&EPA
United States
Environmental Protection
Agency
Environmental Research
Laboratory
Gulf Breeze FL 32561
EPA 600 3 79-031
March 1979
Research and Development
Effects of
Chlorinated
Seawater on
Decapod
Crustaceans and
Mulinia Larvae
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine 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.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-79-031
March 1979
EFFECTS OF CHLORINATED SEAWATER ON
DECAPOD CRUSTACEANS AND MULINIA LARVAE
by
Morris H. Roberts, Jr.
Chae E. Laird
Jerome E. Illowsky
Virginia Institute of Marine Science
Gloucester Point, Virginia 23062
Grant No. R-803872
Project Officer
William P. Davis
Bears Bluff Field Station
Environmental Research Laboratory
U. S. Environmental Protection Agency
Gulf Breeze, Florida 32561
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
GULF BREEZE, FLORIDA 32561
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DISCLAIMER
This report has been reviewed by the Environmental Research
Laboratory, Gulf Breeze,U. S. Environmental Protection Agency, and
approved for publication. Approval does not signify that the contents
necessarily reflect the views and policies of the U. S. Environmental
Protection Agency, nor does mention of trade names or commercial
products constitute endorsement or recommendation for use.
11
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FOREWORD
The protection of our estuarine and coastal areas from damage
caused by toxic organic pollutants requires that regulations restrict-
ing the introduction of these compounds into the environment be formu-
lated on a sound scientific basis. Accurate information describing
dose-response relationships for organisms and ecosystems under varying
conditions is required. The EPA Environmental Research Laboratory,
Gulf Breeze, and its field station, Bears Bluff, contribute to this
information through research programs aimed at determining:
'the effects of toxic organic pollutants on individual species
and communities of organisms;
"the effects of toxic organics on ecosystem processes and
components;
'the significance of chemical carcinogens in the estuarine
and marine environments.
This report presents results of bioassay tests of chlorination-
induced oxidants upon larval crabs, blood physiology of the commercial
blue crab, and toxicity to a small estuarine clam that enters the
crab's food web. In addition to the effects measured with these
organisms, the experimental apparatus described is relevant to future
tests on low levels of stress upon the sensitive life stages of
marine estuarine organisms. These data have appeared in part in
refereed scientific journals and symposia, and should contribute
substantially to our collective analysis of biological risks of
oxidative products from chlorination discharged into marine waters.
Thomas W. Duke
Director
Environmental Research Laboratory
Gulf Breeze, Florida
iii
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ABSTRACT
Eggs and larvae of decapod crustaceans and embryos of Mulinia
lateralis were exposed to chlorinated seawater for varying periods in
continuous flow systems. Mortality, developmental rate, and general
behavior were recorded. Panopeus herbstii zoeae were more sensitive
to chlorine-induced oxldants (CIO) than eggs or adults (96-hr LC50 ca.
2.8 yeq/1 = 0.1 mg/1). The 96-hr LC50 for Pagurus longlcarpus zoeae
was approximately the same as for Panopeus zoeae. The 120-hr LC50 for
Pagurus zoeae was 1.4 yeq/1 (0.05 mg/1).Development was slightly
delayed for Pagurus zoeae at CIO levels as low as 0.6 yeq/1 (0.02
mg/1). Mulinia embryos exposed for 48 hr had an LC50 between 0.3 and
3.0 yeq/1 (0.01 and 0.1 mg/1). Mulinia embryos exposed to chlorinated
seawater for 2 hr had an LC50 of about 2.0 yeq/1 (0.072 mg/1);
subsequent survival rates for larvae in unchlorinated seawater were
unaffected by prior exposure to CIO.
The effects of CIO on serum constituents in Callinectes sapidus
occurred sporadically and appeared unrelated to dose or mortality.
Similar effects were noted for oxygen consumption in whole crabs and
excised gills. It was concluded that there are no physiologically
significant sublethal effects of CIO on serum constituents
(osmoregulation) or oxygen consumption of whole blue crabs or excised
gills. Blue crab antennules are sensitive to sublethal doses of CIO.
Spawning and feeding seem to be inhibited by sublethal doses of CIO.
This effect disappears when CIO is removed.
This report was submitted in fulfillment of Grant No. R-803872 by
the Virginia Institute of Marine Science under the sponsorship of the
U. S. Environmental Protection Agency. This report covers the period
from July 15, 1975 to March 31, 1978.
iv
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CONTENTS
Foreword ill
Abstract iv
Figures vi
Tables viil
Acknowledgments ..... xi
1. Introduction 1
2. Conclusions 5
3. Recommendations 6
4. Developmental Studies 8
General Methods 8
Results 17
5. Avoidance Behavior Experiments . 50
Methods 50
Results 56
6. Serological Effects 66
Methods 66
Results 69
7. Respiratory Effects 90
Methods 90
Results 94
8. Discussion 102
References 107
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FIGURES
Number Page
1 Schematic representation of dilutor system . . 9
2 Test chamber for egg and larval tests 12
3 CIO concentrations measured during decapod egg test. . . 19
4 Survivorship curves for Pagurus longicarpua zoeae,
experiment 9 36
5 Diagrammatic representation of complete avoidance test
apparatus 52
6 Diagrammatic representations of avoidance test chamber.
Upper view, longitudinal cross section through center,
and view from outflow end 53
7 Percent of Pagurus longicarpua stage I larvae In the
upper half of the test chambers versus time (rain)
when exposed to 240 fc in 18 °/oo salinity water ... 58
8 Percent of Pagurus longicarpus stage I larvae in the
upper half of the test chambers versus time (mln)
when exposed to 24 fc in 18 °/oo salinity water. ... 59
9 Percent of Pagurus longicarpus stage 1 larvae in the
upper half of the test chambers versus time (mln)
when exposed to 240 fc and 24 fc in 18 °/oo salinity
water. ........ 60
10 Percent of Pagurus longicarpus stage 1 larvae in the
upper half of the test chambers versus time (mln)
when exposed to 18 °/oo and 24 °/oo at 240 fc 61
11 Percent of Pagurus longicarpus stage I larvae in the
upper half of the test chambers versus time (min)
when exposed to 18 °/oo and 24 °/oo at 24 fc 62
vl
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Number Page
12 Mean percent of Pagurus longicarpue stage I larvae In
the upper half of the test chambers versus time (min)
for all experiments at each light Intensity-salinity
combination 64
13 Composite summary of survival data for Calllnectes
sapidus adults plotted against long CIO concen-
tration. Includes data from 4 experiments. The
LC50 values are considered to be preliminary only. . . 70
14 Effects of CIO exposure (0.27 mg C12/1) and recovery
on whole crab oxygen consumption in Callinectes
sapidus 98
15 Effects of CIO exposure (0.27 mg C12/D and recovery
on oxygen consumption of excised gills in Calllnectes
sapidue. 99
16 Effects of CIO exposure (0.99 mg C12/D and recovery
on whole crab oxygen consumption in Callinectes
sapidus. 100
17 Effects of CIO exposure (0.99 mg C12/1) and recovery on
oxygen consumption of excised gills in Callinectes
sapidus 101
vli
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TABLES
Number Page
1 Summary of flow rate data (diluent and toxicant stock)
and calculated dilution factors for larval experiment
9 10
2 Summary of chlorine-induced oxidant concentration and
water quality data measured during decapod egg
exposure test 18
3 Egg survival, hatching, and immediate post-hatch zoeal
survival 20
A Summary of chlorine-induced oxidant concentrations
and water quality data measured during Panopeus
herbstii zoeal exposure 23
5 Survival records for Panopeus herbstii zoeae for
each CIO exposure test 26
6 Lethal concentrations of chlorine-induced oxidants for
Panopeus herbstii larvae 29
7 Summary of chlorine-induced oxidant concentrations and
water quality data measured during Pagurus longicarpus
zoeal exposures 30
8 Survival records for Pagurus longicarpus zoeae for each
CIO exposure test 33
9 Lethal concentrations of chlorine-induced oxidants for
Pagurus longicarpus larvae ... 35
10 Summary of chlorine-induced oxidant concentrations and
water quality data for Panopeus herbstii juvenile
test 38
11 Survival data for Panopeus herbstii juvenile test. ... 39
viil
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Number Page
12 Summary of chlorine induced oxidant concentrations and
water quality data measured during Mulinia lateralis
embryo tests 41
13 Survival, mortality, and recovery results for 48-hr
Mulinia lateralis embryo exposure tests 44
14 Survival and recovery results for 2-hr Mulinia embryo
exposure tests and subsequent static culture 48
15 Summary of percent photopositive responses for stage I
Pagurus longicarpus larvae at 5-min intervals ar-
ranged according to treatment. (Data for replicate
tests averaged) 63
16 Summary of test system used, doses, sampling intervals,
and serum analyses during Callinectes sapidus sero-
logical studies 72
17 Summary of hydrographic data for Callinectes sapidus
serological studies 74
18 Blood serum parameters for Callinectes sapidus from
experiments 1 and 2 76
19 Blood serum parameters for Callinectes sapidus from
experiments 3-5 77
20 Blood serum parameters for Callinectes sapidus from
experiments 6-12 78
21 Blood serum parameters for Callinectes sapidus from
experiments 13 ...... 81
22 Tukey's modified u>' test for tank size-flow-CIO com-
bination effects on serum TNPS in Callinectes
sapidus from experiment 13 [w1 » qa(p,N2)S;
Steel and Torrie, I960]. Bars underline equal
means (p * 0.05) 83
23 Tukey's modified u>' test for tank size-flow-CIO com-
bination effects on serum chloride in Callinectes
sapidus from experiment 13 [u* - qa(p,N2)S;
Steele and Torrie, I960]. Bars underline equal
means (p » 0.05) 83
ix
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Number Page
24 Tukey's modified GO' test for tank size-flow-CIO com-
bination effects on serum osmotic concentration in
Callinectes sapidus from experiment 13 [S; Steel and Torrie, I960]. Bars under-
line equal means (p = 0.05) 83
25 Comparison of CIO levels in Callinectes dosing
system with and without crabs 87
26 Tukey's w test for tank size-flow-CIO combination
effects on ammonia-nitrogen in tanks from experiment
13. Bars underline equal means (p 0.05) 89
27 Summary of test systems used, doses, sampling intervals
and analyses during Callinectes sapidus respiration
studies 95
28 Summary of hydrographic data for Callinectes sapidus
respiration studies 96
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ACKNOWLEDGMENTS
Dr. William P. Davis (Bears Bluff Field Station) provided several
suggestions regarding methods and apparatus and contributed to our
understanding of various issues related to chlorination practices.
Douglas Middaugh (Bears Bluff Field Station) graciously showed us
his facility for fish studies, which significantly contributed to
facility developments. Jose Castro (Bears Bluff Field Station)
arranged for purchases and shipping of adult female blue crabs from
the North Edisto River, South Carolina, for the serum studies.
Dr. John Kraeuter provided Mulinia adults from the Virginia
Institute of Marine Science Eastern Shore Laboratory for the Mulinia
studies.
We also appreciate the general assistance and support provided by
our colleagues at VIMS during the past two years.
Drafts and final copy of this report were prepared by the VIMS
Report Center.
xi
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SECTION I
INTRODUCTION
Chlorine has been used extensively in the United States and
elsewhere as a disinfectant for treated sewage. More recently it has
come into vogue as an antifouling agent for once-through power plant
cooling systems. The methods of application, disinfectant properties,
and chemistry are extensively reviewed (White, 1972). It has been
considered effective in both applications and was believed to be
environmentally safe because the products formed upon chlorination of
water are highly unstable; hence, according to traditional reasoning,
troublesome concentrations would not be found in receiving waters
under normal operating conditions.
The chemistry of chlorine in fresh water has recently been
reviewed (White, 1972; Brungs, 1974; and Jolley, 1973). The
descriptions provided therein do not, however, apply directly to salt
water. Salt water contains sufficient bromide to allow conversion of
chlorine compounds to analogous bromine compounds (Dove, 1970; Sugam
and Helz, 1977; Johnson, 1977). Reactions with ammonia and organics
can still occur. Macalady et al. (1977) have recently demonstrated
bromate formation in chlorinated seawater exposed to sunlight. There
remains considerable uncertainty concerning the halogen species
present in chlorinated seawater since the analytical methods do not
discriminate between the possible compounds present. Hence one cannot
state positively what chemical species are causing observed toxic
effects. Throughout this report the concentration of measured
toxicant is referred to as chlorine-Induced oxidant (CIO), expressed
as yeq/J. (mg C12/D, in an attempt to avoid confusion regarding the
active material.
The environmental safety of chlorine came into serious question
during the 1960's. Zillich (1970) reported adverse effects of
chlorinated sewage on aquatic life in fresh water systems.
Subsequently, Bellanca and Bailey (1977) (previously reported in Va.
State Water Control Board, 1974) described data which strongly
implicated chlorine residuals emanating from sewage treatment plants
as the causative agent of massive fish kills in the estuarine portion
of the James River in Virginia. Hamilton et al. (1970), Morgan and
Stress (1969), and Carpenter et al. (1972) found effects on natural
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phytoplankton exposed to chlorinated power plant effluents
attributable to chlorine.
An extensive literature, describing the toxicity of chlorine in
both fresh and estuarine waters to phytoplankton, various
invertebrates, and fishes, has been amply reviewed and discussed by
Brungs (1973, 1976). Toxicity studies relating to estuarine species
have been very limited compared to those for freshwater species.
Published studies deal primarily with acute lethal effects.
From our studies in Virginia, oyster larvae (Crassostrea
virginica) and the copepod(Acartia tonsa)appeared to be the most
sensitive estuarine species (48-hr LCSO's of 0.73 and 0.82 yeq/1 -
0.026 and 0.029 mg/1), while Palaemonetes pugio was most tolerant (96-
hr LC50 - 6.20 ueq/1 = 0.22 mg/1). Fishes tested were intermediate in
sensitivity (Roberts et al., 1975; Roberts and Gleeson, 1978).
Phytoplankton exposed to chlorine exhibited marked reductions in
primary production in tests with cultured single-species populations
(Roberts and Diaz, 1976, unpublished manuscript; Bender et al., 1977)
and natural mixed populations (Roberts and Illowsky, unpublished
data).
There has been little or no research on chronic effects of
chlorine on estuarine organisms. Long-term exposure chronic studies
have been carried out in freshwater with Daphia magna, Gammarus
pseudolimnaeus and Pimepheles promelas (Arthur et al., 1975). Such
studies have yet to be performed with estuarine species.
For the acute and sublethal tests in our study, we selected
species representative of the decapod crustaceans and bivalve molluscs
in Atlantic coastal estuaries. From the decapods, we selected the
hermit crab, Pagurus longicarpus, and the mud crab, Panopeus herbstii,
because they are abundant and widely distributed in estuaries, and the
larvae can be readily cultured in the laboratory at the ambient
salinity available to us. Further, _P. longicarpus had previously been
induced to spawn in the laboratory during the winter (Roberts, 1969).
Since these species are characteristic of the estuarine fauna, they
are presumed to be of ecological significance. The shrimp
Palaemonetes pugio was not selected because of the high adult
tolerance (Roberts et al., 1975). The blue crab Callinectes sapidus
was not selected because larval salinity requirements (25 °/oo or
above) could not be readily provided in our laboratory. As a
representative bivalve, the coot clam, Mulinia lateralis, was
selected. M. lateralis is an abundant representative of the estuarine
infauna, capable of completing its life cycle in 6 to 9 months under
appropriate conditions. Its value for toxicological studies has
previously been pointed out by Calabrese (1969b, 1970).
Historically, studies of the physiological effects of exposure of
aquatic animals to chlorine residuals have been limited to fish.
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Typically, the effects have been manifested as changes In properties
of the blood such as methetnoglobin and lactate concentrations, p02>
pH, hematocrit, and plasma water as well as erythrocyte condition and
maturity. Because of basic physiological differences, the
relationship between the effects in fishes and those in invertebrates
is uncertain. Accordingly, we elected to investigate the effects of
chlorine residuals on certain blood and respiratory parameters in the
blue crab, Callinectes sapidus. The blue crab was chosen because it
is a commercially important, common, active inhabitant of the
Chesapeake Bay and its tributaries and is likely to encounter
chlorinated effluents from sewage and power plants. In addition, the
crab offers the availability of a large physiological data base.
Voluminous literature offers useful physiological information
derived from measurements of crab blood parameters. Blue crab blood,
already used to assess crab responses to temperature, salinity and
season, pesticides, disease, and general stress, has provided
information on mechanisms of ionic and osmotic accommodation and
respiration. Changes in blue crab physiology caused by chlorine-
Induced oxidants should also be reflected in the blood and could
probably be observed in the serum constituents.
A portion of this study was conducted to establish the effects of
exposure to chlorine residuals on serum constituents in the blue crab.
Serum constituents were used in order to develop a physiological index
for assessing and estimating the degree of stress imposed on crabs by
different levels of residuals - a possible alternative to long-term
bioassays.
Since the gill is the primary interface across which blue crabs
interact with their environment, it is reasonable to suspect that
physiological effects of chlorine induced oxidants on blue crabs
originate in the gill. Effects on osmoregulatory sites in the gill
should be manifested in the serum constituent studies. In fishes it
has been concluded that the primary mode of action of chlorine is gill
tissue damage resulting in death by asphyxiation. If gill damage
occurs in the blue crab on exposure to chlorine-induced oxidants,
death by asphyxiation is likely. Part of the study was devoted to
this problem. Specific attention was afforded to the effects of
chlorine-induced oxidants on whole crab oxygen consumption, excised
gill oxygen consumption and gill hlstopathology.
The objectives of the present study were;
1. to assess the subacute effects of chlorinated seawater on
Panopeus herbstll and Pagurus longicarpus eggs and larvae, and larvae
of the coot clam, Mulinia lateralis.
2. to determine whether Pagurus longicarpus larvae can avoid
chlorinated seawater,
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3. to examine the effects of exposure to chlorinated seawater on
the blood chemistry of blue crabs at sublethal doses and,
concurrently, to determine the time course of blood serum changes,
4. to examine the effects of exposure to chlorinated seawater on
the oxygen consumption rate^ of whole blue crabs and blue crab gills.
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SECTION 2
CONCLUSIONS
1. The sensitivity to chlorinated seawater for Panopeus herbstii
varies as a function of life history stage. Panopeus herbstii eggs
can develop to hatching at CIO levels up to 5.9 yeq/1 (0.21 mg/1),
whereas the resultant larvae at this dose exhibit less than 10%
survival. The 96-hr LC50 for Panopeus herbstii larvae varied from
1.13 to 3.38 yeq/1 (0.04 to 0.12 mg/1) in most tests. Larvae tested
early and late in the breeding season seemed to be somewhat more
sensitive with a LC50 of ca. 0.68 yeq/1 (0.024 mg/1). In a
preliminary test with field-collected Juveniles, the 96-hr LC50 was
14.1 yeq/1 (0.50 mg/1).
2. The larvae of Pagurus longicarpus exposed to chlorinated
seawater exhibited a 96 hr LC50 of 1.75 to 2.88 yeq/1 (0.062 to 0.102
mg/1), which is similar to the LC50 for Panopeus herbstii larvae.
3. Sublethal doses of chlorinated seawater caused a delay in
development of Pagurus longicarpus larvae.
4. Mulinia lateralis embryos exposed to chlorinated seawater for
the first 48 hr after fertilization exhibited an LC50 between 0.3 and
2.8 yeq/1 (0.01 and 0.10 mg/1).
5. Mulinia embryos exposed to chlorinated seawater for 2 hr
after fertilization exhibited an LC50 of about 2.0 yeq/1 (0.072 mg/1).
During post-exposure culture in static unchlorinated water, larvae
from all treatments exhibited approximately equivalent survival rates.
6. There are no physiologically significant sublethal effects of
CIO on serum constituents (osmoregulation) or oxygen consumption of
whole Callinectes sapidus or excised gills.
7. CIO in water irritates Callinectes sapidus, causing
retraction of the first antennae bearing sensory sites. Further, we
observed reduced feeding and no spawning among crabs exposed to CIO.
Feeding was restored when CIO was removed.
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SECTION 3
RECOMMENDATIONS
1. Studies of both inorganic and organic chemical speciation of
chlorinated saline water are needed in order to identify more
specifically those chemicals responsible for observed biotic effects.
Variations in observed LC50 values expressed in terms of total CIO may
derive from differences in the specific chemicals present at various
pH and ammonia levels.
2. Research should be designed specifically to examine seasonal
changes in CIO tolerance and differences in tolerance that might be
anticipated at estuarine locations of differing salinity.
3. The potential for crabs to recover from short-term exposure
to lethal doses of CIO (i.e. 96-hr LC50 and above) needs to be
evaluated from a physiological and histological perspective.
4. The physiological mechanism of ClO-induced mortality in blue
crabs remains to be ascertained. Knowledge of the mechanism is
Important to the evaluation of possible sublethal physiological
effects.
5. Improved exposure methods and test protocols need to be
developed for Mulinia (and other bivalve) larvae challenged by
chemically unstable pollutants. Attention should be given especially
to culture requirements of volume, larval density, and enumeration
techniques.
6. Quantitative studies of CIO effects on avoidance behavior,
fecundity, spawning, feeding and sensory activities of the blue crab
would be useful to better evaluate the overall ecological impact of
CIO on this species.
7. Histological examinations should be made on selected tissues
of adult crabs in an attempt to identify the organ systems affected by
CIO. Tissues of greatest interest are gills, mid-gut, aesthetascs,
hepatopancreas, and gonads.
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8. The data on biotic effects of CIO at very low concentrations
strongly suggest the need to direct more future research to
alternatives to chlorination, including chlorination/dechlorination,
bromochlorination, and ozonation.
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SECTION 4
DEVELOPMENTAL STUDIES
GENERAL METHODS
Dosing Apparatus
In order to expose all life stages of the crab species and
molluscan larvae to reasonably uniform concentrations of the
chemically unstable toxicants, chlorine fnduced-oxidants, it was
necessary to utilize a continuous flow system. The dosing system
built for the experiments described herein is diagrammatically
represented in Figure 1.
Diluent water was pumped from the York River estuary at
Gloucester Point, Virginia, through PVC pipe to the bioassay
laboratory. The water was passed through 10 ym or 5 ym GAP filter
bags (A) and collected in a reservoir (B). This water was then pumped
(C) through 1 ym Honeycomb filters (PVC core) in parallel or 10 ym and
1 ym filters (E) in series into a diluent header tank (F). Unused
filtered water was returned to the reservoir.
