EPA-600/3-77-131
November 1977
Ecological Research Series
BIOLOGICAL EFFECTS OF PESTICIDES
ON THE DUNGENESS CRAB
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
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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
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The nine series are:
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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-
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This document is available to the public through the National Technical Informa-
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BIOLOGICAL EFFECTS OF PESTICIDES
ON THE DUNGENESS CRAB
by
Richard S. Caldwell
Department of Fisheries and Wildlife
Marine Science Center
Oregon State University
Newport, Oregon 97365
Contract No. 68-01-01!
Project Officer
Marl in E. Tagatz
Environmental Research Laboratory
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 and approved for publication by the
Environmental Research Laboratory, Gulf Breeze, U.S. Environmental Protection
Agency (EPA). Approval does not signify that the contents necessarily relfect
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 restricting the intro-
duction of these compounds into the environment be formulated on a sound
scientific basis. Accurate information describing dose-response relation-
ships for organisms and ecosystems under varying conditions is required.
The Environmental Research Laboratory, Gulf Breeze, contributes to this
information through research programs aimed at determining:
.the effects of toxic organic pollutants on individual species and
communities or organisms;
•the effects of toxic organics on ecosystem processes and components;
.the significance of chemical carcinogens in the estuarine and marine
envi ronments.
This report describes the toxicity of nine pesticides to various life
history stages of the Dungeness crab. The data obtained for each pesticide,
which identify the crab's most sensitive stage and the highest concentration
having no discernible effect on that most sensitive stage during prolonged
exposures, will be useful in establishing saltwater quality criteria.
Thomas W. Duke
Di rector
Environmental Research Laboratory
i i i
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ABSTRACT
The toxicity of nine pesticides to various life history stages of the
Dungeness crab, Cancer magister, was examined to establish the most sensitive
life stage of the crab, and the highest concentration of each pesticide
having no discernible effect on that most sensitive stage during prolonged
exposures. The compounds tested were the insecticides carbofuran, chlordane,
malathion and methoxychlor; the herbicides 2,A-D, DEF, propanil and
trifluralin; and the fungicide captan.
For each pesticide, the zoeal stages were found to be the most sensitive
in long-term tests, approximately 5 to 10 times and 10 to 100 times more
sensitive than juvenile and adult crabs, respectively, and were also affected
at lower concentrations than those that affected egg hatching and prezoeal
development. The maximum acceptable toxicant concentrations for continuous
exposures of C_. magister zoeae to each of the nine pesticides are:
methoxychlor, 0.005 yg/liter; chlordane, 0.015 yg/liter; malathion, 0.02
yg/liter; carbofuran, 0.05 yg/liter; captan, 2 yg/liter; DEF, k yg/liter;
trifluralin, 15 yg/liter; propanil, 80 yg/liter; and 2,4-D, 1000 yg/liter.
The toxicity of each of these pesticides to crabs is compared with
literature reports of their toxicity to other aquatic species.
This report was submitted in fulfillment of Contract No. 68-01-0188 by
Oregon State University under the sponsorship of the U. S. Environmental
Protection Agency. This report covers the period April 28, 1972 to April 28,
197^.
IV
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CONTENTS
Foreword i i i
Abstract iv
Figures vi
Tables xi i
Acknowledgments xiv
1. Introduction 1
2. Conclusions 2
3- Recommendations 3
4. Materials and Methods *f
Pesticides 4
Pesticide analytical methods 5
Experimental animals 5
Egg hatching - prezoeal bioassays 8
96-hr acute toxicity bioassays 9
Chronic toxicity bioassays . 12
Uptake, loss and tissue distribution of methoxychlor. 21
Salinity tolerance experiment 21
Osmotic-ionic regulation experiments . . 22
ATPase assay 22
5. Results and Discussion 23
The fungicide captan 23
The insecticide methoxychlor 36
The herbicides 2,4-D, DEF, propanil and trifluralin . 62
The insecticides carbofuran, chlordane and malathion. 97
Summary of pesticide tolerance 112
References 119
Publications 125
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FIGURES
Number Pagg.
1 Gas chromatogram of technical chlordane standard 6
2 Gas chromatogram of technical chlordane after
extraction from seawater 7
3 Diagram of culture containers illustrating the
relation between the square culture aquarium and
the culture beakers, showing details of the
automatic siphon, and the positioning of the two
screens on the lower side of a culture beaker ......... 1 k
k Percent hatch of Dungeness crab eggs, percent
development of hatched crab larvae through the
prezoeal to the first zoeal stage, and percent of
developed first stage zoeae which are motile as a
function of captan concentration during a 24-hr
exposure period ........................ 2k
5 Effect of continuous exposure to captan on survival
of Dungeness crab zoeae. Data are from the first
zoeal chronic experiment conducted during the spring
of 1973 ..... • ...................... 27
6 Effect of continuous exposure to captan on survival
of Dungeness crab zoeae. Data are from the second
zoeal chronic experiment conducted during the spring
of 197^ ...... . ..................... 28
Effect of continuous exposure to captan on molting
of Dungeness crab zoeae beginning with the first zoeal
molt .............................. 29
Effect of continuous exposure to captan on survival of
juvenile Dungeness crabs. Data are from the first juvenile
chronic experiment which was initiated with newly metamorphosed
first instar crabs ...... . ................ 30
Effect of continuous exposure to captan on molting of
first instar Dungeness crabs to the second instar ....... 31
v i
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Number Page
10 Effect f continuous exposure to captan on survival of
juveh i1? Dungeness crabs. Data are from the second
juvenile chronic experiment which was initiated with
second and third instar crabs 32
11 Effect of continuous exposure to captan on survival of
adult Dungeness crabs 33
12 Percent hatch of Dungeness crab eggs, percent
development of hatched crab larvae through the
prezoeal to the first zoeal stage, and percent
of developed first stage zoeae which are motile
as a function of methoxychlor concentration during
a 24-hr exposure period 36
13 Effect of continuous exposure to methoxychlor on survival
of Dungeness crab zoeae. Data are from the first zoeal
chronic experiment conducted during the spring of 1973 38
14 Effect of continuous exposure to methoxychlor on survival
of Dungeness crab zoeae. Data are from the second zoeal
chronic experiment conducted during the spring of 1974 39
15 Effect of continuous exposure to methoxychlor on molting
of Dungeness crab zoeae beginning with the first zoeal
molt 40
16 Effect of continuous exposure to methoxychlor on survival
of juvenile Dungeness crabs. Data are from the first
juvenile chronic experiment which was initiated with
newly metamorphosed first instar crabs 41
17 Effect of continuous exposure to methoxychlor on survival of
juvenile Dungeness crabs. Data are from the second juvenile
chronic experiment which was initiated with second and
third instar crabs ............. 42
18 Effect of continuous exposure to methoxychlor on molting
of first instar Dungeness crabs to the second instar 44
19 Effect of continuous exposure to methoxychlor on survival
of adult Dungeness crabs ......... 45
20 Whole body concentration of methoxychlor in juvenile crab,
Cancer magister, as a function of time of exposure to the
pesticide in seawater ........... 50
21 Whole body concentration of methoxychlor in adult crabs,
Cancer magister, as a function of time during and after a
15-day exposure to the pesticide in seawater . 51
vi i
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Number Page^
22 Blood osmotic concentration of control and methoxychlor-
treated Cancer magister as a function of medium osmolarity ... 56
23 Blood and urine sodium concentration of control and
methoxychlor-treated Cancer magi ster as a function of
experimental salinity ..... 57
2k Blood and urine potassium concentration of control and
methoxychlor-treated Cancer magister as a function of
experimental salinity . 58
25 Blood and urine magnesium concentration of control and
methoxychlor-treated Cancer magister as a function of
experimental salinity ..... 59
26 Percent hatch of Dungeness crab eggs, percent development of
hatched crab larvae through the prezoeal to the first zoeal
stage, and percent of developed first stage zoeae which are
motile as a function of trifluralin concentration during a
24-hr exposure period ..... 63
27 Percent hatch of Dungeness crab eggs, percent development of
hatched crab larvae through the prezoeal to the first zoeal
stage, and percent of developed first stage zoeae which are
motile as a function of DEF concentration during a 24-hr
exposure period 64
28 Percent hatch of Dungeness crab eggs, percent development of
hatched crab larvae through the prezoeal to the first zoeal
stage, and percent of developed first stage zoeae which are
motile as a function of propanil concentration during a 24-hr
exposure period 65
29 Percent hatch of Dungeness crab eggs, percent development of
hatched crab larvae through the prezoeal to the first zoeal
stage, and percent of developed first stage zoeae which are
motile as a function of 2,4-D concentration during a 24-hr
exposure period 66
30 Effect of continuous exposure to trifluralin on survival of
Dungeness crab zoeae. Data are from the first zoeal chronic*
experiment conducted during the spring of 1973 68
31 Effect of continuous exposure to propanil on survival of
Dungeness crab zoeae. Data are from the first zoeal chronic
experiment conducted during the spring of 1973 69
32 Effect of continuous exposure to DEF on survival of Dungeness
crab zoeae. Data are from the first zoeal chronic experiment
conducted during the spring of 1973 70
v i i i
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Number
33 Effect of continuous exposure to 2,4-D on survival of
Dungeness crab zoeae. Data are from the first zoeal
chronic experiment conducted during the spring of 1973
34 Effect of continuous exposure to trifluralin on survival of
Dungeness crab zoeae. Data are from the second zoeal chronic
experiment conducted during the spring of 1974 72
35 Effect of continuous exposure to propanil on survival of
Dungeness crab zoeae. Data are from the second zoeal chronic
experiment conducted during the spring of 1974 73
36 Effect of continuous exposure to 2,4-D on survival of
Dungeness crab zoeae. Data are from the second zoeal chronic
experiment conducted during the spring of 1974 74
37 Effect of continuous exposure to DEF on survival of Dungeness
crab zoeae. Data are from the second zoeal chronic experiment
conducted during the spring of 1974 75
38 Effect of continuous exposure to trifluralin on molting of
Dungeness crab zoeae beginning with the first zoeal molt .... 76
39 Effect of continuous exposure to DEF on molting of Dungeness
crab zoeae beginning with the first zoeal molt 77
40 Effect of continuous exposure to propanil on molting of
Dungeness crab zoeae beginning with the first zoeal molt .... 78
41 Effect of continuous exposure to 2,4-D on molting of
Dungeness crab zoeae beginning with the first zoeal molt .... 79
42 Effect of continuous exposure to trifluralin on survival of
juvenile Dungeness crabs. Data are from the first juvenile
chronic experiment which was initiated with newly
metamorphosed first instar crabs . 80
43 Effect of continuous exposure to trifluralin on molting of
first instar Dungeness crabs to the second instar 81
44 Effect of continuous exposure to trifluralin on survival of
juvenile Dungeness crabs. Data are from the second juvenile
chronic experiment which was initiated with second and third
instar crabs 82
45 Effect of continuous exposure to DEF on survival of juvenile
Dungeness crabs. Data are from the first juvenile chronic
experiment which was initiated with newly metamorphosed
first instar crabs 83
IX
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Number Page
46 Effect of continuous exposure to DEF on survival of juvenile
Dungeness ^--~bs. Data are from the second juvenile chronic
experiment wnich was initiated with second and third instar
crabs ......... • ................... 84
47 Effect of continuous exposure to DEF on molting of first instar
Dungeness crabs to the second instar .............. 85
48 Effect of continuous exposure to propanil on survival of
juvenile Dungeness crabs. Data are from the first juvenile
chronic experiment which was initiated with newly
metamorphosed first instar crabs ................ 86
49 Effect of continuous exposure to propanil on survival of
juvenile Dungeness crabs. Data are from the second juvenile
chronic experiment which was initiated with second and third
instar crabs .......................... 87
50 Effect of continuous exposure to propanil on molting of first
instar Dungeness crabs to the second instar .......... 88
51 Effect of continuous exposure to 2,4-D on survival of juvenile
Dungeness crabs. Data are from the first juvenile chronic
experiment which was initiated with newly metamorphosed first
instar crabs ............ . ............. 89
52 Effect of continuous exposure to 2,4-D on molting of first
instar Dungeness crabs to the second instar .......... 90
53 Effect of continuous exposure to 2,4-D on survival of juvenile
Dungeness crabs. Data are from the second juvenile chronic
experiment which was initiated with second and third instar
crabs ........... . ................. 91
54 Effect of continuous exposure to trifluralin on survival of
adult Dungeness crabs ..... . ............... 92
55 Effect of continuous exposure to 2,4-D on survival of adult
Dungeness crabs ........................ 93
56 Effect of continuous exposure to DEF on survival of adult
Dungeness crabs .......... . ............. 94
57 Effect of continuous exposure to propanil on survival of
adult Dungeness crabs ..................... 95
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Number Page
58 Percent hatch of Dungeness crab eggs, percent development of
hatched crab larvae through the prezoeal to the first zoeal
stage, and percent of developed first stage zoeae which are
motile as a function of carbofuran concentration during a
24-hr exposure period 97
59 Percent hatch of Dungeness crab eggs, percent development of
hatched crab larvae through the prezoeal to the first zoeal
stage, and percent of developed first stage zoeae which are
motile as a function of chlordane concentration during a
24-hr exposure period 98
60 Percent hatch of Dungeness crab eggs, percent development of
hatched crab larvae through the prezoeal to the first zoeal
stage, and percent of developed first stage zoeae which are
motile as a function of malathion concentration during a
24-hr exposure period 99
61 Effect of continuous exposure to carbofuran on survival of
Dungeness crab zoeae. Data are from the second zoeal chronic
experiment conducted during the spring of 1974 101
62 Effect of continuous exposure to carbofuran on molting of
Dungeness crab zoeae beginning with the first zoeal molt .... 102
63 Effect of continuous exposure to malathion on survival of
Dungeness crab zoeae. Data are from the second zoeal chronic
experiment conducted during the spring of 1974 103
64 Effect of continuous exposure to malathion on molting of
Dungeness crab zoeae beginning with the first zoeal molt .... 104
65 Effect of continuous exposure to chlordane on survival of
Dungeness crab zoeae. Data are from the second zoeal chronic
experiment conducted during the spring of 1974 105
66 Effect of continuous exposure to chlordane on molting of
Dungeness crab zoeae beginning with the first zoeal molt .... 107
67 Effect of continuous exposure to carbofuran on survival of
adult Dungeness crabs 108
68 Effect of continuous exposure to chlordane on survival of
adult Dungeness crabs 109
69 Effect of continuous exposure to malathion on survival of
adult Dungeness crabs 110
XI
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TABLES
Number
1 Concentrations of Pesticides Used in Egg Hatching-
Prezoeal Bioassay 10
2 Concentrations of Pesticides used in 96-hr Acute
Toxicity Bioassays with First Instar Zoeae of Cancer
magister 11
3 Concentrations of Pesticides used in 96-hr Acute
Toxicity Bioassays with First Instar Juveniles of
Cancer magister 12
4 Concentrations of Pesticides used in 96-hr Acute
Toxicity Bioassays with Adults of Cancer magister 13
5 Pesticide Concentrations in Seawater during First
Series of Zoeal Chronic Experiments 15
6 Pesticide Concentrations in Seawater during Second
Series of Zoeal Chronic Experiments 16
7 Pesticide Concentrations in Seawater during First
Series of Juvenile Chronic Experiments 18
8 Pesticide Concentrations in Seawater during Second
Series of Juvenile Chronic Experiments 19
9 Pesticide Concentrations in Seawater during Adult
Chronic Experiments 20
10 Decay Rate of Captan in Seawater of 25 °/00 Salinity
at 13°C 23
11 Acute Toxicity of Captan to First Instar Zoeae, First
Instar Juvenile Crabs and Adult Crabs 26
12 Acute Toxicity of Methoxychlor to First Instar Zoeae,
First Instar Juvenile Crabs and Adult Crabs v . 37
13 The Effect of Methoxychlor on Survival of Molted
Juvenile and Adult Crabs during Continuous Exposure
to the Pesticide for 80 and 85 days, Respectively 43
x i i
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Number Page
14 Whole Body Methoxychlor Concentrations in Adult Crabs
Killed during Continuous Exposure to Methoxychlor for
up to 15 days 48
15 Whole Body Residues of Methoxychlor in Adult Crabs washed
with Water Only or with Water First, followed by Acetone
before Pesticide Analysis 49
16 Distribution of Methoxychlor in Selected Tissue of
Adult Crabs after 15 Days of Continuous Exposure
to 1.8 or 7*5 yg/liter of the Pesticide in Seawater 52
17 Percent Mortality of Adult Crabs Exposed to Methoxychlor in
Dilute Seawater 54
18 ATPase Activity in Gills of Adult Crabs exposed to
Methoxychlor 61
19 Acute Toxicity of Herbicides to First Instar Zoeae, First
Instar Juvenile Crabs and Adult Crabs in 96-hr Tests 67
20 Acute Toxicity of Insecticides to First Instar Zoeae and
Adult Crabs in 96-hr Tests 100
21 Toxic Concentrations of Various Insecticides to Larval Crabs ... 113
22 Relative Sensitivity of Various Life History Stages of C_.
magister to Nine Pesticides 114
23 Estimated Maximum Acceptable Toxicant Concentrations for
Continuous Exposure of Crabs to Each of Nine Pesticides .... 118
XIII
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ACKNOWLEDGMENTS
The author is especially grateful to Mr. David V. Buchanan, Mr. David A.
Armstrong and Mr. Michael H. Mai Ion for substantial contributions in the
design and conduct of various parts of the project studies and also for con-
tributions to the writing of this report. Able technical assistance was
also provided by Mr. Michael J. Myers and Ms. Barbara E. Stone. Professor
Wilbur P. Breese, Mr. Nelson E. Stewart and Dr. Peter Doudoroff often con-
tributed sound advice.
