the selected range for each species. Water of the desired salinity was
prepared by dilution of artificial seawater (Instant Ocean Synthetic Sea
Salts®, Aquarium Systems, Inc.) with tap water. If salinities higher than
the stock solution were required, Instant Ocean Synthetic Sea Salts® were
added. Salinity was measured with a hydrometer to +0.5 °/oo. Temperature
was kept constant at 23-24°C, oxygen at saturation. Test ranges are given
in Table 2.

Reduced Dissolved Oxygen Experiments

Four levels of dissolved oxygen were tested, 10, 30, 75, and 100% satura-
tion. The oxygen dilution systems to produce reduced dissolved oxygen
levels consisted of 1 quart mixing bottles with separate air and nitrogen
inflows monitored by manifold valves and flowmeters. Air from the mixing
bottles was then diffused directly into the water in experimental tanks
through 1 inch airstones. Water in experimental tanks was maintained free
from contact with oxygen-saturated air by closing the plastic liners and
providing an air vent. Oxygen concentration was monitored with a Galvanic
Cell Oxygen Analyzer (Precision Scientific) and Winkler titrations.

Toxaphene Experiments

Toxaphene used in both the fish and crustacean portions of this project
was obtained in two forms from Southern Mill Creek Products Company, Inc.,
of Tampa, Florida. The first form, used only in preliminary screening
experiments, has the trade name of Toxaphene EM-6 and contains 60% Toxa-
phene (chlorine content 67-69%), 35% xylene-range aromatic hydrocarbon
solvent, and 5% inert ingredients. This is called 6 pound per gallon Toxa-
phene.

The second form, which is presently utilized in all acute and interaction
tests, Toxaphene EM-8, contains 71.6% Toxaphene (chlorine content 67-69%),
23% xylene, and 5.4% inert ingredients, and is called 8 pound per gallon
Toxaphene. Only one lot was used throughout this research. No analyses
for Toxaphene were required under the contract. The methods for prepara-
tion of stock and experimental solutions developed by Mahdi (1966) were
followed. Test concentrations for each test are summarized in Table 2.

15.


If synergistic interactions do occur between environmental parameters

as suggested here, then 96 hr Tlsg values provide only a guideline to the
lethal and sublethal levels for the given set of exposure conditions
tested and cannot be applied to situations where environmental levels

are more or less stressful. Single factors rarely play a dominant role
in nature. Therefore bioassay tests should include realistic levels of
both natural and man-made parameters in multiple factor interaction de-
signs to measure the credibility of results when applied to the natural
environment.

Most tests for each species were performed with specimens from a single
geographic location and all specimens in any single test were from a
common source. In subsequent studies, it should be determined whether
there are differences in tolerance to test parameters for animals de-
rived from several geographic locations.

28.


because of the relative differences in developmental complexity and
organogenesis. These stages are less able to move from one area to
another during periods of environmental stresses than are their parent
stocks.

The early life history stages of all four species share several further
important considerations for inclusion in this study: (1) they are
particularly critical stages in development, (2) at least one or more

of these stages occur in areas where interaction effects between environ-
mental factor fluctuation and man-induced environmental pollutants, such
as Toxaphene, can occur (3) methods for laboratory culture of these fishes
for use in research have been and continue to be developed and improved
at Aquatic Sciences, Inc., and (4) all four species are of great economic
value in Florida.

10.


LITERATURE CITED (Continued)

Mahdi, M. A. 1966. Mortality of some species of fish to Toxaphene at
three temperatures. Res. Publ., 11, U. S. Fish Wild Serv., 10 pp.

Nicholson, H. P., A. R. Grzenda, G. J. Lauer, W. S. Cox, and J. I.
Teasley. 1964. Water pollution by insecticides in an agricultural
river basin. I. Occurrence of insecticides in river and treated

municipal water. Limnol. Oceanogr., 9:310-317.

Prosser, C. L. 1971. Temperature. p. 238-284. In: Prosser, C. L.
and F. A. Brown, Jr., Comparative Animal Physiology. 2nd Edition, W.
B. Saunders Co., Philadelphia.

Roberts, M. H., Jr. 197la. Larval development of Pagurus longicarpus
Say reared in the laboratory. II. Effects of reduced salinity on lar-
val development. Biol. Bull., 140:104-116.

Roberts, M. H., Jr. 197ib. Larval development of Pagurus longicarpus
Say reared in the laboratory. III. Behavioral responses to salinity
discontinuities. Biol. Bull., 140:489-501.

Sandoz, M. and R. Rogers. 1944. The effect of environmental factors
on hatching, moulting, and survival of the zoeae larvae of the blue
crab, Callinectes sapidus Rathbun. Ecology, 25:216-228.

Scarratt, D. J. and G. E. Raine. 1967. Avoidance of low salinity by
newly hatched lobster larvae. J. Fish. Res. Bd. Canada, 24:1403-1406.

Sprague, J. B. 1964. Avoidance of copper-zinc solutions by young
salmon in the laboratory. J. Water Pollut. Contr. Fed., 36:990-1004.

Tanner, H. A., and M. L. Hayes. 1955. Evaluation of Toxaphene as a
fish poison. Colorado Coop. Fish. Res. Unit, Quart. Rep., 1:31-39.

Thompson, M. T. 1903. The metamorphosis of the hermit crab. Proc.
Boston Soc. Nat. Hist., 31:147-209.

Thorhaug, A., T. Devany and B. Murphy. 1970. Refining shrimp culture
methods: The effect of temperature on early stages of the commercial
pink shrimp. Gulf Carib. Fish. Inst. Proc., 23:125-132.

VanEngel, W. A. 1958. The blue crab and its fishery in Chesapeake
Bay. Part I. Reproduction, early development, growth and migration.
Comm. Fish. Rev., 20:6-17.

Vernberg, F. J. and W. B. Vernberg. 1964. Metabolic adaptation of
animals from different latitudes. Helgol. Wiss. Meeresunters., 9:476-487.

69.


necrotic spaces. Four of these specimens exhibited intestinal contrac-
tion, one a slight dilation, and five no apparent effect. In stage III,
all specimens exhibited extensive degeneration of the hepatopancreas
and 80% showed contraction of the intestine.

Stage I C. sapidus larvae showed no histological changes compared to
controls at either 5 x 10°* or 1 x 10° ppb Toxaphene. In stage II, he-
patopancreas damage similar to that found in S. cinereum was noted in
50% of the specimens. The exposure concentration for stage II was 1 x
10°2 ppb, several orders of magnitude higher than the exposure level for
stage I.

The same histological effects were observed in stage I, II, and IVR.
harrisii larvae. In stage I, one specimen also exhibited necrosis of
the intestine and sloughing of the stomach epithelium. Stages II and
IV exhibited damage to the nervous tissue with irregular spaces apparent
between the fibers.

In all three species, deposition of orange-brown pigment has been noted
in body spaces, especially along the intestine. This pigment deposition
is not observed in control animals. The pigment is not soluble in wa-
ter, alcohol, or xylene.

No histological changes were observed in other organs of the zoeae.

There was no evidence of bacterial or fungal invasion in the Toxaphene
exposed animals.

54.




TABLE 9 (continued)

Number of
Animals
Species Stage Test Condition Level Examined
Toxaphene
(14 day) 0.033 ppm 2
Trachinotus juvenile Temperature 24°C 4
carolinus
Toxaphene 0.03 ppm 2
Toxaphene 0.25 ppm 6
Toxaphene 0.0035 ppm 2
(14 day)
Toxaphene 0.017 ppm 2
(14 day)
Toxaphene 0.033 ppm 2
(14 day, in food)
Toxaphene/temperature 0.01 ppm/35°C 4
Toxaphene/ reduced 0.1 ppm/30% 4
dissolved oxygen
Toxaphene/salinity 0.1 ppm/48 °/oo 2

No morphological or histological effects were observed in specimens ex-
posed to other experimental variables. Further, no histopathological ef-
fects were observed in T. carolinus exposed to any experimental variable.

25.


GENERAL DISCUSSION

The results of temperature tolerance tests for juvenile fishes and
all decapod larvae were in close agreement (96 hr Tls9 = 35 to 37°C).
As noted above, this coincides with values for numerous other warm
acclimated organisms (Prosser, 1961). In contrast, bass larvae were
Slightly less resistant (96 hr Tlcog = 33.5°C) and bass embryos still
less tolerant (96 hr Tlsg = 31-5°C)-

The heated effluent from fossil fuel plants frequently reaches lev-

els of 32-33°C (and sometimes 39-40°C) near the outfall, and dissi-
pates slowly with distance from the outfall. This temperature is
within the tolerance range of the species tested (at least for temporary
exposures) except for bass eggs and larvae. Effluents closer in
temperature to the lethal limit can be expected to cause considerable
mortality.

Harder (1952a, 1952b) demonstrated that species in several phyla in-
cluding Arthropoda (decapods) and Chordata (fishes) are capable of
responding to gradients of environmental parameters by avoidance.
Most of his work related to avoidance of salinity gradients, but he
did conduct a few experiments with temperature gradients. It is
likely that species used in the present tests have this capability
although no studies of this potentiality were required under the
present contract. Such avoidance of waters of adverse thermal lev-
els would serve to prevent high mortalities but at the same time
would restrict the volume of water available to the species for sup-
port of its populations.

The salinity range tolerated by all species corresponds to the salin-
ity ranges within their respective distribution ranges or, in some
cases, Slightly exceeds the latter range. This latter observation can
be explained in several ways. First, it may reflect the failure to
consider the most sensitive periods for the stages tested as noted in
each of the proceeding sections. Second, it may result from the ina-
bility of the organisms to compete with other animals when living in
waters with salinities approaching the lethal limits. Third, it has
been demonstrated that some decapod larvae avoid water with a salinity
within the range acceptable for complete development but approaching
the lethal limit (Scarratt and Raine, 1967; Roberts, 197lb). It is
reasonable to expect the same thing to be true for species tested
under this contract.