The diluent was delivered to a series of mixing chambers (G) by
means of siphons calibrated for short intervals (hrs) to deliver a
specified flow rate + 1% (approx.) at a given head. Chlorine was
introduced into the mixing chambers by injecting stock solutions of
Ca(OCl)2 (Ii to 15) with a Harvard peristaltic pump (H) at a rate of
1 ml/min. The mixed solution was then delivered to the test chambers
through glass delivery tubes.
A float was installed in each header tank so that if the diluent
water level fell beyond 5-10 mm, a mlcroswitch was opened,
interrupting all power to the toxicant (H) and diluent water (C)
pumps. Heaters located in the header tanks during the winter to
maintain the desired temperature were protected by the same system to
prevent damage to equipment.
Flow rates of toxicant stock and diluent were measured three
times daily during all experiments. Table 1 presents the data from
one experiment which extended over an 18-day period. The mean
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TO B
\ ' i '
t
t t t f
TO DOSE DOSE DOSE DOSE DOSE
CONTROL ABODE
B
RAW
RIVER
WATER
WASTE
Figure 1. Schematic representation of dilutor system.
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TABLE 1.
SU»1ARY OF FLOW RATE DATA (DILUENT AND TOXICANT STOCK) AND CALCULATED
DILUTION FACTORS FOR LARVAL EXPERIMENT 9
Dose Lev
Dace
8 IX
9 IX
10 IX
11 IX
12 DC
13 IX
14 IX
15 IX
16 IX
17 IX
18 IX
19 IX
20 IX
21 IX
22 IX
23 IX
24 IX
25 IX
"5
S
cv
el/ A
Oil. Stock DF
310 l.OS 0.0034
265 1.1 0.0042
295 1.35 0.0046
295 1.35 0.0046
275 1.33 0.0048
285 1.3 0.0046
285 1.33 0.0047
285 1.33 0.0047
265 1.33 0.0050
260 1.33 0.0051
276 1.3 0.0047
245 1.25 0.0051
275 1.25 0.0045
300 1.25 0.0042
315 1.35 0.0043
325 1.15 0.0035
325 1.15 0.0035
315 1.1 0.0035
315 .1 0.0035
315 .3 0.0041
315 .1 0.0035
300 .1 0.0037
285 .1 0.0039
305 1.2 0.0039
300 1.15 0.0038
270 1.1 0.0041
290 1.1 0.0038
275 1.1 0.0040
285 1.1 0.0039
285 1.1 0.0039
280 1.1 0.0039
280 1.1 0.0039
280 1.1 0.0039
295 0.9 0.0031
290 1.15 0.0040
285 1.0 0.0035
280 1.25 0.0045
280 1.25 0.0045
280 1.25 0.0045
295 1.25 0.0043
305 1.15 0.0038
250 1.25 0.0050
255 1.1 0.0043
275 1.25 0.0045
290 1.25 0.0043
290 1.2 0.0041
287.1 1.17 0.0042
18.2 0.16 0.0005
6.3 13.8 12.0
B
Oil. Stock DF
295 1.1 0.0037
280 1.1 0.0039
290 1.45 0.0050
290 .45 0.0050
300 .36 0.0045
300 .3 0.0043
300 .33 0.0044
295 .33 0.0045
275 .37 0.0050
280 1.33 0.0047
290 1.3 0.0045
255 1.25 0.0049
275 1.25 0.0045
305 1.25 0.0041
315 1.35 0.0043
325 1.15 0.0035
325 1.15 0.0035
315 1.15 0.0037
315 1.15 0.0037
310 1.3 0.0042
315 1.1 0.0035
315 1.1 0.0035
315 1.2 0.0038
305 1.13 0.0037
305 1.15 0.0038
285 1.1 0.0039
300 1.1 0.0037
280 1.1 0.0039
280 l.l 0.0039
280 1.1 0.0039
280 1.1 0.0039
275 1.1 0.0040
270 1.1 0.0041
290 1.1 0.0038
290 1.15 0.0040
300 1.1 0.0037
290 1.25 0.0043
280 1.25 0.0045
285 1.25 0.0044
280 1.25 0.0045
295 1.25 0.0042
260 1.25 0.0048
280 1.1 0.0039
280 1.25 0.0045
280 1.25 0.0045
278 1.2 0.0043
294.1' 1.21 0.0041
21.4 1.04 0.0004
7.3 8.5 10.5
C
Oil. Stock DF
300 1.1 0.0037
290 1.1 0.0038
295 1.40 0.0047
290 1.40 0.0048
290 1.42 0.0049
280 1.35 0.0048
280 1.43 0.0051
280 1.43 0.0051
260 1.37 0.0053
270 1.43 0.0053
285 1.4 0.0049
245 1.25 0.0051
250 1.20 0.0048
290 1.25 0.0052
295 1.35 0.0046
305 1.15 0.0038
325 1.15 0.0035
300 1.15 0.0038
295 1.15 0.0039
285 1.3 0.0046
300 1.1 0.0037
300 1.1 0.0037
300 1.2 0.0040
285 1.2 0.0042
290 1.15 0.0040
260 1.1 0.0042
285 1.1 0.0039
275 1.1 0.0040
280 1.1 0.0039
_
285.7 1.23 0.0044
16.9 0.13 0.0006
5.9 10.5 14.0
D
Oil. Stock DF
295 1.1 0.0037
295 1.1 0.0037
300 1.25 0.0042
295 1.20 0.0041
300 1.38 0.0046
300 1.35 0.0045
285 1.4 0.0049
280 1.4 0.0050
275 1.37 0.0049
270 1.41 0.0052
285 1.4 0.0050
245 1.25 0.0051
275 1.25 0.0045
300 1.22 0.0041
300 1.4 0.0047
310 1.15 0.0037
305 1.15 0.0038
300 1.1 0.0037
300 1.1 0.0037
290.5 1.26 0.0043
15.5 0.12 0.0006
5.3 9.7 12.7
E
Dll. Stock DF
310 1.1 0.0036
300 1.1 0.0037
300 1.40 0.0047
300 1.30 0.0043
300 1.30 0.0043
300 1.3 0.0043
300 1.33 0.0044
290 1.33 0.0046
275 1.37 0.0050
275 1.33 0.0048
290 1.3 0.0045
250 1.25 0.0050
275 1.22 0.0044
300 1.25 0.0042
310 1.3 0.0042
325 1.15 0.0035
_
_
293.8 1.27 0.0043
17.8 0.09 0.0004
6.1 7.0 10.3
-------
dilution factors (stock flow rate/diluent flow rate) calculated from
these data were 0.0042 + 0.0005, 0.0041 + 0.0004, 0.0044 + 0.0006,
0.0043 + 0.0006, and 0.0043 + 0.0004. The desired dilution rate was
0.0033. The coefficient of variation ranged from 10.3 to 14.0%.
These results are representative of all experiments.
During the early portion of the study, the fail-safe features
were not included in the system. Nevertheless, difficulties in
maintaining consistent dilution factors were not encountered during
experiments except when power failures occurred or after storms which
caused high sediment loads in the incoming estuarine water.
This basic dilutor system could be used for all tests with
various demands for total flow and concentrations of CIO simply by
changing the diluent flow rate and the stock concentrations.
The chlorine stock solutions were prepared by dissolving Ca(OCl)2
in deionized water. The chlorine demand of the deionized water used
was usually negligible; however, some experiments had to be eliminated
from consideration because stock concentrations declined drastically.
Because of this problem, stock solutions were analyzed daily for
chlorine concentration by titration with thiosulfate to a starch
end-point. Five stock solutions were used to provide the desired
concentration series in the test tanks. Since the seawater diluent
had a chlorine demand, the relationship between tank concentration and
stock concentration at specified flow rates was determined empirically
in order to be able to select the appropriate stock concentration to
produce any desired CIO residual level. Minor adjustments could be
made at the beginning of any given experiment to compensate for
variation in demand by adjusting the toxicant flow rate.
Test Chambers
Two-liter plastic aquaria were used for tests with decapod eggs
and larvae and Mulinia larvae (Fig. 2). Each aquarium (28.5 x 13.4 x
15.0 cm) had a "tidal siphon" drain to produce a fluctuating water
level in the test chamber. At an inflow rate of 250 ml/min, the
"tidal period" was approximately 7 min.
The test animals were placed in a basket constructed from PVC
pipe with a nylon mesh screen glued to the bottom. The basket was
suspended at a fixed level in the aquarium. The change in water level
in the tank served to flush the basket rapidly enough to ensure equal
total chlorine-induced oxidant concentrations inside and outside the
baskets. Maximum water volume in the culture baskets was ca. 500 ml.
The screen used for tests with Mulinia larvae had a mesh size of
26 urn while that used for decapod eggs and larvae was 274 ym. These
11
-------
A-CHLORINATED SEAWATER SUPPLY LINE
B-MIXING BAFFLE
C-LARVAL CULTURE BASKET
D-TIDAL SIPHON
Figure 2. Test chamber for egg and larval tests.
12
-------
sizes were selected as the largest size which would retain 100% of the
animals introduced or, in the case of crab larvae, the size which
would retain Artemia nauplii added as food.
The screen mesh tended to become fouled with aggregations of
particles which passed through the diluent filtration systems. The
accumulations promoted bacterial and fungal growths, especially in
controls and low chlorine doses, with reduced survival of the test
species. This could be alleviated by daily rinsing of the baskets in
a concentrated chlorine solution followed by thiosulfate to remove
residual chlorine while the test animals were removed for counts of
survivors. Control survival then equalled or exceeded survival at the
lowest test concentration(s) .
Chemical Analyses
Various parameters were measured three times daily at
approximately 0800, 1200, and 1600 hr during all tests. Temperature
and dissolved oxygen were measured in the test tanks with a YSI Model
51A oxygen meter fitted with a BOD oxygen probe- pH was measured with
a Perkin Elmer Model 28C pH meter. Salinity of samples from the
header tanks was determined with a Beckman Induction Salinometer Model
RS-7B.
Total chlorine -induced oxidant concentrations were measured by
amperometrlc titration with phenylarslne oxide (PAO) after iodination
at pH 4. A Sargent-Welch Model P Amperometric Titrator was used. A
Fisher Recordall strip chart recorder was used to amplify the signal,
thereby improving recognition of the titration end point. The
electrode system consisted of a rotating platinum-mercury electrode
against a calomel. With this system, the minimum detection level was
0.11 yeq/1 (0.004 mg C12/D, using 0.000564 N PAO and a 50 ml sample
size. Sensitivity could be increased by using a platinum against
platinum electrode combination, an electrode with a larger platinum
surface area (such as a platinum-mercury hook-type electrode) and/or a
polarograph (Andrews and Glass, 1974). However, the instrumentation
used was adequate for these tests.
All PAO solutions used were standardized against 0.0025 N
KH( 103)2* The measured normality of PAO was used in all calculations
of CIO concentrations. CIO concentrations are expressed as yeq/1
(mg/1 Cl2). 1.1 yeq - 0.04 mg/1
Carpenter et al. (1977) have reported that this method
systematically reads less CIO than is actually present. They reported
that results were improved somewhat by buffering samples to pH 2.
Back titration yielded the most accurate results. Their paper did not
appear until the present project was nearing completion, so no change
was made in analytical procedures.
13
-------
General Culture Methods
Ovigeroua Panopeus herbatii were collected exclusively on beaches
around Gloucester Ft., whereas ovigerous female hermit crabs Pagurus
longicarpus were collected either at Gloucester Pt. or from Cedar
Island near Wachapreague, Va.
Hatching of eggs from females collected locally was accomplished
by placing one or more ovigers in a small aquarium receiving a slow
flow of filtered estuarine water. The overflow from the hatching
aquarium was passed through a screened basket immersed in estuarine
water. As eggs hatched, larvae were carried out of the tank and
concentrated in the screened basket. In this way, predation by adults
on the newly hatched larvae was reduced or eliminated.
Ovigerous crabs from Wachapreague (salinity ca. 30 °/oo) with
eggs nearly ready to hatch were held in standing water of 30 °/oo
until hatching, and the larvae were acclimated to York River
salinities (18-26 °/oo). Ovigerous crabs with eggs in an early
developmental stage were first acclimated to York River salinities,
and hatching was carried out as with females from the local
population.
Mullnia lateralis adults were obtained from the VIMS field
station at Wachapreague. At the main laboratory at Gloucester Pt.,
the water in which the clams were maintained was adjusted to the
ambient salinity of ca. 20 °/oo over a 2-3 day period, after which the
clams were maintained in flowing water to which an algal mixture was
added either once daily or continuously. When larvae were needed for
experiments, spawning was induced by thermal shock and addition of
stripped sperm. Eggs were used in experiments immediately after
fertilization. Methods used for spawning induction, fertilization,
and larval handling were as described by Loosanoff and Davis (1963)
and Calabrese (1969a, 1969b, 1970). Static control cultures grown in
parallel to most chlorine tests also utilized the methods in those
papers. Food was not provided, since the larvae would only begin to
feed toward the end of the test period.
Test Protocols
Decapod Egg Development
Eggs at an early stage of development (only yolk evident on gross
examination) were stripped from the pleopods of an ovigerous female
with forceps. The egg clumps were teased apart to yield single eggs
or small clumps which could be accurately counted. Eggs from single
females were divided into groups of approximately 100 and distributed
over the series of concentrations to be tested. Each day, the eggs
were removed from the culture baskets and examined for mortalities,
signs of egg deterioration, and epizootic infestation. Periodically,
14
-------
the eggs were staged to evaluate development. Hatching was recorded
as it occurred and larvae were examined grossly for morphological
anomalies.
Decapod Larval Tests
Newly hatched stage I zoeae were placed in culture baskets, 40
zoeae per basket, and exposed to a logarithmic dose series and a
diluent control. Newly hatched Artemia nauplii were added as food.
At daily intervals, larvae were removed from the baskets and examined
for mortality. The mortality end point was taken to be lack of heart
beat. After enumeration and cleaning of the baskets, larvae were
returned to the test chambers with fresh Artemia nauplii.
Staging of larvae was accomplished periodically during long
experiments. While daily staging would have been desirable, it was
decided not to do this because of the time required and the potential
for damaging larvae in the process. Development to the megalopal and
juvenile stages, readily recognizable without aid of a microscope, was
recorded for all larvae surviving to this stage. Most intact dead
larvae were staged according to descriptions of Costlow and Bookhout
(1961) and Roberts (1969, 1970).
Mulinia Larval Tests
Two test designs were used with Mulinia larvae. In the first
design, larvae were exposed to a series of CIO concentrations in the
flowing water test system for 48 hr. Larvae were in the trochophore
stage at the start of the exposure (about 6 hr after fertilization).
At the end of the test, larvae were in the straight hinge stage. Food
was not provided as the early larval stages do not eat. After 24 hr,
larvae were removed from the baskets briefly so that the baskets could
be cleaned. No counts were made at this time, but larvae were
examined for development. After a further 24-hr exposure, larvae were
removed from the baskets and three quantitative samples counted to
estimate the numbers live and dead. Dead larvae in a 1 ml sample
placed on a Sedgwick-Rafter slide were first counted visually at 100 X
magnification. The sample was then treated with 5% formalin to kill
all larvae, and a second count was made.
To reduce the problem of loss of larvae during exposure, a second
test protocol was used. In this design, Mulinia trochophores were
exposed to chlorinated seawater in the flow-through system with flow
directed into the culture baskets for 2 hr. Larvae were then removed
and counted. Larvae were tabulated as live if any ciliary activity
could be detected; otherwise they were recorded as dead. Each
population was then cultured in static systems for a variable period
and fed with an algal mixture containing equal cell numbers of
Pyramomonas virginica, Chlorella sp. and Pseudoisochrysis paradoxa.
15
-------
Larval population sizes were estimated at 24 hr after exposure and at
48 hr intervals thereafter.
Data Analysis
Mean CIO concentrations for the treatment in each experiment were
calculated along with standard deviations and coefficients of
variation
f std. dev. , nr.,
[ x 100].
mean J
The calculated mean CIO was used in subsequent manipulations to derive
LC50 values. The data were examined for indications of excessive
mortality following major deviations in dose from the mean.
Two methods were used to calculate LC50 values for decapod
larvae. Since control survival in larval experiments was less than
100% .using the best available culture method, the data for survival
were corrected for control deaths by application of Abbott's Formula
(Finney, 1971) for larval experiments 1-6 and 9. In two experiments
noted later, control survival was markedly less than that for animals
exposed to the lowest dose. In those cases, all data was corrected to
assumed 100% survival for animals at the lowest test dose. In all
other tests survival at the lowest dose was comparable to control
survival. The corrected survival percentages were plotted against log
time in hours to determine the LT50. Log LT50 values were then
plotted against log mean CIO concentrations to produce a toxicity
curve from which the LC50 values for various time intervals could be
derived. In cases where control survival was lower than that for the
lowest CIO dose, LT50 values were generally similar to those for the
next to lowest dose. The second method of calculating LC50 values,
used in experiments 7 and 8, Involved plotting corrected survival
rates (derived as above) against log CIO concentration for each
desired time interval and reading the LC50 values from the curves.
This method was used in these cases because of the limited number of
points available for generation of the toxicity curve. Data for
experiment 9 were analyzed by both methods with close agreement in
derived LC50 values.
LC50 values for preliminary acute tests with adults of three
species of crabs were derived by the second method outlined above but
without correction for control mortalities since few or none were
observed.
In the Mulinia embryo tests, recovery of the larvae introduced
was at best less than 100%. Survival percentages were calculated as
the volume adjusted number alive at each dose divided by the volume
adjusted total number of larvae removed after 48 hr (i.e. number alive
divided by number alive plus number dead). The percentages were then
plotted against log CIO concentration to derive the LC50 values.
16
-------
Percent recoveries of larvae were calculated as total number of larvae
recovered divided by number of larvae introduced.
RESULTS
Decapod Egg Test
A marginally successful egg test was accomplished only after the
method for cleaning culture baskets was developed. Earlier tests
yielded data which could not be interpreted because control eggs were
badly Infected with bacteria and fungi, resulting in inadequate
survival and development.
Five measured CIO levels were tested: 1.33 (0.047), 2.23 (0.079),
5.78 (0.205), 25.83 (0.916), and 109.98 yeq/1 (3.90 mg/1) (Table 2 and
Fig. 3). There was a slight trend for measured CIO residuals to
decrease during the test. On two occasions, high CIO levels were
observed for short periods, once on the fourth day of the test and
again on the ninth day. The cause of the high level on the fourth
day, apparent only at the four higher test doses, is unknown. The
second anomaly on the ninth day occurred after a 5-hr power outage
during the night. This experiment was performed prior to the
installation of the fail-safe controls to turn off the toxicant
delivery pump. The system was readjusted in the morning about 3 hr
after the power failure.
Approximately 100 eggs of Panopeus herbstii in an early stage of
development were introduced into each test concentration. Counts of
live and dead eggs were made at irregular intervals for 15 days. Eggs
were considered to be dead when they became uniformly pale purple
(yolk color) without distinct yolk globules, the embryo became opaque
and a hyaline space appeared between the egg membrane and the embryo.
Live eggs on day 15 were well eyed and deemed ready to hatch.
Chlorine dosing was stopped at this time so that zoeae, as they
hatched, would not be exposed to water containing chlorine-induced
oxldants. Hatching actually occurred on days 16-18. On day 19, the
number of live and dead zoeae was determined to evaluate survival of
larvae hatched from chlorine-exposed eggs. The results are summarized
in Table 3.
Maximum percent hatch occurred at 1.33 yeq/1 (0.047 mg/1) CIO
with slightly .lower hatch observed at 0.0 yeq/1 CIO. No hatch
occurred above 2.23 yeq/1 (0.079 mg/1) CIO. All eggs exposed to
109.98 yeq/1 (3.90 mg/1) CIO were deteriorating by day 5, while those
at 25.83 yeq/1 (0.916 mg/1) CIO were deteriorating by day 8.
Twenty-nine percent of the eggs at 5.78 yeq/1 (0.205 mg/1) CIO were
alive on day 15, as indicated by presence of a heart beat; none
hatched.
17
-------
00
TABLE 2. SUFMARY OF CHLORINE-INDUCED OXIDANT CONCENTRATIONS AND WATER QUALITY DATA
MEASURED DURING DECAPOD EGG EXPOSURE TEST.
Applied dose
Mean residual
Standard deviation
Coefficient variation
Number ,of samples
Temperature
Salinity
pH
Dissolved oxygen
yeq/1
««/l
yeq/1
«g/l
Veq/1
»g/l
(°C)
(°/oo)
(g/D
Control
0.000
0.000
0.000
0.000
45
20.3
17.8
7.85
8.11
A
A. 29
0.152
1.33
0.047
0.28
0.010
21.3
45
20.3
17.8
7.93
8.09
B
10.04
0.356
2.23
0.079
0.48
0.017
22.2
45
20.3
17.8
7.93
8.08
C
22.11
0.784
5.78
0.205
1.88
0.049
24.1
45
20.3
17.8
7.94
8.11
D
46.81
1.660
25.83
0.916
9.76
0.346
37.7
45
20.3
17.8
7.88
8.08
E
121.0
4.292
109.98
3.90
16.95
0.601
15.4
14
20.3
17.8
7.99
8.12
-------
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DAYS
Figure 3. CIO concentration* measured during decapod egg test,
19
-------
TABLE 3. EGG SURVIVAL, HATCHING AND
IMMEDIATE POST-HATCH ZOEAL SURVIVAL
Measured Dose (yeq/1)
Day /Treatment
0
5
8
11
15 eggs
18 zoeae
19
% eggs £ heart
beat on day 15
% hatch
% post-hatch
survival
Control
103
65
59
50
41
22
9
39.8
21.3
40.9
1.33
108
56
42
46
31
27
7
28.7
25.0
25.9
2.23
110
(46)*
(61)
67
50
13
3
45.5
11.8
7.7
5.78
106
(43)
(39)
43
31
0
0
29.2
0
0
25.83 109.98
94
10
(0)
2**
0
0
0
0
0
0
94
0
0
0
0
0
0
0
0
0
* Numbers in parenthesis represent clearly erroneous counts. The
reason for these poor counts appears to stem from the difficulty
in dealing with clumps of eggs.