For contributions of pesticide chemicals used in the studies we thank
Eli Lilly and Company; Rohm and Haas Company; the Chemagro Division of
Baychem Corporation; Miller's Products of Portland, Oregon; FMC Corporation;
Chevron Chemical Company; and E. I. DuPont De Nemours and Company.
x iv
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SECTION I
INTRODUCTION
The widespread use of pesticides in recent years has led to concern over
the consequences of such usage for aquatic organisms. Until recently, the
majority of aquatic toxicology studies have dealt with freshwater species,
have emphasized the accumulation of acute toxicity data and have frequently
been limited to studies with adult organisms. In the present study, we have
examined the acute and chronic toxicity of nine pesticides to various de-
velopmental stages of the Dungeness crab, Cancer magister Dana, an important
commercial species of the west coast of North America. The compounds tested
were the fungicide captan; the herbicides 2,4-D, DBF, propanil and
trifluralin; and the insecticides carbofuran, chlordane, malathion and
methoxychlor.
Our objective was to establish for each pesticide the highest concen-
tration having no discernible effect on C_. magister during prolonged expo-
sures on the assumption that these concentrations could be applied as crite-
ria for protection of the species in marine waters. Mount and Stephen (1967)
have recommended procedures for establishing the maximum acceptable toxicant
concentration for aquatic species. These involve exposure of organisms to
constant concentrations of a toxicant throughout an entire life cycle, thus
providing an opportunity for assessment of effects on all stages of the life
history including the potentially sensitive reproductive processes. Compa-
rable studies with C_. magister were not feasible since this species requires
from 3 to k years for completion of its life cycle (Butler, 1961) Our
approach, therefore, was to expose zoeal, juvenile and adult stages of the
crab, in separate experiments lasting up to 80 days, to uniform concentra-
tions of each pesticide in water and determine for each developmental stage
the maximum tolerable concentration of each compound. Tolerable concentra-
tions were those which had no effect on survival and also, in the case of
zoeae, had no effect on the timing of the zoeal molts. In studies with
methoxychlor, additional sublethal criteria having to do with water and salt
balance in adults were also employed. Studies involving short-term exposures
to each pesticide were also used to examine effects on the hatching of eggs
and the metamorphosis of prezoeae to the first zoeal stage.
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SECTION 2
CONCLUSIONS
Tolerance of zoeal, juvenile and adult stages of the Dungeness crab,
Cancer magister, to each pesticide was lower in long-term tests than in acute
tests. The ratios of "no effect" concentrations for zoeae in chronic tests
to the 96-hr LC50s for zoeae ranged from <0.0002 for captan to 0.61 for DEF.
The earliest developmental sequence of C_. mag i ster examined in this
study, encompassing the period from just before egg hatching to just after
development into the first zoeal instar, is not affected by pesticide concen-
trations having an adverse effect on zoeae in long-term exposures. There-
fore, effects on this sequence of development need only be considered when
short-term pesticide exposures are anticipated.
In long-term tests, the zoeal stages of C_. magister were approximately
5 to 10 times more sensitive to the pesticides studied than juvenile stages
and were approximately 10 to 100 times more sensitive than adult stages.
Zoeae should be used, therefore, in deriving water quality criteria for this
species.
Sublethal effects of pesticide exposures were either not observed, or
were noted only at concentrations or exposure times slightly less than those
that affected survival for any stage. Additional sublethal effects studies
do not, therefore, appear to be necessary for the establishment of criteria
for the protection of C_. mag i ster exposed to the nine pesticides examined in
this study.
The lowest toxic concentrations of methoxychlor, chlordane and
carbofuran in long-term tests with larval C_. magister are comparable to those
reported previously for related insecticides in long-term tests with crab
zoeae. Data are lacking with which to make similar comparisons for the
insecticide malathion, the fungicide captan, and the herbicides 2,4-D, DEF,
propanil and trifluralin.
Our studies indicate that the maximum acceptable toxicant concentrations
for continuous exposures of C_. mag i ster to each of the nine pesticides are:
methoxychlor, 0.005 yg/liter; chlordane, 0.015 yg/liter; malathion, 0.02
yg/liter; carbofuran, 0.05 yg/liter; captan, 2 yg/liter; DEF, 4 yg/liter;
trifluralin, 15 yg/liter; propanil, 80 yg/liter; and 2,4~D acid, 1000
yg/liter. As defined in this study, the maximum acceptable toxicant con-
centration is the highest concentration of each pesticide tested individually
for which no lethal or sublethal effects were observed during either acute or
chronic exposures of zoeae, juveniles or adults, or during acute exposures of
eggs and prezoeae.
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SECTION 3
RECOMMENDATIONS
Additional studies to examine the effects of these nine pesticides on
reproduction in Cancer magister would be desirable to ensure that this
process is not more sensitive to pesticides than zoeal development. Such
studies should include exposure of male and female crabs to pesticides prior
to mating and then subsequent exposure of the females and their progeny well
into the zoeal stages. Criteria of effects for these studies should include
viability of the eggs and resulting zoeae and should be evaluated in terms of
the exposure concentrations and residue levels in crabs, eggs and resulting
1arvae.
The toxicity experiments with chlordane suggest that this compound may
be biologically stable and progressively accumulated in crabs. Because of
the high toxicity of chlordane, these properties should be further examined.
Initial evaluations of the effects in crabs of pesticides other than
those examined here should be limited to studies on the survival and molt
inhibition of zoeal stages since, in the absence of further knowledge of
reproductive effects, these experiments seem to provide the most sensitive
assessment of effects.
Based on our results and on our review of the literature for the nine
pesticides examined in this study, we believe that the tolerance of larval
crabs to other pesticides would often be as low or lower than that of other
marine or freshwater organisms. We recommend, therefore, that consideration
be given to the use of larval crab bioassays as a preferred means of estab-
lishing water quality criteria for the protection of other marine species.
We recommend that the concentrations of the nine pesticides in marine
waters not be allowed to exceed the maximum acceptable toxicant concentra-
tions established in this study (Table 23).
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SECTION k
MATERIALS AND METHODS
PESTICIDES
The list of pesticides studied, their sources and the formulations
tested were the following:
1. Trifluralin; a,ot,a-Tr i f1uoro-1,6-d i nitro-N,N-dipropyl -p-toluidine;
Eli Lillv and Company; Technical, Lot No. 5GB70, purity 33%;
Treflan® E.C., Lot No. 8759B, active ingredient kk.$%.
2. DEF ; S ,S ,S, -Tri butyl phosphorotr i thioate; Chemagro Corporation;
Technical, Lot No. 3050106, purity
3- Propanil; 3,^-Dichloropropionani 1 ide; Rohm and Haas; Technical, Lot
No. 2-8262, purity 82%; Stam F-3*t, Lots No. 2-8469 and 2-3078,
active ingredient 35%.
4. 2,4-D; 2,4-D ichlorophenoxyacet ic acid; Aldrich Chemical Company;
Technical, Lot No. 070117, purity 98%.
5- Captan; N- (tr ichloromethylmercapto)-4-cyclohexene-l ,2-dicarboxi -
mide; Chevron Chemical Company; Technical, Lot No. not specified,
purity 92.8%; Orthocide~50W, Lot No. PN1760, active ingredient 50%.
6. Methoxychlor ; 2 ,2-bi s (p-methoxyphenyl ) -1 ,1 , 1 -tr i chlo roe thane;
E. 1. DuPont De Nemours and Company; Technical, Lot No. not
specified, purity 32%.
7 • Carbof uran; 2,3~d i hydro- 2 ,2-d i methyl -7~benzof urany 1 -N-methy 1 -
carbamate; FMC Corporation; Technical, Lot No. ME L514 (C471 7~54-A)
8. Malathion; 0,0-d imethyl phosphorod i thioate of diethyl mercapto-
succinate; American Cyanamid Company; Technical (Cythion grade),
Lot No. not specified, purity 95-0%.
9. Chlordane; 1 ,2,4,5,6,7,8,8-octachloro-2,3,3a,4,7,7a-hexahydro-
4,7~methanoindene; Lot No. not specified.
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PESTICIDE ANALYTICAL METHODS
Gas chromatographic procedures were employed for the analysis of each of
the pesticides in this study. The instrument used was a Hewlett-Packard
Model 5713A gas chromatograph equipped with a 63Ni electron capture detector
and a glass column 1.83 m x k mm. The column packing was 3.8% UCW-98 on
80/100 mesh Chromosorb W-HP- The carrier gas was 5% methane in argon used at
a flow rate of 60 ml/min. Oven and detector temperatures generally were
220°C and 300°C, respectively. The injection volume was 5 yl •
Methoxychlor, chlordane, malathion, propanil, DEF and trifluralin in
seawater were analyzed directly following solvent extraction into hexane.
The solvent extractions of captan and carbofuran employed benzene rather than
hexane. The 2,^-dinitrophenyl ether derivative of carbofuran was prepared by
a modification of the method of Holden (1973) prior to chromatographic
analysis. A satisfactory analytical procedure for 2,^-D in seawater was not
developed and consequently no analyses for this compound were attempted.
Quantitat ion in the case of each pesticide was based upon standards
obtained from EPA (Quality Assurance Section, EPA, Pesticides £ Toxic
Substances Effects Lab., Chemstrand Building, Research Triangle Park, N.C.
27711; formerly the Perrine Lab.), with the exception of trifluralin, which
was obtained from the manufacturer (Eli Lilly & Co.), and chlordane. The
latter, which is a mixture of a-chlordane, y~chlordane, heptachlor and other
related compounds, was quantitated by comparing the height of the a-chlordane
peak with that of a weighed sample of the technical material dissolved in
hexane. There was no evidence that chlordane extracted from water differed
in composition from the technical mixture; chromatograms of the water
extracts were similar to those of the technical chlordane used as a standard
(Figures 1 and 2).
Methoxychlor residues in whole crab and excised tissues were also deter-
mined by gas chromatography following solvent extraction of the homogenized
tissues. The procedure involved a 15~min extraction of 2 g of homogenized
tissue with 10 ml of glass redistilled acetone followed by an additional
15-min extraction with shaking after the addition of 20 ml of glass re-
distilled hexane. The extract was then shaken with 20 ml of hexane-extracted
glass-distilled water. From the hexane layer, 2 ml were taken for further
cleanup on a florisil column (3 g activated florisil contained in a 10 mm
I.D. glass chromatography tube). Methoxychlor was eluted from the column
with 40 ml of 5% ether in hexane and the eluate was chromatographed without
further treatment. A five-fold increase in florisil weight and eluant volume
were needed to effect an adequate cleanup of the fatty hepatopancreas and
gonad tissues. Recoveries estimated by the method of addition averaged 83%
and ranged from 70 to 100%. The data as presented in this report do not
include a recovery correction.
EXPERIMENTAL ANIMALS
Toxicity experiments were conducted utilizing four developmental stages
in the life history of the Dungeness crab (Cancer magister Dana). These were
-------
Figure 1. Gas chroma tog ram of technical chlordane standard.
-------
•
1
1
Q— - — •-•*—<
;
1 .
1
-t
; i •
i
; !
1 I
....
. _- . ^
1 • • '
' ' 1
I 'j '
I i™1 —
;
•
L
.
. : ,
. ' .
_ . ._
; . ' '
. i
.
.
I
*"
Figure 2. Gas chroraatogram of technical chlordane after extraction from seawater. The initial
concentration of the technical material in seawater was 10 yg/liter.
-------
the egg hatching - prezoeal stage, the zoeal stage, the early postlarval or
juvenile crab stage and the adult stage.
Ovigerous female crabs were collected as the source of eggs and zoeae
for tests on the first two developmental stages. The ovigerous female crabs
were collected in the ocean off Newport by commercial fishermen during winter
and early spring and were returned live to the laboratory where they were
maintained in tanks of flowing seawater for variable periods of time depend-
ing on the stage of ova development. The crabs, when maintained in the labo-
ratory in excess of a week, were fed frozen fillets of starry flounder
(Platichthys stellatus). To obtain the eggs for use in the egg hatching
bioassay, unhatched eggs attached to the egg mass of females, in which
initial hatching had been observed, were randomly selected and gently re-
moved from the crab and placed directly in the bioassay containers. The
procedure for obtaining first stage zoeae for use in the zoeal bioassays was
slightly different. For these experiments, at the onset of hatching the
female was transferred to standing, ultraviolet light-sterilized seawater at
13°C and 25 °/0o or 30 °/0o salinity. Within 2k hr, swimming first-instar
zoeae were collected in beakers and used immediately in the bioassay tests.
Large numbers of juvenile crabs at the same developmental stages were
conveniently obtained by collecting megalopae swarming in the bays or near-
shore ocean areas. The megalopae were held in tanks of running seawater in
the laboratory where most metamorphosed into first-instar crabs within 3 to k
days. In the 96-hr bioassay tests and the first chronic toxicity bioassay
series, juvenile crabs were used within k days of metamorphosis. However, in
the second chronic bioassay series the crabs were held in the laboratory for
approximately 2 months in natural flowing seawater prior to use. These crabs
were maintained in a tray with a sand substratum and were fed cockle clams
and rockfish at least weekly.
Adult crabs were collected by trawl from Yaquina Bay, Oregon. These
crabs, which ranged in size from 80 to 100 mm in carapace width, were
probably 10th or llth instars, about 2 years old, and approaching sexual
maturity (Butler, 1961). Slightly larger crabs, averaging 165 9 and 107 mm
in carapace width, were used in the experiments on methoxychlor uptake.
EGG HATCHING - PREZOEAL BIOASSAYS
Buchanan and Millemann (19&9) have described a free prezoeal stage of
5 to 15 min duration in the normal development of C_. mag i ster. The possi-
bility exists that this stage or the egg hatching process itself may have a
greater susceptabi1ity to certain of the pesticides than would the later
developmental stages. A series of experiments were conducted to evaluate
this possibility. Because of the short duration of the prezoeal stage, a
24-hr exposure period was considered adequate.
An ovigerous crab was caught in the ocean off Newport on March 10,
and held for 2 days in the laboratory in a 31~gal tank supplied with flowing
seawater at 11° to 12°C and 25 to 30 °/0o salinity. At the time of initial
hatching, unhatched eggs, with all their cuticular layers intact, were ran-
8
-------
domly selected and then gently removed from the crab and placed approximately
30 to a beaker in 250 ml beakers containing 200 ml of test solution. A loga-
rithmic series of concentrations, spanning at least two orders of magnitude,
were tested for each of the pesticide compounds (Table 1). Each pesticide
concentration was tested in duplicate beakers giving a total of approximately
60 eggs per test concentration. The pesticides were added to seawater by
first dissolving in acetone and then stirring in an appropriate volume of the
acetone stock solution to give a final acetone concentration of 100 yl/liter.
Control incubations were tested with and without acetone. Each control se-
ries consisted of four separate beakers and a total of about 120 eggs. The
numbers of unhatched eggs, prezoeae, and first instar zoeae in the test
vessels, and the percentage of first stage zoeae which were motile, were re-
corded at the end of the 24-hr exposure period. During these exposures, the
dissolved oxygen remained at air saturation, the salinity was 30 °/0o and the
temperature was 12° to 13°C.
96-HR ACUTE TOXICITY BIOA5SAYS
Acute toxicity bioassays lasting up to 96 hr were conducted with each of
the pesticides using first instar zoeae, first instar juvenile crabs and
adult crabs. The acute toxicity experiments were analyzed according to the
straight line graphical interpolation method (American Public Health Associ-
ation et al.,1971)- The results are expressed either as the EC50, the con-
centration of pesticide that produced a nonlethal response in 50% of the test
organisms in 2k, 48 or 96 hr, or the LC50, the concentration of pesticide
that was lethal to 50% of the test organisms during the same time periods.
The criterion of death for juvenile and adult crabs was absence of movement
after stimulation, and for zoeae, opaqueness, an unmistakable indication of
death obvious within 1 to 6 hr. The nonlethal response used for juveniles
was inability to right from an overturned position; for zoeae, it was inhibi-
tion of swimming. A nonlethal response was not employed in the tests with
adult crabs.
In each of two separate series of acute toxicity bioassays with zoeae,
in which different groups of pesticides were examined, the larvae were pooled
from two female crabs that had been held in the laboratory in flowing sea-
water for from 5 to 12 days. First instar juvenile crabs were obtained from
wild megalopae which were allowed to metamorphose in the laboratory in flow-
ing seawater tanks at 13° ± 2°C. The time from collection of magalopae to
use of juvenile crabs in the acute toxicity bioassays was k days. After
collection, adult crabs were acclimated for 5 days to 13° ± 1°C and
25 ± 0.5 °/0o salinity before initiating the acute toxicity bioassays. None
of the stages of crabs were fed in the laboratory prior to initiating the
tests.
All acute toxicity bioassays utilized static water conditions in glass
containers; 250 ml beakers holding 200 ml of test solution for zoeae and
juveniles, and 12-liter glass jars holding 10 liters of test solution for
adults. All bioassays were conducted at 13° ± 1°C and 25 ± 0.5 °/oo salini-
ty. The formulations and concentrations of pesticides tested are given in
Tables 2, 3 & 4. The pesticides were dissolved in acetone to facilitate
-------
TABLE 1. CONCENTRATIONS OF PESTICIDES USED IN EGG HATCHING - PREZOEAL BIOASSAYS
Pest ic ide
Carbof uran
Chlordane
Mai athion
Methoxychlor
Captan
DEF
Propani 1 (Stam F~34)
2,4-D acid
Tr i f 1 ural in
1
0.33
0.33
0.10
0.01
10.
0.10
33-
330.
0-33
Test
2
0.10
0.10
0.033
0.0033
3-3
0.033
10.
100.
0.10
concentrat
3
0.033
0.033
0.010
0.0010
1 .0
0.010
3-3
33-
0.033
ion (yg/1
k
0.010
0.010
0.0033
0.00033
0.33
0.0033
1 .0
10.
0.010
iter) *
5
0.0033
0.0033
0.0010
0.00010
0.10
0.0010
0.33
3-3
0.0033
6
0.0010
0.0010
0.00033
0.000033
-
0.00033
0.10
-
-
...
Nominal concentrations obtained by dilution of stock solutions.