While oxygen availability is a limiting factor only in local areas
and then rarely, it may become limiting in regions of primary and
secondary pollution where BOD and COD are high. Based on the studies
reported here, concentrations below 50 percent saturation at optimal
temperature and salinity are lethal for decapod larvae, larval and

61.


Although the plastic-lined vessels used to maintain fish have been de-
monstrated to have life support characteristics as good as or better

than glass, they are not suitable for bioassay tests involving pesticides.
Plastics rapidly ad- and absorb organic pesticides. Our tests suggest
that 92% is sorbed within 24 hours and virtually 100% within 48 hours.

As has already been pointed out the The gy values obtained for fish are
gross overestimates and hence not reported.

In many interaction tests, synergistic effects between Toxaphene and
all environmental parameters were suggested by the data for crustaceans
but in only a few cases were these effects statistically demonstrable.
The failure to show statistical significance for decapod larvae is at-
tributable to several factors: choice of test levels, number of repli-
cates, number of test levels. More attention should be paid to statis-
tical requirements to demonstrate synergism in experimental design.

63.




Toxaphene Concentrations in Bioassay Tests

No results of tests involving Toxaphene are reported because the method-
ology used was subsequently found to be inadequate for these tests. The
use of plastic for test containers was shown to result in virtually com-
plete removal of Toxaphene from the system. Therefore TL5g values were

grossly overestimated and of no value. .

The results of the analysis of Toxaphene over time in simulated bioassay
tests are presented in Table 7. In both fresh and salt water, the con-
centration at 3 hr. after solution preparation was in all but one case
less than 13% of the calculated concentration. After 48 hours only tra-
ces of Toxaphene were measured in all but one case.

TABLE 7.

ASSAYED TOXAPHENE CONCENTRATION IN SIMULATED BIOASSAY
TEST WITH PLASTIC CULTURE VESSELS

Nominal Calculated
Water Concentration Concentration Assayed Concentration (ppm) on
Type (ppm) (ppm) 0 Day 4
Salt Water 0.50 0.479 0.062 < 0.001 £0.001
30 °/oo 0.10 0.096 0.037 < 0.001 < 0.001
0.05 0.048 0.0056 < 0.001 40.001
Fresh Water 0.5 0.479 0.062 ¢ 0.013 60.001
0.05 0.048 0.001 < 0.001 40.001

In a separate experiment we attempted to account for all Toxaphene in-
troduced. This test was terminated at 24 hours and estimates were made
of the Toxaphene in solution, in solid form, adsorbed to the plastic

and absorbed to the plastic (Table 8). There was a Toxaphene concentra-
tion in solution of 0.0274 ppm at 24 hours. The total Toxaphene in solu-
tion was 132.7 pg. There was 24.1 ge in the distilled water (particulate
fraction). The plastic accounted for most of the Toxaphene with 280.3 yg
adsorbed and 319.0 pg absorbed. The total amount of Toxaphene accounted
for was 756.1 pg or 39.5% of the amount introduced. The Toxaphene unac-
counted for is believed to be associated with the residual undissolved
but liquified plastic after decanting the hexane fraction from the ex-
tract to determine the absorbed fraction; i.e., the absorbed fraction was
underestimated.

22.




TABLE

10.

11.

12.

13,

14.

LIST OF TABLES

TITLE

WATER QUALITY DATA FOR TWO EXPERIMENTS DESIGNED TO
SELECT CULTURE CONDITIONS FOR TESTS INVOLVING JUVEN-
ILE Trachinotus carolinus

RANGES OF TEMPERATURE, SALINITY, REDUCED DISSOLVED
OXYGEN AND CONCENTRATION LEVELS OF TOXAPHENE TESTED
FOR EACH SPECIES AND STAGE

96 hr TLgg VALUES FOR EACH STAGE OF EACH FISH SPECIES

PERCENT MORTALITY AFTER 96 HOURS IN INTERACTION TESTS
FOR Micropterus salmoides

PERCENT MORTALITY AFTER 96 HOURS IN INTERACTION TESTS
FOR Mugil curema

PERCENT MORTALITY AFTER 96 HOURS IN INTERACTION TESTS
FOR Trachinotus carolinus

ASSAYED TOXAPHENE CONCENTRATION IN SIMULATED BIO-
ASSAY TEST WITH PLASTIC CULTURE VESSELS

TOXAPHENE BUDGET AT 24 HR IN SIMULATED BIOASSAY TEST
WITH PLASTIC CULTURE VESSELS

SUMMARY OF FISH SPECIMENS EXAMINED FOR HISTOPATHOLO-
GICAL STUDY

RANGES OF TEMPERATURE AND SALINITY AND CONCENTRATION
LEVELS OF TOXAPHENE TESTED FOR EACH SPECIES AND STAGE

TL5g VALUES AT 96 HR FOR EACH STAGE OF Sesarma ciner-
eum

PERCENT MORTALITY AFTER 96 HOURS IN INTERACTION TESTS
FOR Sesarma cinereum

TL59 VALUES AT 96 HR FOR THE LARVAL STAGES OF Penaeus

duorarum

PERCENT MORTALITY AFTER 96 HOURS IN INTERACTION TESTS
FOR Penaeus duorarum

PAGE

13.

16.

20.

21.

21.

22.

23.

24.

32.

38.

40.

43.

45.


Section

I

II

IIl

IV

VI

VII

VIII

Ix

CONTENTS

CONCLUSIONS
RECOMMENDATIONS
INTRODUCTION

FISHES

INTRODUCTION
MATERIALS AND METHODS
RESEARCH RESULTS
DISCUSSION
CRUSTACEANS
INTRODUCTION
MATERIALS AND METHODS
RESEARCH RESULTS
DISCUSSION

GENERAL DISCUSSION
ACKNOWLEDGEMENTS
REFERENCES

GLOSSARY OF TERMS

iv

11.
19.
27.
29.
29.
31.
37.
SS.
61.
65.
67.

71.


MATERIALS AND METHODS

Micropterus salmoides eggs are demersal and were collected attached
to rocks from Conservation Area II, Broward County, Florida. Eggs at
the late gastrula - neurula stage marked by the first appearance of
melanophores were selected for testing. Other eggs were allowed to
develop and hatch to produce the sac-fry used in testing. Some ad-
ditional sac-fry were obtained from the Federal Fish Hatchery, Welaka,
Florida. Juveniles were obtained from two sources; Federal Fish
Hatchery, Welaka, Florida, and Florida Game and Freshwater Fish Com-
mission Hatchery, Richloam, Florida. Additional juveniles used only
for screening tests were derived from Federal Fish Hatcheries in
Georgia, West Virginia and South Dakota.

Adult Mugil cephalus were collected by cast net at Boynton Beach Inlet,
Palm Beach County, Florida. These adults were then spawned in the
laboratory to produce a limited number of eggs. Juvenile M. cephalus
were collected by seining in Indian River, St. Lucie County, Florida
and Tampa Bay, Hillsborough County, Florida.

Mugil curema juveniles were collected from the same locations as M.
cephalus, the majority at Indian River, Florida.

Trachinotus carolinus juveniles were collected at Cocoa Beach, Brevard
County with limited numbers being derived from Daytona Beach, Volusia
County, Florida and St. Augustine, St. Johns County, Florida.

Aquatic Sciences, Inc. has conducted considerable supportive research
directed toward manipulation of unripe fishes into prespawning condition,
spawning and rearing. This is the only procedure available for obtain-
ing adequate numbers of eggs and larvae of M. cephalus, M. curema, and
T. carolinus for experimentation.

The original proposal (ASI Document Number 103-02) and the Schedule for
Contract 14-12-532 both specify that tests with eggs will begin at the
onset of gastrulation (embryonic shield to primitive streak). Several
problems were encountered in attempting to test eggs at this stage.
First, there is great variability in the number of unfertilized or dead
eggs either collected from the field or produced in Federal hatcheries
or our own laboratory. For M. salmoides, from less than 1% to 60% of
the eggs in a nest were dead or unfertilized. These eggs must be re-
moved prior to testing. Second, embryos are encountered which, regard-
less of source, terminate development because of processes not fully
understood. Arrested development usually occurs during early cleavage
to blastula stages and to a lesser extent in later stages. The propor-
tion of eggs in a given batch which exhibit this phenomenon is highly
variable. These eggs cannot be identified and removed at the early
gastrula stage. These biological anomalies could lead to large errors
in experimental results.

11.


10.

CONCLUSIONS

Synergistic effects were suggested between temperature-reduced
dissolved oxygen and salinity-reduced dissolved oxygen for eggs,
larvae and juveniles of fish and in some cases for larvae of
decapod crustaceans but were not statistically demonstrable.

Synergistic effects have been statistically demonstrated between
Toxaphene and all three environmental parameters for some stages
of decapod species studied. In other cases, the data suggests
synergistic effects.

The earliest stages (embryos) in the life history of fishes were
most tolerant to extremes of salinity. Sac-fry larvae were more
tolerant to this factor than juveniles.

Decapod larvae became more tolerant to Toxaphene at later develop-
mental stages than at earlier stages. In some cases a 10-fold
increase in tolerance was noted from one stage to the next.

Decapod larvae became increasingly tolerant of salinity extremes
in advanced stages but the increase was not as striking as the
increased tolerance of Toxaphene.

Sac-fry of the striped mullet showed enlargement of cells and in-
creased numbers of cells in glandular tissue in the head region
when exposed to high concentrations of Toxaphene.

Sac-fry of largemouth bass exposed to extreme salinities exhibited
deformation of the yolk sac and the notochord. The latter caused
a humpbacked condition.

Larvae of S. cinereum, C. sapidus, and R. harrisii exposed to ex-
treme concentrations of Toxaphene (<96 hr TLs5q) showed destruction
of the hepatopancreas and constriction of the intestine.

Plastic containers should not be used in pesticide bioassays because
of the rate and degree of sorption by the plastic. This phenomenon
led to gross overestimates of 96 hr TLsQ values for all fish studies
in these tests.