** Possibly introduced by accident on day 11.
20
-------
These data suggest that development of the eggs can proceed,
albeit with reduced survival, at CIO concentrations up to about 5.78
ueq/1 (0.205 mg/1). Hatching can occur at CIO levels up to 2.23 yeq/1
(0.079 mg/1) although fewer eggs hatched at 2.23 yaq/1 (0.079 mg/1)
than at lower doses. Usually the larvae were stage 1 zoeae at
hatching; however, one egg at 1.33 yeq/1 (0.047 mg/1) hatched as a
prezoea on day 15, but did not survive. Hatching success at 1.33
yeq/1 (0.047 mg/1) CIO was about equal to that of the control, though
both exhibited poor hatchability. However, survival of larvae hatched
from eggs exposed to 1.33 yeq/1 (0.047 mg/1) was markedly less than
that for control larvae.
There are several possible reasons for poor development and
hatching of control eggs. First, the eggs used in this experiment
were already slightly infested with epizootics, predominantly two
species of stalked ciliated protozoans. The effect of such slight
Infestations is not known, although heavy infestations usually result
in poor hatches. High doses of chlorine killed the protozoans, but
low doses (1.33 and 2.23 yeq/1) did not. Second, water flow around
the eggs in this apparatus was probably less than that provided to
eggs attached to female crabs or those cultured on a shaker table.
Reduced availability of oxygen at the egg surface could have reduced
hatching success. Thirdly, oxygen stress may have occurred during the
5 hr power failure when water flow was Interrupted. The excessive
residuals produced by the power failure do not appear to have had an
immediate effect, since excessive egg mortalities were not observed
after this event; however, this does not rule out a chronic effect*
Larval Development Exposures
The effects of chlorine on decapod larvae were assessed with
larvae from two species, the estuarine mud crab, Panopeus herbstii,
and the hermit crab, Pagurus longlcarpus. Most of the experiments
with these animals were performed during the development of the
fail-safe features of the diluter or prior to development of an
adequate filtration system to provide the desired quantities of sea
water during storm periods when sediment loads are extremely high.
Hence most experiments do not cover the entire period of larval
development.
The experiments presented here are those from which data
unaffected by exposure system break-down could be collected for 48 hr
or longer.
Panopeus herbstii
Six acute toxicity tests with Panopeus herbstii larvae were
performed, varying in duration from two to eleven days. The measured
chlorine-induced oxidant (CIO) concentrations in each exposure level
are summarized for each experiment in tabular form along with mean
21
-------
temperature, salinity, pH, and dissolved oxygen (Table 4). In
general, the coefficient of variation S/X x 100 was between 20 and
50%. The variations are believed to result from variable chlorine
demand of the diluent water which was beyond experimental control.
Daily survival rates are shown in Table 5. Despite the
variations in measured CIO concentrations, the survivorship curves do
not exhibit marked changes in slope corresponding to brief high CIO
concentrations; rather, the survivorship curves are essentially linear
over the periods of chlorine-induced mortality.
In three experiments, survival of control larvae was less than
that for one or more doses during part or all of an experiment. This
result was correlated with the aggregation of detrital material on the
screen baskets in the control tank and was either not observed or less
extensive at the test doses. Bacteria and fungi were associated with
this debris. Dissolved oxygen levels were not affected. Presumably
the control deaths resulted from bacterial and/or fungal invasion of
the larvae. Further support for this interpretation is provided by
the fact that the control animal mortality could be reduced by
exposing the larval baskets to chlorine followed by thiosulfate while
larvae were removed for counting.
The LC50 (lethal concentration for 50% of the population) was
derived from toxicity curves of log LT50 (lethal time for 50% of the
population corrected for control mortality) against log CIO
concentration for 24, 48, and 96 hr (Table 6). In experiments 1, 2,
5, and 6, the 24-hr LC50 ranged from 19.74-28.20 yeq/1 (0.7 to 1.0
mg/1), and 48-hr LC50 from 3.67 to 11.56 yeq/1 (0.13 to 0.41 mg/1) and
the 96-hr LC50 from 1.13 to 3.38 yeq/1 (0.04 to 0.12 mg/1). In
experiment 3 the LCSO's were markedly lower, about 0.28 times the mean
for experiments 1, 2, 5, and 6. LCSO's for experiment 4 could not be
set; the 24-hr LC50 was greater than 32.43 yeq/1 (1.15 mg/1) while the
48-hr LC50 was between 12.69 and 32.43 yeq/1 (0.45 and 1.15 mg/1).
Experiment 3 was performed in October, experiment 4 in early June,
i.e. at the end and the beginning of the normal spawning period,
respectively. Eggs during these periods are generally more heavily
infested with epizootics, which may affect the viability of the
resultant larvae, making them less tolerant of pollutants.
Pagurus longicarpus--
Three experiments with Pagurus longicarpus larvae yielded useful
information. The experiments were of 7, 9, and 21 days duration, the
last experiment covering the entire larval period to the first
juvenile stage.
The chlorine-induced oxidant concentrations measured during each
experiment are summarized in Table 7. The variability at every dose
22
-------
TABLE 4. SUMMARY OF CHLORINE-INDUCED OXIDANT CONCENTRATIONS AND WATER
Experiment 1 QUALITY DATA MEASURED DURING Panooeus herbstil ZOEAL EXPOSURE
10
u>
Treatment
Applied dose
yeq/1 (mg/1)
Mean CIO residual
Stand, deviation
Coeff. variation
No. of samples
Temperature ( ° C)
Salinity (°/oo)
pH**
Dissolved 02 (mg/1)
Control
0.
0.
25.
18.
7.
0
0
2*
793
7
14.
7.
2.
25.
-
-
7.
* These measurements were taken
** pH not taken.
Experiment 2
Treatment
Applied dose*
yeq/1 (mg/1)
Mean CIO residual
Stand, deviation
Coeff. variation
No. of samples
Temperature (°C)
Salinity (°/oo)
pH***
Dissolved 02 (mg/1)
Control
0.
0.
_ "
26.
18.
-
6.
0
0
0
353**
-
35
2.
1.
0.
26.
6.
A
66
33
82
38
4
2*
-
7
one
A
26
13
28
25
8
0
35
(0.52) 22.
(0.26) 11.
(0.10) 2.
25.
-
7.
day only.
-
(0.08) 8.
(0.04) 3.
(0.01) 1.
26.
6.
B
00 (0.78)
56 (0.41)
54 (0.09)
22
4
2*
-
8
B
74 (0.31)
10 (0.11)
13 (0.04)
36
8
0
35
C
30.74 (1.09)
13.25 (0.47)
1.97 (0.07)
14
4
25.2*
7.9
C
20.30 (0.72)
6.49 (0.23)
2.54 (0.09)
39
8
26.0
6.35
45.
17.
2.
25.
-
-
7.
43.
10.
3.
26.
-
6.
D
40 (1.61)
77 (0.63)
26 (0.08)
13
4
2*
-
-
7
D
71 (1.55)
72 (0.38)
67 (0.13)
34
8
0
35
E
80.37
22.56
1.41
6
4
25.2*
7.7
E
87.42
19.18
9.31
49
8
26.0
6.35
(2.85)
(0.80)
(0.53)
(3.10)
(0.68)
(0.33)
* based on measurements from one day.
** taken first day only.
*** pH not taken.
(continued)
-------
Experiment 3
TABLE 4 (continued)
ro
JS-
Treatment
Applied Dose*
yeq/1 (mg/1)
Mean CIO residual
Stand, deviation
Coeff. variation
No. of samples
Temperature (°C)
Salinity (°/oo)
pH
Dissolved 02 (mg/1)**
Control A
0.0 1.41 (0.05)
0.0 0.85 (0.03)
0.56 (0.02)
66
9
26.0** 26.0
17.45 17.45
__
7.0 6.9
B
5.08 (0.18)
2.82 (0.10)
1.69 (0.06)
60
6
26.0
17.45
6.9
C
14.95 (0.53)
5.36 (0.19)
1.97 (0.07)
37
6
26.0
17.45
7.0
D
24.25 (0.86)
12.13 (0.43)
4.23 (0.15)
35
6
26.0
17.45
7.0
E
47.10 (1.
.67)
19.18 (0.68)
11.28 (0.40)
59
7
26.0
17.45
7.0
* based on measurements from only first day.
** taken one day only.
Experiment 4
Treatment
Applied dose
ueq/1 (mg/1)
Mean CIO residual
Stand, deviation
Coeff. variation
No. of samples
Temperature (°C)
Salinity (°/oo)
pH
Dissolved 02 (mg/1)
Control A
0.0 8.45 (0.30)
0.0 2.26 (0.08)
0.56 (0.02)
25
12
23.66 23.66
17.77 17.77
7.83 7.70
8.06 7.97
B
12.97 (0.46)
4.23 (0.15)
0.85 (0.03)
20
12
23.66
17.77
7.83
8.00
C
22.28 (0.79)
6.77 (0.24)
1.41 (0.05)
21
11
23.66
17.77
7.83
8.07
D
35.25 (1.25)
12.69 (0.45)
3.38 (0.12)
27
11
23.66
17.77
7.83
7.97
E
61.20 (2.
32.43 (1.
9.87 (0.
30
7
23.66
17.77
7.80
8.03
17)
15)
35)
(continued)
-------
Experiment 5
TABLE 4 (continued)
Treatment
Applied dose
yeq/1 (mg/1)
Mean CIO residual
Stand, deviation
Coeff. variation
No. of samples
Temperature (°C)
Salinity (°/oo)
pH
Dissolved 02 (mg/1)
Control
0.0
0.0
26.58
20.71
7.71
6.67
A
11.00 (0.39)
1.13 (0.04)
0.28 (0.01)
25
9
26.58
20.71
7.75
6.62
B
17.20 (0.61)
1.69 (0.06)
0.56 (0.02)
33
8
26.58
20.71
7.76
6.68
C
30.17 (1.07)
8.74 (0.31)
7.05 (0.25)
81
7
26.58
20.71
7.76
6.68
D
55.27 (1.96)
14.95 (0.53)
5.92 (0.22)
40
7
26.58
20.71
7.78
6.70
E
92.22 (3
35.53 (1
18.05 (0
51
2
26.58
20.71
7.79*
6.90*
.27)
.26)
.64)
* only one measurement taken.
NJ
U1
Experiment 6
Treatment
Applied dose
yeq/1 (mg/1)
Mean CIO residual
Stand, deviation
Coeff. variation
No. of samples
Temperature (°C)
Salinity (°/oo)
pH
Dissolved 02 (mg/1)
Control
0.0
0.0
29
26.96
20.03
7.81
6.16
A
8.46 (0.30)
0.85 (0.03)
0.28 (0.01)
33
29
26.96
20.03
7.84
6.30
B
15.23 (0.54)
1.97 (0.07)
0.56 (0.02)
28
29
26.96
20.03
7.85
6.28
C
23.69 (0.84)
4.51 (0.16)
1.13 (0.04)
25
29
26.96
20.03
7.88
6.36
D
31.30 (1.11)
8.74 (0.31)
4.51 (0.16)
52
19
26.96
20.03
7.83
6.45
E
60.07 (2
24.82 (0
10.72 (0
43
5
26.96
20.03
7.70*
6.40*
.13)
.88)
.38)
* only one measurement taken.
-------
Experiment 1
TABLE 5. SURVIVAL RECORDS FOR Panopeus herbstii
ZOEAE FOR EACH CIO EXPOSURE TEST
10
Day
0
1
2
Control
n %S
40 100
32 80
26 65
Measured Dose (yeq/1)
7.33
n %S
40 100
23 57.5
12 30
11.56
n %S
40 100
31 77.5
13 32.5
13.25
n %S
40 100
20 50
9 22.5
17.77
n %S
40 100
17 42.5
0 0
Z2.56
n %S
40 100
7 17.5
0 0
Experiment 2
Day
0
1
2
3
4
5
Control
n %S
37 100
27 73
18 49
16 43.2
10 27.0
6 16.2
Measured Dose (yeq/1)
1.13
n %S
33 100
26 78.8
26 78.8
21 63.6
17 51.5
15 45.5
3.10
n %S
34 100
30 88.2
25 73.5
2 5.9
0 0
0 0
6.49
n %S
30 100
23 76.7
19 63.3
0 0
0 0
0 0
10.72
n ZS
35 100
27 77.1
25 71.4
0 0
0 0
0 0
19.18
n %S
30 100
1 3.3
0 0
0 0
0 0
0 0
Experiment 3
Day
0
1
2
3
4
Control
n ZS
40 100
34 85
33 82.5
29 72.5
1 2.5
Measured Dose (yeq/1)
0.85
n %S
40 100
33 82.5
24 60
17 42.5
15 37.5
2.82
n %S
40 100
38 95
0 0
0 0
0 0
5.36
n %S
40 100
19 47.5
0 0
0 0
0 0
12.13
n %S
40 100
0 0
0 0
0 0
0 0
19.18
n %S
40 100
0 0
0 0
0 0
0 0
(continued)
-------
to
-4
Experiment 4
TABLE 5 (continued)
Day
0
1
2
3
Control
n ZS
20 100
18 90
18 90
18 90
Measured Dose (yeq/1)
2.26
n ZS
20 100
19 95
18 90
17 85
4.23
n ZS
20 100
19 95
17 85
15 75
6.77
n ZS
20 100
17 85
17 85
16 80
12.69
n ZS
20 100
17 85
17 85
13 65
32.43
n ZS
20 100
18 90
0 0
0 0
Experiment 5
Day
0
1
2
3
4
5
Control
n %S
20 100
14 70
14 70
13 65
12 60
12 60
Measured Dose (yeq/1)
1.13
n ZS
20 100
18 90
14 70
10 50
8 40
0 0
1.69
n ZS
20 100
17 85
12 60
5 2.5
0 0
0 0
8.74
n ZS
20 100
18 90
13 65
0 0
0 0
0 0
14.95
n ZS
20 100
13 65
0 0
0 0
0 0
0 0
35.53
n ZS
20 100
0 0
0 0
0 0
0 0
0 0
-------
00
Experl
Day
0
1
2
3
4
5
6
7
8
9
10
mont 6 TABLE 5 (continued)
Cout. rol
n ZS
20 100
14 70
13 65
13 65
11 55
8 40
6 30
6 30
4 20
3 15
2 10
Measured Dose (peq/1)
0.85
n ZS
20 100
12 60
11 f)r)
11 55
11 55
9 45
9 45
9 45
9 45
9 45
9 45
1.97
n ZS
20 100
15 75
14 70
13 65
12 60
11 55
11 55
4 20
4 20
4 20
3 15
4.51
n ZS
20 100
13 65
10 50
5 25
1 5
0 0
0 0
0 0
0 0
0 0
0 0
8.74
n ZS
20 100
12 60
6 30
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
24.82
n ZS
20 100
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
-------
TABLE 6. LETHAL COWCENT8ATIONS OF CHLORINE-INDUCED
LARVAE
Exp't.
Ho.
I
2*
3
4
5
6*
Duration
of exp't.
48 hr
144 hr
120 hr
96 hr
144 hr
264 hr
ueq/1
mg/1
ueq/1
mg/1
Ueq/1
fflg/1
ueq/1
mg/1
Ueq/1
«g/l
Ueq/1
«g/l
LC50
24 hr
19.74
0.70
* 19.74
v 0.70
^ 6.77
^ 0.24
>32.43
>1.15
28.20
1.0
25.38-28.20
0.9-1.0
48 hr
11.56
0.41
8.18
0.29
1.69
0.06
12.69
-------
TABLE 7.
Experiment 7
SUMMARY OF CHLORINE-INDUCED OXIDANT CONCENTRATIONS AND WATER QUALITY
(JO
O
Treatment
Applied dose
yeq/1 (mg/1)
Mean CIO residual
Stand, deviation
Coeff. variation
No. of samples
Temperature ( ° C)
Salinity (°/oo)
PH
Dissolved 02 (mg/1)
Control
0.0
0.0
26.93
18.86
7.89
6.94
A
2.82 (0.10)
2.54 (0.09)
1.97 (0.07)
78
21
__
7.83
6.91
_
B
3.67 (0.13)
2.82 (0.10)
1.41 (0.05)
50
16
__
7.87
8.01
*
C
11.84 (0.42)
7.05 (0.25)
4.51 (0.16)
64
16
__
7.83
6.96
D
21.71 (0.77)
18.89 (0.67)
15.51 (0.55)
82
13
__
7.90
7.24
E
33.56 (1.
36.10 (1.
1.92 (0.
5
13
__
7.98
7.23
19)
28)
68)
Experiment 8
Treatment
Applied dose
yeq/1 (mg/1)
Mean CIO residual
Stand, deviation
Coeff. variation
No. of samples
Temperature (°C)
Salinity (°/oo)
pH
Dissolved 02 (mg/1)
Control
0.0
0.0
27.63
20.96
7.87
6.90
A
16.64 (0.59)
0.85 (0.03)
0.28 (0.01)
33
22
7.91
6.90
B
31.30 (1.11)
1.97 (0.07)
0.56 (0.02)
28
22
7.94
6.93
C
40.61 (1.44)
2.82 (0.10)
0.56 (0.02)
20
22
7.94
6.90
D
70.22 (2.49)
7.05 (0.25)
1.41 (0.05)
20
13
7.90
7.10
E
120.98 (4
7.61 (0
0.85 (0
11
11
7.93
7.05
.29)
.27)
.03)
(continued)
-------
u>
Experiment 9
Treatment
Applied dose
yeq/1 (mg/1)
Mean CIO residual
Stand, deviation
Coeff. variation
No. of samples
Temperature (°C)
Salinity (°/oo)
pH
Dissolved 07 (mg/1)
TABLE 7 (continued)
Control
0.0
0.0
23.4
21.21
7.81
7.02
A
3.38 (0.12)
0.56 (0.02)
0.28 (0.01)
50
47
7.82
6.98
B
7.05 (0.25)
1.41 (0.05)
0.56 (0.02)
40
47
7.82
6.97
C
10.72 (0.38)
2.54 (0.09)
0.85 (0.03)
33
31
7.84
6.96
D
25.10 (0.89)
5.08 (0.18)
1.13 (0.04)
22
18
7.80
6.84
E
38.07
10.15
1.41
14
15
7.76
6.82
(1.35)
(0.36)
(0.05)
-------
level in experiment 7 was very high with coefficients of variation
from 53-83%. In the other two experiments, the coefficients of
variation were less than 40%.
The mortality of control animals was relatively uniform during
each experiment. In experiments 7 and 9, control survivorship was
quite similar to survivorship at the lowest dose (Table 8). The daily
point estimates of the survivorship of animals at the lowest dose
generally fell within the 95% confidence interval for the survivorship
of the control animals. In experiment 8, survival of animals in the
lowest dose was unusually low at 192-hr exposure and thereafter, while
control survivorship was similar to that in experiments 7 and 9.
Survival of static control cultured animals was markedly less than
that for control animals in experiments 8 and 9, suggesting that the
flow-through culture system was adequate for culture of these animals.
The LCSO's for experiment 7 and 8 were obtained by plotting
corrected per cent survival against log dose; whereas for experiment 9,
the LCSO's were derived as for Panopeus larvae. The analysis method
used in experiment 7 and 8 was necessary because of the limited number
of points available to prepare a toxicity curve (log LT50 vs. log
dose) . (Values for experiment 9 by both methods were in close
agreement.) The LC50 values are presented in Table 9.
The lethal concentrations derived from these three experiments
for each time interval are generally similar. The 24-^ir LC50 is
approximately 11.28 yeq/1 (0.4 mg/1), the 48-hr LC50 between 4.51 and
8.74 yeq/1 (0.16 and 0.31 mg/1), the 96-hr LC50 between 1.69 and 2.82
yeq/1 (0.06 and 0.1 mg/1), and the 120-hr LC50 about 1.41 yeq/1 (0.05
mg/1).
The survival data for the entire larval period studied in
experiment 9 are shown in Fig. 4 (dose relationship seen here is
characteristic of all decapod larval tests). In this experiment, all
baskets were rinsed with chlorine followed by thiosulfate on a dally
basis after removal of larvae. Survival of the control animals
throughout the test period did not differ significantly from that of
the animals subjected to the lowest CIO dose of 0.56 yeq/1 (0.02 mg
C12/D and was significantly better than that from a control group in
static culture. Larvae exposed to doses of 1.41 yeq/1 (0.05 mg/1) and
above exhibited reduced survival relative to the control animals.