-------
TABLE 2. CONCENTRATIONS OF PESTICIDES USED
OF CANCER MAGISTER
IN 96-HR ACUTE TOXICITY BIOASSAYS WITH FIRST INSTAR ZOEAE
Pest ic ide
Technical Grade
Carbof uran
Chlordane
Malathion
Methoxychlor
Captan
DEF
Propan i 1
2,4-D acid
Tr if lural in
Formulat ions
Orthocide - SOW
Stam F-34
Treflan E.C.
Test concentration (yg/liter) *
1
10
10
10
0.90
10,000
1 ,000
15,000
10,000
110
10,000
100,000
330
2
3.3
3.3
3.3
0.29
3,300
330
4,800
3,300
35
3,300
33,000
100
3
1 .0
1 .0
1 .0
0.090
1 ,000
100
1,500
1 ,000
11
1 ,000
10,000
33
4
0-33
0.33
0.33
0.029
330
33
480
330
3-5
330
3,300
10
5
0.10
0.10
0.10
0.0090
100
10
150
100
1 .1
100
1 ,000
3.3
6
0.033
0.033
0.033
0.0029
33
3-3
48
33
0.35
33
330
1 .0
7 8
_
--
-
0.00090
-
1.0 0.33
-
-
"
_
-
Norn i na 1
concentratjons obtained by dilution of stock solutions.
-------
TABLE 3. CONCENTRATIONS OF PESTICIDES USED IN 96-HR ACUTE TOXICITY BIOASSAYS
WITH FIRST INSTAR JUVENILES OF CANCER MAGISTER
Pesticide Test concentration (yg/liter) *
Methoxychlor
Captan
DEF
Propani 1 (Stam F-34)
2,k-D acid
Tr if 1 ural in
10
1
33
100
1
100
,000
,000
,000
,000
,000
3
10
33
33
,300
330
,000
,000
330
1
3
10
10
,000
100
,300
,000
100
1
3
3.3
330
33
,000
,300
33
1 .0
100
10
330
1,000
10
0.
33
3-
100
330
3-
33
3
3
" Nominal concentrations obtained by dilution of stock solutions.
mixing with seawater. The final acetone concentration was 100 ul/liter
except for the captan tests with adult crabs in which the final acetone con-
centration was 1,000 yl/liter. Control animals were held in untreated sea-
water and in seawater containing acetone. The numbers of animals per test
concentration were 20, 10 and 10 for zoeae, juveniles and adults, respective-
ly. The test solutions were renewed daily after first determining the dis-
solved oxygen levels and pH of the 24-hr-old water. Solutions were aerated
only in the adult tests. Dissolved oxygen levels always exceeded 7-0 and
5-5 mg/liter in the zoeal and juvenile tests, respectively, and averaged
6.0 mg/liter in tests with adults. The averages of pH for the same three
groups was 7-8, 7-8 and 7-5- The photoperiod approximated that occurring
naturally during the time the tests were conducted. These were 9 hr of light
- 15 hr of darkness for the zoeal tests, 15 hr of light - 9 hr of darkness
for the juvenile tests, and 12 hr of light - 12 hr of darkness for the adult
tests.
CHRONIC TOXICITY BIOASSAYS
Long-term exposures of zoeae, juvenile crabs and adult crabs to pesti-
cides were conducted in flowing-water culture systems. Pesticides were
introduced into the culture water by metering acetone stock solutions of the
chemicals into the upper ends of cascading mixing troughs. The final sea-
water concentration of acetone in all tests was 100 yl/liter. Brinkmann
multi-channel peristaltic metering pumps were used to deliver the acetone
stock solutions. The flow of seawater into the mixing troughs was regulated
by using adjustable hose clamps and constant level headtanks. Careful moni-
toring and periodic adjustment of seawater flow rates and acetone metering
rates was necessary to provide uniform pesticide concentrations in seawater.
Failsafe devices were employed to ensure that the metering of pesticides were
halted if the supply of seawater failed. All seawater was filtered through
large quartz sand filter beds before use in the bioassays. Additional fil-
tration and UV sterilization were employed in all of the juvenile crab and
12
-------
TABLE k. CONCENTRATIONS OF PESTICIDES USED IN 96-HR ACUTE TOXICITY
BIOASSAYS WITH ADULTS OF CANCER MAGISTER
Test concentration (yg/liter) *
Pesticide —-—
1 23^5
Carbofuran
Chlordane
Malathion
Methoxychlor
Captan
DEF
Propan i 1
2,4-D acid
Tr i f 1 ural in
1 ,000
3,300
3,300
920
100,000
1 ,000
26,000
-
9,300
330
1 ,000
1 ,000
520
33,000
330
8,200
-
3,000
100
330
330
290
10,000
100
2,600
-
930
33
100
100
160
3,300
33
820
-
300
10
33
33
92
1 ,000
10
260
-
93
-
-
-
29
330
3-3
82
-
30
" Nominal concentrations obtained by dilution of stock solutions.
zoeal tests. During the 197^ zoeal tests, a third filter, capable of re-
moving 10 ym particles, and a second UV sterilization treatment step were
also employed. The latter precautions were taken to further reduce the
possibility of infections of zoeae by microorganisms.
Zoeal Experiments
Although the basic bioassay technique used in all of the chronic tox-
icity experiments was essentially the same, the practical problems of cultur-
ing the organisms differed for the various developmental stages and required
separate solutions. In previous long-term toxicity studies with brachyuran
larvae (Bookhout et al., 1972; Buchanan et al., 1970; Epifanio, 1971) inves-
tigators have employed static culture vessels which required periodic renew-
al of the test solutions by manual procedures. In order to utilize a flowing-
water system, we developed the culture apparatus illustrated in Figure 3-
Crab larvae were contained in each of several 250 ml glass beakers modified
with a 15 mm diameter hole on the side near the bottom of the beaker. Nylon
screen, 360 ym mesh "Nitex," was cemented, by using silicone rubber sealant,
across the opening on the inside of the beaker. A second screen, 210 ym mesh
"Nitex," was cemented in the same manner across the opening on the outside of
the beaker so that the two screens were separated by a distance of alx>ut 3 to
k mm. As many as eight culture beakers could be contained in each 28 cm by
28 cm by 10 cm deep glass aquarium. Seawater containing toxicant of the
desired concentration entered each aquarium through a k mm inside diameter
glass tube. By means of an automatic siphon, the water level in the aquarium
was made to fluctuate, causing seawater to enter and exit the beakers through
the screened holes.
Two series of chronic toxicity experiments were conducted with zoeae.
The first utilized the progeny of a single female collected on April 12,
1973- At the onset of hatching 1 day later, the female was placed in
13
-------
Stock S Wottr Flow
From Mixing Box
Fluctuoting
V
I
To Corbon
Rlttr
Figure 3- Diagram of culture containers illustrating the relation between
the square culture aquarium and the culture beakers, showing
details of the automatic siphon, and the positioning of the two
screens on the lower side of a culture beaker.
standing UV treated seawater at 13° ± 1°C and 25 °/00 salinity. First instar
zoeae were collected 5 to 10 hr after hatching and placed 10 each in the 250-
ml culture beakers. The pesticides used and their concentrations are given
in Table 5- A total of 80 larvae were tested with each pesticide concentra-
tion. Twice per week, following transfer to clean culture beakers, zoeae
were fed 700 to 1,000 newly hatched San Francisco brine shrimp (Artemia
sal ina) naupl i i per beaker. The seawater salinity during the first 15 days
of the test was 25 °/oo and afterwards was 15 °/oo or higher Temperature
was maintained at 13° ± 1°C. The dissolved oxygen levels always exceeded
8.0 mg/liter and the pH ranged from J.6 to 7-9. The photoperiod was 12.5 hr
of light - 10.5 hr of darkness.
The second series of chronic toxicity tests was also conducted with the
progeny of a single female which was obtained from a commercial fisherman on
March 10, 1974. The ovigerous female was held in the laboratory in flowing
seawater at 11° to 12°C for 2 days. At the onset of hatching the female was
transferred to standing UV sterilized seawater at 30 °/0o salinity and, after
10 hr, swimming first stage zoeae were collected for use in the bioassays.
For each pesticide concentration used (Table 6), 10 zoeae were placed in each
of four 250 ml beakers giving a total of kO larvae per pesticide concentra-
tion. Zoeae were fed first naupl iar stages of Artemia sal ina three times
-------
TABLE 5. PESTICIDE CONCENTRATIONS IN SEAWATER DURING FIRST SERIES OF
ZOEAL CHRONIC EXPERIMENTS
Pesticide
Methoxychlor
Captan
DEF
Propan i 1 (Stam F~34)
2,4-D acid
Tri f 1 ural in
Norn i na 1
concentrat ion
(yg/1 iter)
0.1
0.01
0.001
0.0001
200.
20.
2.
0.2
2.
0.2
0.02
0.002
800.
80.
8.
0.8
10,000.
1 ,000.
100.
10.
150.
15-
1.5
0.15
Measured
concentration
(yg/1 iter)
0.06
0.007
240.
20.
0.18
1 .2
0.70
1 ,400.
150.
<2.
480.
53-
4.1
± 0.02"
± 0.004
-
-
± 90.
± 5-
-
± 0.3
± 200.
± 0.
-
-
-
-
-
± 40.
± 15-
± 0.6
~
Number
of
analyses
4
3
-
-
4
3
-
1
2
1
-
-
3
2
3
-
_
-
-
-
2
3
4
—
--• Mean ± one standard deviation.
per week at a feeding density of 700 brine shrimp per beaker for the first to
third instar zoeae, and 1,000 brine shrimp per beaker for later zoeal stages.
Zoeae were transferred to clean beakers three times a week. The photoperiod
was adjusted monthly to approximate ambient conditions and was 11 hr of
light - 13 hr of darkness, 13-5 hr of light - 10.5 hr of darkness, and 15 hr
of light 9 hr of darkness for the months of March, April and May, respec-
tively. The mean and standard deviation of culture temperatures recorded
daily was 12.3° and 0.5°C. The salinity recorded daily was 28.8 ± 1.4 °/00
and the dissolved oxygen and pH recorded weekly were 8.7 ± 0.3 mg/liter and
8.1 ±0.1, respectively. Mortality and molt data were recorded daily.
15
-------
TABLE 6. PESTICIDE CONCENTRATIONS IN SEAWATER DURING SECOND SERIES OF
ZOEAL CHRONIC EXPERIMENTS
Pest icide
Carbofuran
Chlordane
Mai athion
Methoxychlor
Captan
DEF
Propani 1 (Stam F-34)
2,4-D acid
Tr if lural in
Nom i na 1
concentrat ion
(yg/1 i ter)
5-
0.5
0.05
15-
1.5
0.15
0.015
2.
0.2
0.02
0.002
0.5
0.05
0.005
200.
20.
2.
4.
0.4
0.04
800.
80.
8.
10,000.
1 ,000.
150.
15-
1.5
Measured
concentrat ion
(yg/1 iter)
7-3 ± K3*
0.63 ± 0.32
—
62.
1.70 ± 0.28
0.17 ± 0.09
"•
-
-
-
-
0.91 ± 0.39
0.074 ± 0.019
—
450. ± 80.
30. ± 11.
3-1 ± 2.7
6.9 ± 2.4
0.95 ± 0.24
-
1 ,700. ± 500.
-
9-6 ± 3-6
-
220. ± 50.
26. ± 5-
3.1 ± 0.3
Number
of
analyses
2
8
~
1
2
11
""
-
-
-
—
6
13
—
6
8
14
13
13
-
12
-
14
-
2
11
14
* Mean ± one standard deviation.
16
-------
Juvenile Experiments
Two series of chronic toxicity bioassays were done with juvenile crabs,
the first lasting 36 days and the second 80 days. Wild megalopae collected
in Yaquina Bay, Oregon between June 12 and June 15, 1973 were held in the
laboratory in flowing seawater at 15° ± 2°C and at variable salinities
exceeding 25 °/oo until they had molted into first instar crabs. Some of the
crabs were used immediately in the first series of experiments. The remain-
ing crabs, which were used in the second series of tests, were held in flow-
ing seawater in the laboratory for approximately 2 months and fed cockle
clams (C1 inocardiurn nuttalli) and rockfish (Sebastodes sp.) at least once a
week.
In the first experiments, crabs were reared in the same apparatus used
in the long-term tests with zoeae except that each beaker was divided by a
glass partition into two separate chambers. Each chamber contained one crab
which was thus protected from cannibalism. As the first crabs began to molt
in this exper iment, we noticed that the animals were experiencing difficulty
in completing the molt. In earlier experiments, Buchanan et al. (1970) found
that crabs of this age were able to molt with good survival when a sandy sub-
stratum was present. On day 15, therefore, all the crabs were removed from
the glass beakers and allowed to intermix in the rearing troughs, to which
was added clean beach sand to a depth of 1.0 cm.
Four concentrations of each pesticide were used in the first series of
experiments (Table 7)- Control animals received either untreated seawater or
seawater containing 100 yl/liter of acetone. During the experiment, seawater
temperatures recorded daily were 13° ± 2°C, and the salinity was 3-1 to J>k °l
Daily determinations of dissolved oxygen and pH gave values ranging from 8.0
to 8.7 mg/liter and from 7.3 to 8.1, respectively. The photoperiod was 14 hr
of light - 10 hr of darkness.
Juvenile crabs used in the second series of tests were mostly in the
third crab instar by the time the tests were initiated. Fewer concentrations
of some of the pesticides were used (Table 8), and acetone and seawater con-
trols were employed as before. In these experiments, the crabs were reared
directly in the large exposure troughs that contained 2 cm of beach sand as a
substratum. By using Teflon-coated screens, each aquarium was, in these ex-
periments, divided into 30 cm2 compartments each containing one crab. The
sand was replaced and the screens cleaned on test days 28 and 58. Dissolved
oxygen levels monitored daily ranged from 8.0 to 8.7 mg/liter, and the daily
pH reading ranged from 7-3 to 8.1. Seawater temperatures and salinittes
recorded daily were 13° ± 1°C and 32 to 34.5 °/00, respectively. The photo-
period used was the same as in the first series of juvenile tests. Crabs in
both the first and second series of tests were fed to repletion small pieces
of cockle clam on alternate days and daily observations were made on their
survival and molting.
Adult Experiments
In an initial series of adult chronic toxicity experiments, lasting 85
days, crabs were exposed to from two to four concentrations of the pesticides
17
o o-
-------
TABLE 7- PESTICIDE CONCENTRATIONS IN SEAWATER DURING FIRST SERIES OF
JUVENILE CHRONIC EXPERIMENTS
Pest icide
Methoxychlor
Captan
DEF
Propanil (Stam F-34)
2,4-D acid
Tr i f 1 ural in
Norn i na 1
concentrat ion
(yg/1 iter)
4.
0.4
0.04
0.004
200.
20.
2.
0.2
150.
15-
1.5
0.15
800.
80.
8.
0.8
10,000.
1 ,000.
100.
10.
150.
15-
1-5
0.15
Measured
concentrat ion
(yg/1 iter)
4.9
0.45
0.033
510.
51.
1.5
0.20
260.
25-
2.7
1 ,200.
130.
8
190.
19-
2.6
± 1.3'
± 0.24
± 0.017
-
± 160.
± 14.
± 0.4
± 0.14
± 70.
± g.
± 0.6
—
± 200.
± 80.
-
-
-
-
-
± 40.
± 2.
± 0.7
Number
of
analyses
3
3
3
-
3
2
2
2
3
3
3
~
3
3
1
-
-
-
-
—
2
2
3
Mean ± one standard deviation.
methoxychlor, 2,4-D, trifluralin, DEF, propanil (as Stam F-34) and captan
(Table 9). Ten crabs, five males and five females, were exposed to each
pesticide concentration. Each of the two control groups, one exposed to un-
contaminated seawater and the other to seawater containing 100 yl/liter of
acetone, consisted of 30 crabs, half male and half female. The crabs were
held in 47-liter aquaria which were partitioned sequentially into five
interconnected compartments. The dimensions of each compartment were 30 x
21 x 15 cm, and each contained one crab. Seawater entered each aquarium at
a rate of 1.6 to 2.0 liters/min giving a weight specific replacement rate of
about 2.6 1iters/g/day.
18
-------
TABLE 8. PESTICIDE CONCENTRATIONS IN SEAWATER DURING SECOND SERIES OF
JUVENILE CHRONIC EXPERIMENTS
Pest ic ide
Methoxychlor
Captan
DEF
Propanil (Stam F-34)
2,4-D acid
Tri f 1 ural in
Nom i na 1
concentration
(yg/1 iter)
4.
0.4
0.0**
0.004
200.
20.
150.
15-
1.5
0.15
800.
80.
10,000.
1 ,000.
150.
15-
Measured
concentrat ion
(yg/1 iter)
6.5
0.49
0.043
290.
38.
270.
24.
1.9
2,000.
88.
590.
47-
+
+
±
-
±
±
±
±
±
-
±
±
-
-
±
±
j~
4.7"
0.11
0.020
290.
16.
90.
10.
1 .2
600.
46.
210.
24.
Number
of
analyses
6
10
10
-
8
11
7
9
11
-
9
10
_
-
11
9
Mean ± one standard deviation.
Salinity during the exposure period averaged 32.4 °/00 (22.9 to
34.0 °/00), temperature was 12.6°C (11.0° to 15-0°C), the dissolved oxygen
was 8.2 mg/liter (6.8 to 8.5 mg/liter) and the pH was 7-9 (7-0 to 8.3).
were fed portions of starry flounder, Platichthys stellatus, or English
Parophrys vetulus, every other day, and uneaten food was removed after
, A photoperiod of 11 hr of light and 13 hr of darkness was used
Crabs
sole,
24 hr,
throughout.
In a second series of adult chronic bioassays, lasting 90 days, four
concentrations each of carbofuran, chlordane and malathion were tested
(Table 9)- The numbers of crabs per concentration and in the control groups
were the same as in the preceding tests. During these tests, which were run
during the late fall and early winter, the salinity occasionally dropped to
low levels during low tide periods not exceeding 5 hr. The salinities, as
measured, averaged 25.4 °/0o (11.4 to 32.7 °/0o) , the temperatures averaged
10.0°C (6.6° to 11.8°C), and the dissolved oxygen and pH levels and the
photoperiod were the same as in the preceding tests.