Glass containers also exhibit some degree of sorption to pesticide
so that the 96 hr Tlsq is overestimated (10 to 25%). Chemical assays
for the pesticide should be made during all bioassay tests.




Upon completion of the Toxaphene bioassay tests, a contract modification
was negotiated to determine the concentration of Toxaphene in simlated
bioassay tests without animals. Three concentrations were prepared in
the same way as for bioassay tests in the polyethylene-lined styrofoam
boxes. Samples were removed for analysis at 3 hr., 48 hr., and 96 hr.
after solution preparation. The samples were then extracted with hexane,
and analyzed by gas chromatographic methods.

Since the recovery of Toxaphene was very low, a subsequent experiment
was conducted to develop a Toxaphene budget for the system at a single
concentration. The solution was prepared at 0.5 ppm in the usual manner.
A sample of the solution was taken at 3 hr. and 24 hr., extracted with
hexane, and analyzed. The remaining solution was then drawn off care-
fully so that any precipitate would not be removed. The container was
then rinsed with distilled water to remove particulate Toxaphene. The
wash was extracted with hexane and analyzed. The container was then
washed briefly with hexane to remove adsorbed Toxaphene and analyzed.
That portion of the bag exposed to the solution was then cut into small
pieces and extracted with hexane for twenty-four hours to remove absorbed
Toxaphene. The hexane partially dissolved the plastic but this did not
interfere with the gas chromatograph analysis. It was not possible to re-
extract the plastic; therefore the completeness of recovery of absorbed
Toxaphene is not known.

Interaction Tests

Five combinations of factors were tested: temperature-reduced dissolved
oxygen, salinity-reduced dissolved oxygen, temperature-Toxaphene, salin-
ity-Toxaphene, and reduced dissolved oxygen-Toxaphene. Conditions were
established and maintained as described above for each parameter.

Levels to be tested were selected to be sublethal based on the results
of the acute tolerance tests, ranging from an optimal level to just be-
low the 96 hr TLeg level.

In acute and interaction tests involving eggs and sac-fry, there were 4
replicates at each test level or a total of about 100 individuals at each
test level. For juveniles there were 2 replicates at each test level or
approximately 50 individuals.

96 hr TLs5g values were determined by the graphic method of Douderoff et
al. (1951, Anon, 1965) with the data for replicates pooled.

Specimens from each test series were preserved for examination of gross
morphology and histopathology. Two hundred ninety-two specimens from
tests involving Toxaphene were sectioned at 6 » and stained with hema-
toxylin-eosin for histological study of gill, pharyngeal region, digestive
tract, liver, kidney, and epithelial tissues to determine if Toxaphene af-
fects these tissues.

17.


EPA-R3-73-035
April 1973 Ecological Research Series

Environmental Effects on

Toxaphene Toxicity to
Selected Fishes and Crustaceans

TED S74
Ss "&s

2
% 3

be,
YY “S
74 prove?

MV AGENCY

Office of Research and Monitoring

U.S. Environmental Protection Agency
Washington, D.C. 20460


megalopae are anomalous. In this case mortality was significantly grea-
ter at 100% saturation than 75% saturation regardless of Toxaphene con-
centration which suggests an antagonistic effect. Further, mortality
was significantly higher when Toxaphene was absent than at a Toxaphene
concentration of 5 ppb. This result may be due to an error in data col-
lection md the experiment should be repeated. The data for all inter-
action tests are summarized in Table 12.

TABLE 12.

PERCENT MORTALITY AFTER 96 HOURS IN INTERACTION
TESTS FOR Sesarma cinereum

Stage I
Temperature (°C) Salinity (°/00)
25 31 34 25 | 31 | 34
Reduced 75 |i 1.25 | 6.75 | 6.25 2.50 | 6.25 | 8.75
Dissolved : H i H
Oxygen hoo |! 3.75 | 6.75 2.50 |} 2.50 | 5.00 | 5.00
(% sat.) ‘ | |
: i |
Reduced
Temperature Salinity Dissolved
_ (°C) (2/00) Oxygen (% sat.)
25 35 25 34 75 | 100
|
Toxaphene 0.0 I 0.0 | 10.0 10.0 | 27.5 Oo; 0
i i i
(ppb) 0.05} 0.0 | 62.5 |! 17.5 |100.0]} 51 0
jo Oo

Stage II
IW, Temperature °c) Salinity (°/o0)
25 31 34 30 in
q
Reduced 75 25.0 16.25 | 38.75 5.0 117.5
Dissolved |
Oxygen 100 20.75 {| 13.75 © 35.00 10.0 ! 20.0
(% sat.) :

40.




RECOMMENDATIONS

Continued research is recommended on these species to elucidate
the effects of Toxaphene and environmental parameters on stages
not covered in this report.

It is believed that the 96 hr TLcog is higher than the TL 0 ulti-
mate. In the case of fishes, sensitivity seems to be related to
development of gills, not included in the test period. In the case
of crustaceans, the most sensitive phase in development is the molt
between stages. The contract did not call for study at these points
in the life history. Further study is recommended to elucidate these
points.

Since significant synergistic effects of Toxaphene with environmen-
tal parameters were noted, consideration must be given to the envi-
ronmental parameters of receiving waters in determining maximum per-
missible concentrations of Toxaphene.

Exposure to extreme conditions has a marked effect on behavior and
feeding of both fish and decapods. Further study is recommended to
elucidate the role of starvation as a possible cause of death.

The synergistic effect between Toxaphene and salinity suggests that
Toxaphene interferes with osmoregulation and perhaps ionic regula-
tion. Studies are recommended to examine this hypothesis.


TABLE 21.

SUMMARY OF CRUSTACEAN SPECIMENS
EXAMINED FOR HISTOPATHOLOGICAL STUDY

Number of Number of Toxaphene
Control Animals Experimental Concentration
Species Stage Examined Animals Examined (ppb)
Sesarma I 3 2 0.03
cinereun
2 0.04
Il 6 10 0.04
IIt 6 5 0.04
Callinectes I 3 1 0.0005
sapidus
1 0.00001
II 7 6 0.01
Rhithropanopeus I 8 3 10.0
harrisii
II 7 2 100.0
2 200.0
IIl 10 2 400.0
Iv 10 3 200.0

Histopathology

Table 21 specifies the species, stage, number of animals, and exposure
levels for those slides examined. In stage I of S. cinereum, the he-
patopancreas had vacuolated granulated cells in one specimen coupled
with contraction of the intestine. The other specimens showed no no-
ticeable effects. In stage II, 20% of the specimens showed no effects,
50% had vacuolated granulated hepatopancreas cells, and 30% exhibited

53.




TABLE 9.

SUMMARY OF FISH SPECIMENS EXAMINED
FOR HISTOPATHOLOGICAL STUDY

Number of
Animals
Species Stage Test Condition Level Examined
Micropterus larva Toxaphene 0.000 ppm 6
salmoides
Toxaphene 0.001 ppm 2
Toxaphene 0.1 ppm 2
Toxaphene/temperature 0.05 ppm/33°C 3
Toxaphene/salinity 0.01 ppm/9 °/oo 4
0.05 ppm/9 °/oo 6
0.05 ppm/12 °/oo 9
Toxaphene/reduced
dissolved oxygen 0.01 ppm/75% 3
0.05 ppm/75% 3
juvenile Toxaphene 0.00 ppm 3
Toxaphene/temperature 0.01 ppm/35°C 2
0.05 ppm/35°C 4
oO
Toxaphene/salinity 0.05 ppm/12 /oo 4
Toxaphene/reduced
dissolved oxygen 0.5 ppm/75% 5
Mugil Toxaphene
curema juvenile (14 day) 0.033 ppm 2
Mugil egg Toxaphene 0.00 ppm 13
cephalus
Toxaphene 0.5 ppm 15
juvenile Salinity 0.00 ppm 2

24.


Toxaphene

(ppb)

Reduced
Dissolved
Oxygen

% sat.)

Toxaphene

(ppb)

TABLE 12

(continued)

| Reduced
Temperature Salinity Dissolved
(2C) (2/00) Oxygen (% sat.)
/ \ 25 | 35 | 30 40 75 100
0.0 2.5 ‘ o0.5 | 7.5 0.0 20.0 2.5
Ss | 15-9 70.0 | 20.0 |15.0 87.5 | 22.5
Stage IIT
Temperature (°c) Salinity (9/00)
25 35 30 | 40
75 25.0 77.5 10.0 57.5
100 10.0 30.0 7.5 | 25.7
i
| i
x Temperature Salinity Reduced Dissolved
(Pc) H Chee} Oxygen (% sat.)
25 | 35 | 30 40 | 75 | 100
| 0.0 2.50 | 40.00 | 2 2.5 | 32. 5) 27.5 22.5
‘0.5 2.50 | 67.75 | 22.5 4.0! 32.8 | 10.0
aoe it

Al.



TABLE 8.

TOXAPHENE BUDGET AT 24 HR IN SIMULATED BIOASSAY
TEST WITH PLASTIC CULTURE VESSELS

Fraction 2g
soluble 132.7
particulate 24.1
adsorbed 280.3
absorbed* 319.0
TOTAL 756.1

pg Toxaphene introduced 1916 yg
% recovered 39.5%

*extraction not complete

Histopathology

The species, stage, test condition, and number of specimens sectioned is
summarized in Table 9. Morphological anomalies were noted among embryos
of Micropterus salmoides and Mugil cephalus exposed to test situations.
The majority of the larvae of M. salmoides which hatched from eggs exposed
to 20 °/oo salinity were deformed in four ways: (1) curvature of the no-
tochord causing a "humpback" condition; (2) shortened trunk region in se-
veral fry apparently resulting from lack of several embryonic somites; (3)
distortion in the shape of the yolk sac in some fry; and (4) forward dis-
placement of the yolk sac in others. As these anomalies did not occur

in other newly-hatched fry of the same age from the same test series ex-
posed to lower levels of salinity nor in those of the control series, these
anatomical deformations are attributed to the higher salinity.