In addition to the effect of CIO on survival, there was a marked
effect on the rate of development even at the lowest dose where no
effect on survival was observed. The molt from zoea I to zoea II was
complete after day 4 or 5 in the control, 0.56, 1.41, and 2.54 yeq/1
(0.02, 0.05, and 0.09 mg/1) cultures; but no zoea II were observed at
5.08 and 10.15 yeq/1 (0.18 and 0.36 mg/1) prior to death. The larvae
exposed to 2.54 yeq/1 (0.09 mg/1) died in zoea II. Those larvae
exposed to 1.41 yeq/1 (0.05 mg/1) which reached the megalopal instar
32
-------
co
to
Experiment 7
TABLE 8. SURVIVAL RECORDS FOR Pagurus longicarpus
ZOEAE FOR EACH CIO EXPOSURE TEST
Day
0
1
2
3
4
5
6
Control
n ZS
20 100
18 90
17 85
10 50
10 50
10 50
10 50
Measured Dose (ueq/1)
2.54
n ZS
20 100
18 90
16 80
16 80
16 80
14 70
5 25
2.82
n ZS
20 100
20 100
17 85
13 65
6 30
0 0
0 0
Experiment 8
Day
0
1
2
3
4
5
6
7
8
Static Control
n ZS
40 100
35 87.5
23 57.5
22 55
13 32.5
8 20
8 20
8 20
8 20
Control
n ZS
40 100
38 9b
37 92.5
34 85
34 85
29 72.5
29 72.5
28 70
27 67.5
7.05
n ZS
20 100
20 100
15 75
12 60
0 0
0 0
0 0
18.89
n ZS
20 100
0 0
0 0
0 0
0 0
0 0
0 0
36.10
n ZS
20 100
0 0
0 0
0 0
0 0
0 0
0 0
Measured Dose (yeq/1)
0.85
n ZS
40 100
36 90
35 87.5
32 80
31 77.5
25 62.5
1 2.5
1 2.5
1 2.5
1.97
n ZS
40 100
37 92.5
32 80
27 67.5
7 17.5
1 2.5
1 2.5
1 2.5
1 2.5
2.82
n ZS
40 100
39 97.5
32 80
28 70
18 45
5 12.5
1 2.5
1 2.5
1 2.5
7.05
n ZS
40 100
39 97.5
28 70
17 42.5
0 0
0 0
0 0
0 0
0 0
7.61
n ZS
40 100
32 80
7 17.5
0 0
0 0
0 0
0 0
0 0
0 0
(continued)
-------
Experiment 9
TABLE 8 (continued)
Day
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Static
Control 1
n %S
40 100
39 97.5
36 90
34 85
32 80
23 57.5
18 45
15 37.5
15 37.5
11 27.5
9 22.5
9 22.5
7 17.5
5 12.5
5 12.5
5 12.5
4 10
4 10
4 10
2 5
Static
Control 2
n %S
40 100
39 97.5
38 95
36 90
31 77.5
19 47.5
12 30
12 30
11 27.5
7 17.5
7 17.5
6 15
6 15
6 15
6 15
6 15
3 7.5
3 7.5
3 7.5
3 7.5
Control
n %S
40 100
38 95
38 95
38 95
35 87.5
34 85
30 75
30 75
27 67.5
26 65
20 50
20 50
20 50
20 50
20 50
20 50
19 47.5
16 40
14 35
12 30
9 22.5
9 22.5
Measured Dose (jieq/1)
0.56
n %S
40 100
40 100
39 97.5
39 97.5
38 95
36 90
36 90
35 87.5
34 85
29 72.5
26 65
23 57.5
22 55
21 52.5
21 52.5
21 52.5
20 50
17 42.5
17 42.5
9 22.5
8 20
8 20
1.41
n %S
40 100
40 100
39 97.5
35 87.5
32 80
29 72.5
28 70
24 60
23 57.5
23 57.5
13 32.5
11 27.5
9 22.5
6 15
5 12.5
5 12.5
5 12.5
5 12.5
4 10
4 10
4 10
3 7.5
2.54
n ZS
40 100
40 100
40 100
37 92.5
31 77.5
21 52.5
18 45
15 37.5
11 27.5
4 10
0
5.08
n ZS
40 100
40 100
38 95
33 82.5
18 45
7 17.5
0
0
10.15
n ZS
40 100
39 97.5
34 85
23 57.5
10 25
0
"
-
U)
-------
TABLE 9. LETHAL CONCENTRATIONS OF CHLORINE-INDUCED
OXIDANTS FOR Paeurus loneicarous LARVAE
Exp't. No.
7
8
9
Exp'tal
Duration
144 hr
192 hr
504 hr
yeq/1
mg/1
yeq/1
mg/1
yeq/1
mg/1
24 hr
11.56
0.41
(11.84)
(0.42)
>11.28
> 0.40
48 hr
8.74
0.31
7.61
0.27
4.51
0.16
96 hr
2.82
0.10
2.82
0.10
1.69
0.06
120 hr
1.41
0.05
1.41
0.05
( ) » extrapolated to 50% survival; no dose had less than 50%
survival during the first 24 hr.
35
-------
CONTROL
0.02
0.05
0.09
0.18
0.36
STATIC CONTROL
\
24 48 72 96 120 144 168 192 216 24O 264 288 312 336 360 384 408 432 456 480 5O4
TIME (HOURS)
Figure 4. Survivorship curves for Pagurus longicarpus zoeae, experiment 9.
-------
(3 animals) did so 5-6 days later than the control animals. The
larvae exposed to 0.56 yeq/1 (0.02 mg/1) reached the megalopal instar
about 1 day later than the controls.
Juvenile Crab Tests
An exploratory experiment with Panopeus herbstii juveniles was
carried out with feral specimens. The purposes of this experiment
were to determine an appropriate dose range for further study and to
explore what problems might arise in long term exposures (feeding
requirements, cannibalism, disease potential) and what might be done
to compensate.
The doses tested ranged from 0.85 to 45.97 ueq/1 (0.03 to 1.63
mg/1) (Table 10). Although concentrations were very consistent for
periods of a week or more, storms and equipment problems temporarily
interrupted diluent flow and prevented maintenance of consistent
concentrations for the entire period. Overall, coefficients of
variation for all treatments ranged from 29 to 66%. The most variable
treatment was the highest dose, which caused complete mortality within
72 hr.
During the first 22 days of the experiment, no substrate was
provided to the crabs. Survival rates were high for the first 10 days
(80-100%) except for the highest doses (Table 11). Thereafter,
survival rates decreased markedly in all treatments including the
control. Observation indicated that most if not all deaths resulted
from agonistic behavior and cannibalism. Therefore, it was concluded
that some type of substrate was necessary to allow crabs to hide,
especially during molting.
Two substrates were tested: a layer of sand about 2 cm thick and
an assemblage of oyster shells. One tank of each pair at a given dose
was provided with the shell substrate (Treatment I), the other with
sand substrate (Treatment 11). Mortality rates declined following
provision of substrates, with some slight advantage seemingly provided
by the shell substrate. With the shell substrate, crabs utilized the
entire bottom of the aquarium, spending considerable time hiding under
shells. Crabs with no substrate or the sand substrate tended to
aggregate along the edges of the tank, leading to local high density.
With the sand substrate, wandering was reduced as crabs dug into the
substrate somewhat, thereby reducing the likelihood of agonistic
encounters.
The food provided in this experiment consisted of chopped frozen
fish or squid. No attempt was made to quantify food provided. The
control crabs and those at low CIO doses fed actively on this food.
At higher doses, the crabs were generally less active and were less
aggressive in attacking food. No attempt was made to quantify these
observations.
37
-------
TABLE 10. SUMMARY OF CHLORINE-INDUCED OXIDANT CONCENTRATIONS AND WATER QUALITY DATA
FOR Panopeus herbstii JUVENILE TEST
00
Control
I
Mean CIO residual peq/1 0.00
(mg/1)
Std. dev. ueq/1
(mg/1)
Coef. var.
No. samples
Temperature °C
Salinity, °/oo
pH*
Dissolved 02, mg/1
II
0.00
19.7
16.8
7.9
7.9
A
I
0.85
(0.03)
0.28
(0.01)
33
42
7
7
II
0.85
(0.03)
0.56
(0.02)
66
42
.9
.9
I
1.69
(0.06)
0.56
(0.02)
33
42
7
7
B
II
1.97
(0.07)
0.85
(0.03)
43
42
.8
.9
I
4.79
(0.17)
2.26
(0.08)
47
42
7
7
C
II
4.79
(0.17)
1.41
(0.05)
29
42
.9
.9
I
7.90
(0.28)
3.67
(0.13)
46
42
7
7
D
II
7.61
(0.27)
3.10
(0.10)
41
42
.9
.9
E
I
45.97
(1.63)
30.46
(1.08)
66
5
N.D.
N.D.
N.D. - no data
* - no data for first 19 days due to instrument malfunction
CIO expressed as yeq/1 (mg/1)
-------
TABLE 11. SURVIVAL DATA FOR Panooeus herbstii JUVENILE TEST
Measured Dose (ueq/1)
Day
0
1
2
3
4
5
6
7
8
9
10
.11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
Control
I
10
10
10
10
10
10
10
10
10
10
8
6
6
4
4
4
4
4
4
4
4
4
4
4
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
II
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
9
9
9
9
9
9
9
9
9
9
9
9
9
8
8
8
8
8
8
8
6
6
6
6
6
0.85
I
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
8
0.85
II
10
10
10
10
10
10
10
10
10
10
10
9
9
8
8
8
8
7
7
7
7
6
5
5
5
5
5
5
5
5
5
5
5
4
4
4
3
3
3
3
3
1.69
I
10
10
10
10
10
10
9
9
9
9
9
9
9
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
1.97
II
10
10
10
10
9
9
9
9
9
9
9
9
9
9
9
9
9
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
4.79
I
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
4.79
II
10
10
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
8
8
8
8
8
8
8
6
5
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
7.90
I
10
10
10
10
10
10
10
10
10
10
10
10
10
9
9
9
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
7.61
II
10
10
10
10
10
9
9
9
9
9
9
9
9
8
8
8
8
6
5
4
4
4
4
4
3
3
3
3
2
2
2
1
1
1
1
1
1
1
1
1
1
45.97
I
7
5
1
0
~
39
-------
Deaths at the highest dose level of 45.97 yeq/1 (1.63 mg/1) were
attributable to the chlorinated seawater. An approximate 96-hr LC50
of 14.10 eq/1 (0.50 mg/1) was estimated. No deaths at any other dose
were clearly attributable to the chlorine although the exposure
continued for 40 days. Juvenile crabs can tolerate exposure to at
least 7.90 yeq/1 (0.28 mg/1) for extended periods.
Exploratory tests were also conducted with Pagurus longlcarpus
adults and Calllnectes sapidus Juveniles in order to determine the
approximate toxic concentrations for each species. The 24-hr, 48-hr
and 96-hr LCSO's for Pagurus adults were estimated to be 16.07, 14.10,
and 5.92 yeq/1 (0.57, 0.50, and 0.2 mg/1) respectively. The 24-hr, 48~
hr, and 96-hr LCSO's estimated for Callinectes Juveniles were 12.41,
11.84, and 9.02 yeq/1 (0.44, 0.42, and 0.32 mg/1)frespectively. These
values must be considered preliminary pending more careful and
detailed experiments.
Mulinla lateralls Experiments
Several experiments were performed with Mulinia lateralls larvae
exposed for the 48 hr immediately following fertilization. The
exposure conditions for the larval experiments are summarized in Table
12. In experiment 1, test condition A had a higher mean CIO residual
than condition B although the range was essentially the same. At
these doses and below, it becomes increasingly difficult to maintain
consistent residuals, and analytical error Increases greatly. This is
obvious in experiment 2 in which the lowest dose was 0.25 yeq/1 (0.019
mg/1) with a coefficient of variation of about 100%. The temperature
in experiment 1 was low (18°C), but sufficient to allow development to the
straight hinge.
Experiment 1 was conducted before institution of the basket
cleaning process. Control survival was less than that for the lowest
dose as was total percent recovery of the animals introduced (Table
13). Control survival and recovery were also markedly less than the
same parameters for a static control culture.
Only the lowest dose tested (0.99 yeq/1 or 0.035 mg/1) permitted
survival. At 1.04 yeq/1 (0.037 mg/1), all dead animals had failed to
reach the straight hinge stage. Recovery percentage declined with
Increasing dose. This has been characteristic of tests with oyster
larvae as well. It Is not clear whether chlorine simply falls to
preserve the larvae, or whether the dead larvae are oxidized by the
chlorine.
Poor survival in the control and survival at only one test dose
preclude determination of an LC50. It would appear, however, that the
LC50 is less than 1.41 yeq/1 (0.05 mg/1) and probably greater than
0.85 yeq/1 (0.03 mg/1) since survival and recovery of larvae at the
lowest dose approximated results from the static culture.
40
-------
TABLE 12. SUMMARY OF CHLORINE-INDUCED OXIDANT CONCENTRATIONS AND WATER QUALITY
Experiment 1
DATA MEASURED DURING Mulinia lateralis EMBRYO TESTS
Treatment
Applied dose
ueq/1 (mg/1)
Mean CIO residual
Stand, deviation
Coeff. variation
No. of samples
Temperature ( ° C)
Salinity (°/oo)
pH
Dissolved 02 (mg/1)
Static
Control
0.0
0.0
j
"
Control
0.0
0.0
_
18.33
17.96
7.85
8.43
A
5.41 (0.192)
1.04 (0.037)
0.71 (0.025)
68
7
._
7.86
8.47
B C
6.66 (0.236) 26.37 (0.935)
0.99 (0.035) 1.78 (0.063)
0.59 (0.021) 0.37 (0.013)
60 21
7 7
JIL,
7.86 7.83
8.40 8.43
D
30.54 (1.083)
2.48 (0.088)
0.31 (0.010)
13
7
._ ^ ,
7.83
8.43
Experiment 2
Treatment
Applied dose
ueq/1 (mg/1)
Mean CIO residual
Stand, deviation
Coeff. variation
No. of samples
Temperature ( ° C)
Salinity (°/oo)
PH
Dissolved 0. (mg/1)
Control
0.0
0.0
23.5
18.21
8.00
7.65
A
1.69 (0.
0.25 (0.
0.25 (0.
100
7
8.05
7.05
B
060) 13.87 (0
009) 0.76 (0
009) 0.23 (0
30
7
8.00
7.70
C D
.492) 5.33 (0.189) 9.73 (0.345)
.027) 1.33 (0.047) 2.17 (0.077)
.008) 0.45 (0.016) 0.59 (0.021)
34 27
7 7
8.00 " 8.00
7.80 7.75
E
15.81 (0.561)
3.86 (0.137)
0.82 (0.029)
21
7
8.05
7.75
(continued)
-------
Experiment 3
TABLE 12 (continued)
NJ
Treatment
Applied dose
peq/1 (mg/1)
Mean CIO residual
Stand, deviation
Coeff. variation
No. of samples
Temperature (°C)
Salinity (°/oo)
PH
Dissolved 02 (mg/1)
Static
Control Control
0.0 0.0
0.0 0.0
__ _
28.0
20.0
8.27
7.25
A
1.78 (0.063)
0.53 (0.019)
0.11 (0.004)
20
6
X_IM
8.37
7.26
B
2.48 (0.088)
0.96 (0.034)
0.45 (0.016)
46
6
^
8.37
7.47
C
6.26 (0.222)
1.72 (0.061)
0.51 (0.018)
29
6
-_ _,
8.30
7.30
D
10.01 (0.355)
3.47 (0.123)
2.03 (0.072)
59
6
__
8.30
7.18
E
16.75 (0.
7.33 (0.
0.93 (0.
13
6
__
8.33
7.23
594)
260)
033)
Experiment 4
Treatment
Applied dose
yeq/1 (mg/1)
Mean CIO residual
Stand, deviation
Coeff. variation
No. of samples
Temperature (°C)
Salinity (°/oo)
PH
Dissolved 02 (mg/1)
Static
Control Control
0.0 0.0
0.0 0.0
25.8
22.8
7.67
7.19
A
1.64 (0.058)
0.28 (0.010)
0.39 (0.014)
146
6
__
7.60
7.17
B
2.62 (0.093)
0.53 (0.019)
0.11 (0.004)
22
6
__
7.53
7.22
C
4.94 (0.175)
1.18 (0.042)
0.31 (0.011)
27
6
-__
7.60
7.20
D
10.41 (0.369)
2.26 (0.080)
0.45 (0.016)
20
6
__ _
7.60
7.20
E
18.89 (0.
5.64 (0.
0.62 (0.
11
6
_
7.63
7.20
670)
200)
022)
(continued)
-------
Experiment 5
TABLE 12 (continued)
Static
Treatment Control
Applied dose 0.0
yeq/1 (mg/1)
Mean CIO residual 0.0
Stand, deviation
Coeff. variation
No. of samples
Temperature (°C)
Salinity (°/oo)
pH*
Dissolved 02*(fflg/l)
Control A B C D
0.0 1.40
0.0 0.45
0.09
19
6
21.0
15.0
«* r
(0.049) 2.78
(0.016) 0.90
(0.003) 0.09
9
6
__
(0.099) 5.04
(0.032) 1.24
(0.003) 0.37
28
6
ilium
(0.179) 9.08 (0.322)
(0.044) 2.40 (0.085)
(0.013) 0.25 (0.009)
10
6
~
-_^
E
16.79
5.64
2.23
39
6
_.,_
(0.556)
(0.200)
(0.079)
No data.
-------
t>
C-
TABLE 13., SURVIVAL, MORTALITY AND RECOVERY RESULTS
Experiment 1 FOR 48-flR Mulinia lateralis EMBRYO EXPOSURE TESTS
CIO
concentration
ueq/1 (mg/1)
0.00 0.000
0.99 0.035
1.04 0.037
1.78 0.063
2.48 0.088
Static Control
Experiment 2
CIO
concentration
yeq/1 (tng/1)
0.00 0.000
0.25 0.009
0.76 0.027
1.33 0.047
2.17 0.077
3.86 0.137
Initial
number
6090
5250
5670
4830
4830
3780
Live @ 48 hr
(straight hinge)
%
N survival
3100 89
3550 82
0 0
0 0
0 0
2800 92
Initial
number
4074
4912
4340
4774
4466
4453
Live @ 48 hr
(straight hinge)
%
N survival
240 86
160 77
198 69
119 37
0 0
0 0
Dead @ 48 hr
(straight hinge) (Embryo)
N N
400 0
800 0
0 1267
0 417
0 100
250 0
%
recovery
57
83
22
9
2
81
Dead @ 48 hr
(straight hinge) (Embryo)
N N
40 0
47 0
90 0
203 0
299 1131
0 210
%
recovery
7
4
7
7
32
5
(continued)
-------
Experiment 3
TABLE 13 (continued)
CIO
Concent rat ion
peq/1 (mg/1)
0.00 0.000
0.53 0.019
0.96 0.034
1.72 0.061
3.47 0.123
7.33 0.260
Static Control
Initial
Number
8165
7495
6994
10472
8175
7228
8342
Live @ 48 hr
Z
N Survival
113 81
644 58
1380 90
287 56
292 55
0 0
4806 95
Dead @ 48 hr
N
27
468
160
230
240
0
267
Z
recovery
2
15
22
5
7
0
61
Experiment 4
CIO
Concent ration
yeq/1 (mg/1)
0.00 0.000
0.28 0.010
0.53 0.019
1.18 0.042
2.26 0.080
5.64 0.200
Static Control
Initial
Number
4464
4096
3869
5320
3735
4785
4512
Live @ 48 hr
%
N Survival
840 72
208 34
209 42
116 35
0 0
0 0
3239 88
Dead @ 48 hr
N
322
399
285
213
15
0
430
Z
recovery
26
15
13
6
0.4
0
81
(continued)
-------
Experiment 5
TABLE 13 (continued)
CIO
Concentration
yeq/1 (mg/1)
0.00 0.000
0.45 0.016
0.90 0.032
1.24 0.044
2.40 0.085
5.64 0.200
Static Control
Initial
Number
5205
5175
5236
5440
4940
5553
5260
Live @ 48 hr
2
N Survival
2728 100
116 100
240 71
0 0
0 0
0 0
4660 89
Dead @ 48 hr
N
0
0
100
100
121
234
0
%
recovery
52
2
6
2
2
4
89
-------
Overall recovery of larvae In experiment 2 was poor (4 to 7% In
most treatments) because larval baskets with 37 ym mesh were used
instead of baskets with 26 ym. Mulinia embryos are larger than 37 ym,
but may be deformed to pass through 37 m at the 1-, 2-, and 4-cell
stages.
Experiment 2 was carried out after the basket cleaning process
was instituted. Control survival exceeded survival at all dose levels
(Table 13). The static control was inadvertently discarded before
counting. Therefore, we cannot evaluate how much of the observed
mortality was attributable to culture conditions versus viability of
the larval batch.
Survival was reduced progressively with increasing dose beginning
with the lowest dose (0.25 yeq/1 or 0.009 mg/1). No embryos survived
at 2.17 yeq/1 (0.077 mg/1) or above. At 2.17 yeq/1 (0.077 mg/1) some
embryos developed to the straight hinge stage prior to death, whereas
at 3.86 yeq/1 (0.137 mg/1) all animals died as embryos. The 48-hr
LC50 determined from these data was 1.07 yeq/1 (0.038 mg/1) within the
range suggested by the first experiment.
Experiments 3, 4, and 5 were conducted with water entering the
culture baskets directly rather than using the tidal siphons. Percent
recoveries of larvae were again low. The LCSO's for these experiments
cannot be precisely estimated statistically. The LC50 for experiment
3 appears to be over 2.82 yeq/1 (0.1 mg/1). For experiment 4, the
LC50 is much lower near 0.28 yeq/1 (0.01 mg/1). In experiment 5, the
,LC50 was about 0.93 yeq/1 (0.033 mg/1). The low recovery rates for
larvae and the difficulties in maintaining consistent CIO levels over
a 48 hr test at these low concentrations do not allow a stronger
statement than that the LC50 for 48 hr continuous exposure lies
between 0.28 yeq/1 and 2.82 yeq/1 (0.01 mg/1 and 0.1 mg/1).
When larvae were exposed for only 2 hr in the continuous flow
system (experiments 6 and 7), larval recoveries were generally better
than 50% and often around 100% (Table 14). Survival rates were also
high except at CIO levels above 2.82 yeq/1 (0.1 mg/1). The 2 hr
LCSO's for these experiments were 2.06 yeq/1 (0.073 mg/1) and 1.97
yeq/1 (0.070 mg/1), respectively. The survival curves during the
subsequent static culture period were approximately parallel
indicating no residual mortality effects after removal from
chlorinated water. Larvae did not grow significantly in these 9 day
experiments even in control cultures and hence did not metamorphose.
This probably reflects the inadequacy of small culture volumes and
high larval densities for culture of bivalve larvae (Dupuy et al.,
1977; Windsor, 1977).
47
-------
TABLK
SURVIVAL AND RHCOVKRY RKSU1.TS FOR 2-HRMullnia KMRRYO
oo
Tlm.
he fore
exposure
after
exposure
2/« hr.
72 hr.
120 hr.
168 hr.
N
N
Z S
Z R
N
Z S
N
Z S
N
Z S
N
Z S
0.00
9453
9072
96
96
6536
72
4933
54
1067
12
660
7
0.25
(0.009)
9372
7728
82
82
6467
84
2811
36
1353
18
1276
17
CIO Concent
Ueq/l (m«
0.51
(0.018)
9675
8064
83
83
7397
92
6650
82
2920
36
1133
14
rn t 1 on
/D
0.79
(0.028)
10234
9406
92
92
8900
95
6673
71
2520
27
2210
23
1.5?
(0.054)
10361
7666
74
74
*
H
__
_
~~
2.99
(1.00)
10133
1995
20
52
**
1603
80
1650
82
550
28
Stat Ic
Control
9322
9430
101
101
5394
57
5647
60
2532
27
1325
14
* This culture was spilled when the 24 hr. count was attempted.
** Culture volume was not recorded so N could not be estimated.
(continued)
-------
VO
Experiment 6
TABLE 14 (continued)
Time
before
exposure
after
exposure
24 hr.