19
-------
TABLE 9. PESTICIDE CONCENTRATIONS IN SEAWATER DURING ADULT CHRONIC
EXPERIMENTS
Pesticide
Carbofuran
Chlordane
Malathion
Methoxychlor
Captan
DEF
Propanil (Stam F-34)
2,4-D acid
Tr if lural in
Nominal Measured Number
concentration concentration of
(yg/liter) (yg/liter) analyses
250.
25-
2.5
0.25
100.
10.
1 .
0.1
1,500.
150.
15-
1.5
40.
4.
0.4
0.04
200.
20.
2,000.
200.
20.
2.
4,000.
400.
40.
10,000.
1 ,000.
100.
10.
1 .
210.
23-
1.7
130.
13.
1 .1
0.14
2,400.
180.
15-
1 .2
75-
7.4
0.73
0.053
340.
19-
2,600.
380.
35-
3.0
6,600.
590.
34.
300.
33-
2.6
±
±
±
-
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
+
±
±
±
±
-
~*
±
±
±
100."
14.
0.7
30.
4.
0.5
0.12
1 ,600.
40.
5-
0.5
36.
2.4
0.21
0.028
40.
4.
1,500.
140.
14.
0.7
3,100.
170.
42.
110.
10.
0.8
6
9
7
—
10
15
15
15
2
16
14
15
7
7
10
7
10
10
7
10
10
10
9
9
10
-
9
10
9
Mean ± one standard deviation.
20
-------
UPTAKE, LOSS, AND TISSUE DISTRIBUTION OF METHOXYCHLOR
Rates of uptake of methoxychlor by second and third instar juvenile
crabs exposed to 0.04 and 2.0 yg/liter of pesticide were determined by ana-
lyzing subsamples consisting of. 10 juvenile crabs each at 3 to 6 day inter-
vals over an 18-day period. Adult crabs, 165 9 and 107 mm in mean weight and
carapace width, were exposed for 15 days to 1.8 and 7-5 yg/liter of methoxy-
chlor, and individuals were analyzed for whole-body pesticide residues peri-
odically during the exposure period and also during a 15-day depuration
per'iod. In addition, 10 adult crabs were taken from each exposure concentra-
tion at the end of the 15-day exposure period for the analysis of residues in
specific tissues. The tissues from each group of 10 crabs were pooled for
analysis. The measured methoxychlor concentration in each tissue, times the
mean weight of each tissue for a 165 g crab, gave the total amount of pesti-
cide in each tissue and this value divided by the sum of total, calculated
methoxychlor from all tissues was used to calculate the percentage of total
whole-body methoxychlor found in each tissue. The mean wet weight of each
tissue was determined by dissecting and weighing the tissues from eight un-
exposed crabs. The weight of the blood was estimated as the difference
between the whole-body weight and the sum of individual tissue weights.
Water conditions during these tests with both juveniles and adults were:
temperature, 11 .0°C (9-0° to 11.5°C); salinity, 31-0 °/00 (30.2 to
32.7 °/oo); dissolved oxygen, 9.0 mg/liter (7-8 to 9.4 mg/liter); and pH,
7-9 (7-0 to 8.3)- All other maintenance and exposure conditions were as
described previously for the chronic toxicity experiments.
To determine whole-body residues in crabs after death from exposure to
methoxychlor, adults, exposed to 1.8, 7-5, 18 and 32 yg/liter for up to 15
days under the conditions described previously, were removed from the water
and analyzed for pesticide residues within 12 hr of death. Unless indicated
otherwise, residue data are based on the wet weights of the crabs and their
various tissues.
SALINITY TOLERANCE EXPERIMENT
In salinity tolerance tests with adult C_. mag i ster, crabs were pre-
exposed in flowing water aquaria for 10 days to 10 yg/liter methoxychlor or
acetone only. During this period the temperatures ranged from 12° to 14°C,
the salinities from 30 to 33 °/oo and dissolved oxygen was maintained at air
saturation by vigorous aeration. The crabs were then transferred to*glass
troughs containing various seawater dilutions and 10 yg/liter methoxychlor or
acetone supplied in a flowing bioassay system, and deaths were recorded
daily during a 7~day exposure period. Temperature and dissolved oxygen were
the same as during the pre-bioassay period. Frequent analysis of the sea-
water in these and later tests showed that the methoxychlor concentration was
maintained in the flowing water systems to within ± 10% of the desired value.
-------
OSMOTIC - IONIC REGULATION EXPERIMENTS
In orde to examine the effects of methoxychlor on osmotic and ionic
regulation, "ji'lt crabs were pre-exposed for 1 4 days to 10 yg/liter of
methoxychlor or to acetone alone, as described previously for the salinity
tolerance experiment. The crabs were then transferred to the same glass
troughs used in the salinity tolerance experiment and exposed for 48 hr to
various seawater dilutions with continued pesticide exposure. After 48 hr,
blood and urine was collected for determination of the osmotic concentrations
and the concentrations of Na+, K+, and Mg++ ions. Following the 14 day ex-
posure period, another group of crabs was taken for the determination of gill
ATPase act ivi t ies.
Blood was obtained by puncture of the thin membrane at the base of the
second or third walking leg by Pasteur pipettes, transferred to polyethylene
vials, and frozen for storage. Upon thawing, the clots were broken up with a
glass stirring rod and aliquots of serum were used undiluted for measurements
of osmotic concentration or after dilution for the analysis of Na+, K+, and
Mg++ ions. Urine was collected into 0.25 ml syringes by careful insertion of
a blunted 24~gauge needle into the nephropore, and was stored frozen in small
polyethylene vials. Osmotic concentrations were determined with a Wescor
Model 5100 vapor pressure osmometer. A Perkin-Elmer Model 403 atomic ab-
sorptio: spectrophotometer was used in the determination of Na+, K+, and
Mg++ ions.
ATPASE ASSAY
ATPase activity was determined in whole homogenates of C_. magister gill.
Gills from freshly killed crabs were homogenized in 0.05 M glycylglycine
buffer, pH 7-4, at high speed in a Sorvall omnimixer. The whole homogenate
was then centrifuged for 10 min at 30 x g to sediment coarse materials.
Total ATPase activity was measured in a 2~ml reaction medium containing
100 mM NaCl, 15 mM KCL, 5 mM MgCl2, 6> mM ATP and 50 mM glycylglycine buffer,
pH 7.4. In some assays, Mg ATPase was determined by adding 1 mM ouabain to
the reaction medium to inhibit the NaK activated enzyme. NaK ATPase was
calculated as the difference between total ATPase and Mg ATPase. The re-
action was allowed to proceed for 30 min at 15°C and was terminated by
pipetting 0.5 ml aliquots of the reaction medium into 0.5 ml 10% TCA. Phos-
phate was determined by the method of Fiske and SubbaRow (1925). Protein
was estimated by the Lowry procedure (Lowry et al., 1950- Specific activity
was expressed as pinoles P. hydrolyzed/mg protein/hr.
22
-------
SECTION 5
RESULTS AND DISCUSSION
THE FUNGICIDE CAPTAN
Captan is reported to have a short half-life in aqueous solution; the
rate of its hydrolysis to tetrahydrophthalimide and tetrahydrophthalic acid
is related directly to pH and temperature (J. N. Osperson and D. E. Pack,
Chevron Chemical Company, Richmond, Calif., personal communication cited by
Hermanutz et al., 1973). Hermanutz et al. (1973) reported that the half-life
of captan in Lake Superior water at a pH of 7.6 is about 7 hr at 12°C and
about 1 hr at 25°C. Since data are lacking on the half-life of captan in
seawater, we examined the rate of its breakdown at several concentrations
under conditions prevailing in the acute toxicity bioassays. After 2k hr,
the concentrations of captan remaining in seawater were from 48 to 74% of the
initial concentrations, which ranged from 23 to 2,300 yg/liter (Table 10).
TABLE 10. DECAY RATE OF CAPTAN
AT 13°C *
IN SEAWATER OF 25 °/0o SALINITY
Initial
measured
concentrat ion
(yg/1 i ter)
2,300
1 ,400
300
220
23
Measured
concentrat ion
after 2k hr
(yg/1 iter)
1 ,100
900
200
150
17
Percent
of initial
concentration
after 2k hr
48
6k
67
68
Ik
Est imated
half-1 ife
(hr)
23
37
41
k3
5k
* pH ranged from 7-6 to 7-9-
The half-life of captan estimated from these data ranged from 23 to $k hr.
The effects of captan on egg hatching success, on success of molting
from prezoeae to first stage zoeae, and on the activity of first stage zoeae,
determined at the end of a 24-hr exposure period, are summarized in Figure k.
23
-------
KX) b:~r-$±7.
20
\
/ 1
0\
1
"x^
KX) 1,000
Captan Conetntration (^ug/liter)
10,000
Figure 4. Percent hatch of Dungeness crab eggs ( Q - O}> percent develop
ment of hatched crab larvae through the prezoeal to the first
zoeal stage ( A — - — A), and percent of developed first stage
zoeae which are motile (^ ---- O)as a function of captan con-
centration during a 2k hr exposure period.
The mean and standard deviations of hatching successes in both seawater con-
taining acetone (shown simply as zero captan concentration in Figure k} and
untreated seawater controls, in replicate tests, were 39 ± 7% and 36 ± 16% of
the initial numbers of eggs, respectively. Hatching success in the presence
of all concentrations of captan tested exceeded this value, averaging 76 ± 1%
in ten separate cultures; these results are similar to those reported previ-
ously by Buchanan et al. (1970) for eggs exposed to the pesticide Sevin. Of
those prezoeae hatching, more than 90% molted successfully to first stage
zoeae even at 10,000 yg/liter, the highest captan concentration tested. The
developed first stage zoeae, however, were largely immobilized at only 3,300
pg/liter. A morphological abnormality was also observed in the zoeae devel-
-------
oping at 3»300 and 10,000 ug/liter of captan. The abnormality was a lateral
enlargement of the newly formed carapace following the prezoeal molt to a
width about twice that found for the control larvae.
In 96-hr acute tests with first stage zoeae, the concentrations causing
50% immobilization of zoeae (EC50) in 2k . 48 and 96 hr were 5,600, 3,500 and
1,500 yg/liter of technical captan, respectively (Table 11). A 50% wettable
powder formulation of captan appeared to be slightly more toxic in a compara-
ble series of tests; the 2k-, 48- and 96-hr EC50s were 3,200, 700 and 360
ug/liter, respectively. The 96-hr LC50 for technical captan exceeded 10,000
yg/liter, but the 96-hr LC50 for the 50% wettable formulation was 8,000
yg/liter (Table 11). In 96-hr acute toxicity tests with the juvenile and
adult stages of the crabs, we did not observe any deaths at the highest con-
centrations tested, which were 10,000 and 100,000 yg/liter, respectively. At
these highest concentrations of technical captan, the solubility of the fun-
gicide in seawater had clearly been exceeded, since we observed the formation
of a heavy white precipitate. The solubility of technical captan in water is
reported to be 3,300 yg/liter at 25°C (Lhevron Chemical Company, Ortho Divi-
sion, San Francisco, Calif. Orthocide^ Experimental Data Sheet, April,
1970)- In seawater at 20°C, the solubility has been estimated to be 3,500
yg/liter (G.W. Newell, Stanford Research Institute, Palo Alto, Calif., per-
sonal communication).
First stage zoeae exposed to 200 yg/liter captan were rapidly killed in
the chronic toxicity tests. In the first experiment lasting only 18 days,
the time to 50% mortality at this exposure concentration was 6 days, and that
to 100% mortality was 10 days (Figure 5). In the second test, the time to
50% mortality was about 9 days, and total mortalities had occurred by day 21
(Figure 6). In the latter test, none of the zoeae exposed to 200 yg/liter
captan molted successfully to second stage zoeae, even though some of these
larvae survived beyond the first molt period, which occurred between days 12
and 16 in control groups (Figure 7). Zoeae exposed to only 20 yg/liter
captan and less survived as well as controls until the termination of the two
tests on days 18 and 69, respectively. In the longer test, survival at these
lower fungicide concentrations and in the control cultures still exceeded 80%
of the original numbers of zoeae by day 50, at which time molting to the
fifth zoeal stage was occurring. After this period, however, the mortality
rates of all of the treatment groups, including the controls, increased
greatly (Figure 6). Exposure of zoeae to 2 and 20 yg/liter of captan did not
appear to affect the duration of the first two zoeal stages, but by the time
of the third zoeal molt, larvae exposed to both of the fungicide concentra-
tions showed a molting delay of about 3 days when compared with controls
(Figure 7). A delay was no longer apparent, however, in the 2 yg/liter group
by the time of the fourth molt.
The first chronic toxicity test with juvenile crabs was continued for a
period of 36 days; at the end, most of the crabs had molted to the second
instar, and survival in controls remained above 80% of the original number of
crabs. At this time, survival of crabs exposed to the highest captan concen-
tration, 200 yg/liter, was as high as that of the controls (Figure 8). Fur-
thermore, exposure of juvenile crabs to captan in these tests did not appear
to delay the initiation of the first molt (Figure 9)•
25
-------
TABLE 11. ACUTE TOXICITY OF CAPTAN TO FIRST INSTAR ZOEAE, FIRST INSTAR JUVENILE CRABS, AND
ADULT CRABS
Stage
of
crab
Zoeae
Juven i 1 e
Adult
Pest icide
formulation
Techni cal
.50% wettable
Technical
Techn ical
Toxic
24-hr
EC50 LC50
5,600 >10,000
3,200 >10,000
>10,000 >10,000
>100,000
concentrations (yg/1
48-hr
EC50 LC50
3,500 >io,ooo
700 >io,ooo
>10,000 >10,000
>100,000
iter)
96-hr
EC50 LC50
1,500 >10,000
360 8,000
>10,000 >10,000
>100,000
-------
100
80
60
40
20
10
Days
15
20
Figure 5- Effect of continuous exposure to captan on survival of
Dungeness crab zoeae. Data are from the first zoeal chronic
experiment conducted during the spring of 1973-
In a second chronic exposure test with juveniles, the crabs were exposed
only to 20 and 200 yg/liter captan. These crabs had been reared in the labo-
ratory in uncontaminated seawater for 2 months following their metamorphosis
from the magalopae stage and were mostly third instars at the time the test
was initiated. The mortality rates of crabs exposed to the fungicide appear-
ed to be higher than those of the controls during the initial month, but by
the end of the test on day 80, the survival of the fungicide-exposed crabs
was as high as that of the acetone-exposed control crabs (Figure 10).
Adult crabs exposed to 20 and 200 yg/liter captan suffered no mortali-
ties over a 75-day exposure period (Figure 11). Also, there was no apparent
effect of the fungicide on the behavior or the feeding activity of either
juvenile or adult crabs.
In acute toxicity tests, Dungeness crab zoeae exhibited a lesser sensi-
27
-------
100
80
60
OO
40
20
10
20
30 40
Days
50
60
70
Figure 6. Effect of continuous exposure to captan on survival of Dungeness crab zoeae. Data are
from the second zoeal chronic experiment conducted during the spring of 1974.
-------
lOOr
ho
20
CAPTAN
10
A Control
• Acotorw Control
A 2 /ig/IHer
O 20 "
IV
20
30
Days
40
50
6O
Figure 7- Effect of continuous exposure to captan on molting of Dungeness crab zoeae beginning with
the first zoeal molt. The molt success for each stage is the percentage of the original
number of zoeae. Roman numerals indicate the zoeal instar at each stage in the zoeal se-
quence. Data are from the second zoeal chronic experiment conducted during the spring of
-------
100
80
60
.240
20
CAPTAN
A Control
• Acttorw Control
A O2 /ig/ftar
O 2 "
0 20 ••
O 200 •
10
20
Days
30
40
Figure 8. Effect of continuous exposure to captan on survival of
juvenile Dungeness crabs. Data are from the first
juvenile chronic experiment which was initiated with
newly metamorphosed first instar crabs.
tivity to captan than that reported for another crustacean of similar size.
The 26-hr LC50 of the freshwater Daphnia magna was found to be 1,300 yg/liter
(Frear & Boyd, 1967)• ln our studies, the 24-hr EC50 of technical captan for
zoeae was 5,600 yg/liter and the 96-hr EC50 was 1,500 yg/liter (Table 11).
LC50 values all exceeded 10,000 yg/liter, the highest captan concentration
employed in the tests.
Our data, and those of Frear and Boyd, suggest that crustaceans may be
slightly less susceptible to captan intoxication than are fish. The 90-min
LC50 for zebrafish larvae was 670 yg/liter (Abedi & McKinley, 196?) and the
acute lethal threshold for fathead minnows, bluegill sunfish and brook trout
were 64, 72 and 29 yg/liter, respectively (Hermanutz et al., 1973)- However,
the 72-hr LC50 of captan~50W for rainbow trout was 320 yg/liter (Holland
et al . , I960) , which value is close to that for Daphnia magna estimated by
30
-------
r CAFTAN
S
o
Control
• Acetone Control
A CX2
O 2
0 20
O 200
Figure 9- Effect of continuous exposure to captan on molting of first
instar Dungeness crabs to the second instar. The molt success
is expressed as the percentage of the original number of first
instar crabs.
extrapolation of the data of Frear and Boyd to 72 hr.
Of all the crab life history stages examined, the zoeal stage was the
most sensitive to captan intoxication in both acute and chronic toxicity
tests. In the chronic exposure experiments, survival of this stage appeared
unaffected at 20 yg/liter of captan but was clearly reduced at 200 yg/liter
(Figures 5 & 6). In addition, at 20 yg/liter, there was an indication that
the molting of later stages of crab larvae was delayed, although the results
were somewhat equivocal (Figure 7)- In similar, long-term experiments with
juvenile and adult crabs, both of these stages were unaffected by continuous
exposure to 200 yg/liter, the highest concentration tested. The same re-
lationship between age of C. magister and sensitivity to the carbamate
31
-------
100
80
60
.1 40
I
20
0
CAPTAN
A Control
• Acetone Control
A 20
O 200 «
10
20
30
40
Days
50
60
70
80
Figure 10. Effect of continuous exposure to captan on survival of juvenile Dungeness crabs. Data
are from the second juvenile chronic experiment which was initiated with second and
third instar crabs.