Histopathological effects were found in the head region of Mugil cephalus
embryos exposed in a preliminary Toxaphene 96 hr TLs5g test. The maximum
Toxaphene concentration was 0.5 ppm in this series. Glandular structures
in the optic region of those embryos exposed to the highest concentrations
of Toxaphene were larger and more numerous than those exposed to lower
concentrations and in the control series. These glandular structures ap-
pear to be mucous glands and their anomalous condition may be due to epi-
thelial irritation by Toxaphene.

Histopathological effects were also noted in larvae of Micropterus sal-
moides. Necrosis was found in kidney tissues and the lining of the diges-
tive tract of larvae which had been exposed for a period of 14 days to

10% of the 96 hr TLsg for Toxaphene administered in the food. Necrosis of
kidney tissues was severe with near total destruction of the kidney tubules.
There was no significant destruction of other organs detected.

23.




TABLE

15.

16.

17.

18.

19.

20.

21.

LIST OF TABLE (continued)

TITLE

TL., VALUES AT 96 HR FOR THE FIRST FOUR ZOEAL
sTRCES OF Callinectes sapidus

PERCENT MORTALITY AFTER 96 HOURS IN INTERACTION
TESTS FOR THE FIRST THREE ZOEAL STAGES OF Callinec-

tes sapidus

TL5q VALUES AT 96 HR FOR EACH STAGE OF Rhithropano-
peus harrisii

PERCENT MORTALITY AFTER 96 HOURS IN AN INTERACTION
TEST FOR Rhithropanopeus harrisii

ASSAYED TOXAPHENE CONCENTRATION IN SIMULATED BIO-
ASSAY TEST WITH GLASS CULTURE VESSELS

TOXAPHENE BUDGET AT 24 HR IN SIMULATED BIOASSAY TEST
WITH GLASS CULTURE VESSELS

SUMMARY OF CRUSTACEAN SPECIMENS EXAMINED FOR HISTO-
PATHOLOGICAL STUDY

vi

PAGE
47.

48.

49.

$1.

52.

82.

$3.


Fertilized eggs and sac-fry were placed in specially designed "protec-
tors" during exposure for ease of handling, examination, feeding, and
removal of dead individuals. The "protectors" consist of cylindrical
baskets of fine mesh netting supported by cylindrical PVC frames and
floated from a styrofoam collar. The collar is inexpensive and dispos-
able.

In standard bioassay tests, experimental animals are not fed during ex-
posure. Food deprivation is suggested to reduce the number of deaths
resulting from breakdown products of non-ingested food materials and
from increases in metabolites from experimental animals (Anon. 1960).
While these factors may be real problems in bioassays, food deprivation
is an unnatural situation and in itself becomes a stress factor. Fur-
thermore, larval and juvenile fishes of the sizes used in this project
cannot be maintained for 96 hours without feeding.

Therefore, all experimental animals were fed according to the following
procedure: three times daily food was added until the fish ceased feed-
ing, usually 15 to 20 minutes. Excess food was then removed. Microp-
terus salmoides larvae were fed Artemia nauplii and juveniles were fed
Tive Daphnia, M. cephalus and M. curema were fed live Cyclops, and T.
carolinus were fed live adult Artemia (when live Artemia was not avail-
able, either frozen Artemia or, rarely, Tetramin® was substituted).
Chemical monitoring of the water at the end of each experiment rarely
showed significant metabolite concentrations.

Twenty-five fish in each "protector" is the optimal number for adequate
survival. There are many problems associated with counting very small,
live, active fishes and consequently several individuals fewer or more
than the desired number were frequently introduced into "protectors".
In addition some cannibalism was observed. Cannibalism occurred at a
variable rate which could not be precisely determined in every case.

Because of potential counting errors in placing fish in the "protectors"
and variable amounts of cannibalism during tests, the initial n (number
of experimental animals) is taken to be the number of dead recorded and
preserved at each observation time plus the number of animals alive at
the termination of an experiment. The number dead was recorded at the
end of each 24 hour period in the test. Thus, if only 23 fish can be
accounted for in a given "protector" at the end of the experiment (total
dead and live), percent mortality is based on this number. This proce-
dure excludes the effect of cannibalism on the mortality data. The fact
that the cannibalistic individual(s) may survive an exposure better than
others because of this ready source of food is, however, masked.

The procedures followed here effectively cancel out mortality resulting
from predation both by other animals and by cannibalism which together
probably represent the major cause of death in natural populations. This
still leaves the "natural mortality syndrome" of inexplicable deaths of
animals maintained under "optimal" conditions. The procedure for appor-
tioning animals in the several conditions of each test did not completely

14.


ficantly better at 34 700, 100% saturation than at any other salinity
and at the same salinity in the acute test for salinity.
TABLE 18.

PERCENT MORTALITY AFTER 96 HOURS IN AN
INTERACTION TEST FOR Rhithropanopeus harrisii

Salinity (°/oo)

25 31 34
Reduced 75 10.00 7.50 11.25
Dissolved
Oxygen 100 6.25 3.75 0.00
(% sat.)

Some preliminary screening tests were completed in addition to the above
tests. A preliminary test of the effect of salinity on stage IV zoeae
showed that the lower salinity 96 hr TL5g does not exist. The salinity
range tested did not include the upper salinity 96 hr TL., which exceeds
35 °/oo. Two preliminary tests of Toxaphene on stage II°zoea give 96 hr
TL5q values of 288 ppb and 345 ppb. These tests are summarized in Table
17.

In interstage comparisons for R. harrisii, temperature is the only pa-
rameter for which a complete series of comparisons is possible. There
was no significant difference in the response to temperature for any
stage.

The data for the analysis of Toxaphene over time in simulated bioassay
tests are presented in Table 19, The concentration at 3 hr was approxi-
mately 29.2% of the calculated value at a nominal concentration of 0.5
ppm and 89.6% at 0.1 ppm. In the other concentration (0.05 ppm) the as-~
sayed concentration exceeded the expected by about 100%. The reason for
this aberrant result is not apparent.

In a separate experiment we determined the distribution of Toxaphene

in the test system after 24 hr at a concentration of 0.50 ppm. The data

is presented in Table 20. At 3 hr the Toxaphene concentration was 0.359

ppm, and at 24 hr it was 0.475 ppm. This increase in concentration at

24 hr was noted in several duplicate experiments to the one reported here.
The explanation seems to be that the Toxaphene went into solution slowly.
The previous test indicates that the concentration decreased to a level

51.


Subject Mold & Group

SELICTED ‘. SCURCES ADSTRACTS
INPUT TRANSACTION FORM

i

5 Aquatic Sciences, Inc.
2624 N.W. 2nd Avenue
Boca Raton, Florida 33432

{Title
S45 Environmental Effects on Toxaphene Toxicity to Selected
Fishes and Crustaceans

| Project Beaegnation
EPA, WQO Contract 14-12-532 18080DLR
Noto

1G | Auchor(ay
-—— Courtenay, Walter R., Jr.
Roberts, Morris H., Jr.

GD} Chtatioa

|

Environmental Protection Agency report
number, EPA-R3-73-035, April 1973.

2g | Desctiptors (Starred Pies)

*Pesticide Toxicity, *Chlorinated Hydrocarbon, *Salinity, *Temperature,
“Dissolved Oxygen, Bass, Mullets, Crabs, Pink Shrimp, Pathology of Pollutants,
Larvae, Juvenile Fishes, Florida

2 51 Tdentifiors (Starred First) i 5 . . .
2) *Toxaphene, *Interaction, *Synergism, Micropterus salmoides, Mugil cephalus,
Mugil curema, Trachinotus carolinus, Callinectes sapidus, Penaeus duorarum,
esarma cinereum, Rhithropanopeus harrisii, Zoea, Megalopa, Mysis, Protozoea, Naup]ius

97 | Abstract
27,

Laboratory studies were conducted to determine lethal limits (96 hr TL, ) for Toxaphene,
salinity, temperature, and dissolved oxygen and their interaction effeees on develop-
mental stages of selected warm-temperate and subtropical fishes and crustaceans. Spe-
cies tested were Micropterus salmoides (largemouth bass), Mugil cephalus (striped mul-
let), Mugil curema (silver mullet), Trachinotus carolinus (pompano), Callinectes sapi-
dus (blue crab), Penaeus duorarum (pink shrimp), Sesarma cinereum (drift line crab),
and Rhithropanopeus harrisii (mud crab). Histopathological and gross morphological
studies were conducted on all early life history stages of the species included.

Earliest developmental stages of the fish species treated are more resistant to high
levels of salinity, and to low levels of dissolved oxygen, but more sensitive to high
temperatures than are later stages. Decapod larvae showed increasing tolerance to
Toxaphene with increasing developmental age. Synergistic effects between Toxaphene and
the three environmental factors were suggested in the species tested. Some histopath-
Ology was noted in fry of bass and mullet, and in larvae of S. cinereum, C. sapidus,
and R. harrisii.

Abstactor af institution 5 7
Walter R. Courtenay, Jz. Aquatic Sciences, Inc
ar i F MENT, TO: WATER RESO CIENTIFIC INFORMATION CENTER
Tan TREY Suny 19037 SEND, WITH COPY OF DOCUMENT, Tor HATER ReOURCES SCENT IEIG NEO

WASHINGTON, D.C, 20240
GPor 1970 - 407-201


No tests were conducted on the interaction effects of temperature and
salinity. Costlow, Bookhout and Monroe (1960, 1966) have shown that
there are such effects for all stages of S. cinereum and R. harrisii.
Costlow (1967) reported the same types of effects for the megalopa of
C. sapidus.

The present results indicating a synergistic interaction between two
test parameters could reflect one of three processes. The simultaneous
action of two parameters may accelerate the rate at which the response
is achieved with no change in the final degree of response (ultimate
lethal dose). Alternatively, the simultaneous action of two parameters
may lead to a change in the ultimate lethal dose which exceeds the sum
of independent responses to each parameter with no change in the time to
achieve the ultimate lethal dose. Finally, both rate and ultimate le-
thal dose may be affected. These alternatives cannot be distinguished
from the present tests because the ultimate lethal dose cannot be es-
timated from the data. It is doubtful that the TL., equals the ultimate
lethal dose, especially since the most sensitive period (ecdysis) in
the larval period generally does not occur within the test period.