72 hr.
120 hr.
168 hr.
216 hr.
N
N
Z S
Z R
N
Z S
N
Z S
N
Z S
N
Z S
N
Z S
0.00
10368
11100
107
107
6033
54
4067
37
1867
17
966
9
633
6
0.23
(0.008)
13321
7033
53
53
1600
23
933
11
633
9
100
1
367
5
CIO Concent rat ion
yeq/1 («g/l)
0.59 1.10
(0.021) (0.039)
9635
11333
118
118
6533
58
3967
35
1833
16
333
3
0
0
12352
6933*
56
90
8567
124*
6000
87
1833
26
1900
27
833
12
1.69
(0.060)
12413
7067*
57
79
8200
116*
6633
94
2133
30
967
14
833
12
4.23
(0.150)
10346
1233
12
91
533
43
433
35
500
40
567
46
67
5
Static
Control
11936
11867
107
107
9433
79
6767
57
3700
31
2900
24
1767
IS
* Tito number alive was underestimated baaed on the criteria of ciliary activity.
Therefore the percent survivals based on those rotmta arc overeat invited. However
the scope of the survivorship curvet* (not shown) was unaffected.
-------
SECTION 5
AVOIDANCE BEHAVIOR EXPERIMENTS
METHODS
Larval Culture
Pagurus longicarpus zoeae for avoidance experiments were obtained
from laboratory populations. Adults were obtained from Cedar Island
near Wachapreague, Va. or Gloucester Point, Va. The adult crabs were
maintained on a sea table receiving a continuous supply of unfiltered
or 10 ym filtered estuarine water. A thin layer of subtidal sand was
provided as a food-containing substrate. Small pieces of frozen fish
were added every other day. Purina Marine Chow was added periodically
as a food supplement. A minimum 14-hr light10 hr dark photoperiod
was maintained by means of a timer connected to two 30 W fluorescent
tubes (daylight white) located directly above the sea table. The
photoperiod was lengthened during summer months by natural lighting
from windows adjacent to the sea table. Water temperature was
maintained at 20°C or above by passing the incoming water through a
thermo-regulated water bath.
Each week the adult populations were examined for ovigerous
females. Ovigerous crabs were placed in an aquarium receiving 1 pm
filtered estuarine water. The discharge water flowed through a tube
into a screened basket (254 ym mesh) immersed in a water bath. Larvae
released in the tank were removed with the effluent water and
collected in the screened basket containing freshly hatched Artemia
nauplii which were replaced daily. The basket was examined daily for
newly hatched zoeae which were then placed in 20.3 cm diameter glass
bowls until used in experiments.
Larvae were maintained in static cultures according to methods
described by Roberts (1969, 1970). The culture medium was 1 um
filtered estuarine water. Newly hatched Artemia nauplii were provided
daily as food.
Experimental Apparatus
The apparatus used to evaluate the response of larvae to
chlorinated estuarine water was designed specifically to deal with a
50
-------
nonconservative material, chlorine-induced oxidants, and to
accommodate decapod larvae which respond primarily in the vertical
plane. In principle, the apparatus provides a flowing two-layered
water column with chlorine in the upper layer. Larvae are introduced
into the lower layer and induced to swim upward by a directed light
source. In this way, the expected avoidance response, vertically
downward, can be elicited. To maintain consistent CIO levels, each
layer is continuously replenished.
The final apparatus design settled on as providing the necessary
conditions is shown in Figures 5 and 6. Figure 5 depicts
diagrammatically the complete apparatus. Water was supplied from a
header tank (a) to a flow splitter tank (b) which in turn supplied the
constant level header tanks (c and d). Tanks c and d were placed on
laboratory jacks so that flow rates could be finely adjusted by
changing their heights relative to the test chambers. Gross flow
adjustment was achieved with screw clamps. Tank c supplied water for
the upper layers of each test chamber while tank d supplied the lower
layers. Inflow rates were measured with flow meters (i). The outflow
rates were regulated adjusting the height of tubes attached to the
outflow ports (k and p). The flow rates were selected to provide a
horizontal velocity of approximately 0.167 cm/sec for each layer which
is sufficiently slow to prevent larvae being trapped against the nylon
mesh screens. Outflow rates for each layer must equal the inflow
rates for that layer in order to maintain a sharp discontinuity. A
Vycor heater and temperature probe In tank c with thermostat (j)
maintained the temperature slightly above that in tank d (0.5 to 1°C)
which helped insure discrete layers in chambers e and f. A mechanical
stirrer in tank c (not shown) kept the water temperature homogeneous.
In order to maintain the test water temperatures during passage from
the header tanks (c and d) to the test chambers, much of the tubing
was passed through water jackets (k) receiving water from the same
header tanks as the test chambers they supplied. The two test
chambers were enclosed in a darkened box. Two 20 W fluorescent lights
were located above the test chambers to provide a directional stimulus
for the larvae. Two holes were provided in one side of the box to
allow visual or photographic observations of the larvae (not shown).
The test chamber (Figure 6) was located inside a water bath to
provide thermal insulation. Water entered the water bath through a
port (A) at one end and exited through a port (B) at the same end
after passing around the test chamber. Water entered the test chamber
through two inflow ports (C and D), one for the upper layer and one
for the lower layer. Incoming water to the upper layer passed from
chamber E through a diffuser plate (F) into a second chamber (6) and
thence into the test chamber through a diffuser plate and nylon mesh
screen (H, 254pm mesh size). Incoming water to the lower layer
followed a similar path from chamber L, through diffuser plate M into
chamber N and then through the diffuser plate and nylon mesh screen
(H). The layers flowed out through a nylon mesh screen and diffuser
51
-------
10
Figure 5. Diagrammatic representation of complete avoidance test apparatus.
-------
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ooo
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J 1
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J
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Figure 6. Diagrammatic representations of avoidance test chamber. Upper
view, longitudinal cross section through center, and view from
outflow end.
-------
Key to Figure 6. Diagram of avoidance test chamber
A. inflow port for water bath surrounding test chamber
B. outflow port for water bath surrounding test chamber
C. upper layer inflow port
D. lower layer inflow port
E. upper layer mixing chamber
F. upper layer diffuser plate
G. upper layer propagation chamber
H. diffuser plate for chambers g & n
I. diffuser plate for chambers j & o
J. upper layer outflow chamber
K. upper layer outflow port
L. lower layer mixing chamber
M. lower layer diffuser plate
N. lower layer propagation chamber
0. lower layer outflow chamber
P. lower layer outflow port
Q. separator plate between outflow chambers
54
-------
plate (I) into chambers J (upper layer) and 0 (lower layer) and out
ports K and P respectively.
A peristaltic pump (Fig. 5, g) was used to inject a Ca(OCl)2
solution into the upper layer inflow tube just downstream of the flow
meter (i). Injection may be made into either chamber. The resultant
CIO concentration in the upper layer was measured amperometrically
just before and Just after each avoidance test. Larvae were injected
into the apparatus through a port located centrally in the bottom of
each test chamber.
Dye Studies
In order to determine the discreteness of the two layers,
rhodamine B and fluorescene dye were injected into the upper and lower
layers, respectively. Dye distribution was observed for periods in
excess of one hour.
In a typical dye experiment, the two layers were well delineated
over the test period. Under certain conditions, colored water from
the upper layer infiltrated the lower layer along a narrow strip
immediately adjacent to the side walls. This problem has been
overcome by adding holes in the diffusor plate close to the sides of
the test chambers which improved flow along the sides of the tank.
Experimental Protocol
For preliminary experiments with chlorine applied to the upper
layer, two test chambers were set up and flow rates adjusted. The
upper layer of one chamber received the desired concentration of
chlorine; the other chamber served as a control. After a period for
equilibration, the CIO level of the upper layer was determined.
For each test 20-40 stage I zoeae were introduced into the lower,
unchlorinated layers of both chambers with a large-bore syringe.
Direct visual observations were made every five minutes with notes on
the observable behavior (direction of swimming, telson flip response,
passive sinking, etc.).
Preliminary Experiments
Preliminary experiments without chlorine were designed to
evaluate three areas of concern: 1) do larvae behave in the same way
within the two test chambers, 2) what is the effect of light intensity
on the number of larvae exhibiting a positive response, and 3) does
salinity affect the number of larvae exhibiting a positive response?
To answer the first question, each chamber was operated without
chlorine injection with water of 18% salinity. Maximal light
intensity of 240 fc was used in one test, 24 fc (achieved by
55
-------
introducing neutral density filters between the light source and test
chamber) in a second experiment. Larvae introduced into each chamber
were observed for over 1 hr.
The effect of light intensity was evaluated under the same
conditions except that 240 fc was applied to one chamber, 24 fc to the
other. In a second experiment, the light intensities were switched
for the two chambers. Larval responses were observed for over 1 hr.
Salinity effects were observed at 240 fc and 24 fc in separate
experiments. The salinities tested were 18 °/oo, at which positive
responses were expected to be low, and 24 °/oo, at which positive
responses were expected to be high. This salinity range brackets
approximately the usual salinity range for York River water entering
the laboratory. If the difference in larval response was large, we
felt it would be necessary to add a means of adjusting salinity to
some uniform level for these tests.
Data Analysis
Photographic observations, as originally planned, were not
feasible because of low light intensities. Instead, the number of
larvae in each horizontal quarter section of the test chamber was
determined by visual observations.
Observations were collected in a time series, and hence any
numerical data analysis must consider the time factor in some way.
For each observation time, the number of larvae in the upper layer
(nu) and in the lower layer (n^) were tablulated for each experiment.
The proportion of the larvae in the upper layer was then calculated as
follows:
JHi
x 100
nu + nL
We desired to test the null hypothesis that there was no
difference in the level of response during the test period. Therefore
we performed a Wilcoxon Sign Rank test (Steele and Torrie, 1960) on
the differences between p in the test and control chambers. Although
this approach ignores the magnitude of response differences, no truly
appropriate statistical procedure was Identified for analysing the
magnitude of response differences. Those test conditions producing
significantly different responses were thus identified, and the
directions of the responses were determined by inspection.
RESULTS
Preliminary Experiments
At both light intensities, there was no difference in the number
of P. longicarpus larvae in the upper layers of the two chambers in
56
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any experiment. The level of response was significantly different
between replicate experiments at a given light intensity in some
cases. This difference between experiments is attributed to the fact
that larvae from different females were used. At 240 fc the larvae
responded rapidly, whereas at 24 fc the number of larvae responding
increased gradually during the first 35 min of the experiment (Fig.
7, 8).
In experiments comparing response to a light Intensity of 240 fc
versus 24 fc, conflicting results were obtained. In one experiment,
the larvae exhibited a significantly greater response at 24 fc than at
240 fc (based on a Wilcoxon Sign Bank Test). In a replicate test, the
responses were not significantly different at the 95% level, but again
the stronger response was observed at 24 fc (Fig. 9). In the first
test, the mean response at 24 fc was 46%, whereas In the second test
it was 79%; i.e. the number of positive responses was high in the test
showing no significant differences.
Larvae exposed to 240 fc gave a stronger positive response in
18 °/oo water than In 24 °/oo water. In two cases the response was
significantly greater throughout the experiment. In a third
experiment, there was no significant difference overall, but larvae in
18 °/oo water gave a stronger positive response than those In 24 °/oo
water for the first 38 min (Fig. 10). In one experiment, the mean
percent responding positively was extremely low (7.1 and 23.9%).
Larvae used in this test were 48-hr-old and were probably ready to
molt to stage II.
At 24 fc, larval responses as a function of salinity were
inconsistent. In two experiments, larvae gave a significantly greater
response at 18 °/oo than at 24 °/oo (Fig. 11). In a third experiment,
the responses were not significantly different although larvae in 18
°/oo water gave a stronger positive response than those in 24 °/oo
water for the first 28 min.
To summarize these experiments, the mean response was determined
at each time interval for all tests performed under identical light
Intensities and salinities despite the high variability in responses
between tests. The high variance prevents demonstration of
statistically significant differences. Graphically it can be seen
that there was no consistent overall response of larvae to light
intensity. The larvae at 240 fc showed a higher positive response up
to 35 min and thereafter a lower response than the larvae at 24 fc.
At 24 °/oo the larval response to 240 fc was generally lower than the
response to 24 fc. Larvae tested at 18 °/oo salinity gave greater
responses than those tested at 24 °/oo at both light Intensities.
Thus, the levels of response were not as greatly affected by light
intensity as by salinity (Table 15, Fig. 12).
57
-------
100-
0-
eo-
60
50
- 50
p 20
u
a.
10 -
g
S
10 15 20 2% 10 35 40 48 50
TIME (mm)
60 69
Figure 7. Percent of Pagurue j'-mgf' arpua stage I larvae Jn the upper half
of the test chanters versus time (min) when exposed to 240 fc In
18 °/oo salinity water.
58
-------
lOCh
0
80-
70
O 60
oc
4°
z 30-
20-
10-
10 18 20 25 30 38 40 46 60
TIME (mm)
60 66
Figure 8. Percent of Pagurue longlcarpus atage I larvae In the upper half
of the teat chanfcera veraua time (min) when expoaed to 24 fc in
18 °/oo aalinity water.
59
-------
100-
90-
(E
HI
U
V)
8:
60-
50-
40-
30-
20-
8
g
o- IOH
10 15 20 25 30 35 40 45 50 55 60 65
TIME (mln)
Figure 9. Percent of Pagurus longicarpuB stage I larvae in the upper half
of the test chambers versus time (min) when exposed to 240 fc and
24 fc in 18 °/oo salinity water.
60
-------
5
&
ac
ui
a.
o
K
100-
90
80-
70-
60-
50-
40-
30-
20-
10-
Exp.
8
9
10
Chamber Sal.
L
R
L
R
L
R
18
24
24
18
24
18
5 10 15 20 25 30 35 40 45 50 55
TIME (min)
60 65
Figure 10. Percent of Pagurus longicarpus stage I larvae in the upper half
of the test chambers versus time (min) when exposed to 18 °/oo
and 24 °/oo at 240 fc.
61
-------
100-
90-
in
fc
ui
a:
ui 60
UJ
a.
a.
50
40
30.
20
10
0-
/-
Ex*
II
12
f
13
Chamber
R
L
R
L
R
804.
18
24
24
18
18
24
\
10 10 20 25 30 35 40 45 50 55 60 65
TIME (mln)
Figure 11. Percent of Pagurua longicarpus stage I larvae in the upper half
of the test chambers versus time (min) when exposed to 18 °/oo
and 24 °/oo at 24 fc.
62
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o<
TABLE 15. SUMMARY OF PERCENT PHOTOPOSITIVE RESPONSES FOR STAGE 1
Pagurus longicarpus LARVAE AT 5-MIN INTERVALS ARRANGED
ACCORDING TO TREATMENT. (DATA FOR REPLICATE TESTS AVERAGED)
Salinity (°/oo) -
Light Intensity (fc)
Observation
tine (ndn )
5
10
15
20
25
30
35
40
45
50
55
60
Ho. of tests averaged
1
24
"x SD
40.4 12.2
40.1 10.1
46.6 13.1
47.5 17.2
54.5 18.2
56.4 19.5
57.6 19.3
59.3 20.1
59.9 20.9
59.5 24.0
58.5 24.1
58.9 24.1
9
B
240
"x SD
34.1 22.5
46.9 22.1
55.0 24.6
58.4 22.0
56.7 21.5
58.3 23.7
57.1 24.5
53.9 25.3
55.1 23.9
53.2 21.3
53.6 20.9
54.8 21.6
9
24
24
x SD
19.7 9.1
27.7 23.7
25.0 19.4
28.3 22.8
30.5 24.0
31.2 22.6
31.2 16.8
32.2 9.6
34.2 8.0
38.2 11.3
34.0 11.4
32.2 8.1
3
240
x SD
9.3 0.6
13.2 5.4
17.0 7.8
16.0 5.3
20.7 12.2
23.0 13.1
22.7 16.1
24.2 21.8
28.8 22.0
33.5 23.8
35.7 24.6
34.2 24.5
3
-------
UJ
ffl
2
<
x
o
UJ
100-
90-
80-
70
o 60-
u
e
UJ
Q.
0.
a
50-
40-
30-
LU 20H
ioH
o
240 fc 18%
24 f c 18 %
24 fc 24%
240 fc 24%
10 15 20 25 30 35 40 45 50 55 60
TIME (mln )
Figure 12. Mean percent of Pagurua longlcarpus stage I larvae in the upper
half of the test chambers versus time (min) for all experiments
at each light intensity-salinity combination.
64
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Responses to Chlorinated Seawater
Larvae of P_. longicarpus were not available for tests of the
effect of a chlorinated seawater layer scheduled to be initiated in
January. As the preliminary tests were being completed, the
laboratory population ceased reproduction as a result of reduced
salinity. Salinity of the incoming water dropped to 13 °/oo, which is
below the level permitting egg development to hatching (Roberts,
1969). At this time of year ovigerous females are not available in
the field at any location within the geographic range of the species.
A trial experiment was carried out earlier with Palaemonetes
pugio larvae. The primary purpose of the test was to test the larval
introduction system and the feasibility of photographic observations.
Hence no quantitative response data were collected. The test
conditons were 240 fc, 20 °/oo salinity, and 70.5 yeq/1 (2.5 mg/1) in
the upper layer. Larvae were observed visually to determine the
type(s) of responses produced. Three types of larval responses as
described by Roberts (1969, 1971; telson flips, downward swimming, and
passive sinking), were observed when larvae encountered the
chlorinated layer. Larvae in the control chamber entered the upper
layer without responding to the two water layers.
These studies will be continued by one of the authors (J.E.I.)
as a thesis project.
65
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SECTION 6
SEROLOGICAL EFFECTS
METHODS
Experimental Animals
Adult blue crabs were obtained from two sources. At the
beginning of the study crabs were obtained from the Chesapeake Bay
potting fishery. The severe crab shortage in Chesapeake Bay during
the winter of 1976-77 forced us to import crabs by air from the North
Edisto River in South Carolina for several subsequent tests. This
source was used until July 1977 when Chesapeake Bay crabs were once
again available.
The nature of the crab fisheries in both localities makes it
impossible to be precise about the temperature and salinity regimes
from which the crabs were taken, but it can be assumed that the
Chesapeake Bay crabs came from salinities (20-25 °/oo) 5-10 °/oo
lower than the North Edisto River crabs (^30 °/oo). Local crabs
were taken from summer water temperatures (20-30°C) while those from
Bears Bluff were collected during early spring and summer (water
temperatures ^15-25°C). No attempt has been made to compare
physiological measurements between the two populations, but crabs
from only one locality have been used for a given series of tests.
All crabs were held for 7-14 days prior to their use. The
Initial Chesapeake Bay crabs were held in flowing York River water.
The South Carolina and later Chesapeake Bay crabs were held in a
recirculating sea water system heated to room temperature
(20.5-27°C). The water was continuously filtered to 5-10 ym.
Approximately one-third of it was replaced daily with 5 ym filtered
York River water (17.4-24.6 °/oo). All crabs were fed squid daily,
and, although their claws were bound to prevent cannibalism, they had
no apparent problems in feeding.
Exposure Systems
The basic dilutor system was similar to that described in detail
in Section 4.
66
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A crude estimate of CIO LC50 for adult blue crabs was obtained
using three doses and a control. The exposure system consisted of
four aerated 18-liter (5-gallon) aquaria receiving chlorinated York
River water directly from individual mixing boxes. Chlorine stock
solutions of various concentrations were pumped into a diluent flow
of approximately 300 ml/mln. The tanks each held five adult crabs.
The exposure system used for long-term (six-week) serologlcal
studies consisted of six tanks, each with eight 3-liter compartments
for the crabs. This system was designed to allow exposure of eight
physically Isolated crabs to each of five exposure levels and one
control level without cannibalism. Each tank was supplied through a
header box which received chlorinated water from a mixing box. Each
animal compartment received water through a calibrated siphon (100
ml/min). Appropriate exposure levels were obtained by pumping stock
solutions of varied concentrations into a mixing box with a diluent
flow of approximately 800 ml/min.
This 48-compartment dosing system proved too cumbersome to
maintain* It was discovered that the crabs were depleting their
ambient 02 supply by up to 25%. Doubling flow rates through the
system did not solve the problem, and aeraton of the system was
impractical. In addition, the number of crabs available from the
system for blood analysis was shown to be too small given the high
variances observed for the blood parameters.
These reasons, coupled with our growing suspicions that crab
responses were more rapid than previously anticipated, led us to use
a system of eight 37-liter (10-gallon) aquaria with higher flow rates
(1 liter/min) and vigorous aeration to maintain oxygen levels at near
saturation* There was virtually no effect of aeration on the CIO
concentrations. The tanks were arranged in two groups of four so
that a given test consisted of one dosed group and a control group.
Each group of four tanks received water from a single header.
Toxicant was mixed as before, except that diluent flow was increased
to ca. 4800 ml/min. The flow rates and aeration of this system
allowed ten crabs per tank. Coupled with the new experimental
design, the sample size at each interval was increased from the four
crabs in the previous system to ten crabs.
An additional system was used to determine whether tank size and
flow rates might have influenced early tests. The experimental group
included one 37-liter (10 gallon) and two 18-liter (5-gallon) tanks.
The 37-liter and one 18-liter tank were supplied by a single header
with a diluent flow of ca. 4800 ml/min. Tank flows were ca. 1000
ml/min and ca. 500 ml/min, respectively. The remaining 18-liter tank
received a flow of ca. 250 ml/min from a separate header receiving
diluent at a rate of ca. 1100 ml/min. The control group had similar
tanks with similar flows drawn from a single header. In each group,
the 37-liter tank contained ten crabs while each 18-liter tank
contained five.
67
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Experimental Protocols
The LC50 tests were performed by exposing five adult crabs to
each of three doses and a control. Doses were obtained by diluting
stock solutions of varying concentrations determined to give the
desired CIO levels in the tanks. Stable CIO levels were established
before crabs were put into the tanks. CIO concentrations and crab
mortalities were monitored twice daily, and flow rates into the tanks
were determined daily. Each test was terminated at the end of 96 hr;
any remaining crabs were bled.