-------
U)
100
80
60
40
20
0
CAPTAN
A Controi
$ Acetone Controi
A 20 /ig/liter
O 200 "
0 10 20 30 40 50 60 70 80 90
Days
Figure 11. Effect of continuous exposure to captan on survival of adult Dungeness crabs.
-------
insecticide Sevin was noted by Buchanan et al. (1970).
Exposures of the egg and prezoeal stages to captan were shorter than
those in the acute toxicity experiments with the older stages; therefore,
comparisons between these age groups are difficult. Nevertheless, it appears
that the zoeae may be more sensitive to captan than either the eggs or pre-
zoeae. Concentrations as high as 10,000 yg/liter did not inhibit the hatch-
ing of eggs nor the transformation of prezoeae into zoeae, but by using
activity as a criterion of toxic affect, the 50% effective level for those
zoeae that developed in the test was found to be as low as 2,000 yg/liter
(Figure 4). Similar results were obtained by Buchanan et al. (1970) with
Sevin. In their studies, the 50% effective level for inhibition of prezoeal
development ranged from 6 to 30 yg/liter in three experiments, but activity
of developing zoeae was largely inhibited at only 5 yg/liter.
The apparent resistance of prezoeae to captan is probably attributable
to the very short duration of this stage. The newly hatched prezoeae nor-
mally molt into the first zoeal stage within only 5 to 15 min of hatching
(Buchanan & Millemann, 1969)- If the egg membrane effectively resists
penetration of captan, the newly hatched prezoeae may have very low tissue
levels of the fungicide and these may not be greatly increased by a further
5 to 15 min exposure to captan in water. Subsequent additional accumulation
of the fungicide by the newly metamorphosed first instar zoeae might then
result in impaired swimming behavior attributable to a higher body burden of
captan.
We are uncertain of the reasons for the low hatch of eggs in the sea-
water and acetone control groups compared with the 76% average hatch found in
all captan treatments. In a similar experiment with C. magister eggs exposed
to the insecticide Sevin, Buchanan et al. (1970) found that a higher hatching
success also occurred in eggs exposed to Sevin than in untreated eggs, al-
though the difference between the treated eggs and untreated eggs was not as
great as reported here with captan. In addition, Buchanan and Millemann
(1969) earlier showed that reducing the salinity from 30 °/00 to 15 °/oo
nearly doubled the hatching success of Dungeness crab eggs in a 3&-hr period.
These results indicate that the application of stressful conditions may ac-
celerate the egg hatching process.
Although a captan concentration as high as 10,000 yg/liter did not pre-
vent the development of prezoeae to the first zoeal stage, the zoeae produc-
ed at 3,300 and 10,000 yg/liter did exhibit a deformity. Zoeae developing at
these concentrations of captan were seen to have a lateral enlargement of the
carapace. We did not observe similar deformations after molting of zoeae
exposed to captan in the chronic toxicity experiments even though the highest
concentration employed in those tests, 200 yg/liter, proved to be lethal
within 21 days to first instar zoeae. It should be noted, however, that none
of the larvae exposed to 200 yg/liter molted to second instars. Zoeae ex-
posed chronically to only 20 yg/liter of captan survived as well as control
larvae and developed from first instar zoeae through the later zoeal stages
to the fifth instar without any evidence of morphological abnormality. Pro-
duction of the zoeal deformity apparently requires exposure of molting zoeae
to a higher concentration of captan than used in the chronic tests, but such
34
-------
concentrations can only be survived by zoeae in short-term exposures.
Interestingly, a gross morphological malformation has been observed in
at least one other aquatic species exposed to captan. Abedi and McKinley
(1967) reported a deformity to the head in larvae of zebrafish Brachydan io
rer'\o, acutely exposed to captan concentrations of 500 to 1,000 ijg/liter.
The injury was always associated with the death of the fish larvae. A common
characteristic of the captan-caused morphological lesions in both zebrafish
larvae and crab larvae is an enlargement or swelling of the affected region.
Captan is chemically similar to the teratogenic compound thalidomide.
This fact has lead several investigators, in studies with vertebrates, to
compare the teratogenic potential of captan and other N-substituted phthali-
mide compounds with thalidomide. The results, however, are inconclusive.
Captan did not produce thaiidomide-1ike embryopathic effects in two studies
with rabbits (Fabro et al., 19&5; Kennedy et al., 1968), but did in two later
investigations, one with rabbits (Mclaughlin et al., 1969) and the other with
chicks (Verrett et al., 1969).
It is tempting to suggest that the abnormalities observed in this study
with crab larvae and in studies with vertebrates, including those with fish
larvae (Abedi and McKinley, 1967), have a common etiology. Unfortunately,
the reasons for the abnormal development are not clearly understood. Fur-
ther, it is known that captan interacts with both soluble and insoluble thiol
groups in biological material (Owens and Blaak, 1960b; Richmond and Somers,
1966) and is thus likely to have broad-spectrum effects on biological sys-
tems. Such effects have been shown to include increased mutagenesis in E_.
coli and inhibition of mitosis and DMA synthesis and increased incidence of
chromosome breaks in mammalian cell cultures (Legator et al., 1969). Also,
captan interferes with oxidative phosphorylation and mitochondrial structure
(Nelson, 1971a; 1971b) and with citrate synthesis from acetate and oxaloace-
tate (Owens and Blaak, 1960a).
The results of our work with crabs indicate that the environmental con-
sequences for the use of captan near marine or estuarine waters are likely to
be minimal. Even direct application of the fungicide at the manufacturer's
highest recommended rates (10-12 Ib/acre for Orthocide-50W) to an estuary of
2 meters average depth would result in an average seawater concentration of
only 336 yg/liter. If maintained, this concentration would probably be toxic
to crab zoeae within a few days, but because of the high rate of degradation
of the fungicide in seawater, the concentration resulting from a single ap-
plication could be expected to rapidly fall below the levels that have
demonstratable chronic toxicity.
Although the morphological effects of captan, observed during the trans-
formation of prezoeae to zoeae are of considerable interest in connection
with the question of the teratogenic potential of captan, the ecological sig-
nificance of this effect is probably minimal. It did not occur when eggs
were exposed to captan concentrations lower than those that result in death
of first instar zoeae during 96-hr acute exposures. Whether longer exposure
of eggs would or would not modify this response is unknown.
35
-------
THE INSECTICIDE METHOXYCHLOR
Tox i c i ty
Methoxychlor exposures of up to 10 yg/liter did not reduce the percent-
age of eggs that were able to hatch during a 24-hr period. In fact, as was
noted with captan, an increase in hatching rate of all pesticide-exposed eggs
was noted over that found in the controls (Figure 12). Larvae from nearly
OJ I
Methoxychlor Concentration tyig/liter)
10
Figure 12. Percent hatch of Dungeness crab eggs (Q O)> percent devel-
opment of hatched crab larvae through the prezoeal to the first
zoeal stage (A— •—A) > and percent of developed first stage
zoeae which are motile ( <£> <^>)as a function of methoxychlor
concentration during a 24-hr exposure period.
36
-------
all of the eggs that hatched in the control vessels, successfully completed
development through the brief prezoeal stage and were first instar zoeae by
the end of 2k hr. At increasingly higher concentrations of methoxychlor, the
successful completion of this developmental sequence decreased until at 1 to
10 yg/liter only 70% of hatching eggs became first instar zoeae (Figure 12).
Of the zoeae that developed, a reduction in motility of 50 and 90% occurred
at methoxychlor concentrations of about 0.18 and 1.0 yg/liter, respectively,
whereas 100% of the controls were motile (Figure 12).
Acute toxicity of methoxychlor to later developmental stages was
inversely related to crab age. The 96-hr LC50s for zoeae, juveniles and
adults were 0.42, 5-10 and 130 yg/liter, respectively (Table 12). The latter
value exceeds the estimated solubility of methoxychlor of 50 yg/liter in sea-
water of 25 °/oo salinity at 13°C and is based on the amount of methoxychlor
added to the bioassay containers. A similar relationship between develop-
mental stage and methoxychlor sensitivity was found by using sublethal
teria with zoeae and juvenile crabs (Table 12).
cr i -
TABLE 12. ACUTE TOXICITY OF METHOXYCHLOR TO FIRST INSTAR ZOEAE, FIRST
INSTAR JUVENILE CRABS, AND ADULT CRABS
Toxic concentrations (yg/liter)
UCVCIUplllCIILcll
stage
Zoeae
Juven i 1 e
Adult
24-hr
EC50
0.50
7-20
"
LC50
>0.92
12.0
>920
48-hr
EC50
0.23
5-50
LC50
0.92
8.20
>920
96-hr
EC50
0.05
5.10
LC50
0.42
5-10
130
In the -initial chronic experiment with zoeae, of 18 days duration,
methoxychlor did not affect the survival of larvae even at 0.1 yg/liter, the
highest concentration employed (Figure 13)- In the second experiment, zoeae
were reared in the presence of methoxychlor for 69 days by which time they
had reached the fifth zoea1 stage of development. Survival of controls and
of zoeae exposed to the lowest test concentration of 0.005 yg/liter exceeded
85% for 50 days (Figure 14). Thereafter, survival of zoeae in all groups,
including the controls decreased markedly, possibly due to increased inci-
dence of bacterial infection or to a nutritional deficiency. Of the larvae
exposed to 0.5 yg/liter, 50% were dead by day 5 and all were dead by day 24.
Survival of larvae exposed to the intermediate concentration of 0.05 yg/liter
was slightly less than that found in the controls, and all were dead by day
65. This group, in addition, experienced a slight delay of the onset of
molting at each zoeal stage (Figure 15)- Survivors of the 0.5 yg/liter
methoxychlor exposure also were delayed in molting to the second zoeal stage.
37
-------
KX)
80
60
1 40
20
METHOXYCHLOR
A Control
• Acetone Control
A 0.0001 /ig/liter
O 0.001 "
0 0.01
O O.I ••
10
Days
15
20
Figure 13- Effect of continuous exposure to methoxychlor on survival of
Dungeness crab zoeae. Data are from the first zoeal chronic
experiment conducted during the spring of 1973-
Juvenile crabs were markedly affected by chronic exposures to 0.4 and
4.0 yg/liter of methoxychlor. During the first test, lasting 36 days,
survival of crabs at these concentrations was similar to that of the controls
until day 20, but declined rapidly thereafter (Figure 16). In the second
test, which lasted for 80 days, 50% of the crabs exposed to these same con-
centrations had died by days 27 and 12, respectively, and all were dead by
days 80 and 40 (Figure 17)- Survival of crabs exposed to the two lowest con-
centrations in the second test (0.04 and 0.004 pg/liter) was similar to that
of controls and averaged JB% by day 60. Thereafter, there was a gradual de-
cline in the survival of these crabs; but the relationship of survival of the
exposed crabs to that of the controls did not change (Figure 17)-
38
-------
OJ
METHOXYCHLOR
Control
Acetone Control
A 0.005 /ig/liter
O 0.05
0 0.5
Days
Figure 14. Effect of continuous exposure to methoxychlor on survival of Dungeness crab zoeae.
from the second zoeal chronic experiment conducted during the spring of
Data are
-------
100
I
vt
i
o
80
x
0>
o
60
20
METHCKYCHLOR
Control
Ac0ton6 Control
0.005 jug/liter
IV
10
20
3O
4O
50
60
Days
Figure 15. Effect of continuous exposure to methoxychlor on molting of Dungeness crab zoeae beginning
with the first zoeal molt. The molt success for each stage is the percentage of the origi-
nal number of zoeae. Roman numerals indicate the zoeal instar at each stage in the zoeal
sequence. Data are from the second zoeal chronic experiment conducted during the spring of
-------
100
80
60
§40
*
20
i
. METHOXYCHLOR
0
A Control
• Acetone Control
A 0.004 /ig/liter
O 0.04
0 0.4
O 4
H
II
10
20
Days
30
40
Figure 16. Effect of continuous exposure to methoxychlor on survival of
juvenile Dungeness crabs. Data are from the first juvenile
chronic experiment which was initiated with newly metamor-
phosed first instar crabs.
Sensitivity of juvenile crabs to methoxychlor was greatest during molt-
ing or shortly thereafter. Of second and third instar crabs molting at Q.k
and 4.0 yg/liter, 38% and 100%, respectively, died within 72 hr after ecdysis
in the 80-day test (Table 13)- Deaths of crabs at these same concentrations
in the 36-day tests also appeared to occur at, or slightly following, the
period of molting (Figures 16 and 18).
Methoxychlor also delayed the onset of molting of crabs in the 36-day
test (Figure 18). The controls began to molt to second instars on about day
15, and by day 30 this molt was essentially complete. Molting was delayed in
all crabs exposed to methoxychlor, but this effect was most extreme among
crabs exposed to 4.0 yg/liter. We were unable to unequivocally measure
possible effects of methoxychlor on molting of juveniles in the second,
-------
100
METHOXYCHL
A Control
Acetone Control
0.004 /ig/IHer
0.04
0.4
4
Figure 17- Effect^ of continuous exposure to methoxychlor on survival of juvenile Dungeness crabs.
Data are from the second juvenile chronic experiment which was initiated with second and
thi rd instar crabs.
-------
TABLE 13. EFFECT OF METHOXYCHLOR ON SURVIVAL OF MOLTED JUVENILE AND ADULT
CRABS DURING CONTINUOUS EXPOSURE TO THE PESTICIDE FOR 80 AND 85
DAYS, RESPECTIVELY
Methoxychlor Original number Percentage Percentage of molting
concentration of crabs of crabs crabs that died within
(yg/liter) per treatment which molted 72 hr (juveniles) or
during experiment 2k hr (adults) after
molt i ng
Juveni1e crabs
Control 54 100 18
Acetone control 54 81 19
0.004 18 100 0
0.04 18 83 18
0.4 18 89 38
4.0 18 83 100
Adult crabs
Control
Acetone control
0.04
0.4
4.0
40.0
30
30
10
10
10
10
43
67
70
80
60
20
0
0
14
0
100
100
80-day, test because molting of these older crabs was largely out of synchro-
ny by this time. However, some crabs in the control groups and each concen-
tration of methoxychlor in this test had molted by day 7 of exposure, and
approximately 75% of crabs in all groups had molted by day 19 so that no
evidence for an effect of the pesticide on molting was evident in this ex-
per iment.
The increase in size of methoxychlor-exposed juvenile crabs that molted
during the 80-day test was less than that of the controls. The mean carapace
widths of fourth instar crabs in control and 4.0 yg/liter groups were 19.1 mm
and 16.6 mm, respectively, or greater by 34% and 16% than the mean width of
third instar crabs (14.3 mm). These differences were found to be statisti-
cally significant (a = 0.01) by the method of least significant differences.
All adult crabs exposed to 40.0 yg/liter of methoxychlor had died by day
47 and by the end of the test period, 60% of those exposed to 4.0 yg/liter
were dead also (Figure 19)- Survival of controls and of crabs exposed to the
two lower concentrations of 0.4 and 0.04 yg/liter was about 85% at the test's
end. As was true of the juvenile crabs, sensitivity of the adults to
methoxychlor increased during ecdysis. None of the controls that molted
43
-------
100
80
METHOXYCHUDR
A
O
0
O
Central
Acetone Control
O.OO4 /ig/lfter
0.04 "
0.4
4
N
Days
Figure 18. Effect of continuous exposure to methoxychlor on molting of
first instar Dungeness crabs to the second instar. The molt
success is expressed as the percentage of the original number
of first instar crabs.
during the 85-day test died. However, ] 00% of the crabs that molted during
exposure to 4.0 and 40.0 yg/liter died within 2k hr (Table 13)-
Adult crabs affected by methoxychlor were hyperactive and moved Ijjieir
mouth parts and chelipeds much more frequently than did the control animals.
The more severely affected crabs were unable to maintain an upright posture
and some remained flipped on their dorsal surface for several weeks, until
death. Feeding was active, except during the molting period, among control
crabs and those exposed to the two lower concentrations of methoxychlor, but
crabs exposed to 40 yg/liter did not eat after test days 6 to 8. Crabs ex-
posed to 4.0 yg/liter had difficulty in locating food, and would usually
shred and scatter it rather than consume it entirely.
-------
100
80
60
I 40
w,
rj
CO
20
0
METHOXYCHLOR
A Control
• Acetone Control
A 0.04
O 0.4
0 4
O 40
10
20 30 40 50 60
Days
70 80
90
Figure 19- Effect of continuous exposure to methoxychlor on survival of adult Dungeness crabs.
-------
The 96-hr LC50 values of methoxychlor for larval, juvenile, and adult
stages of £. magister were 0.42, 5-10 and 130 -yg/liter, respectively (Table
12), whereas the toxic levels for these same stages for chronic exposure were
only 0.05, 0.4 and 4.0 yg/liter (Figures 14, 17 and 19, respectively). An
estimate of the toxic concentration of methoxychlor based only on short-term
tests with adult crabs would have, been 2,600 times higher than the concentra-
tions found to be toxic in chronic tests with larval stages of this animal.
These differences in toxicity values clearly show the desirability of testing
pesticides against several life history stages of an animal with prolonged
exposure, or at least against that stage thought to be the most sensitive.
The concentrations of methoxychlor that we found to be toxic to C_.
magister are among the lowest reported for insecticides tested against this
and other species of crab. Poole and Willis (1970) reported high mortalities
of C_. magi ster zoeae exposed to endrin and DDT concentrations of 0.25 and
0.50 yg/liter, respectively, during chronic tests. The lowest level of
methoxychlor that we found to be toxic to larvae was 0.05 yg/liter. Buchanan
et al. (1970) reported that Sevin concentrations as low as 0.32 yg/liter de-
layed molting of C_. mag i ster larvae, but did not significantly affect sur-
vival. In a study by Epifanio (1971), dieldrin was toxic to larvae of the
crabs Leptodius floridanus and Panopeus herbst i i at concentrations of 1.0 and
5.0 yg/liter, respectively, during chronic tests. Bookhout et al. (1972)
found that survival of various larval stages of the crabs Ri thropanopeus
harr i s i i and Menippe mercenaria was reduced in concentrations of the insecti-
cide mirex down to 0.01 yg/liter; in this instance lower than our toxicity
values for methoxychlor.