The evidence presented here indicates that in establishing permissible
levels of Toxaphene in natural environments, consideration must be given
to the effects of environmental factors such as temperature, salinity,
and oxygen concentration, especially at the extremes of the range toler-
ated by the species which will be exposed to the pesticide. On the
basis of available data and the refractory nature of C. sapidus larvae
in culture, one would expect this species to be least tolerant of these
tested with significant synergistic effects.

The analyses of Toxaphene concentration indicate that 75 to 90% of the
Toxaphene was in solution (excluding suspect assays). The concentration
of Toxaphene in solution increased from 3 hr to 24 hr after solution
preparation and then decreased slowly until the end of the test. This
would indicate that TLsy estimates may be 10 to 25% higher than the
actual TLeg's. Correction of the TL.. values is not warranted as the
basis of the available analysis data?» These results point out the neces-
sity of frequent chemical analyses with every bioassay test on the actual
test solutions.

The only histological changes observed in preliminary studies for Toxa-
phene-exposed S. cinereum and C. sapidus were a contraction of the intes-
tine and progressive vacuolation and necrosis of the hepatopancreas.
These conditions may develop more rapidly in older than younger zoeae;
however, an inadequate number of larvae have been examined to be certain
on this point. The same conditions were observed with R. harrisii. In
addition, R. harrisii showed some deterioration of the nervous system.

During exposure tests, it was observed that animals in high Toxaphene

concentrations became disoriented, swam slower, and frequently flexed
and extended the telson. This telson flip response is a common response

58.


MATERIALS AND METHODS

Ovigerous females of each brachyuran species were collected locally in
south Florida and placed in aquaria until the eggs hatched. Sesarma

cinereum were collected at Boca Raton, Palm Beach County, Florida, C.

sapidus from the St. Lucie Estuary, Martin County, and R. harrisii from
drainage ponds on the University of Miami Campus, Dade County, Florida.
The females were maintained at 25°C, and a salinity comparable to that
of their natural habitat (C. sapidus, 30 °/oo; R. harrisii, 25 °/oo; S Ss
cinereum, 25 °/oo). Hatching occurred within a week after placing in
hatching tanks. The adults were not fed during incubation of the eggs.

Larvae were maintained in the laboratory under standardized conditions
until the desired stage was reached. Callinectes sapidus larvae were
kept at 25 °/oo or, recently, 30 °/oo in 20.3 cm finger bowls and fed

sea urchin blastulae-gastrulae and Artemia nauplii. In some recent ex-
periments, Dunaliella salina was provided as food for zoea stage I and

II. Sesarma cinereum larvae were maintained at 25°C, 25 °/oo, R. harrisii
at 250C, 25 ©7oo. Both species were fed Artemia nauplii. The shrimp, P
duorarum, larvae were purchased at the desired stage (nauplius, protozoea,
or mysis) from Seafarms, Inc., Key West, Florida. They were fed with the
motile chlorophytan Dunaliella salina in the protozoeal stages and with
Artemia nauplii during the mysis stage. The nauplii are non-feeding
stages.

All experiments were conducted in 11.4 cm finger bowls. Bowls were ex-
amined daily for deaths and molts. The food organisms were provided as
needed to maintain adequate food concentrations.

Temperature Experiments

In experiments with temperature as the experimental variable, the cultures
were maintained in BOD boxes. Over the range of temperature to be tested,
cultures were maintained at 5°C increments. Salinity was kept constant

at the culture salinity used for each species. The range tested for each
species is given in Table 10.

Salinity Experiments

In salinity experiments, tests were conducted at 5 °/oo intervals over
the selected range for each species. Water of the desired salinities
was prepared by dilution of artificial seawater (Instant Ocean Synthetic
Sea Salts®, Aquarium Systems, Inc.) with distilled water. The stock so-
lution had a salinity of 35 °/oo. To prepare medium with a higher sa-
linity than stock, Instant Ocean Synthetic Sea Salts® were added to the
stock solution. Salinity was measured with a hydrometer to +0.5 °/oo.
Temperature was kept constant at 25°C. The test ranges for each species
are given in Table 10. Both upper and lower salinity 96 hr TLsog values
were determined.

31.


GLOSSARY
acclimation: the compensatory change in an organism under maintained
deviation of a single environmental factor.

acclimatization: the compensatory changes in an organism undergoing
multiple natural deviations of milieu.

antagonism: the interaction effect of two (or more) toxicants which
is less than the sum of the effect of each acting separately.

blastula: that embryonic stage containing a cavity, the blastocoel
with an associated layer of cells, the blastoderm.

brackish water: referring to waters of a salinity between 0 and 10 °/oo.

epithelium: any sheet of cells which covers and lines free body sur-
faces, internal or external.

eury-: prefix meaning wide as in eurytolerant.

gastrula: that embryonic stage consisting of two tissue layers,
ectoderm and entoderm.

gravid: refers to female animals possessing ripe eggs.

hepatopancreas: the organ of the digestive system of crabs producing
digestive enzymes; also called "liver".

histopathology: the study of tissues and disease.

interaction effect: effects produced by two factors acting on an
organism together (see antagonism and synergism).

juvenile: that stage in the life history when an animal has adult
morphology but is not sexually mature.

larva (e): that stage in the life history transitional from egg to
adult. It is an embryonic stage. In present usage, it refers
only to free-living forms.

megalopa (e): the last larval stage of brachyuran and anomuran deca-
pods possessing larval and juvenile characteristics.

melanophore: pigment-bearing and pigment-producing cells having the
black pigment melanin.

metabolite: any of a variety of biologically active compounds produced
by enzymatic reactions.

71.


TABLE 10

RANGES OF TEMPERATURE AND SALINITY AND CONCENTRATION
LEVELS OF TOXAPHENE TESTED FOR EACH SPECIES AND STAGE

Temperature (°c)
Sesarma cinereum all stages 25 - 40
Penaeus duorarum all stages 25 - 40
Callinectes sapidus SI- Iv 25 - 40
Rhithropanopeus harrisii all stages 25 - 40

Salinity (°/00)
Sesarma cinereum sl 10 - 40

S II, § III 5 - 45
SIV 5 - SO
Meg 0 - SS
Penaeus duorarum Nauplius 20 - 50
Protozoea 15 - 50
Mysis 20 - 5S
Callinectes sapidus SI, SII 15 - 40
S Il 10 - 45
s Iv 15 - 45
Rhithropanopeus harrisii sI 0 - 40
S II, $ III 0 - 45
Toxaphene (ppb)
Sesarma cinereum SI 0, 0.4, 0.5, 0.6
S II 0, 0.01, 0.05, 0.1,
0.5, 1.0
S III, S IV 0, 0.5, 1.0, 5.0, 10.0
Meg 0, 5, 10

32.


survival rates of early larval stages are believed to be representative
of survival rates in nature and not solely a reflection of inadequacies
of culture technique.

The 96 hr temperature Tle, was 35.7 to 37°C for the first four zoeal
stages of C. sapidus. There was no significant difference in mortality
from 25 to 35 C except in Stage III. The lower salinity TLog at 96 hr
was 17.5 to 19.0 °/oo for stages I, II, and IV, but only 14. 58 ©/oo for
stage III. This latter value is lower than expected. The upper salinity
TLso at 96 hr ranged from 37.0 °/oo for stage I and II zoeae to 41 to 42
°/oo for stage III and IV zoeae respectively. There was no significant
difference in mortality from 20 to 35 °/oo for stage I and II, from 20
to 40 °/oo for stage III and from slightly above 30 to 40 2/00 for stage
IV. The 96 hr reduced dissolved oxygen 96 hr TLsg was 56.0 to 60.5% sa-
turation for all stages tested. There was no significant difference in
mortality between 75 and 100% saturation except in stage I. The 96 hr
TLsg values are summarized in Table 15.

TABLE 15.

VALUES AT 96 HR FOR THE FIRST FOUR ZOEAL

TL
50 STAGES OF Callinectes sapidus

Reduced
Lower Higher Dissolved
Temperature Salinity Salinity Oxygen
Stage (°C) (2/00) (P/o0) (% sat.)
I 37.0 17.5 37.0 56.0
Il 36.0 18.3 37.0 60.5
Til 35.7 14.0 41.0 59.0
IV 36.3 19.0 42.0 60.5

Interaction tests were completed for stage I through III zoea (exclud-
ing pairs involving Toxaphene). For stage I and II zoeae there was a
synergistic effect between elevated temperature and reduced oxygen le-
vels. For stage III, there was an effect noted only for temperature.
In salinity-reduced dissolved oxygen tests, there was a synergistic ef-
fect in stage I but only an effect of salinity in stage III. The data
for interaction tests are summarized in Table 16.

47.


TABLE 4.

PERCENT MORTALITY AFTER 96 HOURS IN INTERACTION
TESTS FOR Micropterus salmoides

Sac-fry Larvae

Temperature (°C) Salinity (°/oo)
t -
é i t
4 22-24 | 30 | 33 0: 9 12
Reduced. i {
Dissolved| 7s * 13.6 } 88.0 | 40.0 | 100.0
Oxygen i :
100 ]] 8.3 ' 17.8 |
(% sat.) 1 '
{ i
Juveniles
Salinity (°/o0)
0 9 12
Reduced 75 40.0 58.0
Dissolve
Oxygen 100 87.0
(% sat.)

* - not tested

The 96 hr temperature TL,, for M. cephalus was 26.4°C for embryos and
36.5°C for juveniles. A 36 hr lower salinity TLg was not observed for
juveniles but a 96 hr upper salinity TL, occurred at 62 foo. The 96
hr reduced dissolved oxygen They for juveniles was established at 49%

saturation. These results are’ Summarized in Table 3.