The protocol for the 48-compartment system called for eight
mature female crabs sampled at time zero as a baseline, and a maximum
of 24 experimental (dosed) and 24 control (undosed) crabs. In the
tests, four experimental and four control crabs were randomly
sacrificed for serum analysis at selected intervals until all were
utilized or had died. CIO concentrations, mortalities, dissolved
oxygen, temperature, and salinity were monitored three times daily.
Flow rates and pH were determined once per day.
The protocol for the 37-liter (10-gallon) aquarium system
required that ten maturg female crabs be sampled at time zero as a
baseline. Forty crabs (ten per tank) were dosed with a single CIO
level, and 40 more crabs served as controls. At the end of each day
of the four day test, ten dosed and ten control crabs, representing
the entire contents of randomly selected tanks, were sacrificed for
analysis. CIO levels, temperature, salinity, dissolved oxygen, and
mortality were measured three times daily; pH and flow rates were
recorded once daily.
In the final test using the 18- and 37-liter tanks, ten baseline
crabs were sampled at time zero. Twenty crabs (ten per 37-liter
tank; five per 18-liter tank) were dosed, matched by twenty controls.
Surviving crabs were sampled after three days. CIO levels,
temperature, salinity, dissolved oxygen, mortality, pH and flow rates
were monitored as before. In addition, NH3~N levels were determined
daily for each tank and for the diluent water by the method of
Solorzano (1969).
Methods of Serological Analysis
Six or seven milliliters of blood vere usually taken from each
crab by withdrawal from the sinus at the base of the fifth pereiopod
by means of a plastic syringe with an 18 gauge needle. Samples were
placed in capped test tubes in an ice water bath. The samples were
allowed to chill for at least 30 minutes, after which the clot in
each was broken up and centrifuged out (2000 g for 30 min). The
decanted serum was then divided into at least two portions and
frozen. Analyses were performed on samples frozen and thawed only
once.
68
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Constituent analyses were made using procedures described in
detail for blue crab serum by Lynch and Webb (1973a) for TNPS (total
ninhydrin positive substances), Lynch and Webb (1973b) for protein,
Lynch and Webb (1973c) for glucose and Lynch, Webb and Van Engel
(1973) for chloride and osmotic concentration.
Data Analysis
Preliminary 24-, 48- and 96-hr LCSO's were determined from a
plot of percent survival versus log CIO concentration representing a
composite of data from four different tests for lethal limits. Serum
constituent values from two of these tests were subjected to one-way
analysis of variance.
Statistical analysis of the data from the 48-compartment
system/six-week tests was unproductive because of the small sample
sizes and the large variances encountered. The overlap between
standard errors precluded detection of significant differences.
Time course and concentration effects on serum from the four-day
tests were evaluated by factorial analysis. The analysis was
performed on individual 2x4 factorial tests where the factors were
dose (one dose plus control) and time (one, two, three and four
days), respectively.
The final-3 day test was evaluated by one-way analysis of
variance and a modified Tukey's u>'-procedure (Steel and Torrie, 1960)
for effects of tank size-flow-CIO combinations on crab serum
constituents. A separate one-way analysis of variance and standard
Tukey's u> -procedure were used to determine effects on tank NH3~N
levels.
RESULTS
Lethal Limits
Groups of five mature crabs were exposed to twelve different CIO
concentrations ranging from 3.1 to 315.8 veq/1 (0.11 to 11.20 mg/1)
and a control for 96 hr at approximately 20°C. Total mortality was
observed at 141 yeq/1 (5.0 mg/1) after 24 hrs, 71.3 Weq/1 (2.53 mg/1)
after 48 hrs and 45.1 yeq/1 (1.6 mg/1) after 96 hrs. Total survival
was observed after 96 hrs at 18.3 yeq/1 (0.65 mg/1) and less. The 96-
hr LC50 was 28.8 yeq/1 (1.02 mg/1); the 48-hr LC50 was 30.2 yeq/1
(1.07 mg/1); the 24-hr LC50 was 91.7 yeq/1 (3.25 mg/1) (Fig. 13).
Mortality estimates from other four day tests were roughly comparable
to these data, although direct comparisons are difficult because of
differing experimental conditions. Mortality after 96 hr was 0% at
10.2, 11.8, and 14.1 yeq/1 (0.36, 0.42 and 0.50 mg/1), 38.3% at 13.3
yeq/1 (0.47 mg/1), 30.9% at 13.5 yeq/1 (0.48 mg/1) and 24.2% at 17.5
yeq/1 (0.62 mg/1). Mortality after 48 hr was 23.4% at 29.3 yeq/1
(1.04 mg/1).
69
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100 -
90 -
O3
0.4 OJS OuB OJ 08 O9 1JO
CHLORINE-INDUCED OXIDANT (mtf)
2JO
ao
4.0
ao
Figure 13. Composite summary of survival data for Callinectes sapidus
adults plotted against log CIO concentration. Includes data
from 4 experiments. The LC50 values are considered to be
preliminary only.
-------
Time Course and Concentration Effects on Serum
Serum from thirteen different experiments was analyzed for
glucose (first experiment only), TNPS, protein, chloride and total
osmotic concentrations (Table 16). CIO levels and other hydrographic
data appear in Table 17. Mean serum constituent values and other
pertinent data can be found in Table 18 for experiments 1 and 2,
Table 19 for experiments 3 through 5, Table 20 for experiments 6
through 12 and Table 21 for experiment 13.
Glucose
Glucose levels were not significantly affected by CIO levels up
to 18.3 yeq/1 (0.65 mg/1) (ANOVA, p > 0.10, Table 18). Measured
glucose levels at all dose levels including controls were low,
approaching the detection limit of the analytical method, and were
not comparable to levels reported by Lynch and Webb (1973c). Careful
analysis of the method failed to explain the discrepancy. Values
were consistently low regardless of method modifications or age of
reagents. The discrepancy may have been the result of the high
sensitivity of blood glucose to stress. Florkin (1960) cited blood
glucose values of 175-182 mg % (cf. Lynch and Webb, 1973c) for
freshly caught C. sapidus and values of 9.6-19.0 mg % (cf. Table 18)
after one day of fasting. The low glucose values observed coupled
with the reported high sensitivity of this blood constituent to any
general stress indicated to us that blood glucose was not a suitable
parameter to use in our laboratory study. Therefore blood glucose
was not measured in subsequent experiments.
TNPS
TNPS concentration was unaffected in the first experiment up to
18.3 yeq/1 (0.65 mg/1) (ANOVA, p > 0.10; Table 18). In the second
test, TNPS was unaffected at low doses but increased over 2000% at
27.4 yeq/1 (0.97 mg/1), a dose very close to the 96-hr LC50 (ANOVA,
p < 0.005, Table 18). In the fifth test, statistical analysis of the
data was unproductive because of the small sample sizes and large
variances encountered. The overlap between the standard errors
precluded detection of significant differences. In spite of this,
TNPS (Table 19) showed a slight increase in both controls and
experimentals after two week exposure to 12.4 yeq/1 (0.44 mg/1), the
experimentals tending to have higher concentrations throughout the
test.
In experiment 6, TNPS varied with time (ANOVA, p < 0.005; Table
20) but not with dose (10.2 yeq/1 0.36 mg/1). Similar results were
found for experiment 7 (11.8 yeq/1 - 0.42 mg/1) (ANOVA, p < 0.01).
In both tests an increase in TNPS was seen between days one and two,
and, as in test 5, the experimental crabs seemed to maintain slightly
higher TNPS levels than the control crabs.
71
-------
TABLE 16. SUMMARY OF TEST SYSTEMS USED, DOSES, SAMPLING INTERVALS AND SERUM ANALYSES
DURING Callinectes saoidus SEROLOGICAL STUDIES
Experiment
no.
1
2
3
4
5
"
6
7
System
18-liter
(5-gallon)
aquaria
48 compart-
ment
37-liter
(10-gallon)
aquaria
Mean
measured CIO
(mgCl2/liter)
with crabs
0.00, 0.11,
0.51, 0.65
0.00, 0.17,
0.30, 0.97
0.00, 0.75
0.00, 0.71
0.00, 0.44
0.00, 0.36
0.00, 0.42
Sampling
interval
4 days
4 days
1 week
1 week
1 week
1 day
1 day
No. crabs /sample
(design)
5
5
8 baseline;
4 control;
4 experimental
8 baseline;
4 control;
4 experimental
8 baseline;
4 control;
4 experimental
10 baseline;
10 control;
10 experimental
10 baseline;
10 control;
10 experimental
Analyses
Glucose, TNPS, Protein, Chloride,
Osmotic
TNPS, Protein, Chloride, Osmotic
Protein, Chloride, Osmotic
Protein, Chloride, Osmotic
TNPS, Protein, Chloride
TNPS, Protein, Chloride, Osmotic
TNPS, Protein, Chloride, Osmotic
(continued)
-------
Experiment
no.
8
9
10
11
12
13
System
37-liter
(10- gallon)
aquaria
37-liter
(10- gallon)
1000 ml/min
18- liter
(5-gallon)
aquaria
500 ml/min
18-liter
(5-gallon)
aquaria
250 ml/mLn
Mean
measured CIO
(mgCl2/liter)
with crabs
0.00, 0.47
0.00, 0.50
0.00, 0.48
0.00, 0.62
0.00, 1.04
0.00, 0.82
0.00, 0.74
0.00, 0.58
TABLE 16
Sampling
interval
1 day
1 day
1 day
1 day
1 day
3 days
3 days
3 days
(continued)
No. crabs /sample
(design)
10 baseline;
10 control;
10 experimental
10 baseline;
10 control;
10 experimental
10 baseline;
10 control;
10 experimental
10 baseline;
10 control;
10 experimental
6 baseline;
10 control;
10 experimental
10 baseline;
10 control;
10 experimental
5 control
5 experimental
5 control
5 experimental
Analyses
TNPS, Protein, Chloride, Osmotic
TNPS, Protein, Chloride, Osmotic
TNPS, Protein, Chloride, Osmotic
TNPS, Protein, Chloride, Osmotic
TNPS, Protein, Chloride, Osmotic
TNPS, Chloride, Osmotic
TNPS, Chloride, Osmotic
TNPS, Chloride, Osmotic
-------
TABLE 17. SUMtf&KY OF HYDROGRAPHIC DATA FOR Callinectea sapidus SERDLOGICAL STUDIES
Experiment
No.
1
2
3
4
5
6
7
8
9
Applied dose
weq/1 mg/1
HD
HD
HD
HD
HD
HD
63.5
60.6
54.7
36.4
43.7
46.2
55.3
HD
HD
HD
HD
ND
ND
2.5
2.15
1.94
1.29
1.55
1.64
1.96
Mean
measured
CIO level
yeq/1 mg/1
3.1
14.4
18.3
4.7
8.4
27.4
21.2
(25.1)
20.0
(23.1)
12.4
(13.3)
10.2
(7.6)
11.8
(9.0)
13.3
(9.6)
14.1
(10.7)
0.11
0.51
0.65
0.17
0.30
0.97
0.75
(0.89)
0.71
(0.82)
0.44
(0.47)
0.36
(0.27)
0.42
(0.32)
0.47
(0.34)
0.50
(0.38)
Measured CIO
standard
deviation
yeq/1 mg/1
0.56
0.85
3.95
0.85
0.56
0.77
7.61
(11.84)
3.38
(23.1)
1.97
(3.67)
0.85
(1.13)
1.13
(0.56)
1.97
(1.69)
0.56
(0.85)
0.02
0.03
0.14
0.03
0.02
0.24
0.27
(0.42)
0.12
0.82
0.07
(0.13)
0.03
(0.04)
0.04
(0.02)
0.07
(0.06)
0.02
(0.03)
Temperature
^ SD
21.3
11.8
18.4
20.2
27.2
20.9
21.11
23.0
21.8
0.80
0.76
0.77
0.94
0.81
0.21
0.32
0.62
0.68
Salinity
x SD
17.0
17.0
18.5
18.4
20.3
19.3
18.6
17.1
19.4
1.3
ND
0.28
0.34
0.87
1.3
0.72
0.42
0.42
Mean
D.O.
(«g/D
ND
ND
6.2
6.7
4.8
6.7
6.7
6.6
6.4
Mean
PH
ND
ND
7.6
7.8
7.7
7.8
7.8
8.0
7.9
(continued)
-------
TABLE 17 (continued)
Experiment
Mo.
10
11
12
13
Applied dose
lieq/1 mg/1
69.4
93.6
120.7
102.9
102.9
129.2
2.46
3.32
4.28
3.65
3.65
4.58
Mean
measured
CIO level
ueq/1 fflg/1
13.5
(12.4)
17.5
(23.1)
29.3
(39.8)
23.1
(32.4)
20.9
(32.7)
16.4
(30.5)
0.48
(0.44)
0.62
(0.82)
1.04
(1.41)
0.82
(1.15)
0.74
(1.16)
0.58
(1.08)
Measured CIO
standard
deviation
yeq/1 ing/1
1.41
(2.26)
2.26
(5.92)
9.87
(3.95)
5.92
(4.51)
4.23
(4.79)
1.97
(2.54)
0.05
(0.08)
0.08
(0.21)
0.35
(0.14)
0.21
(0.16)
0.15
(0.17)
0.07
(0.09)
Temperature
(°C)
x SD
29.0
27.6
26.7
25.0
25.0
25.0
0.60
0.81
0.65
0.62
0.62
0.62
Salinity
( /oo)
x SD
21.9
22.8
24.3
24.0
24.0
24.0
0.43
0.21
0.14
0.09
0.09
0.09
Mean
D.O.
Ong/1
5.6
6.0
6.4
6.6
6.6
6.6
Mean
PH
7.6
7.7
7.8
7.8
7.8
7.8
37-1
tank
18-1
high
flow
18-1
tank
low
flow
ND No data
( ) Without crabs
-------
TABLE 18. BLOOD SERUM PARAMETERS FOR Callinectes sapidus FROM EXPERIMENTS 1 AND 2.
Temp.
Experiment °C
1 21.3
21.3
21.3
21.3
2 11.8
11.8
11.8
11.8
CIO
(mg/1)
0.00
0.11
0.51
0.65
0.00
0.17
0.30
0.97
Glucose
(mg/1 00 ml)
x SD
7.3
8.8
8.9
13.8
ND
ND
ND
ND
1.5
2.5
6.3
14.7
ND
ND
ND
ND
Protein
(mg/ml)
x SD
71.4
85.1
63.7
53.9
92.3
66.8
59.4
58.5
24.7
15.4
32.1
32.0
23.2
46.7
43.0
23.3
Chloride
(meq/1)
x SD
392.1
404.1
386.8
334.0
387.4
392.8
376.4
259.8
20.8
8.5
19.7
18.4
13.6
121.7
23.3
13.5
TNPS
( moles /ml)
x SD
2.9
2.6
2.8
2.9
0.4
0.4
0.2
8.1
1.4
1.3
1.2
2.2
0.3
0.3
0.1
1.0
Osmotic Cone.
(milliosmols)
x SD
864.2
883.0
834.0
767.0
775.0
785.1
797.2
613.8
15.0
17.9
45.9
88.6
19.9
21.4
7.0
35.7
ND = No data
-------
TABLE 19. BLOOD SERUM PARAMETERS FOR Callinectes sapidus FROM EXPERIMENTS 3-5.
Experiment
3
4
5
Temp.
°C
18.4
18.4
20.2
20.2
27.2
27.2
27.2
27.2
TNPS
CIO (ymoles/ml)
(mg/1) Weeks x SD
0.00
0.75
0.00
0.71
0.00
0.00
0.44
0.44
1
1
1
1
1
2
1
2
ND
ND
ND
ND
1.5
3.0
2.6
2.3
ND
ND
ND
ND
0.7
0.7
1.0
0.9
Protein
(mg/ml)
x SD
73.5
75.3
33.2
54.8
26.4
6.6
44.3
21.7
27.3
24.3
10.0
16.0
18.3
3.7
32.4
17.1
Chloride
(meq/1)
x SD
372.4
301.6
416.3
300.6
387.2
558.5
373.2
311.7
18.6
52.6
66.1
52.6
13.5
34.2
59.7
26.8
Osmotic Cone.
(milliosmols)
x SD
845.0
740.3
748.3
699.0
ND
ND
ND
ND
82.2
88.7
98.1
59.3
ND
ND
ND
ND
ND = No data
-------
TABLE 20. BLOOD SERUM PARAMETERS FOR Callinectes sapidus FROM EXPERIMENTS 6-12.
00
Temp. CIO
Experiment °C (mg/1)
6 20.9 0.00
20.9
20.9
20.9
20.9 0.36
20.9
20.9
20.9
7 21.1 0.00
21.1
21.1
21.1
21.1 0.42
21.1
21.1
21.1
8 23.0 0.00
23.0
23.0
23.0
23.0 0.47
23.0
23.0
23.0
TNPS
(umoles/ml)
Days x SD
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
2.0
2.7
2.4
2.3
1.6
2.9
2.8
2.6
2.6
3.3
3.2
3.4
2.8
3.5
3.2
4.0
3.0
3.4
2.6
2.9
2.5
3.2
2.5
2.4
0.3
0.7
0.4
0.6
0.4
0.9
0.6
0.6
0.7
0.6
0.9
0.7
0.5
1.3
0.9
1.3
0.4
1.0
1.4
1.0
0.8
1.5
1.1
1.2
Protein
(mg/ml )
x SD
61.3
68.0
65.3
65.0
56.4
67.1
56.1
63.0
67.4
69.6
63.6
72.5
67.5
72.4
61.8
78.2
52.0
56.7
52.5
47.3
42.2
56.2
56.9
37.4
15.6
16.0
15.2
21.6
16.1
22.7
13.0
24.7
19.9
29.0
16.0
15.0
15.3
21.0
20.3
22.7
20.6
27.4
10.4
9.5
16.1
23.8
21.8
16.7
Chloride
(meq/1)
x SD
397.2
382.2
380.1
365.6
398.9
388.7
381.2
371.7
370.0
364.5
364.8
363.9
382.2
373.8
369.7
364.7
361.1
384.4
373.5
393.2
388.0
371.6
383.8
390.6
8.5
10.4
17.8
15.8
18.4
5.5
12.7
11.9
12.8
9.7
10.9
9.9
11.4
14.5
18.1
10.9
24.1
21.2
16.4
33.7
48.4
28.0
15.0
48.2
Osmotic Cone .
(milliosmols)
x SD
786.4
799.2
791.6
775.3
804.6
831.5
811.8
788.1
739.3
757.6
746.4
769.8
762.2
763.0
745.8
763.1
774.0
776.7
737.5
733.6
762.5
775.0
755.2
748.0
23.2
25.7
20.4
16.6
27.2
28.9
25.9
11.3
18.8
19.1
13.7
12.0
22.6
22.2
27.7
19.3
21.2
24.5
25.3
52.2
35.5
41.0
41.2
40.7
-------
Table 20. (continued)
Experiment
9
10
11
Temp*
°C
21.8
21.8
21.8
21.8
21.8
21.8
21.8
21.8
29.0
29.0
29.0
29.0
29.0
29.0
29.0
29.0
27.6
27.6
27.6
27.6
27.6
27.6
27.6
27.6
TNPS
CIO (ymoles/ml)
(mg/1) Days x SD
0.00 1
2
3
4
0.50 1
2
3
4
0.00 1
2
3
4
0.48 1
2
3
4
0.00 1
2
3
4
0.62 1
2
3
4
2.2
2.9
2.4
2.2
1.9
3.2
2.4
2.5
3.1
2.7
3.3
1.8
2.9
2.8
2.7
2.9
3.0
2.7
4.3
3.2
2.5
3.7
2.8
3.4
1.4
1.0
0.9
1.1
0.5
1.2
1.0
1.3
0.5
1.0
1.0
0.9
0.7
0.5
0.5
1.0
0.8
0.7
1.4
0.8
0.8
2.1
0.8
2.7
Protein
(mg/ml)
x SD
50.0
45.0
54.8
45.4
44.8
57.6
46.6
53.4
49.2
50.7
61.2
50.4
46.8
51.1
55.4
47.4
38.4
35.3
45.7
34.5
32.1
33.6
34.3
39.0
23.3
20.8
24.8
27.0
17.7
20.8
9.2
20.0
15.5
17.5
19.0
22.8
17.3
20.3
15.9
19.4
21.5
11.5
25.3
9.3
15.7
13.9
12.7
25.7
Chloride
(meq/1)
x SD
401.9
382.0
379.0
377.9
420.3
386.1
382.3
381.6
418.7
371.8
381.5
369.5
425.8
395.7
390.3
377.0
389.0
380.1
371.1
379.5
398.5
388.5
377.4
371.0
34.0
14.7
10.5
5.4
17.9
10.4
25.8
16.2
47.5
11.5
19.9
12.5
42.2
13.3
10.4
8.1
7.4
12.0
8.7
15.5
6.3
7.3
17.4
14.4
Osmotic Cone.
(milliosmols )
x SD
769.2
761.1
774.4
775.0
799.2
799.3
804.1
789.2
776.6
774.5
765.9
775.5
798.5
808.5
787.9
817.2
782.2
804.5
798.7
786.1
803.4
818.8
814.0
794.6
18.4
10.9
26.1
28.8
43.8
35.7
26.9
30.6
32.6
23.1
37.9
16.2
34.9
45.6
29.0
51.6
33.7
60.1
23.3
20.2
22.5
33.6
32.3
27.1
-------
Table 20. (concluded)
TNPS
Experiment
12
Temp.
°C
26.7
26.7
26.7
26.7
CIO
(mg/1)
0.00
1.04
(y moles /ml)
Days
1
2
1
2
X
2.9
2.6
3.2
3.1
SD
1.3
1.2
1.0
0.6
Protein
(rag/ml )
X
22.0
32.9
26.0
34.4
SD
7.6
10.7
12.5
14.1
Chloride
(meq/1)
X
396.4
397.6
385.8
383.1
SD
10.8
10.9
15.3
13.8
Osmotic
Cone.
(milliosmols)
X
775.6
792.1
796.9
780.1
SD
17.9
25.4
32.0
14.4
00
o
-------
TABLE 21. BLOOD SERUM PARAMETERS FOR Callinectes sapidus FROM EXPERIMENT 13.
oo
Tank; flow
37
18
18
37
18
18
liter;
liter;
liter;
liter;
liter;
liter;
high
high
low
high
high
low
flow
flow
flow
flow
flow
flow
Temp.