A sublethal effect of methoxychlor observed with zoeae was delay of
molting. Only 7% of larvae exposed to 0.05 yg/liter in the chronic experi-
ment had molted to second stage zoeae by day 13, whereas 46% of the controls
and those exposed to 0.005 yg/liter had molted by this time (Figure 15)-
This same trend continued for the next two molts to the fourth zoeal stage,
but was not as evident when treatment groups molted to the fifth stage.
Delays in molting of crab larvae exposed to pesticides have been previously
observed by Buchanan et al. (1970), Epifanio (1971). and Bookhout et al.
(1972).
Delay of molting during exposure to methoxychlor was also noted with
juvenile crabs but was apparent only in the first test. This 36-day experi-
ment was begun with newly metamorphosed first instar crabs, that were sub-
sequently exposed to methoxychlor for the entire intermolt period. Onset of
molting of crabs in the highest concentration of 4.0 yg/liter was delated by
more than 10 days compared with that of the controls (Figure 18). By day 30
of exposure, only 10% of the initial number of crabs held in 4.0 yg/liter had
molted, although survival was 70% at this time; about 77% of both control
groups had molted within 30 days.
There was no evidence during the second test, lasting 80 days, that
methoxychlor delayed molting in older juvenile crabs. Most of these crabs
probably were exposed to the pesticide for less than the latter half of their
intermolt phase, suggesting that the effect of methoxychlor on molting may
depend upon the length or timing of exposure of the crabs during an intermolt
46
-------
period.
A possible explanation for a relationship between duration of intermolt
exposure to methoxychlor and incidence of molt delay is related to the
neuroendocrine control of molting. The interrelationship of the X- and Y-
organs in crustacean ecdysal processes has been well documented (Passano,
I960; Lockwood, 1967). The neurosecretory X-organ-sinus gland complex pro-
duces a^molt-inhibiting hormone that acts directly on the Y-organ to depress
synthesis or release of its molting hormone. At the time in the molt cycle
when inhibition ceases, the Y-organ releases the molting hormone, and pro-
ecdysis is initiated and quickly becomes irreversible (Lockwood, 1967)- If
methoxychlor affected the X-organ in such a way that production or release of
the molt-inhibiting hormone continued beyond the normal time, the effect
would be to delay initiation of the molting process. Another possibility is
that methoxychlor directly delayed the production or release of molting hor-
mone by the Y-organ, but this hypothesis is less attractive because methoxy-
chlor is believed to be a neurotoxin (O'Brien, 19&7), and molting hormone
production by the Y-organ is not a neurosecretory process (Passano, I960).
The hypothesis that action of methoxychlor on the X-organ affects molt-
ing may not explain molting delays observed with £. magister larvae exposed
to 0.05 yg/liter. Costlow (1966) has produced evidence that the control of
molting in larval crustaceans may not involve X-organ inhibition in some in-
stances, because eyestalk ablation in Rhithropanopeus zoeae did not result in
anticipated acceleration of molting.
Another possible cause of delay of molting may be starvation of crabs
(Passano, I960). We observed that larval and juvenile crabs exposed to
methoxychlor did eat, but we did not make quantitative determinations of food
consumption during the tests. The amount eaten or the efficiency of food
utilization may have been reduced by the high pesticide concentrations and
molting thereby delayed.
There was an apparent increase in sensitivity of the crabs to methoxy-
chlor during or shortly after ecdysis. A similar observation has been re-
ported by Duke et al. (1970) and Nimmo et al. (1970 for crustaceans exposed
to the PCB, Aroclor 1254. All of the juvenile crabs that molted during ex-
posure to 4.0 yg/liter of methoxychlor died within 72 hr after ecdysis
(Table 13). Similarly, 100% of the adult crabs that molted in 40.0 and 4.0
yg/liter died within 24 hr after ecdysis. Bookhout et al. (1972) suggested
that release of stored mirex from fat reserves could account for increased
deaths of crab larvae during molting to megalopae. A similar release of
methoxychlor from the lipid storage sites of our exposed adult and juvenile
crabs may explain their increased sensitivity at ecdysis. Hepatopancreatic
lipid reserves are highest as proecdysis is initiated, and levels decline
through molting until active feeding is resumed (Passano, I960).
Possibly, increases in blood volume during ecdysis contributed to higher
circulating levels of methoxychlor at this time, a result that could lead to
higher levels of the pesticide being transported to affected tissues. The
blood volume of Cancer can increase nine-fold during ecdysis (Passano, I960)
due to active uptake of water through the gut (Lockwood, 1967)- Addition-
47
-------
ally, blood levels of lipids rise during ecdysis (Passano, I960), and this
also could increase the carrying capacity of the blood for lipid soHIble pes-
ticides such as methoxychlor.
Finally, the increased death rate of methoxychlor exposed crabs after
molting may occur because pesticides are more readily absorbed by the animal
at this time. The integument of newly molted crabs is thinner and in all
probability more permeable to the pesticide than after extensive mineraliza-
tion has occurred. If this hypothesis is correct, then one would not an-
ticipate an increase in death rate of zoeae during the molting periods
because even during intermolt of the larval stages, the exoskeleton is not
thick or heavily mineralized. In fact, we noted that the mortality of zoeae
which was attributable to methoxychlor exposure was constant throughout the
exposure period and was not apparently correlated with the time of molting
(Figures 14 and 15)•
Uptake, Loss, and Tissue Distribution of Methoxychlor
Although the range of pesticide residue concentrations in adult crabs
was wide, there was a tendency for animals killed at the highest methoxychlor
concentrations to have higher whole body residues than did those killed at
the lower concentrations (Table 14). Crabs killed after exposure to 32.0
pg/liter had an average methoxychlor concentration of 1.59 mg/kg in whole-
body tissues, whereas crabs killed after exposure to 7-5 yg/liter had whole-
body residues averaging only 0.57 mg/kg. Most of the crabs killed after
exposure to the lower concentrations had recently molted, whereas none of the
animals exposed to 32.0 yg/liter molted (Table 14). The higher residue
levels in the latter group may reflect greater quantities of pesticide ab-
sorbed to, or otherwise associated with, the exoskeleton and its epiflora,
TABLE 14. WHOLE BODY METHOXYCHLOR CONCENTRATIONS IN ADULT CRABS
KILLED DURING CONTINUOUS EXPOSURE TO METHOXYCHLOR FOR
UP TO 15 DAYS
Methoxychlor
concentrat ion
in exposure water
(yg/1 i ter)
1.8
7-5
18.0
32.0
Number
of
an imal s
analyzed
1
5
6
14
Whole body
concentration
of methoxychlor
(mg/kg)*
0.11
0.57
(0.46 - 1.3)
0.95
(0.45 - 1.3)
1.59
(1.10 - 2.5)
Percentage
of ki 1 led crabs
which had
just mo 1 1 ed
100
80
67
0
Mean concentration with range in parentheses.
48
-------
rather than a significant difference in the levels of pesticide residing in
the affected internal tissues.
We estimated the amount of methoxychlor which associated loosely with
the exoskeleton and its epiflora, by measuring the whole-body residues in two
groups of crabs; one having received the routine washing with water only, and
the second having also been thoroughly rinsed with acetone. Whole-body resi-
dues in crabs exposed for 72 hr to 7.5 yg/liter were 33% greater when acetone
was not used as a rinse than when it was (Table 15). Microscopic examination
of crabs washed only with water revealed the presence of diatoms still on the
exoskeleton. It is probable that much of the pesticide removed from crabs
rinsed with acetone had been associated with these microorganisms.
TABLE 15. WHOLE BODY RESIDUES OF METHOXYCHLOR IN ADULT CRABS
WASHED WITH WATER ONLY OR WITH WATER FIRST, FOLLOWED
BY ACETONE BEFORE PESTICIDE ANALYSIS*
Exposure Whole body residues (mg/kg)t
period ——-—• —
(hr) Water-washed crabs Water + acetone-washed crabs
12
24
48
72
0.08
0.15
0.19
0.23
0.07
0.10
0.12
0.14
Crabs were exposed for up to 3 days to a pesticide concentration
in seawater of 7-5 yg/liter.
+ Each value is the methoxychlor level in a pooled sample of five
crabs.
Uptake of methoxychlor by juvenile crabs was linearly related to time,
but not proportional to the exposure concentration (Figure 20). After 12
days, the whole-body pesticide concentrations in crabs exposed to 0.04 and
2.0 yg/liter were 0.11 and 0.88 mg/kg wet weight, respectively, and repre-
sented concentration factors of 2,725 and 440 above the exposure levels.
After 12 days, many crabs exposed to the higher concentration had died, indi-
cating that lethal whole-body levels of methoxychlor had been attained.
In contrast to the apparent zero order uptake curves for juvenile crabs,
those for adults approximated first order kinetics (Figure 21). At 15 days,
residues in crabs exposed to 1.8 and 7-5 yg/liter were about 0.11 and 0.48
mg/kg, respectively, with the uptake curves approaching plateaus at this
time. The loss of methoxychlor was also a first order relationship, and only
5% to 6% of the residues present at day 15 remained by day 30, 15 days after
the termination of methoxychlor exposures. The 12-day concentration factors
for adult crabs exposed to 1.8 and 7.5 yg/liter were 58 and 60; this, unlike
the situation with juvenile crabs, indicates that the uptake rate was
49
-------
0.04 yug/liter
O 2.0
Figure 20. Whole body concentration of methoxychlor in juvenile crab,
Cancer magister, as a function of time of exposure to the
pesticide in seawater.
approximately proportional to the exposure concentration.
The highest concentrations of methoxychlor in individual tissues of ex-
posed adult crabs were found in the exoskeleton, gill, and hepatopancreas, in
decreasing order (Table 16). The concentration factors (tissue
concentration/exposure concentration) for these three tissues in crabs ex-
posed to 1.8 yg/liter were 183, 122, and 78, respectively, and for crabs ex-
posed to 7-5 yg/liter were 89, 56, and kO. Dividing the sum of methoxychlor
recovered in all tissues at each exposure concentration by the total weight
of the crabs gives a whole-body residue of 0.16 mg/kg and 0.33 rng/kg for
50
-------
A 1.8/jg/IHer
O 7.5 »
Figure 21 .
24 27 30
Whole body concentration of methoxychlor in adult crabs,
Cancer magister, as a function of time during and after a
15~day exposure to the pesticide in seawater.
water exposures of 1.8 and 7-5 ug/liter, respectively, values which compare
favorably to those determined in the experiments with whole crabs (Figure
21). The concentrations in the exoskeleton were about 83 times greater than
those in the blood, which contained the lowest levels of all tissues assayed.
The weight of the exoskeleton is about 33% of the wet weight of whole crabs,
and about 81% of the total methoxychlor was calculated to have been present
in this tissue at both water exposure levels (Table 16). Most of the remain-
ing pesticide was found to be concentrated in the hepatopancreas and gill
tissues. When the calculated tissue levels of methoxychlor were based on dry
weights, the gills were found to have had the highest pesticide concentra-
tion, about three times higher than those in the exoskeleton.
There is little information on the uptake and loss of methoxychlor by
aquatic invertebrates. Kapoor et al. (1970) found that levels of methoxy-
chlor in larvae of the mosquito, Culex, and a snail were 0.48 and 15-7 mg/kg,
respectively, after 7 and 23 days of exposure in a model ecosystem.
51
-------
TABLE 16. DISTRIBUTION OF METHOXYCHLOR IN SELECTED TISSUES OF ADULT CRABS
AFTER 15 DAYS OF CONTINUOUS EXPOSURE TO 1.8 OR 7.5 yg/LITER OF
THE PESTICIDE IN SEAWATER
Mean
water content
Tissue of
t i ssue
Exoskel eton
Gill
Hepatopancreas
Gonad
Muscl e
Heart
Blood
40
88
74
60
76
84
93
Mean
wet weight
t i ssue
(g)
64
5
14
6
45
0
28
.5
.9
.4
.0
.0
.7
.5
Tissue concentration
of methoxychlor
(yg/g)
1
0
0
0
0
0
0
0
.8 yg/1 i ter
• 330
.220
.140
.070
.014
.006
.004
083)#
(122)
(78)
(39)
(7-7)
(3-3)
(2.2)
7
0
0
0
0
0
0
0
• 5 yc
.670
.420
.300
.320
.044
.014
.008
I/I iter
(89)
(56)
(40)
(43)
(5-9)
(1.9)
(1.1)
165.0
Mean of 8 individual determinations.
Based on a mean whole body weight of 165 g-
# ,. , , r r , TISSUE CONCENTRATION
Numbers m parentheses are Concentration Factors =ExpOSURE CONCENTRATION
Juvenile crabs exposed to 2.0 yg/1iter had whole-body levels of 0,48 mg/kg
after 6 days in our experiments. Uptake of the pesticide by Culex, however,
was through their food rather than directly from water. Reinbold et al.
(1971), using dry weight as a basis for estimating whole-body levels o£
pesticides, reported that Daphnia magna and snails, Physa sp. , exposed to 3
yg/1iter of methoxychlor for 3 days had tissue levels of 24 and 22 mg/kg,
respectively; their levels represent concentration factors of 8,000 and
7,300. In our study, juvenile crabs exposed to 2.0 yg/1iter of methoxychlor
had whole-body levels of 0.3 mg/kg after 3 days of exposure. Even when
corrected for dry weight, this value would only be 1.2 mg/kg, giving a con-
centration factor of 600. The relatively low accumulation of methoxychlor in
C_. mag i ster suggests that this species may efficiently metabolize the pesti-
cide or not absorb it as rapidly.
Concentration of methoxychlor in body tissues was more rapid in juvenile
52
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TABLE 16. CONTINUED
Total of methoxychlor Percent of total
recovered recovery
(%)
1-8 yg/liter 7-5 yg/liter 1.8 yg/liter 7.5 yg/liter
21.3
1.30
2.02
0.42
0.63
0.004
0.11
43.2
2.48
4.32
1 .92
1 .98
0.010
0.23
82.6
5-0
7-8
1.6
2.4
0.02
0.43
79-9
4.6
8.0
3-5
3-7
0.02
0.43
25.8 54.1 99-9 100.2
crabs than in adults. Body size of the two stages may account for this
difference in uptake rates. Juvenile and adult crabs averaged, respectively,
14 and 107 mm in width and 0.8 and 165 g in wet weight. Juvenile crabs
exposed to 2.0 yg/liter methoxychlor for 12 days had whole-body levels of
0.88 mg/kg, whereas adults exposed to 1.8 yg/liter had whole-body levels of
only 0.10 mg/kg by this same time.
Wildish and Zitko (1971) reported that the rate of uptake of Aroclor
1254 by an amphipod increased as the ratio of body or branchial surface
area to body weight increased. If there is an inverse relationship between
rate of methoxychlor accumulation in tissues and the size of crabs, then the
larvae may have had the highest rate of pesticide uptake, although this was
not measured, and this could explain the increasing sensitivity to methoxy-
chlor we observed for successively smaller stages.
53
-------
The highest concentrations of methoxychlor were in different tissues
than have been reported in other Crustacea. Nimmo et al. (1970) found that
the hepatopancreas contained most of the DDT found in two species of shrimp;
the levels being 200 times higher in that organ than in whole-body samples.
The PCB, Aroclor 1254, was concentrated most in the hepatopancreas of pink
shrimp and least in muscle and exoskeleton (Nimmo et al., 1970- We found
that concentrations of methoxychlor were highest in the exoskeleton, gill,
and hepatopancreas, in decreasing order, with less than a two-fold difference
in methoxychlor levels between these tissues. The gills contained the high-
est concentration of methoxychlor calculated on the basis of dry weight, but
even so, computed levels were only about three times higher than those in the
exoskeleton.
It was calculated that the exoskeleton accounted for some 81% of the
methoxychlor found in whole crabs. A large portion, at least 33%, of this
value was shown to be associated with the surface of the exoskeleton. The
extent to which the pesticide was taken up by the epiflora or adsorbed to the
epicuticle is not known. Johnson et al. (1973) reported that uptake of
methoxychlor by bacteria was rapid and reached biomagnification levels of
3,400x in 2k hr. Certainly adhering algae and bacteria could account for a
large portion of whole-body pesticide levels given in the literature for
other Crustacea.
Salinity Tolerance of Methoxychlor Exposed Crabs
A preliminary indication of the effect of exposure of adult crabs to
methoxychlor on osmoregulatory responses was obtained by studying the rela-
tive survival of treated and control crabs exposed to low salinities. There
was a clear indication that the crabs treated with pesticide were less re-
sistant to the low salinity exposures (Table 17)- Although no deaths
TABLE 17- PERCENTAGE MORTALITY OF ADULT CRABS EXPOSED TO
METHOXYCHLOR IN DILUTE SEAWATER
Salinity exposure (°/0o)
Treatment
26.5 17-1 9.7 5-5
2 days
Control 0 0 29 43
Methoxychlor 0 0 47 73
(10 yg/1iter)
7 days
Control 17 7 64 100
Methoxychlor 13 7 100 100
(10 yg/1 iter)
54
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occurred at the higher two salinities tested, within two days mortalities
were occurring in C_. magister at 9-7 and 5-5 °/00 salinity- The percentage
mortality of crabs exposed to methoxychlor at this time was nearly twice that
for the controls. By seven days 100% mortality was noted in both groups of
crabs exposed to 5.5 °/00 salinity and also in the methoxychlor treated crabs
at 9-7 °/oo> but only 64% mortality was noted in the controls at this
salinity. Alspach (1972) reported that adult C. magister held in 25% sea-
water were unable to survive for longer than 4F hr, and that crabs exposed to
50% seawater survived for only three days before becoming moribund.