No interaction tests or long term exposures were conducted with M. ce-

phalus.

Juveniles of Mugil curema were utilized in interaction and long-term expo-
sures. Highest mortalities were experienced in test series with 36 C in
temperature-reduced dissolved oxygen exposures, with 60% salinity in sa-
linity-dissolved oxygen tests (Table 5).

20.


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50.


of decapod larvae to stressful conditions (Roberts, 1971b) and not a
specific response to Toxaphene. There was no histological evidence of
damage to the nervous system except in R. harrisii.

The evidence presently available suggests that the site of action of
Toxaphene is the digestive system, in particular the hepatopancreas.

It is desirable to examine additional specimens exposed for long periods
to lower concentrations or specimens exposed to high concentrations for
shorter times. In this way it would be possible to follow the course of
hepatopancreas degeneration.

59.


EPA Review Notice

This report has been reviewed by the Environmental
Protection Agency and approved for publication.
Approval does not signify that the contents neces-
sarily reflect the views and policies of the
Environmental Protection Agency, nor does mention
of trade names or commercial products constitute
endorsement or recommendation for use.

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39


RESEARCH RESULTS

There is considerable variability in the number of deaths per day under
given conditions. This variability is infrequently significant at the
95% level when tested by %2-tests except in Penaeus. In most cases where
significant differences were obtained, these differences were signifi-
cant on one day only, indicating differences in timing of death rather
than different responses. This reflects the problems of infrequent obser-
vations (relative to the duration of the test) and small sample size. In
the case of Penaeus, under extreme conditions in certain tests, nauplii
and protozoea showed significant differences in mortality between repli-
cates that cannot be discounted as a product of observation procedure and
sample size. In summary, there are only a few cases where replicates dif-
fered in response to a given set of conditions and these may be examples
of the 1 in 20 chance that a difference will occur within a single popu-
lation. Therefore, the data for replicates has been pooled before analysis
for differences in mortality between different test conditions.

The 96 hr temperature TLsq for all stages of S. cinereum ranged from 36.3
to 37°C. For stage II and Meg there was no significant difference between
the mortality rate at 25 and 35 C although the rate of mortality was al-
ways higher at 35 C than at 25 C. The lower salinity TL5g for 96 hr ranged
from 7.3 - 12.8 °/oo for all zoeal stages and was less than 1 °/oo for the
megalopa, while the upper salinity TLcg for 96 hr ranged from 36.5 to 52.0
°/oo (including megalopa). There was no consistent trend in lower salinity
96 hr TL. but the upper salinity 96 hr TLs increased during development
from stage I zoea to megalopa. The range oe salinity over which no signi-
ficant difference in mortality occurred was 15 to 35 °/oo for zoea I, 10
to 40 °/oo for zoea II and III and 5 to 50 °/oo for the megalopa. For stage
IV all comparisons were significant, but the range of acceptable salinities
for high survival is 10 to 40 S700. Thus while there was no uniform trend
in 96 hr TLyg with advancing development, there was an increase in the op-
timal salinity range. The 96 hr TLsg for reduced dissolved oxygen concen-
tration ranged from 49.0 to 57.5% saturation. These values are of low pre-
cision because the test interval (30% sat. to 75% sat.) was rather broad.
There was no significant difference between the mortality rates at 75 and
100% saturation except in stage II. At 30% saturation mortality was total
after 24 hours in stages I and IV, after 48 hours in stage III, after 96
hours for the megalopa. Only two zoeae survived after 96 hours at 30% sa-
turation in stage II. The 96 hr Toxaphene TLs5g was 0.054 ppb for stage I
zoea, increased about tenfold to 0.76 ppb for stage II zoea and 0.74 ppb
for stage III zoea and increased about tenfold again to 6.8 ppb for stage
IV zoea and 8.4 ppb for the megalopa. Percent survival versus concentra-
tion at 96 hr is shown for each stage tested in Figure 1. In stage I

there was no significant difference in mortality at all concentrations

from 0.00 to 0.05 ppb, in stages II and III (significant on day 3 only)
from 0.0 to 0.5 ppb and in stage IV and megalopa, from 0.0 to 5.0 ppb.
However there was in every case higher mortality in the presence of Toxa-
phene than in the control. The 96 hr TLs59 values are summarized in Table 11.

37.


juvenile bass, and juvenile mullet. Bass embryos and juvenile pompano
tolerate oxygen levels of 15.3% and 18% saturation respectively. There
is a trend for species eurytolerant to other stresses to be slightly
more tolerant of reduced oxygen concentrations than less eurytolerant
species. There are significant synergistic effects with temperature
and salinity which probably reflects the increased metabolic demands

in meeting the challenge of extreme temperatures and salinities.

Avoidance of low oxygen concentrations has been tested in four species
of fish by Whittmore, Warren, and Doudoroff (1960). Included in this
study were late juvenile - early adult largemouth bass (Micropterus sal-
moides, 50-90 mm total length). This species avoided water with an
oxygen concentration of 1.5 mg/l (15.8% saturation) but did not avoid
concentrations of 3.0 mg/1 (32.6% saturation) or above. Two other spe-
cies tested (chinook salmon and coho salmon) exhibited avoidance at 4.5
mg/1 (20.6% saturation) at 22.8°C and 3.0 mg/1 (28.3% saturation) at 13.2 ion
Bluegills responded in the same manner as largemouth bass. These authors
concluded that these fishes avoided waters with oxygen concentrations
higher than that concentration causing mortality within 24 hours. This
point merits further investigation.

The fishes studied at all stages were much less tolerant of Toxaphene

than either Sesarma cinereum, Rhithropanopeus harrisii or Penaeus duorarum.
We suspect that Callinectes sapidus is much less tolerant than any other
species tested. The degree to which fish are less tolerant than decapod
larvae is much greater than indicated by direct comparison of TL5q values
defined here because the values for fish are gross overestimates.

It has been shown that the sheepshead minnow (Cyprinodon variegatus) avoided
water containing certain pesticides (Hansen, 1969) and salmon avoided heavy
metal ions (Sprague, 1964). No similar studies have been reported in the
literature for decapod larvae. It seems reasonable that some avoidance ca~-
pability exists for the fish and decapods studied although tests of this hy-
pothesis were not required under this contract. If this is true, it im-
plies that habitat limitation might result from pesticide presence rather
than increased mortality. Obviously this would not be possible for fish em-
bryos and perhaps also sac-fry larvae. However, these stages are more tol-
erant than juveniles of largemouth bass.

Toxaphene has very low solubility in aqueous media. It enters the environ-
ment in a xylene carrier which is non-miscible with water. In preparing
our test solutions a Toxaphene-xylene solution was used. The maximum amount
of xylene which could have been present in the final test solutions was less
than 5 x 10-8 ml/1 at the highest Toxaphene concentration tested (50 ppb
Toxaphene) assuming xylene remained in the aqueous solutions. No xylene
control groups were tested. Xylene residues (and volatile hydrocarbons)
evaporate rapidly and hence could have had little or no effect on our re-
sults. Better solution of Toxaphene would probably have been achieved by
including a surfactant in solution preparation as suggested by Mount and
Brungs (1966).

62.


(4) these species could be cultured at Aquatic Sciences, Inc. Informa-
tion on these organisms is provided in the introduction for the fish
and crustacean sections of this report.


Ovigerous R. harrisii were scarce during the course of this study,
perhaps as a result of the bi-annual severe drought in southern Flo-
rida. Temperature tests were completed for all stages. The 96 hr
temperature TL.,. was 37.0 to 37.3°C in every case. There was no sig-
nificant difference in mortality between 25 and 35°C except in stage
III. Salinity tests were completed only for stages I to III. The
lower salinity TLgo at 96 hr does not exist for stage I zoea and lies
somewhere between 0 and 5 °/oo for stages II and III. Insufficient
test concentrations in this salinity range preclude a more exact de-
termination of the lower salinity 96 hr TLsg. The 96 hr upper sal-
inity TL.,. was 37 °/oo for stage I, 47.5 °/oo for stage II and 42.5
°/oo for stage III. There was no significant difference in mortal-
ity between 0 and 35 °/oo in stage I and between 5 and slightly less
than 40 °/oo in stages II and III. The 96 hr reduced dissolved oxygen
TLeg was 45% for stage I, and 41.0% for stage II, the only stages tes-
ted? There was no significant difference in mortality between 75 and
100% saturation for stage I, but significant differences were found be
tween 30 and 75% saturation and 10 and 30% saturation for both stages.
The 96 hr Toxaphene TLsg for stage I was 43.75 ppm with no other tests
completed (Figure 3). There was no significant difference in mortal-
ity over the range 0.0 to 40.0 ppb Toxaphene. The 96 hr TL509 values
are summarized in Table 17.

TABLE 17.

TL5g VALUES AT 96 HR FOR EACH STAGE OF Rhithropanopeus harrisii

Reduced
Lower Upper Dissolved
Temperature Salinity Salinity Oxygen Toxaphene
Stage (2c) (2/00) (°/00) % sat.) (ppb)
T 37.0 does not 37.0 45 43.75
exist
It 37.3 (2.5) 47.5 41.0 288-345*
III 37.0 (2.5) 42.5 290-430*
Iv 37.0 does not 35
exist

Meg 37.0

*Based on preliminary screening tests

The only interaction test completed was salinity-reduced dissolved oxy-
gen for stage I. There was no evidence of combined action of these pa-
rameters. These data are summarized in Table 18. Survival was signi-

49.


DISCUSSION

The 96 hr temperature TLyQ for all stages of all species tested ranged
from 35.7 to 37°C. This range is comparable to upper lethal tempera-
ture limits for a variety of warm acclimated organisms (see for example
Prosser, 1961). In the experiments described here the acclimation tem-
perature was in every case 25°C, or about the optimal temperature for
each species. No data are available in the literature on the upper
lethal limits for animals acclimated at lower or higher temperatures.
One would expect the values for those stages of Callinectes sapidus not
tested to fall within the above range.