°C
25
25
25
25
25
25
CIO
(mg/D
0.00
0.00
0.00
0.82
0.74
0.58
TNPS
(y moles /ml)
x SD
2.9
2.9
2.7
3.6
3.3
1.9
0.5
0.7
1.3
0.7
0.5
0.7
Chloride
(meq/1)
x SD
391.6
402.6
403.2
371.0
360.9
398.6
9.3
9.0
7.5
14.9
6.8
8.3
Osmotic Cone.
(milliosmols)
x SD
783.4
786.4
780.8
738.9
741.8
797.3
16.2
25.0
17.4
3.2
21.6
10.4
-------
TNPS was unaffected by either dose or time in experiments 8
(13.3 yeq/1 - 0.47 mg/1) (ANOVA, p > 0.10) and 9 (14.1 yeq/1 - 0.50
mg/1) (ANOVA p > 0.10). However, trends with time seemed similar to
those noted for experiments 6 and 7, and the experimental crabs in
experiment 8 seemed to maintain slightly lower TNPS levels than the
control crabs.
TNPS was affected by time but not dose (13.5 yeq/1 0.48 mg/1)
in experiment 10 (ANOVA, p < 0.025). The overall trend was not
readily obvious and may reflect fluctuations in control values, even
though there was no significant interaction between dose and time
(p > 0.05).
TNPS was not influenced by dose or time in experiments 11 (17.4
yeq/1 - 0.62 mg/1) (ANOVA, p > 0.10) and 12 (29.3 yeq/1 - 1.04 mg/1)
(ANOVA, p > 0.10). However, as in some of the previous tests,
experimental crabs in the twelfth test seemed to maintain slightly
higher TNPS levels than control crabs.
TNPS levels did not vary with the tank size-flow-CIO
combinations of experiment 13 (ANOVA, p > 0.10; Table 21). the ANOVA
was supported by Tukey's modified u)1 (Table 22).
Protein
Protein in the first two tests appeared to decrease with
increasing CIO level (Table 18), but the decrease was not significant
(ANOVA, p > 0.10). In addition, there was no effect at the higher
CIO doses of tests 3 (21.2 yeq/1 = 0.75 mg/1) and 4 (20.0 yeq/1 -
0.71 mg/1) (t- -0.10, -2.26 respectively; p > 0.05). In the fifth
test serum protein showed a tendency to be elevated by exposure to
CIO levels (12.4 yeq/1 - 0.44 mg/1) (Table 19). However, as with
TNPS, the small sample sizes and high variability in the data
precluded meaningful statistical analysis. The apparent decreases in
protein over the two-week period in this test and over the four-day
period in tests 1 and 2 may be attributable to inadequate feeding.
Serum protein in experiments 6 through 12 varied with neither dose
nor time (ANOVA, p > 0.05; Table 20). Therefore, protein was not
determined for experiment 13.
Chloride
At low doses in the first two experiments serum chloride levels
showed a slight increase in concentration, but as the dose was
increased above 8.5-14.1 yeq/1 (0.3-0.5 mg/1), the serum chloride
level dropped markedly (Table 18). The effect on serum chloride was
highly significant in spite of high variance in the data. Similar
trends were apparent in the third and fourth experiments (t 2.54,
2.74, respectively; p < 0.05) and, although not statistically
verifiable, in experiment 5 (Table 19). In experiment 3 chloride was
82
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TABLE 22. TUKEY's MODIFIED u' TEST FOR TANK SIZE-FLOW-CIO
COMBINATION EFFECTS ON SERUM TNPS IN Callinectes aapidus
FROM EXPERIMENT 13. [a)'-qa(P,N2)S; STEEL AND LORRIE, 1960]
tank size (liters)
flow rate (ml/min)
CIO level (mgC!2/l)
TNPS x (ymoles/ml)
Equal means
18
250
0.58
1.92
18
37
250 1000
0.00
2.70
0.00
2.89
18
500
0.00
2.91
18
37
500 1000
0.74
3.34
0.82
3.56
TABLE 23. TUKEY's MODIFIED o>' TEST FOR TANK SIZE-FLOW-CIO
COMBINATION EFFECTS ON SERUM CHLORIDE IN Callinectes
sapidus FROM EXPERIMENT 13. [a>'-qa(P,N2)S; STEEL AND TORRIE,
19601 BARS UNDERLINE EQUAL MEANS (a-0.05)
tank size (liters)
flow rate (ml/min)
CIO level (mgCl2/D
chloride (meq/liter)
Equal means
18 37
500 1000
0.74 0.82
360.9 371.0
37 18 18 18
1000 250 500 250
0.00 0.58 0.00 0.00
391.6 398.6 402.6 403.2
TABLE 24. TUKEY's MODIFIED w' TEST FOR TANK SIZE-FLOW-CIO
COMBINATION EFFECTS ON SERUM OSMOTIC CONCENTRATION IN
Callinectes sapidus FROM EXPERIMENT 13. [u>'-qa(P,N2)S;
STEEL AND TORRIE, 1960]
tank size (liters)
flow rate (ml/min)
CIO level (mgC!2/l)
osmotic concentration
(milliosmols)
Koiifll mpano
37
1000
0.82
738.9
BARS UNDERLINE EQUAL MEANS (a=
18
500
0.74
741.8
18
250
0.00
780.8
37
1000
0.00
783.4
18
500
0.00
786.4
0.05)
18
250
0.
797.
58
3
83
-------
reduced from the baseline value of 390.0 meq/1 to 301.7 meq/1 in the
experimental compared to a control reduction to 372.4 meq/1 at 21.2
yeq/1 (0.75 mg/1) CIO. In experiment 4 the chloride in the
experimental was reduced from 393.7 meq/1 to 300.6 meq/1, while the
controls showed an increase to 416.3 meq/1 at 20.0 yeq/1 (0.71 mg/1)
CIO. Similarly, while chloride concentrations in the hyper-
regulating control crabs of experiment 5 conformed to changes in
chloride ion in the medium and increased after two weeks, the levels
in the experimental crabs dropped markedly at 12.4 yeq/1 (0.44 mg/1).
Though rarely significant, serum chloride levels in most
subsequent tests showed a tendency to be elevated in crabs exposed to
CIO. Serum chloride levels in the sixth test were affected only by
time (ANOVA, p < 0.005; Table 20), decreasing over the four-day
period. Those in the seventh and eighth experiments were unaffected
by either dose or time (ANOVA, p > 0.10). In experiment 9, chloride
levels were affected by time (ANOVA, p < 0.005), decreasing from the
first to the second day before leveling out in both experlmentals and
controls. Serum chloride in the tenth test varied with both dose and
time (ANOVA, p < 0.005). Chloride levels decreased from the first to
the fourth day and were consistently higher in the dosed crabs.
Serum chloride in experiment 11 changed only with time (ANOVA,
p < 0.05), showing a gradual decrease from day 1 to day 4. In
experiment 12 serum chloride was affected only by dose (ANOVA,
p < 0.01), experimental crabs having lower levels than the controls.
Serum chloride levels varied with tank size-flow-CIO
combinations in experiment 13 (ANOVA, p < 0.005; Table 21).
Essentially, chloride levels in crabs from the 37-liter-1000 ml/min-
23.1 yeq/1 (0.82 mg/1) and 18-liter-500 ml/min-20.9 yeq/1 (0.74 mg/1)
combinations were equivalent to each other and lower than those in
all other combinations (Table 23).
Osmotic
Serum osmotic concentrations, not surprisingly, followed trends
similar to serum chloride. In the first two tests, osmotic
concentrations showed a slight increase at low chlorine residual
doses, and then dropped radically at doses above 8.5-14.1 yeq/1
(0.3-0.5 mg/1) (Table 18). The effect of the CIO levels on serum
osmotic concentration was highly significant. It was surprising,
therefore, that at the higher doses of tests 3 (21.2 yeq/1 = 0.75
mg/1) and 4 (20.0 yeq/1 = °*71 mg/1) no effect was evident (t = 1.73,
0.86, respectively; p > 0.05). Samples for determining serum osmotic
concentrations for the fifth test were lost because of a
malfunctioning osmometer.
Like serum chloride, serum osmotic concentrations in subsequent
tests showed usually nonsignificant but consistent increases in crabs
exposed to CIO. Serum osmotic concentrations in the sixth experiment
84
-------
were dependent on both dose and time (10.2 yeq/1 = 0.36 mg/1) (Table
20). The experimental crabs had consistently higher values than the
controls as all values increased from the first to the second day and
then steadily declined. In the seventh test, osmotic concentration
was dependent only on time. Apparent minor fluctuations with time
were found significant, probably because of the unusually low
variability of the data. With the exception of experiment 9 osmotic
concentration varied with neither dose nor time from experiments 8
through 12 (ANOVA, p > 0.05). In the ninth test osmotic
concentration was affected by dose, showing higher levels in the
experimental crabs than in the controls on all days (ANOVA,
p < 0.005).
The effect of the tank size-flow-CIO combinations of experiment
13 on osmotic concentration (ANOVA, p < 0.005; Table 21) was similar
to that observed for serum chloride levels. Osmotic concentrations
in crabs from the 37-liter-1000 ml/min-23.1 yeq/1 (0.82 mg/liter) and
18-liter-500 ml/min-20.9 yeq/1 (0.74 mg/liter) combinations were
equivalent to each other and lower than those in all other
combinations (Table 24).
Behavioral and Other Responses
Feeding
Crabs were fed throughout experiments 5 through 12 with either
an experimental Purina Marine Chow or squid squares. Disintegration
of the marine chow in the fifth test after wetting and manipulation
or trampling by the crabs made it difficult to be certain whether
there were feeding differences between experimentals and controls.
It seemed, however, that the experimentals were feeding less. This
was confirmed during tests 6 through 12 in which experimental crabs
obviously ate less squid than the controls. Whether this feeding
response was the result of ill health in the experimental crabs or an
inability on their part to sense the food in chlorinated water is
unclear. A clue may have been offered in experiment 9 in which
experimental crabs began feeding immediately (1-2 hr) after chlorine
input to the dosing system was accidently shut off.
Spawning
In the fifth experiment of this section, several of the crabs
spawned in the 48-compartment system. Virtually all of the observed
spawning occurred in the controls. Retention of eggs on the pleopods
was poor probably because of the confinement.
Activity
Effects of CIO levels on crab motility were unobservable because
the dosing system was too confining. However, it was noticed that
85
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dosed crabs tended to keep their antennules withdrawn, making only a
few tentative movements into the water. This reaction seemed more
pronounced at higher doses and contrasted with the control crabs
whose antennules seemed to be always extended.
Chlorine Demand
During tests using the 48-compartment system, it was noticed
that introduction of crabs to the system seemed to alter the measured
CIO concentrations. Specifically, at high CIO concentrations
introduction of crabs lowered the measured level. Such a demand was
present in the fifth test where the CIO concentration was reduced
from 13.3 yeq/1 (0.47 mg/1) to 12.4 yeq/1 (0.44 mg/1) when the crabs
were present (t - 2.46, < 0.02). It also occurred in tests 3 and 4
but was significant only in test 4 where the CIO level was reduced
from 23.1 yeq/1 (0.82 mg/1) to 20.0 yeq/1 (0.71 mg/1) (t - 3.99,
< 0.001). This phenomenon occurred also in the tests for lethal
limits but was attributed to varying demand in the Incoming water.
In the 48-compartment system, however, the crabs were implicated
because the pattern was not immediately visible except at the higher
doses. In one test, measured CIO levels in tanks with and without
crabs were compared statistically for a series of doses ranging from
approximately 0.85 to 14.1 yeq/1 (0.03 to 0.5 mg/1) (Table 25).
Analysis revealed a demand only at the highest level where crabs
reduced the measured CIO concentrations from 13.8 to 11.8 yeq/1 (0.49
to 0.42 mg/1). This demand was thought to be the result of the
excretion of ammonia or organics by the crabs. It was thought
unusual, therefore, and perhaps anomalous that at the next lower dose
(5.6-7.6 yeq/1 - 0.20-0.27 mg/1) introduction of crabs raised the
measured CIO level.
This latter phenomenon turned out not to be unusual at all. A
retrospective look at Table 25 shows that, although differences were
not significant at individual doses, compartments with crabs
consistently showed higher measured CIO levels at the lower doses
than those without. Also, in tests 6 through 10 introduction of
crabs to the system caused a marked increase in the measured CIO
level that persisted throughout the tests (Table 17). Differences
were significant (t - 11.2, 11.5, 8.5, 14.6 and 2.5, respectively;
p < 0.02). The effect was reversed in experiment 11 (t - 4.7;
p < 0.001) and apparently in experiments 12 and 13, although
simultaneous comparisons among tanks with and without crabs were not
possible in these latter tests.
It seems as if crabs increase the measured CIO level at low
doses but decrease it at high doses approaching and exceeding the
level of no effect (18.3 yeq/1 - 0.65 mg/1) derived from the 96-hr
LC50 tests. Determination of the mechanisms responsible for this
effect is beyond the scope of this study, but we hypothesize that
ammonia produced by the crabs ties up the CIO as monochloramine,
86
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TABLE 25. COMPARISON OF CIO LEVELS IN Callinectes DOSING SYSTEM
oo
Compartment
A
B
C
D
E
F
G
H
without crab
^
SD
CV
with crab
^
SD
CV
t
Applied Dose
1
0.03
0.03*
0.04
0.04*
0.04*
0.04
0.05*
0.03
0.033
0.005
15
0.04
0.008
20
1.5666 ns
30.8 mg/1
WITH AND
2
0.07*
0.07
0.08*
0.08
0.05
0.07
0.07*
0.07*
0.068
0.013
18
0.073
0.005
7
0.7385 ns
61.6 mg/1
WITHOUT CRABS
Tank
3
0.12
0.11*
0.11
0.11
0.11*
0.12*
0.12*
0.10
0.110
0.008
7
0.115
0.006
5
0.9999 ns
92.4 mg/1
4
0.23*
0.30*
0.19
0.20
0.28*
0.19
0.25*
0.20
0.195
0.006
3
0.265
0.031
12
4.43**
123.2 mg/1
5
0.50
0.50
0.41*
0.48
0.43*
0.45*
0.37*
0.46
0.485
0.019
4
0.415
0.034
8
3.575**
154.0 mg/1
6
0.00*
0.00
0.00*
0.00
0.00
0.00
0.00
0.00*
0.000
0.000
0
0.000
0.000
0
0
* with crab
-------
which in turn contributes to higher measured CIO levels. Positive
demand at higher doses could represent bounding of chlorine residuals
to organic compounds released by stressed crabs.
Ammonia Nitrogen
Tank NH3~N levels were dependent on the tank size-flow-CIO
combinations of experiment 13 (ANOVA, p < 0.005). As would be
expected the highest levels occurred in the tanks with the lowest
flow per crab (18-liter-250 ml/min) (Table 26). The remaining tanks,
all with equivalent flows per crab, had roughly comparable NH3~N
levels within each of the control and experimental groups. Measured
NH3~N levels were greatly reduced in tanks containing CIO. The mean
diluent NH3-N level (x » 0.10 mg/1; SD - 0.04) was less than half the
lowest level of the control group (x - 0.25 mg/1; SD - 0.06).
88
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TABLE 26. TUKEY's u-TEST FOR TANK SIZE-FLOW-CIO COMBINATION
EFFECTS ON AMMONIA-NITROGEN IN TANKS FROM EXPERIMENT 13
BARS UNDERLINE EQUAL MEANS (oc-0.05)
tank size (liters) 37 18 18 37 18 18
flow rate (ml/min) 1000 500 250 1000 500 250
CIO level (mgCl2/l) 0.82 0.74 0.58 0.00 0.00 0.00
NH^-N (fflg/1) 0.01 0.03 0.12 0.25 0.32 0.49
Equal means
89
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SECTION 7
RESPIRATORY EFFECTS
METHODS
Experimental Analysis
Mature female blue crabs for study of the short-term effects of
CIO on oxygen consumption were obtained from the Chesapeake Bay
potting fishery. Mature females for all other studies came from the
Chesapeake Bay dredge fishery. Again, the nature of the fisheries
makes it difficult to determine the precise temperature and salinity
regimes from which the crabs were taken. Potted crabs probably came
from salinities between 20 and 25 °/oo and temperatures of 15 to 20°C.
Dredged crabs came from higher salinities (20-30 °/oo) and lower
temperatures (< 5°C). No attempt has been made to compare
physiological measurements between the two populations, but crabs from
only one group have been used for a given series of tests.
Prior to their use all crabs were held for 4-7 days in the
recirculating sea water system used in the serological study. Crabs
were held at room temperature (18-20°C for those used in the
short-term test; 14-17°C for those used in subsequent tests) and York
River salinities (19.5 to 21.8 °/oo and 15.1 to 19.4 °/oo,
respectively). All crabs were fed squid daily, and their claws were
bound to prevent cannibalism.
Exposure Systems
The basic dilutor system was similar to that described in detail
in Section 4.
Short-term effects of CIO on blue crab oxygen consumption were
determined directly in a continuous-flow respirometer. Each of two
cylindrical, three-liter chambers for individual crabs was fitted at
the top with a large rubber stopper containing a tapered opening for a
self-stirring B.O.D. oxygen probe, an incurrent tube and an excurrent
tube. Rate of flow through each plexiglas chamber could be adjusted
by raising or lowering the excurrent tube. Incurrent water was
supplied from a header box which received water from a mixing box.
Appropriate CIO levels were obtained by pumping stocks of varied
90
-------
chlorine concentrations into the mixing box and by adjusting the diluent
flow (2040 ml/tnin for high doses, 3000 ml/min for low doses).
Crabs in all remaining tests were exposed to CIO in 37-liter tanks.
Crabs in six aquaria served as controls while crabs in another six
aquaria were dosed with CIO.
Experimental Protocols
Short-term effects of CIO on the oxygen consumption of whole
crabs were determined by monitoring oxygen levels (mg/1) in the 3-liter
chambers while individual crabs were being exposed to CIO. Ten crabs
each were exposed to a high and a low dose. After crabs were introduced
to the chambers, excurrent flow was monitored and adjusted to ^100 ml/
min. Oxygen levels in the chambers were monitored continuously, using
YSI 5420A oxygen probes and YSI 54 oxygen meters attached to a YSI 81A
dual channel recorder. The crabs were exposed to unchlorinated water
for 2 hr from the time the oxygen concentration in both chambers became
relatively constant. The water was then chlorinated for 2 hr. At the
end of this period, chlorination was stopped; CIO levels in the chambers
were allowed to decrease to undetectable levels; and the crabs were
exposed to unchlorinated water for a final 2 hr. Throughout each test,
ambient oxygen levels in the header tanks were monitored at 15-mln
intervals. Excurrent flow rates were determined every 30 min. Tem-
perature was measured at the beginning and end of each test along with
salinity and at the beginning and end of each chlorination period. CIO
was measured at 7.5-min intervals during and after chlorination until
levels became undetectable. After each test, the crabs were blotted
dry and weighed.
Remaining longer-term tests used twelve 37-liter tanks. The design
called for sixty crabs (ten per tank) to be subjected to a single CIO
level with sixty others serving as controls. Total exposure time was
four days followed by a recovery period of four days. At two, four, six,
and eight days, preselected random samples of nine dosed and nine control
crabs were extracted for determination of oxygen uptake. In one test
employing only one experimental and one control tank, preselected samples
of five crabs were taken after one day. CIO levels, temperature, salinity,
dissolved oxygen, and mortality were measured three times daily; pH and
flow rates were monitored once daily.
Respirometry
Respirometry for the continuous-flow system has already been
described.
The respirometer used to determine whole animal oxygen con-
sumption with the longer-term tests was a static system. Ten
cubical plexiglas animal chambers (volume x = 2930 ml; SD - 8.8) were
91
-------
submerged in a water bath. To maintain acclimation conditions the
water bath was continuously supplied with unchlorinated diluent water
from the dosing system. Each animal chamber was fitted at the top
with a rubber stopper containing an opening fashioned from a B.O.D.
bottle neck.
At the beginning of each test, a crab from the preselected random
sample of nine was put into each chamber and was allowed to adjust to
the apparatus for 30 min. A tenth chamber was used as a blank. The
chambers were isolated with dark plastic partitions. During the
adjustment period,the chambers were aerated and open to the water
bath. After the adjustment period, a rubber stopper was inserted into
the first chamber along with a YSI 5720 self-stirring B.O.D. oxygen
probe attached to a YSI 54 oxygen meter. A dissolved oxygen (mg/1)
reading was taken following equilibration by the probe. The probe was
then moved to the second chamber following Insertion of its stopper.
The first chamber was plugged with a B.O.D. bottle stopper. The
procedure was repeated until rubber stoppers werfe in all the chambers.
Subsequent readings from each chamber required only the removal of the
B.O.D. bottle stopper and insertion of the probe. Readings were made
in sequence through five cycles. Cumulative time from the beginning
of each test was recorded at each reading. After each test, the crabs
were blotted dry and weighed.
Following each determination of whole crab oxygen consumption,
the same crabs were used to determine gill respiration on a Gilson
differential respirometer. The second gill from the posterior
(pleurobranch) was removed from the right side of each crab. Each
gill, rinsed in 1 ym-filtered York River water, was placed in a 15 ml
flask containing 5 ml unchlorinated 1 ym-filtered York River water in
the bottom and ^ 1 ml of 10% KOH in the side arm. After attachment
to the respirometer, flasks were allowed 30 min to equilibrate at
16°C. A shaker frequency of 80 strokes per minute was used.
Following equilibration, standard Gilson techniques were used and
cumulative readings of pi of oxygen consumed were made every 10
min for 90 min. At the end of each test, the gills were blotted
and' dried to constant weight.
Histology
When gills were removed for the determination of their oxygen
consumption, the opposing gill from the left side of each crab was
preserved in Dietrich's fixative for sectioning and histological
examination. Simultaneously, miscellaneous gills were examined fresh
under 430 X magnification.