Effects of Methoxychlor on Osmotic and Ionic Regulation
Decreased survival at low salinities could be caused by a reduced
osmoregulatory response in the pesticide treated crabs. Kinter et al. (1972)
have shown that the osmolarity and the Na+ and K+ concentrations in Angui1 la
rostrata serum are significantly elevated following a 6 hr exposure to 1 ppm
DDT in seawater, a concentration of the pesticide which is fatal in 10 hr.
In additional studies with killifish, Fundulus heteroclitus, both DDT and
Aroclor 1221 significantly elevated serum osmolarity but only Aroclor 1221
exposure caused elevation of serum Na+ levels (Kinter et al., 1972). To
determine whether similar effects on osmotic and ionic regulation occurred in
crabs, we exposed adult C_. mag i ster to 10 yg/liter methoxychlor in seawater
for 14 days and subsequently held the crabs for two more days in a series of
reduced salinities. The osmoregulatory response is illustrated in Figure 22.
Blood osmotic concentrations of the pesticide exposed crabs did not differ
from that of the controls at any of the salinities examined. The results
confirm the observations of Alspach (1972) that C_. mag i ster is a weak hyper-
osmoregulator at low seawater salinities. Examination of two of the pesti-
cide group of crabs confirmed that the treated crabs had methoxychlor resi-
dues similar to those reported earlier in methoxychlor killed crabs (Table
14), an indication that the pesticide concentration employed in this experi-
ment was only slightly sublethal.
Blood Na+ regulation was also unaffected by methoxychlor treatment
(Figure 23)- The pattern of regulation of this ion was essentially the same
as that observed for osmotic concentration, consistent with the fact that the
Na+ ion is the major cation found in the blood. The pattern of urine Na+
concentrations at salinities less than 30 °/0o was comparable to that of the
blood in both groups, confirming the extrarenal regulation of this ion at
salinities less than full strength seawater (Alspach, 1972; Engelhardt and
Dehnel, 1973)- In full strength seawater salinities and higher, some regu-
lation of blood Na+ by reabsorption of this ion from the urine has been
demonstrated (Alspach, 1972; Engelhardt and Dehnel, 1973). Our crabs exposed
to 30 °/oo salinity had urine Na+ levels less than that of the blood, con-
firming this relationship. Although this response was observed in both the
methoxychlor exposed and control crabs, the differences between blood and
urine Na+ concentrations were greatest in the control animals.
Blood K+ regulation (Figure 24) appeared to be poorer at low salinities
than that reported by Engelhardt and Dehnel (1973) but similar to the results
reported by Alspach (1972). Our results with blood K+ levels were highly
variable and exhibited certain spurious responses. For example, blood K+ in
55
-------
1000-
^800-
Jsooh
§400
s
200
Concur m««i>t«r
• MctkoqrcMor treated crab*
A Caitrol erobs
200 400 600 800
Mtdium (mOsm/kg)
1000
Figure 22. Blood osmotic concentration of control and methoxychlor treated
Cancer magister as a function of medium osmolarity. Each point
represents the mean of analyses on from 7 to 11 crabs.
response was reversed at about 28
crabs had lower blood K levels than those found
control crabs was significantly lower (p<0.05; Student's t test) at 5 °/oo
salinity than that found in methoxychlor-treated crabs. However, this
°/oo salinity where methoxychlor-treated
in the control animals. At
the higher salinities used, the urine to blood ratio was less than unity sup-
porting the contention of renal K regulation in high salinities (Alspach,
1972; Engelhardt and Dehnel , 1973)- However, except for the spurious obser-
vations already mentioned, blood and urine K+ levels were not significantly
different in comparisons between the control and methoxychlor-treated groups
at any of the salinities examined, indicating that the pesticide exposure had
no effect on the regulation of this ion.
As previously reported in £. mag i ster (Alspach, 1972; Engelhardt and
Dehnel, 1973) and in several other crustacean species (Gross, 1964; Gross and
Capen, 1966), blood Mg++ was strongly regulated to levels below that of the
seawater medium (Figure 25)- The only indication that methoxychlor treatment
affected the blood Mg++ concentration was seen at 28 °/0o salinity where the
pesticide-treated crabs had significantly higher (p<0.05) blood Mg++ levels
56
-------
600
MtttioxycMor trootod
• Blood
O Urin*
Control crabs
A Blood
A Urln*
10 20
Salinity of medium (%•)
Figure 23- Blood and urine sodium concentration of control and methoxychlor
treated Cancer magister as a function of experimental salinity.
Each point represents the mean of analyses on from 7 to 11 crabs.
than that seen in the controls. However, this difference was not observed in
crabs exposed to 30 °/0o salinity.
Control animals exposed to the higher salinities produced urine in which
the Mg concentrations were appreciably elevated over that of the blood and
the medium (Figure 25)- However, crabs treated with methoxychlor failed to
produce a urine concentrated in Mg++. In view of our inability to demon-
strate that methoxychlor affected the regulation of Na and K , this obser-
vation is of particular interest. The regulation of blood Mg by crabs has
been shown to be a function of the excretory organs, specifically the bladder
wall (Gross and Capen, 1966). Although the highest urine Mg++ concentrations
57
-------
Ul
E
c
o
o
o
•o
o
•o
o
_o
CD
Mcthoxychtor tr«ot«d erobt
Blood
OUrin*
Control crobi
A Blood
A Urin*
10 20
Solfctity of m«4ium (%•)
Figure 24. Blood and urine potassium concentration of control and methoxy-
chlor-treated Cancer magister as a function of experimental
salinity. Each point represents the mean of analyses on from
7 to 11 crabs.
have been found in crabs immersed in normal and concentrated seawater, Gross
and Marshall (I960) demonstrated that, in Pachygrapsus crassipes, the total
amount of Mg++ excreted per day may be highest when crabs are placed in* 50%
seawater; since the rate of urine production is appreciably increased in
dilute media. The rate of Mg transport across the bladder wall appears to
be a direct function of the blood Mg level (Gross and Capen, 1966),
although this property seems to be better developed in Carcinus maenas than
in £_. crass i pes (Lockwood and Riegel, 1969). Associated with the secretion
of Mg"1"1", Na"1" is reabsorbed from the urine, presumably by direct exchange in
order to achieve electrochemical balance (Gross and Capen, 1966). We have
observed that urine Na+ concentration is depressed relative to blood Na+ at a
salinity of 30 °/0o (Figure 23)- Although the urine Na+ concentration of
58
-------
200-
kJ
E
o
o
I
100
I
m
MotfcMyeMor trootod crabs
• Mood
OUriM
Control erabs
A Hood
AUrhw
f
i
I
i
*
10 20
Salinity of medium (%0)
30
Figure 25. Blood and urine magnesium concentration of control and
methoxychlor-treated Cancer magister as a function of experi-
mental salinity. Each point represents the mean of analyses on
from 7 to 11 crabs.
59
-------
methoxychlor-treated crabs at this salinity is not significantly less than
for the control crabs, the urine to blood ratio is lower, possibly a conse-
quence of the effect of the pesticide on Mg++ transport across the bladder
wal 1 .
Although the most plausible explanation of the effect of methoxychlor on
urine Mg++ concentration is that the pesticide inhibits the active transport
of the ion across the bladder wall, another explanation can be considered.
The rate of urine production in crabs is inversely related to the salinity of
the medium because in low salinities excess water is absorbed by the animal.
As a result, when crabs are in low salinity seawater, the bladder contents
are frequently voided, reducing the possibility of concentrating Mg++ in the
urine (Gross and Capen, 1966). An alternative explanation, therefore, for
our observations that urine Mg++ concentrations were low in crabs exposed to
methoxychlor is that the rate of urine production in high salinities was
much higher in the pesticide-exposed crabs than in the control crabs.
Although we did not measure the rates of urine production at the experimental
salinities used, it seems highly unlikely that urine production by pesticide-
exposed crabs would be unusually high since we did not see any significant
difference between control and treated crabs in the urine Na+ and K+ concen-
trations at high salinities. Also, it is well known that the flow rate of
urine in hyperosmoregulating crabs is related to the osmotic gradient between
the blood and the medium (Lockwood, 1967), a difference which increases as
salinity decreases. It seems most likely, therefore, that our results were
caused by a direct inhibition of transbladder Mg++ transport by methoxychlor.
Interpretation of the specific effects of methoxychlor on Mg++ transport
in crab bladder is difficult because little information is available con-
cerning the biochemical nature of this process. There is considerable evi-
dence that different types of transport enzymes exist (Oxender, 1972) so that
it is reasonable to expect that the Mg++ transport process in crab bladder
may be quite different from the more thoroughly studied NaKMg ATPase trans-
port system. Indeed, Gross and Capen (1966) have shown that crabs treated
with the Na+ transport inhibitor, ouabain, were still able to transport Mg++
into the urine,discounting the possibility that Mg++ secretion is enzymati-
cally coupled to active Na+ uptake from the urine. Similar evidence for the
independent nature of divalent cation transport from the NaKMg ATPase trans-
port system was obtained by Schatzmann and Vincenzi (1969)- They have shown
in their studies that the active transport of Ca++ in human red blood cells
is also unaffected by ouabain, as well as oligomycin and Na+ and K+ ions.
Although we have obtained evidence that the Mg++ transport system *in the
bladder wall was inhibited by methoxychlor, no evidence was found, in the
form of impaired osmotic or Na+ or K+ ionic regulation to suggest that the
ion transport functions of the gills were affected. Since the gill NaKMg
ATPases are presumed to function in the Na+ transport process, these results
suggested that the NaKMg ATPases in crab gills may not have been inhibited by
the pesticide. Davis et al. (1972) have shown that the ATPase activities of
rainbow trout gill are inhibited by methoxychlor in vi tro, but the amount of
inhibition produced was considerably less than that found with a number of
other organochlorine insecticides. To determine the effect of methoxychlor
60
-------
on crab gill ATPases, gills were excised from adult C_. magister which had
been held in 10 yg/liter methoxychlor for a 14-day period. The control group
was treated in an identical fashion, but was exposed only to acetone. The
gill tissue from two animals.was pooled and activity was measured in whole
homogenates (Table 18). NaKMg ATPase activity was significantly (P<0.0$)
inhibited by methoxychlor treatment to the extent of 37%. Inhibition of both
the Mg and NaK components of the enzyme appeared to be similar, 36 and 50%,
respectively, but the differences were not significant.
TABLE 18. ATPASE ACTIVITY IN GILLS OF ADULT CRABS EXPOSED TO
METHOXYCHLOR
ATPase activity (ymoles Pj/mg protein/hr)*
Treatment
NaKMg Mg NaK
Control 2.13 ± 0-32 1.83 ± 0.60 0.34 ± 0.35
(4) (4) (4)
Methoxychlor 1.34 ± 0.34 1.17 ± 0.37 0.17 ± 0.25
(10 yg/liter) (5) (5) (5)
Data are the mean ± two standard errors of the mean. The
numbers in parentheses indicate the number of individual
analyses conducted per treatment.
The reasons for the apparent contradiction between the effect of
methoxychlor on gill ATPase activity and the lack of effect of the pesticide
on blood osmotic regulation and on regulation of blood Na+ and K+ in the
crab are difficult to understand. The activity of the Na+ transport system
in crustacean gills is influenced by both the blood and medium concentrations
of this ion (Lockwood, 1962). It is possible that the degree of inhibition
of the enzyme was insufficient to significantly affect its function in
osmotic and ionic regulation of the blood if the requirement for activity was
less than the maximum capacity of the enzyme in normal crabs. This is
plausible since the capacity for enzyme catalyzed reactions often greatly
exceeds the rates required in vivo (Chance and Hess, 1959)- The correlation
between gill ATPase inhibition and gross toxicologica1 effects is equivocal
in other studies as well. Davis et al. (1972) in comparing literature values
of pesticide chemicals, reported that the chlorinated hydrocrabon insecti-
cides were more lethal and also more effective inhibitors of gill ATPases
than the herbicides, whereas PCB's were intermediate in effect. However, the
same direct relationship was not found within the insecticide group alone,
which suggests that the inhibition of gill ATPase is coincidental to a prima-
ry lethal lesion. If the primary lesion is associated with ion distributions
across axonal membranes, as has been previously suggested (O'Brien, 1967),
possibly due to an effect on the membrane NaKMg ATPases of the axon, then a
61
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partial but imperfect correlation with gill ATPases is not surprising. Davis
et al. (1972) also reported that trout given lethal doses of DDT orally ac-
cumulated high tissue residue levels in gills, brain and kidney; but that no
differences in ATPase activities of these tissues were observed between
treated and control groups of fish, lending further support to this suppo-
sition.
Our initial assumption in this study was that methoxychlor, as is the
case with other organochlorine pesticides and PCB's examined (Davis et al.,
1972), would inhibit the NaKMg ATPase activity of gills and consequently
impair osmotic and ionic regulation in the crabs, an effect which should
ultimately reduce the tolerance of the animals to low salinity exposures.
Our results with gill ATPase in C_. magi ster confirmed that the pesticide
administered to crabs at barely sublethal levels resulted in inhibition of
the enzyme (Table 18). However, with the exception of Mg++ regulation, we
have been unable to show any adverse effect on osmotic and ionic regulation.
Yet, despite this apparent lack of effect on osmotic and ionic regulation,
the crabs, after exposure to the pesticide, exhibited a decreased tolerance
to low salinity exposures (Table 17)- Although the role of Mg++ regulation
is uncertain, these results suggest that impairment of osmotic and ionic
regulation in crabs by organochlorine pesticides is not the principle effect
leading to decreased survival at low salinities.
Exposure to low salinities, even in normal crabs, is accompanied by a
reduction in blood osmotic and ionic concentrations (Alspach, 1972; Dehnel,
1962; Dehnel and Carefoot, 1965; Engelhardt and Dehnel, 1973). It is con-
ceivable, therefore, that the normal reduction of osmotic and ionic content
of the blood may be potentiating the known physiological effects of organ-
ochlorine pesticides on the nervous system. The familiar symptoms of
poisoning by this class of compounds, which were also seen in the present
study in methoxychlor-poisoned crabs, begins with the hyperactive state and
at high dosages is eventually followed by loss of motion and paralysis.
Individual nerve axons of diverse species poisoned by DDT are characterized
by uncontrolled volleys of impulses (Yeager and Munson, 19^5)- It is postu-
lated that the DDT-like organochlorines exert their effects on nerves by
blocking the membrane Na+ and K* channels and by inhibiting the nerve ATPases
(Matsumura and Narahashi, 1971; Matsumura and Patil, 1969; Narahashi and
Hass, 1967)- It is reasonable to suppose that normal nerve function occurs
within a finite range of blood ionic concentrations with optimum conditions
near the center of that range. We postulate that damage to nerve axons,
resulting from sublethal exposure to methoxychlor, serves to reduce the range
of blood ionic concentrations compatible with continued nerve function wTth
the result that survival of crabs at extreme salinities is reduced.
THE HERBICIDES 2,4-D, DEF, PROPANIL AND TRIFLURALIN
Exposure of eggs to trifluralin, DEF and propanil accelerated hatching
compared with controls at all concentrations tested (Figures 26-28). The
hatching success during 2k hr for each of these compounds was in the range of
55 to 70% while that for controls was only 39%- The response to 2,4~D ex-
posure differed from that of the other herbicides (Figure 29). At the two
62
-------
lowest test concentrations, 3,300 and 10,000 yg/liter, eggs hatched with a
success of 80% or higher. At 33,000 and 100,000 pg/liter 2,4-D, hatching
success was comparable to that found with the other herbicides, 60 to 70%,
but at the highest concentration tested, 330,000 yg/liter, the hatching of
eggs was almost completely inhibited; only one egg out of 58 hatched. At the
latter concentration all of the unhatched eggs appeared larger and were more
opaque than were unhatched control eggs.
80
60
p
40
20
10 100
Triflurdin Concentration (/ig/lrfcr)
Figure 26. Percent hatch of Dungeness crab eggs (Q O)> percent
development of hatched crab larvae through the prezoeal to the
first zoeal stage ( A — - —A)> and percent of developed first
stage zoeae which are mo tile ( <£> <£>) as a function of
trifluralin concentration during a 2^-hr exposure period.
63
-------
KX)
80
60
£.
40
20
\
\
\
0\
I 10
DEF Concentration (/ig/literi
100
Figure 27- Percent hatch of Dungeness crab eggs (Q - O)» percent
development of hatched crab larvae through the prezoeal to the
first zoeal stage (A — •— 'A)> and percent of developed first
stage zoeae which are motile(<^> ---- O)as a function of DEF
concentration during a 2^-hr exposure period.
At all test concentrations of trifluralin, DEF and propanil, more than
80% of hatching prezoeae successfully molted into the first zoeal stage
(Figures 26-28). At the highest DEF concentration, 100 yg/liter, thSre was
an indication that the success of this development was being affected. As
was the case for egg hatching, the effect of 2,4-D treatment was more pro-
nounced. No effect was observed at 33,000 yg/liter, but at 100,000 yg/liter
less than 20% of the hatching prezoeae successfully developed into first
stage zoeae (Figure 29)-
Another criterion of the effect of these compounds on early develop-
mental stages was the motility of developing first stage zoeae. No effect
-------
100
80
60
0>
o
40
20
\
100 1,000 10,000
Proponil Concentration (/*g/liter)
Figure 28.
Percent hatch of Dungeness crab eggs (Q O)> percent
development of hatched crab larvae through the prezoeal to the
first zoeal stage (A — •—A)> and percent of developed first
stage zoeae which are motile ( <^> ^>)as a function of
propanil concentration during a 24-hr exposure period.
on zoeal motility was observed with trifluralin exposure (Figure 26), but
effects were noted with both DEF and propanil (Figures 27 and 28). The
percentage of zoeae motile after exposure to DEF gradually declined with
increasing concentration up to 10 yg/liter at which point only 80% of the
zoeae were motile. Motility appeared to be generally unaffected with
propanil exposures up to 10,000 yg/liter, but at 33,000 yg/liter only 20% of
first stage zoeae were motile. Exposure to 2,4-D did not affect motility of
developing first-stage zoeae at concentrations up to and including 33,000
yg/liter. However, most of the few larvae that metamorphosed into first
stage zoeae at 100,000 yg/liter were also non-motile.