Costlow, Bookhout, and Monroe (1960) obtained, complete development of

S. cinereum at 30°C, 20 and 25 °/oo, but not at salinities above and
below this range. Even at these salinities, the percent reaching the
juvenile stage at 30°C was less than 10 percent. A similar result was
obtained for R. harrisii though the range of salinities permitting com-
plete development and the level of success, were both much greater (Cost-
low, Bookhout and Monroe, 1966). This is not surprising in this very
eurytolerant species.

The effect of temperatures on larvae of P. duorarum has recently been
studied by Thorhaug, Devany, and Murphy (1970). Development of nauplii
to protozoea occurred only from 24 to 31.5 C although survival was high
for a 10 hour period from 15 to 37°C. The median lethal limit for pro-
tozoea I and III was 37.0 to 37.8 C and 35.7 to 36.7 C respectively
which agrees favorably with our results although their tests were of
short duration (equal to the intermolt duration of each stage, generally
18 to 24 hours). The upper lethal limit for the mysis stage was 36.9 to
37.4 C, again very similar to our result.

The species tested differ markedly with respect to the salinity range
in their respective habitats. S. cinereum is an estuarine species which
spends a major portion of its life above the waterline though near salt
water. However, like other semi-terrestrial crabs, it must return to
water for reproduction (Williams, 1965). The larvae have a lower 96 hr
TLs5g ranging around 10 °/oo which is near the level at the upstream
limit of natural distribution of adults. The upper salinity limit in-
creased from 36.5 °/oo to 52 °/oo from stage I zoea to the megalopa.
Larvae flushed from an estuary into the sea would therefore be capable
of surviving and developing and could reinvade the estuary as megalopae
or juveniles. Complete development to the juvenile for S. cinereum has
been observed at 20 and 26.5 °/oo but not at 12.5 and 31.1 °/oo at 20,
25 and 30°C (Costlow, Bookhout, and Monroe, 1960).

The life cycle of P. duorarum has been reviewed in Williams (1965).

Early juveniles migrate into estuaries where growth is rapid. The adults
migrate out of the estuary and spawn in open waters. The larval stages
are passed in the lower reaches of the estuary or offshore. The lower
salinity limit was 19.5 °/o0 or above, commensurate with the normal dis-

55.


FISHES

INTRODUCTION

Four species of fishes were chosen for this study. The largemouth bass,
Micropterus salmoides (Lacepede), was included because (1) it is the
most important freshwater sport fish in the southeastern United States,
and particularly Florida, where millions of dollars are expended annually
by sportsmen (J. Walter Dineen, personal communication), (2) the species
inhabits waters which adjoin or flow through agricultural areas, and (3)
largemouth bass spawn and live in fresh waters in the coastal plain and
in the upper reaches of estuaries where fluctuations of temperature and
salinity and, on occasion, dissolved oxygen (when eutrophication is ad-
vanced) occur. With or without the presence of pesticides, these fluc-
tuations can cause stress.

Two species of mullet, Mugil cephalus Linnaeus, the striped mullet, and
Mugil curema Valenciennes, the white mullet, were chosen as being rep-
resentative of estuarine fishes. They provide an important commercial
fishery in Florida with an annual value exceeding $2.4 million (Anon.,
1970). Both species spawn at sea, the late larvae and early juveniles
returning to inshore waters at a standard length of between 14 and 20 m.
This life history stage coincides with a major change in the feeding me-
chanism and a departure from a zooplankton - harpacticoid copepod diet
to filter feeding on detrital material, primarily of plant origin, in
bottom sediments (Cowart, 1971). At this point, however, the two species
diverge in their habitat preferences. Mugil cephalus juveniles enter in-
tracoastal waterways, canals, and rivers, into fresh waters such as Lake
Okeechobee inhabited by largemouth bass and other freshwater fishes. Ju-
veniles of M. curema migrate into low salinity waters of intracoastal wa-
terways, canals, and estuaries but typically do not invade waters of less
than 15 °/oo salinity.

Trachinotus carolinus (Linnaeus), the Florida pompano, was chosen as a
representative marine species for this study. From an economic stand-
point, T. carolinus is the most valuable commercial food fish along the
Atlantic coast of the United States and represents a $1 million annual
fishery in Florida alone. Mariculture interests have placed Florida pom-
pano as the high priority species for commercial culture.

Although the entire life history of T. carolinus has not been thoroughly
studied, adults apparently spawn at sea. Early juveniles return to in-
shore waters at a standard length of 10 to 20 mm where they are often
found in the surf zone along beaches (Cowart, 1971).

Three early life history stages of each species were studied, gastrula,
10-12 day post hatch larva, and juveniles of 20-30 mm standard length.
Gastrulae hatched during the 96 hour exposure period, and reached a late
sac-fry stage by the end of the test period. These stages were chosen


EPA-R3-73-035
April 1973

ENVIRONMENTAL EFFECTS ON TOXAPHENE TOXICITY

TO SELECTED FISHES AND CRUSTACEANS

By
Walter R. Courtenay, Jr.

Morris H. Roberts, Jr.

Contract No. 14-12-532
Project 18080 DLR

Dr. Roy J. Irwin
Office of Permit Programs
Environmental Protection Agency
Washington, D.C. 20460

Prepared for
OFFICE OF RESEARCH AND MONITORING

U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460

thot Fae. Phys,

For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402
Price $1 domestic postpaid or 75 cents GPO Bookstore




GLOSSARY (continued)

molt: the process of shedding the exoskeleton necessary for growth
in anthropods including decapods.

mysis (es): that larval stage in the development of penaeids in
which propulsion is by thoracic appendages and the abdominal
appendages appear (corresponds to the zoeae of other decapods
in part).

nauplius (i): the larval stage in the development of penaeids in
which propulsion is by the antenual appendages. Only the anten-
nules, antennae, and mandibles are functional.

necrosis: localized death and disintegration of tissue.

neurula: that embryonic stage in which organ systems are differen-
tiated; characterized by neural tube formation.

notochord: a long, flexible, rodlike structure found in all verte-
brate embryos.

organogenesis: the process of organ formation.

ovigerous: refers to female crabs bearing developing eggs on the
abdominal appendages.

pelagic: of or pertaining to the habitat of free-floating or free-
swimming organisms (plankton and nekton, respectively).

protozoea (e): that larval stage in the development of penaeids pre-
ceding the mysis in which propulsion is by thoracic appendages
and there are no abdominal appendages (corresponds to the zoeae
of other decapods in part).

sac-fry: the just-hatched larvae of fishes in which the external
yolk sac is still present.

screening test: a preliminary test to determine the range of concen-
tration within which lies the 96 hr TL,9.

standard length: the length of fishes measured from the mouth to the
end of the peduncle.

static test: a test in which conditions were maintained constant
throughout the test period.

sublethal: refers to a concentration of toxicant (or other adverse

condition) less than the 96 hr Tlsq which causes less than 50%
mortality.

72.


All Toxaphene tests were conducted in completely independent laboratory
modules located outside of the main building complex at Aquatic Sciences,
Inc. These research spaces were provided to prevent contamination of
culture, life support, and other research areas in the main complex.

18.


RESEARCH REPORTING SERIES

Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:

1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research

4. ‘Environmental Monitoring

5. Socioeconomic Environmental Studies

This report has been assigned to the ECOLOGICAL
RESEARCH series. This series describes research
on the effects of pollution on humans, plant and
animal species, and materials. Problems are
assessed for their long- and short-term
influences. Investigations include formaticn,
transport, and pathway studies to determine the
fate of pollutants and their effects. This work
provides the technical basis for setting standards
to minimize undesirable changes in living
organisms in the aquatic, terrestrial and
atmospheric environments.


TABLE 12 (continued)

Stage IV
Temperature (°C) Salinity (°/o0)
25 35 30 40
Reduced 75 32.5 77.5 35.0 47.5
Dissolved
Oxygen 100 17.5 30.0 37.5 22.5
(% sat.)
Reduced
Temperature Salinity Dissolved
(OC) (2/00) Oxygen (% sat.)
25 35 30 45 75 100
Toxaphene | 0 35.0 | 47.5 27.5 | 37.5 32.5] 22.5
(ppb) 5 27.5 | 80.0 40.0 | 85.0 100.0} 32.5
Megalopa
Temperature (°C) Salinity (°/oo)
25 35 30 50
Reduced 75 15 30 12.5 47.5
Dissolved
Oxygen 100 | 10 30 17.5 45.0
(% sat.) IL
Reduced
Temperature Salinity Dissolved
(Cc) (2/00) Oxygen (% sat.)
25 35 30 50 75 100
Toxaphene | 0 17.5 17.5 10.0 40.0 7.5 25.0
5 2.5 10.0 17.5 77.5 5.0 15.0

42.




cant differences between the sum of the effects for each variable acting
alone and the effect of the variables acting simultaneously. The per-
cent mortality data was transformed by the sin-1JM/100 function as sug-
gested by Bartlett (1957) for probabilities or proportions with binominal
variance. Percent mortalities of 0 and 100 were transformed by the method
described in Snedecor with Cochran (1968) to correct for bias at extreme
probabilities.

Synergism is defined here as an excess of mortality when the variables
are acting together versus when acting alone, i.e. the effect is more than
additive.

35.


TABLE S.

PERCENT MORTALITY AFTER 96 HOURS IN
INTERACTION TESTS FOR Mugil curema*

Juveniles
Temperature (°C) Salinity (°/o0)
T
23-24 | 24 | 33 36 35 54 57__| 60
Reduced i | ,
Dissolved) 75 | 141.2 |44.0 21.0 24.3 56.0
Oxygen i | H
(4 sat.) | 100} 31.3 | 24.2 | ! 31.5
{
i

A 96 hr temperature TL, 9 of 36.2°C was established for juvenile T. caro-
linus. A 96 hr lower SQiinity TLeg was not observed for juveniles but

a 96 hr upper salinity Tl5g of 50. °/oo was determined. The 96 hr re-
duced dissolved oxygen TL. for juveniles was 18% saturation. These data
are summarized in Table 3°

In interaction exposures, highest mortalities among juvenile T. carolinus
were observed at 30% saturation of dissolved oxygen in the temperature-
reduced dissolved oxygen and salinity-reduced dissolved oxygen. These
data are reported in Table 6.