92
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Data Analysis
Ambient oxygen levels were plotted on individual recordings (mg/1
02 vs. time) from the continuous-flow respirometers. Each recording
was then divided into five sections including the initial
unchlorinated period, periods of rising CIO, steady CIO and declining
CIO and a final unchlorinated period of recovery. Areas between
ambient and chamber oxygen levels were then determined for each
section on a Numonics model 237 graphics calculator (electronic
planimeter). Oxygen consumption rate was computed for each section
from the following formula:
r ,/ / wv i 41940 A C F
02 consumption [ yl/g(wet)/hr] - ^
A (cm^): section area from planimeter
C [(mg) (min)/1000 ml]: recording units of section area
(calibration factor)
F (ml/min): flow rate through chamber
W (g): crab wet weight
T (min): total section minutes
41940 - conversion factor (0.699 ml/mg x 1000 yl/ml x
60 mln/hr)
Within each dose level all possible comparisons were made among
the initial unchlorinated (control) period, the level CIO period and
the unchlorinated recovery period using Student's t-teat for paired
observations.
Regressions of chamber oxygen levels (mg/1) versus time (min)
were used to determine oxygen consumption rates for individual crabs
in the static respirometers according to the following formula:
, , , , , 41940 [S(Vt-Vc)-B]
02 consumption [yl/g(wet)/hr] - -
W
S (mg/1000 ml/min): slope of regression: x-min;
y-mg/1000 ml
Vt (ml): chamber volume with stopper inserted
Vc (ml): calculated crab volume (Laird, unpublished)
B (mg/min): rate of change of oxygen content of blank
chamber
W (g): crab wet weight
Differences between experimental and control crabs were
determined for each sampling period by using Student's t-test for
independent observations.
Regression of oxygen consumed (ul) versus time (hr) were used to
determine rates of oxygen consumption for Individual gills. Slopes
(yl/hr) were converted to ul dry gas at 760 mm Hg and inserted into
the following formula:
93
-------
02 consumption [ yl/g(dry)/hr] - §_
Sg (yl/hr): slope of regression for gill: x-hr; y-yl
Sjj (yl/hr): slope of regression for blanks: x-hr; y-yl
W (g): gill dry weight
Differences in oxygen consumed by gills of experimental and
control crabs were determined for each sampling period by using
Student's t-test for independent observations.
RESULTS
Short-Term Effects
A summary of test systems can be found in Table 27. CIO levels
and other hydrographic data appear in Table 28.
At the low dose (17.8 yeq/1 - 0.63 mg/1) in the first experiment
oxygen consumption did not change significantly from the initial
unchlorinated period (135.5 yl/g/hr; SD - 29.3) to the CIO plateau
period (119.0 yl/g/hr; SD - 30.8) (t - 2.53; p > 0.02) but showed a
decrease from both of these to the recovery period (97.3 yl/g/hr;
SD - 17.0) (t - 6.27, 3.62, respectively; p < 0.01). '
At the high dose (22.3 yeq/1 - 0.79 mg/1) in the second
experiment oxygen consumption decreased from the initial period (134.7
yl/g/hr; SD - 20.8) to the plateau period (114.9 yl/g/hr; SD - 14.7)
(t - 3.11; p < 0.02) and to the recovery period (114.4 yl/g/hr; SD -
17.6) (t - 4.12; p < 0.01). There was no difference, however, between
values from the plateau and recovery periods (t - 0.09; p > 0.5).
Several tests were performed without chlorination to determine
whether time alone was responsible for observed changes in oxygen
consumption. Oxygen consumption did not vary significantly among time
periods equivalent to those compared in the chlorinated tests
(p > 0.05).
Longer-Term Effects
In the third (one day) test, using the static respirometers,
crabs were exposed to 8.7 yeq/1 (0.31 mg/1). Oxygen consumption of
exposed crabs (44.7 Vl/g/hr; SD - 10.4) was not significantly
different from that of the controls (59.8 y1/g/hr; SD - 14.2) (t -
1.92; p > 0.05).
In the fourth and fifth experiments crabs were exposed to 7.6
yeq/1 (0.27 mg/1) and 27.9 yeq/1 (0.99 mg/1), respectively. In the
fourth experiment oxygen consumption did not differ between
experimental and control crabs either during the exposure period (t -
0.29, 1.09 for the two sampling days, respectively; p > 0.2) or during
94
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TABLE 27. SUMMARY OF TEST SYSTEMS USED, DOSES, SAMPLING INTERVALS
AND ANALYSES DURING Callinectes saoidus RESPIRATION STUDIES
Experiment
No.
1
2
3
4
5
System
(Respirometer)
flow-through
static
static
(whole crab)
Gilson
(Gill)
Mean
Measured
CIO
(MgCl2 /liter)
0.63
0.79
0.31
0.27
0.99
Sampling
Interval
continuous
continuous
1 day
2 days
2 days
No. crabs /sample
(Design)
10
11
5 experimental
5 control
9 experimental
9 control
9 experimental
9 control
Analysis
whole crab
oxygen consumption
whole crab
oxygen consumption
whole crab
oxygen consumption
whole crab and
excised gill
oxygen consumption
whole crab and
excised gill
oxygen consumption
-------
TABLE 28. SUMMARY OF HYDROGRAPHIC DATA FOR Callinectes aanidus RESPIRATION STUDIES
Experiment
No.
1
2
3
4
5
Me
appl
peq/1
88.6
124.1
33.0
31.3
79.5
an
led
mg/1
3.14
4.40
1.17
1.11
2.82
Mean
meas
CIO ]
peq/1
17.8
22.3
8.7
7.6
27.9
ured
.evel
mg/1
0.63
0.79
0.31
0.27
0.99
Measured CIO
star
devlf
Peq/1
1.2
1.7
0.6
1.2
4.2
idard
ition
mg/1
0.04
0.06
0.02
0.04
0.15
Tempe
(
x"
21.5
21.2
17.3
15.8
14.5
rature
°C)
SD
0.78
0.95
0.71
0.58
0.69
Sail
(°/
Tc
20.5
19.5
18.4
13.8
14.6
nity
DO)
SD
0.41
0.25
0.29
0.88
0.36
Mean
D.O.
(mg/1)
7.8
(Header)
7.9
(Header)
8.5
8.8
8.8
Mean
PH
ND
ND
7.8
7.7
7.8
o*
ND - No data.
-------
the recovery period (t - 0.05; -0.65; p > 0.5) (Fig. 14). Gill
respiration was also unaffected (t 0.13, 1.25, 0.22, -0.83,
respectively, for the four sampling days; p > 0.2) (Fig. 15). In the
fifth test, no sample was taken on the fourth recovery day because of
high mortalities among the experimental crabs. Experimental crabs
from the first sample taken during chlorination showed higher whole
crab oxygen consumption (52.0 Pl/g/hr; SD = 6.3) than did the controls
(35.5 Wl/g/hr; SD - 9.3) (t = 4.39; p < 0.001) (Fig. 16). There was,
however, no effect on the last day of chlorination (t = 0.38; p > 0.5)
or on the second day of recovery (t = -1.26; p > 0.2). Gill
respiration was unaffected throughout the test (t = -1.63, -0.63,
1.04, respectively, for the three sampling days; p > 0.1) (Fig. 17).
Histopathology
Fresh Material
Gills from crabs exposed to 27.9 ueq/1 (0.99 mg/1) had a blanched
appearance after two days. Microscopic examination of individual
platelets showed an optically denser, possibly thickened area at the
center. Extent of this area varied somewhat among gills. In
addition, the normally reticular pattern of cells in the outer
portions of the platelets seemed disrupted. These effects persisted
into the recovery period.
Fixed Material-
Gill tissues were fixed in Dietrich's fixative, stained with
Mayer's hematoxylin, Astra blue and Eosin Y, and sectioned.
Examination of gill tissues with a light microscope revealed no
pathological changes.
Activity
The continuous-flow tests allowed a crude estimate to be made of
the amount of CIO required for crabs to withdraw their antennules.
Permanent withdrawal was caused by 6.2 yeq/1 (0.22 mg/1) at 21.5 +
97
-------
NO
00
CONTROL
10 20 30 40 SO 60 70 80 '90 100 110 120 130 140 150 160 170 180 190 200
I I
100
90 .
~ 80 .
jC
^^
f 70 .
a
7; 60
o
'"5.
90
Z
O
t 40 .
lao .
8
£20 .
10 .
0
~sa«-
oconu'
-KM
-KM.
^
-
L
r
.
,
'
, »
"1
t
.-
.
EXPOSURE RECOVERY
1 I 1 1
10 20
30
I
1
EXPERIMENTAL
I 1 1 1 I 1 1 I ' I I
40 50 60 70 80 90 100 110 120 130 140
i i i i i i
150 160 170 180 190 200
TIME (HOURS)
Figure 14. Effects of CIO exposure (0.27 ing C12/1) and recovery on whole
crab oxygen consumption in Callinectes sapidus.
-------
CONTROL
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200
NO
1300
1200 -
1100 -
s 1000 -
o
3
o
fc 800 -
CO
I
CM
O
700 -
600 -
500 -
400
EXPOSURE
RECOVERY
75m ...
PERCENTILE
25th _
PERCENTILE
^
1
MEAN
[MEDIAN
I
4
RANGE
EXPERIMENTAL
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200
TIME (HOURS)
Figure 15. Effects of CIO exposure (0.27 og Cl-2/1) and recovery on oxygen
consumption of excised gills in Callinectes sapidus.
-------
o
o
CONTROL
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200
I
» »
100-,
90-
80.
.-. 70-
I »-
^
6* 50-
co
I 20
CM
O
10
75th _
PERCENTILE
25th _
PERCENTILE
"
MEAN
-MEDIAN
'
RANGE
EXPOSURE
RECOVERY
EXPERIMENTAL
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200
TIME (HOURS)
Figure 16. Effects of CIO exposure (0.99 ng d.2/1) and recovery on whole
crab oxygen consumption in Callinectes sapidus.
-------
CONTROL
0 10 20 30 40 SO 60 70 80 90 100 110 120 130 140 160 160 170 180 190 200
1200.
1100.
1000.
900.
O
~ 800^
O
t 700.
S 600J
500.
400.
EXPOSURE
RECOVERY
75th _.
PERCENTILE
ZStk _
PERCENTILE
^
MEAN
[-MEDIAN
4
RANGE
EXPERIMENTAL
Till
w
10 20 30 40 50 60 70 80 90 100 110 120 130 140 160 160 170 180 190200
TIME (HOURS)
Figure 17. Effects of CIO exposure (0.99 ag ClWl) and recovery on oxygen
consumption of excised gills in Callinectes sapidus.
-------
SECTION 8
DISCUSSION
Considerable effort was necessary to develop a flow-through test
system for use with invertebrate larvae. Earlier work with decapod
larvae has been carried out in static systems of varying dimensions.
As outlined earlier, the experiments described herein required a
flow-through system to continuously maintain the toxicant
concentration. The design of the system used here is similar in
principle to that described by Buchanan et al. (1975) for decapod
larvae. The test system used in the present studies, as finally
applied, yielded better survival of control decapod larvae than larvae
from the same batch grown in static culture. Survival and recovery of
Mulinia larvae was good in one experiment, but data are not available
for a parallel static control. In most Mulinia experiments, however,
recovery of larvae was too inadequate to allow statistical treatment
of the data. This basic type of flow-through system, using a tidal
siphon to insure exchange of water within the animal chambers without
physical damage to the test animals seems well suited for experiments
with small planktonic invertebrates such as decapod larvae, while
improved systems need to be developed for bivalve larvae.
From the data collected, it would appear that for any given
decapod species, eggs are more tolerant of CIO than larvae, while
mature adults are most tolerant of any stage. This is the case for
Panopeus herbstil, for which we have the most complete survey of life
stages. For Pagurus longicarpus comparison of larval test data with
the exploratory test data for adults yields the same conclusion.
Calllnectes Juveniles (see section 4) seem to be less tolerant than
mature adults (see section 6).
The concept that early stages are more sensitive than adults is
not especially new, although we are unaware of any specific published
data which demonstrates this point. The greater tolerance of eggs
compared to zoeae of crabs is in contradiction to the basic premise.
However, crab eggs, which develop while attached to the pleopods
of mature females, have thick membranes which are rather impermeable.
These membranes serve to protect the developing embryo from changes
in the external medium. Zoeal stages have a relatively poorly
calcified exoskeleton which may be more permeable than the egg
membrane. Further, during the molting process the exoskeleton is
102
-------
shed, and water is rapidly taken in before the new exoskeleton
hardens. During this period larvae would be especially sensitive to
any toxicant. The gut also represents an avenue of entry into larvae
for toxicants.
There is an apparent seasonal change in the acutely toxic dose
for Panopeus herbstii zoeae, with larvae produced early or late in the
normal breeding season being more sensitive than those produced during
the rest of the breeding season. Tatem et al. (1976) has demonstrated
a seasonallty in the LC50 of Palaemonetes adults exposed to dodecyl
sodium sulphate.
Mulinia embryos are less tolerant of CIO than decapod larvae of
either species tested. The LC50 for Mulinia while ranging from 0.3 to
2.8 yeq/1 (0.01 to 0.1 mg/1) was approximately 1.0 yeq/1 (0.035 mg/1)
in several tests. This value is similar to that derived for oyster
larvae (0.73 yeq/1 * 0.026 mg/1) using the same test system (Roberts
and Gleeson, 1978). For those species tested to date, molluscan
larvae appear to be more sensitive to CIO than decapod larvae. Among
the crustaceans, only Acartla tonsa seems to be as sensitive as
molluscan larvae (Roberts and Gleeson, 1978).
Avoidance tests with decapod larvae have not previously been
attempted in a flowing-water system, but rather in static systems.
The chemical nature of chlorine-induced oxidants necessitated
development of a flowing-water test system.
Larvae, when tested under identical conditions of light and
temperature in both chambers, respond in the same way, indicating that
there is no systematic chamber effect. Salinity does significantly
affect the level of response with more positive responses at 18 °/oo
than.24 °/oo salinity. This is contrary to previous observations
(Roberts, 1971). One would expect the greater response at the higher
salinity since 18 °/oo is close to the salinity tolerance limit for
larvae. No explanation of the observations can be given at this time.
For chlorine avoidance tests, one need not have an explanation.
Rather it is only necessary to perform all tests at a controlled
salinity(ies).
The effect of light intensity is rather small, with a slightly
higher response at 24 fc than at 240 fc. This agrees with the results
of Latz and Forward (1977) for Rhlthropanopeus harrlssii larvae.
Light should be retained as a variable in chlorine avoidance tests,
however, since the measure of response does not measure strength of
the response. The slight delay in achieving the response at the lower
light intensity suggests that the strength of the response at this
intensity Is less.
The single test to date yielding information on response to
chlorinated seawater, performed with Palaemonetes puglo, indicates
103
-------
that larvae can detect CIO at 70.5 yeq/1 (2.5 mg/1) as evidenced by
telson flips and downward swimming. After a few minutes, however, a
few larvae swam Into the upper chlorinated layer and stayed there,
although they still exhibited signs of distress. This may indicate
that the CIO level destroyed or interfered with the chemoreceptors of
the larvae. Latz and Forward (1977) reported a similar situation with
Rhithropanopeus harrissii larvae challenged by a sudden salinity
change. They called this reversal from negative initial responses to
positive final response, habituation. However in the study of Latz
and Forward (1977) there was no higher salinity layer underlying the
salinity layer into which the larvae could retreat. In tests where a
refuge layer was provided in previous studies (Lance, 1962; Roberts,
1971), no evidence of habituation in this sense was observed.
The validity of the observation on response to a chlorinated
upper layer can only be determined by further study. The minimum CIO
concentration which larvae can detect remains to be determined. If
habituation is a real phenomenon, it will be necessary to determine
whether this occurs at CIO concentrations above the LC50 to assess
whether this behavior represents an environmental risk.
Statistically significant as well as apparent trends in serum
constituents observed In preliminary tests (experiments 1 through 5)
using small sample sizes suggested that exposure to CIO affects the
ability of the blue crab to osmoregulate. Effects were measured only
at CIO levels at or above the lowest level causing mortality In the
test population. Wherever significant effects or apparent trends
occurred, chloride and osmotic concentrations decreased and TNPS
levels Increased in crabs exposed to CIO. Apparent trends for protein
were more variable but also tended to decrease overall. All such
changes are expected results of osmoregulatory failure in the
hyperregulating crabs. Similar effects in the blue crab have been
attributed to osmoregulatory failure by Block (1977). Increases in
TNPS, in particular, have been related to osmotic stress In Eriocheir
sinensis (Vincent-Marique and Gilles, 1970) and Panulirus longlpes
(Dall, 1975).
Inhibition of osmoregulatlon was not observed in subsequent
tests. Significant effects of CIO on serum constituents occurred
sporadically and only in chloride and osmotic concentration. These
effects did not occur concommltantly and appeared unrelated to CIO
levels. Apparent trends were also highly variable and seemed
unrelated to CIO levels. In addition, the majority of trends and
statistical effects were in the contrary direction to that which would
be expected with failure of osmoregulation in the crabs. This was
true even in experiment 12 in which the crabs were originally
hyporegulatlng as a result of the high salinity. Finally, the
magnitude of trends and statistical effects was of doubtful
physiological significance. Even statistically significant
differences in blood chloride and osmoconcentratlon were rarely over
104
-------
5% and never more than 10% of control levels.
It is possible that the observed effects were merely secondary
manifestations of the impending death of the crabs. Although effects
of CIO on serum constituents were of no greater magnitude in
experiments with mortalities than in other tests, more pronounced
effects were found in moribund crabs.
Since CIO had no clearly apparent direct inhibitory effect on
osmoregulation, we felt that the most obvious alternative effect
mechanism would be inhibition of oxygen uptake. Initial short-term
tests proved promising, showing a general decrease in oxygen
consumption of whole crabs with no apparent recovery. However, based
on further tests, this effect was probably a short-term reaction to
sensing the presence of CIO.
In the remaining tests with whole animals a difference between
oxygen consumption of experimental and control animals was observed in
only one sample group. In this case exposure to CIO seemed to
stimulate oxygen consumption rather than inhibit it after 2 days at
27.9 vteq/1 (0.99 mg/1). This effect was ultimately assumed to be
spurious, since there were no effects on whole crabs after other
exposure intervals or on excised gills from crabs in any tests.
Death of blue crabs exposed to CIO does not seem to be related to
asphyxiation. Despite mortalities in the test population exposed to
27.9 ueq.l (0.99 mg/1) CIO, there was no effect on oxygen consumption
of surviving whole crabs or excised gills. In addition, the gill
damage apparent in fresh material was not confirmed on examination of
fixed tissue. Therefore, there is no reason to believe that the
ability of blue crab gills to transport oxygen is permanently
inhibited by exposure to CIO.
There appeared to be no relationship between the occurrence of
mortalities among test populations and effects of CIO on serum
constituents and respiration of crabs from these populations.
Mortalities in many tests occurred in the absence of effects on serum
constituents and respiration. This fact coupled with the magnitude
and the sporadic nature of effects observed in some tests leads to the
conclusion that there are no physiologically significant sublethal
effects of CIO on the selected serum constituents or oxygen
consumption of whole blue crabs or excised gills.
We observed an obvious sensitivity of blue crab antennules to CIO
and inhibition of spawning and feeding In dosed crabs. The effect on
spawning occurred at CIO levels as low as 12.4 yeq/1 (0.44 mg C12/D,
and the effect on feeding was at CIO levels as low as 10.2 yeq/1 (0.36
mg C12/1). Although the effects seem to be transient (as observed in
antennule activity and feeding response), disappearing when CIO is no
longer present, they have long term implications and should be
105
-------
considered in determining safe levels of CIO for blue crabs. It would
be useful to test whether these effects ever become permanent and to
quantify more precisely their relationships to dose and time.
106
-------
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Dall, W. 1975. The Role of Ninhydrin-Positive Substances in
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Johnson, J. D. 1977. Analytical Problems in Chlorination of Saline
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*U.S.GOVIRNMENTPRINTINGOFFICE:1979-6«tO-OOV 986 REGION NO. 4
110
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing!
1. REPORT NO.
EPA-600/3-79-031
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Effects of Chlorinated Seawater on Decapod
Crustaceans and Mulinia Larvae
5. REPORT DATE
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Morris H. Roberts, Jr., Chae E. Laird,
and Jerome E. Illowsky
8. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Virginia Institute of Marine Science
Gloucester Point, Virginia 23062
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
R-803872
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
13. TYPE OF REPORT AND PERIOD COVERED
Final, 9/14/75-3/31/78
14. SPONSORING AGENCY CODE
EPA-ORD
16. SUPPLEMENTARY NOTES
16. ABSTRACT
Eggs and larvae of decapod crustaceans and embryos of Mulinia lateval'ie were
exposed to chlorinated seawater for varying periods in continuous flow systems.
Mortality, developmental rate, and general behavior were recorded. Panopeue herbetii-
zoeae were more sensitive to chlorine-induced oxidants (CIO) than eggs or adults
(96-hr LC50 ca. 2.8 yeq/1 = 0.1 mg/1). The 96-hr LC50 for PagianiB langioaypus zoeae
was approximately the same as for Panopeus zoeae. The 120-hr LC50 for Pagurus zoeae
was 1.4 ueq/1 (0.05 mg/1). Development was slightly delayed for Pagtams zoeae at CIO
levels as low as 0.6 yeq/1 (0.02 mg/1). Mulinia embryos exposed for 48-hr had an
LC50 between 0.3 and 2.8 yeq/1 (0.01 and 0.1 mg/1). Mulinia embryos exposed to
chlorinated seawater for 2-hr had an LC50 of about 2.0 yeq/l(0.072 mg/1); subsequent
survival rates for larvae in unchlorinated seawater were unaffected by prior ex-
posure to CIO.
The effects of CIO on serum constituents in Callinectes eapidus occurred
sporadically and appeared unrelated to dose or mortality. Similar effects were
noted for oxygen consumption in whole crabs and excised gills. It was concluded
that there are no physiologically significant sublethal effects of CIO on serum
constituents (osmoregulation) or oxygen consumption of whole blue crabs or excised
gills. Blue crab antennules are sensitive to sublethal doses of CIO. Spawning and
feeding are inhibited by sublethal doses of CIO. This effect disappears when
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Toxicity
chlorine
Mulinia lateralis
Crustacea
larvae
blood
respiration
8. DISTRIBUTION STATEMENT
Unlimited
b.lOENTIFIERS/OPEN ENDED TERMS
decapod larvae
19. SECURITY CLASS (This Kcport/
unclassified
20 SECURITY CLASS tlhispagei
unclassified
c. COSATI Held/Group
21. NO. OF PAGES
110
22. PRICE
EPA FC-TI 2220-1 (Rt*. 4-77) PREVIOUS ei'
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