TABLE 6.

PERCENT MORTALITY AFTER 96 HOURS IN INTERACTION
TESTS FOR Trachinotus carolinus

Juveniles
Temperature (°c) Salinity (2/00)
22 32 35 33 42 | 45 48
|
Reduced 30 |__| 72.0 | 100.0 ___| 53.0 | 76.0} 75.0
Dissolved 1 | |
Oxygen 75 —_ ' 4.0 | 6.0 __| 23.0 18.0 | 28.0
| i i
Kt ‘
(% sat.) 100 | 2.0 | 2.0} |

21.


INTRODUCTION

For several decades there has been increasing concern among scientists
and fishermen (sport and commercial) that aquatic organisms and their
population dynamics are being affected adversely through the addition
of various chemicals to the aquatic ecosystem. Pesticides, particularly
the persistent chlorinated hydrocarbons, have been implicated in many
“fish kills" in the United States and elsewhere. These chemical pollu-
tants, along with many others, can be passed through the food chain and
may be accumulated to lethal levels in those organisms at the top of the
chain through the process of biological magnification (Egler, 1964).

The chlorinated hydrocarbon insecticides generally enter aquatic eco-
systems by runoff during periods of rain or may be directly introduced
through carelessness during applications. Highest concentrations occur
in and near agricultural areas where these pesticides are most often
used. Once these chemicals are in aquatic situations, they are distri-
buted downstream into lakes, estuaries, and eventually into marine wa-
ters (Nicholson, Grzenda, Laver, Cox, and Teasley, 1964).

Dilution occurs from the site of entry of pesticides into the aquatic
ecosystem and continues throughout the downstream drift of these chemi-
cals. High concentrations at the source of entry may produce a fish kill.
Similar effects may not occur downstream as the chemicals undergo dilu-
tion to sublethal levels. Pesticides typically enter the lower portions
of inland waterways, estuaries, and marine waters in sublethal concentra-
tions.

The coastal regions of streams, rivers, canals, and inshore marine waters
are generally shallow, often associated with marshes and swamps, and un-
dergo wide fluctuations in hydrographic conditions with tidal, climatic,
and seasonal changes. Variations in such characteristics as salinity

and temperature are often dramatic in these areas. Variations in tempera-
ture are now being produced by man as new electrical power plants are con-
structed. Many of these are thermonuclear and require vast quantities of
water to cool the reactors, and return the heated effluent to the environ-
ment.

Coastal regions are very productive in terms of aquatic organisms. Shal-
low areas of fresh waters, estuaries, and inshore marine habitats provide
vast breeding and spawning grounds for the species present. Moreover,
these same factors are of economic and recreational importance to man be-
cause large commercial and sport fisheries depend upon these areas of
stock renewal for the fisheries.

Fishery biologists have long recognized the importance of water quality
in these areas to renewal of fishery stocks although until recently re-
latively few biologists have investigated water quality with respect to
chemical pollutants (Holden, 1966; Johnson, 1968; Butler, 1966). While
they cite declining stocks of animals to support a fishery, and pollution

5.


ranges for temperature and salinity derived from our 96 hr TLz, deter-
minations are broader than those based on the ability of larvae to com-
plete development. This supports the above contention.

The reduced dissolved oxygen TL, at 96 hr was near 50% saturation for

S. cinereum and P. duorarum whieh are moderately tolerant species with
Yegard to many parameters. C. sapidus, the least tolerant species had

a reduced dissolved oxygen 96 hr TL<g approaching 60% saturation, where—
as the most tolerant species, R. harrisii, had a value 41-45% saturation
for all stages tested. The trend of decreasing tolerance of larvae to
reduced dissolved oxygen with increasing fragility of the larvae is

quite reasonable. No data are available in the literature for comparison.

S. cinereum was very sensitive to Toxaphene in zoeal stage I, increased
in tolerance by ten fold in stages II and III, and ten fold again in
stages IV and megalopa. The same trend is suggested by the data for R.
harrisii. However, R. harrisii was approximately 1000 times more tol—
erant than S. cinereum in stage I and about 400 times more tolerant in
stages II and III. Complete data are not available for C. sapidus; how-
ever a few preliminary experiments suggest that this species is much less
tolerant than S. cinereum. Values for P. duorarum in all stages were
slightly higher than values for stage IIT and III S. cinereum. There was
a slight decrease in tolerance as the larvae developed from the nauplius
to the mysis stage.

In the temperature-reduced dissolved oxygen interaction tests, there was
no synergistic effect for any stage of S. cinereum. For C. sapidus, how-
ever, there was a synergistic effect in stages I and I1, though surpris-
ingly, not in stage III. A synergistic effect of temperature-reduced
dissolved oxygen was observed only in the mysis stage of P. duorarum.

The results for S. cinereum in salinity-reduced dissolved oxygen tests
showed the same trend with no synergistic effects for any stage. For C.
sapidus there were large synergistic effects for stage I only. The only
data for R. harrisii showed no synergistic effect for stage I. There
was no statistically significant evidence of synergistic action on all
stages of P. duorarum.

Data for interaction tests involving Toxaphene are available for S. cin-
ereum and P. duorarum. There was a significant interaction of tempera—
ture and Toxaphene for all stages of P. duorarum and for stages I and IV
of S. cinereum. Similarly, there was a synergistic effect of elevated
salinity for stage I of S. cinereum and the mysis stage of P. duorarua.
There was also a synergistic effect of reduced dissolved oxygen for zoeal
stages II and IV of S. cinereum. As noted above, the results for the
megalopa are exactly the opposite. This antagonistic effect is suspect.
It is suggested that this test be repeated. A synergistic effect of re-
duced dissolved oxygen was also noted for the mysis of P. duorarum but
not earlier stages. ~

57.


GLOSSARY (continued)
synergism: the interaction effect of two (or more) toxicants which
is greater than the sum of the effects of each acting separately.

TLs5o: median tolerance limit, i.e., the concentration of toxicant
causing 50% mortality in a population at specified times, e.g.
96 hours.

vacuolated: possessing hollow spaces within cells.

yolk sac: that outpocketing of the gut containing yolk. Fishes gen-
erally have both an internal and an external yolk sac.

zoea (e): the planktotrophic larval stage of decapod crustaceans in
which propulsion is provided by the thoracic appendages;
shrimp-like.

© US, GOVERNMEN? PRINTING OFFICE 1973—514-154/258

73.


Interstage comparisons for S. cinereum revealed that there was a sig-
nificantly different response to salinity and temperature for stage

I versus all other stages and for stages III, IV and megalopa. The
response to Toxaphene was significantly different for stage I versus
all stages and stages II and III versus stage IV and megalopa.

The 96 hr temperature TLog for P. duorarum was 36.3 - 36.5°C for naup-
liar, protozoeal and mysis stages. There was no significant difference
in mortality between 25 and 35°C in the mysis. The lower salinity TL 50
at 96 hr was 19.5 - 23.5 °/oo, the upper salinity Theo at 96 hr was

47.0 - 51.0 °/oo with a distinct increase from protozoea to mysis. There
was no significant difference in mortality from 25 to 45 °/oo for the
nauplius, from slightly above 20 to 45 °/oo for the protozoea, and from
25 to 50 °/oo for the mysis. The 96 hr reduced dissolved oxygen TLsg
was 43.5 - 54.8% saturation. There was no significant difference in mor-
tality between 75 and 100% saturation. Mortality at 30% saturation was
not total but always greater than 75%. The 96 hr Toxaphene TLeq decreased
from 2.2 ppb for nauplii to 1.4 ppb for mysis. Percent survival versus
concentration at 96 hr is shown in Figure 2 for each stage tested. There
was a significant difference in mortality between controls and experimen-
tal animals at all Toxaphene concentrations. These TLs5g values are sum-
marized in Table 13.

TABLE 13.

TLoo VALUES AT 96 HR FOR THE LARVAL
STAGES OF Penaeus duorarum

Reduced
Lower Upper Dissolved
Temperature Salinity Salinity Oxygen Toxaphene
Stage (2c) (2/00) __(/oo) _(% sat.) (ppb)
nauplius 36.5 22.8 47.0 48.0 2.2
protozoea 36.3 19.5 47.3 43.5 1.8
mysis 36.5 23.5 51.0 54.8 1.4

The temperature-reduced dissolved oxygen test showed distinct synergis-
tic effects in the mysis, and no synergism in the other stages. Sig-
nificant interaction of salinity and reduced dissolved oxygen was not
observed; however there was an increase in mortality at high salinity-
reduced dissolved oxygen in every stage.

There was a significant interaction at every stage in temperature-Toxa-
phene tests. A significant synergism of salinity and Toxaphene was ob-

43.


served in the mysis stage. The nauplius stage showed significant mor-
tality only when Toxaphene was present. At reduced oxygen levels a
synergistic effect was not observed. The data for interaction tests
are summarized in Table 14.

TABLE 14.

PERCENT MORTALITY AFTER 96 HOURS IN
INTERACTION TESTS FOR Penaeus duorarum

nauplius
1
Temperature (°C) Salinity (°/o00)
25 35 35 45
Reduced 75 0.0 77.5 7.5 15.0
Dissolved
Oxygen 100 5.0 42.5 0.0 10.0
(% sat.)
Reduced
Temperature Salinity Dissolved
(°C (2/00) Oxygen (% sat.)
25 35 35 45 75 100
Toxaphene | 0.0 10.0 32.5 0.0 0.0 0.0 | 0.0
(ppb)
1.0 20.0 100.0 17.5 15.0 2.5 | 5.0
protozoea
Temperature (°C) Salinit: (°/oo)
25 35 35 45
Reduced 75 30.0 40.0 12.5 25.0
Dissolved
Oxygen 100 7.5 32.5 12.5 17.5
(% sat.)

45.