65
-------
10,000 100,000
2,4-D Concentration (/ig/liter)
Figure 29- Percent hatch of Dungeness crab eggs ( O O)> percent
development of hatched crab larvae through the prezoeal to the
first zoeal stage (A A)> and percent of developed first
stage zoeae which are motile ( <^> <^>) as a function of 2,4-D
concentration during a 24-hr exposure period.
The results of 96-hr acute toxicity tests for each of the herbicides
tested against first instar zoeae, first instar juvenile crabs and adult
crabs are summarized in Table 19- The sensitivity of first stage zoeae to
the four herbicides increased in the series 2,4-D, propanil, trifluralin
and DEF. No acute lethal effects were seen with 2,4-D at a concentration of
10,000 yg/liter, the highest concentration tested against zoeae. Concentra-
tions of propanil lethal to 50% of the test organisms in 96-hr were 7,300
yg/liter and 1,500 yg/liter, respectively, for the technical and the Stam
F-34 formulation. The 96-hr LC50 for technical trifluralin against first
instar zoeae exceeded 110 yg/liter, but with the Treflan EC formulation the
96-hr LC50 for trifluralin was 250 yg/liter. Only 6.6 yg/liter of technical
DEF was lethal to 50% of the test organisms in 96 hr. For zoeae the same
66
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TABLE 19. ACUTE TOXICITY OF HERBICIDES TO FIRST INSTAR ZOEAE, FIRST
INSTAR JUVENILE CRABS AND ADULT CRABS IN 96-HR TESTS. DATA
ARE EXPRESSED IN yg/LITER
Zoeae Juvenile Adult
Pesticide
Tr if lural in
Technical 60 >110 >1 ,000 >1,000 - >9,300
Treflan E.C. 140 250 - -
DEF
Techn i cal
Propani 1
Techn ical
Stam F-3^
2,4-D acid
Techn i cal
1
2,200
560
>10,000
6.6 170 190
7,300
1,500 5,600 6,000
>10,000 >100,000 >100,000
>1,000
>26,000
-
relative order of toxicity was noted with these four herbicides as judged by
the EC50 criteria, absence of swimming, except that the EC50s were about 18
to 50% of the respective LC50 values.
Juvenile and adult crabs were considerably less sensitive to the herbi-
cides in acute tests than were first insta.r zoeae (Table 19)- In most of
these tests with older stages no acute effects were seen even at the highest
concentrations tested. Juvenile crabs were 170 and 29 times less sensitive
to technical DEF than were first instar zoeae judging from the 96-hr EC50 and
LC50s, respectively. Juvenile crabs were 10 times and 4 times less sensitive
to propanil (as stam F~3*0 than were first instar zoeae as judged by the same
tests.
Two chronic toxicity experiments were conducted with zoeae and each of
the four herbicides. During the first experiment widespread infection of the
cultures with the fungus Lagen id i urn, coinciding roughly with the molt to
second instar between days 10 and 15, led to early termination of the experi-
ment on day 18. However, even in this short test? the highest trifluralin and
propanil concentrations, 150 and 800 yg/liter, respectively, clearly reduced
the survival of the first instar larvae (Figures 30 and 30- No effects of
either of these compounds were noted at lower concentrations. Neither 2
yg/liter of DEF nor 10,000 yg/liter of 2,4-D, the highest test concentra-
tions, respectively, had an effect on survival of larvae during this test
67
-------
100
80
60
5 40
20
TRFLURAUN
Acetone Control
10
Days
15
20
Figure 30. Effect of continuous exposure to trifluralin on survival of
Dungeness crab zoeae. Data are from the first zoeal chronic
experiment conducted during the spring of 1973-
series (Figures 32 and 33)-
During the second experiment survival of zoeae in control cultures and
in cultures exposed to low concentrations of the herbicides generally exceed-
ed 85% until day 50 (Figures 3^~37)• Beyond this time increased mortalities
occurred in all of the cultures including the controls until the tests were
terminated on day 69- Survival in control cultures averaged about ^0% at
this time. In good agreement with the results of the first experiment,
larvae exposed to 150 yg/liter of trifluralin were rapidly killed; no larvae
survived beyond day 8 (Figure 3*0- Similarly, larvae exposed to 800 yg/liter
of propanil were rapidly killed as in the first experiment. Survival at this
concentration was only 10% on day 20, but some of the remaining larvae sur-
vived an additional 35 days (Figure 35)- As in the first test, larvae
68
-------
100
I
Acetone Control
20
Figure 31- Effect of continuous exposure to propanil on survival of
Dungeness crab zoeae. Data are from the first zoeal chronic
experiment conducted during the spring of 1973-
exposed to lower concentrations of these two herbicides were entirely un-
affected during the entire experiment. Larval survival was also clearly
affected by exposure to 10,000 yg/liter of 2,4-D in this experiment (Figure
36). Approximately 50% of the zoeae exposed to this concentration survived
to day 20, but less than 10% survived 50 days of exposure. Survival of
larvae exposed to 1,000 yg/liter 2,4-D was essentially the same as that in
controls. At the highest DBF concentration, k yg/liter, no effects were
noted on the survival of zoeae (Figure 37)-
During the second chronic toxicity experiment we attempted also to
determine whether molting was affected in crabs not killed by herbicide ex-
posure. First stage zoeae exposed to 150 yg/liter trifluralin had all died
69
-------
80
60
40
20
0
DEF
A Control
• Acetone Control
A 0.002 /jg/liter
O 0.02 "
0 0.2 «
O 2 «
10
Days
15
20
figure 32. Effect of continuous exposure to DEF on survival of
Dungeness crab zoeae. Data are from the first zoeal chronic
experiment conducted during the spring of 1973-
by day 8 (Figure 3^), four days before the controls had molted to the second
zoeal stage (Figure 38). However, zoeae exposed to 15 yg/liter, which sur-
vived as well as controls, also seemed to molt in synchrony with the«control
zoeae (Figure 38) indicating that this herbicide does not affect molting.
For zoeae exposed to the highest DEF concentration, k yg/liter, the first
and second molts each occurred about 1 day behind those of control larvae
(Figure 39), but by the time of the fourth molt larvae at this concentration
were molting as soon or sooner than the controls. These results suggest that
DEF also does not affect molting. At sublethal concentrations of propanil,
80 and 8 yg/liter, molting occurred in synchrony with controls, but larvae
exposed to a lethal concentration, 800 yg/liter, either failed to molt or
molted much later than controls (Figure kO). At this latter concentration,
only three first stage zoeae molted, two on day 2k and one on day 32, and
70
-------
100
80
60
40
20
2,4-D
Control
Acetone Control
10 yug/ltar
A
O 100
0 1,000
O K)tOOO
10
Doyt
15
Figure 33- Effect of continuous exposure to 2,4-D on survival of
Dungeness crab zoeae. Data are from the first zoeal chronic
experiment conducted during the spring of 1973-
none of these molted to the third instar even though one larva survived
until day 5&-
Exposures to 2,4-D at both 1,000 and 10,000 yg/liter clearly resulted in
some delay of molting during the larval development period (Figure 41).
Larvae exposed to 1,000 yg/liter were about two to three days delayed in the
time to the first molt compared to controls,and this interval was maintained
for each of the following three molts. An even greater delay was observed in
the first molt of larvae exposed to 10,000 yg/liter. An initial molt was not
noted until day 15 at which time essentially all of the control animals had
completed the first molt, and of those larvae eventually completing this
molt, the median time was about 18 to 19 days, a delay of 5 to 6 days from
that of the controls. Because of continued mortalities of the larvae exposed
71
-------
TRIFLURALIN
Control
Acetone Control
A 1.5 /ig/liter
O 15
0 150 "
Figure 3**. Effect of continuous exposure to trifluralin on survival of Dungeness crab zoeae.
from the second zoea1 experiment conducted during the spring of
Data are
-------
PROFttNIL
A
Control
Acetone Control
A 8 yug/liter
O 80
0 800
Days
Figure 35- Effect of continuous exposure to propanil on survival of Dungeness crab zoeae.
from the second zoeal chronic experiment conducted during the spring of
Data are
-------
100
80
60
.> 40
^
CO
20
Control
Acetone Control
1,000 yug/liter
10,000
10
20
30 40
Days
50
60
70
Figure 3&- Effect of continuous exposure to 2,^-D on survival of Dungeness crab zoeae. Data are from
the second zoeal chronic experiment conducted during the spring of ^^7t^•
-------
A Control
• Acetone Control
A 0.04 /Ag/Hter
O 0.4
0 4
Figure 37- Effect of continuous exposure to DEF on survival of Dungeness crab zoeae,
the second zoeal chronic experiment conducted during the spring of
Data are from
-------
100
§80
o
o>
s
« 60
TJ
V
"5 40
e
0)
I
20
III
TRFUJRAUN
A Cortr*
w Actions Control
A 1.5 /*g/H*r
10
20
30
40
50
60
Days
Figure 38. Effect of continuous exposure to trifluralin on molting of Dungeness crab zoeae beginning
with the first zoeal molt. The molt success for each stage is the percentage of the
originat number of zoeae. Roman numerals indicate the zoeal instar at each stage in the
zoeal sequence. Data are from the second zoeal chronic experiment conducted during the
spring of 197**-
-------
Days
Figure 39- Effect of continuous exposure to DBF on molting of Dungeness crab zoeae beginning with the
first zoeal molt. The molt success for each stage is the percentage of the original number
of zoeae. Roman numerals indicate the zoeal instar at each stage in the zoeal sequence.
Data are from the second zoeal chronic experiment conducted during the spring of 197^-
-------
00
lOOr
|80
isl
4)
^40
o>
I
20
PRORVML
ii
A Control
w AcitOM Control
A 8 /ig/lfor
O 80
0 800 «
IV
10
20
30
40
50
60
Days
Figure ^0. Effect oftcontinuous exposure to propanil on molting of Dungeness crab zoeae beginning with
the first zoeal molt. The molt success for each stage is the percentage of the original
number of zoeae. Roman numerals indicate the zoeal instar at each stage in the zoeal
sequence. Data are from the second zoeal chronic experiment conducted during the spring
of 19?11*.
-------
2,4-D
A Oortral
Conlrai
A 1,000
O KXOOO
Days
Figure 41. Effect of continuous exposure to 2,4-D on molting of Dungeness crab zoeae beginning with
the first zoeal molt. The molt success for each stage is the percentage of the original
number of zoeae. Roman numerals indicate the zoeal instar at each stage in the zoeal
sequence. Data are from the second zoeal chronic experiment conducted during the spring
of
-------
to this concentration of 2,4-D only a few larvae were successful in. com-
pleting the second zoeal molt, and at least one of these occurred as long as
20 days after that of the controls.
Two chronic toxicity experiments were conducted with juvenile crabs for
each of the herbicides: The first lasted 36 days and began with first instar
juvenile crabs; the second lasted 80 days in which most crabs were initially
third instars. Survival of juvenile crabs exposed to 0.15 yg/liter triflura-
lin was less than for control crabs after 20 days in the first experiment,
but since this effect was absent in crabs exposed to higher concentrations of
trifluralin, the response cannot be attributed to the herbicide exposure
(Figure 42). Also trifluralin exposure did not influence the time of occur-
rence of the first juvenile molt (Figure 43). In the second experiment with
100
80
60
.1 40
20
TRIFLURALIN
A Control
• Acetone Control
A 0.15 /ig/|jter
O 1.5 «
0 15 »
O 150 «
10
20
Days
30
40
Figure k2. Effect of continuous exposure to trifluralin on survival of
juvenile Dungeness crabs. Data are from the first juvenile
chronic experiment which was initiated with newly metamor-
phosed first instar crabs.
80
-------
loo r TRIFLURALIN
80
Acetone Control
35
Days
Figure 43-
Effect of continuous exposure to trifluralin on molting of
first instar Dungeness crabs to the second instar. The molt
success is expressed as the percentage of the original number
of first instar crabs.
trifluralin, crabs exposed continuously to 15 and 150 yg/liter of the herbi-
cide (the second highest and highest concentrations tested) survived as well
as controls (Figure 44). Juvenile crabs exposed to 150 yg/liter of DEF ex-
hibited poorer survival than controls in both the first and second experi-
ments (Figures 45 and 46) but were not affected by exposure to 15 yg/liter.
Molting of crabs in the first experiment with DEF was not affected at any
concentration; of the crabs surviving to day 35 in each treatment, approxi-
mately half had molted by day 24 (Figure 47).
The highest concentration of propanil, 800 yg/liter, did not affect sur-
vival of crabs in the first experiment (Figure 48) but was associated with a
reduced survival- of crabs in the second experiment after day 20 (Figure 49).
81
-------
100
80
60
CO
NJ
£
"b
.> 40
20
TRIFLURALIN
A Control
• Acetone Control
A 15 /*g/Hter
O 150
10
20
30
40
Days
50
60
70
80
Figure kb. Effect of continuous exposure to trifluralin on survival of juvenile Dungeness crabs,
Data are from the second juvenile chronic experiment which was initiated with second
and third instar crabs.
-------
100
80
60
I 40h
i«
CO
20
DEF
A
A
O
0
O
Control
Acetone Control
0.15 /tg/lfter
1.5
15 »
150 »
10
20
Days
30
40
Figure 45- Effect of continuous exposure to DEF on survival of juvenile
Dungeness crabs. Data are from the first juvenile chronic
experiment which was initiated with newly metamorphosed first
instar crabs.
In the second experiment, crabs exposed to propanil at 80 yg/liter survived
as well as controls, at least until day 60, but the mortality rate after this
time increased substantially. Propanil did not delay the time to the first
molt in the first juvenile chronic experiment (Figure 50). Juvenile crabs
exposed to 10,000 yg/liter 2,^-D in the first experiment exhibited poorer
survival after day 10 than controls and crabs exposed to low 2,4-D concen-
trations (Figure 50. but molting exhibited a tendency to be advanced rather
than delayed in this group (Figure 52). In the second juvenile chronic ex-
periment 2,4-D had no effect on survival of crabs at either 1,000 or 10,000
yg/1i ter (Figure 53)•
In long-term experiments with adult crabs, neither trifluralin nor
2,4-D acid at concentrations as high as 100 y/liter and 10,000 yg/liter,
respectively, affected survival (Figures 5^ and 55)- Adult crabs exposed
83
-------
CO
-p-
Control
Acetone Control
0.15 >ig/liter
1.5
15
150
Figure 46. Effect of continuous exposure to DEF on survival of juvenile Dungeness crabs. Data
are from the second juvenile chronic experiment which was initiated with second and
third instar crabs.
-------
100
r DEF
80
60
40
20
10
A Control
• Acetone Control
A 0.15 /ig/Kter
O 1.5
0 15
O 150
15
20
Days
30
35
Figure 47. Effect of continuous exposure to DEF on molting of first
instar Dungeness crabs to the second instar. The molt
success is expressed as the percentage of the original
number of first instar crabs.
chronically to DEF showed reduced survival both at 200 and 2,000 yg/liter
when compared with controls (Figure 56). The median survival time at 2,000
yg/liter was about 32 days; at 200 yg/liter, about 50 days. Propanil
exposure of 4,000 yg/liter reduced survival of adult crabs compared with
controls, but concentrations of 400 yg/liter and lower had no effect (Figure
57)- Even at 4,000 yg/liter, half of the animals survived until the end of
the test on day 85-
Relatively little data are available on the toxicity of DEF to aquatic
organisms. The 96-hr LC50 for the freshwater crustacean, Gammarus lacustris,
was found to be 100 yg/liter by Sanders (1969) and the 48-hr EC50 for mortal-
ity or paralysis of the marine shrimp Penaeus aztecus was 28 yg/liter
(Butler, 1965). In 96-hr tests the concentration of DEF causing a 50%
85
-------
100
80
60
40
CO
20
PROFftNIL
A Control
• Acetone Control
A 0.8 yug/Kter
O 8 «
0 80
O 800 "
10
20
Days
30
40
Figure 48. Effect of continuous exposure to propanil on survival of
juvenile Dungeness crabs. Data are from the first juvenile
chronic experiment which was initiated with newly metamor-
phosed first instar crabs.
reduction in shell growth of the oyster Crassostrea virginica was 100
yg/liter; for Leiostomus xanthurus, a marine fish, the 48-hr LC50 was 240
yg/liter (ButleT^1965)• We have found that DEF has a comparable toxicity to
first stage juvenile crabs; the 96-hr LC50 for this stage is 190 yg/yter
(Table 19)- In long-term tests survival was affected upon exposure to 150
yg/liter, but not by exposure to 15 yg/liter (Figures 45 and 46). Adult
Dungeness crabs were substantially more resistant to this herbicide. We were
not able to demonstrate a 50% mortality of this stage during 96 hours of
exposure to 1,000 yg/liter, but survival of adult crabs was reduced during
chronic exposures to 200 yg/liter (Figure 56). As has been found with other
pesticides, the first stage zoeae were the most sensitive to DEF of the
stages tested in our experiments. The 50% toxic concentration for zoeal
swimming in 96-hr was found to be only 1 yg/liter and the 96-hr LC50 was
86
-------
100
80
oo
60
40
20 K
I- PROFANIL
A Control
• Acetone Control
A 80 >ug/liter
O 800 «
10
20
30
40
Days
50
60
70
80
Figure 49• Effect of continuous exposure to propanil on survival of juvenile Dungeness crabs.
Data are from the second juvenile chronic experiment which was initiated with second
and third instar crabs.
-------
100
80
60
PROFANIL
A
O
0
Control
Actions Control
0.8 /ig/liter
8
80
O 800
Figure 50. Effect of continuous exposure to propanil on molting of first
instar Dungeness crabs to the second instar. The molt success
is expressed as the percentage of the original number of first
instar crabs.
6.6 yg/liter. These concentrations represent the lowest concentrations
having an adverse effect on the larvae since we were unable to demonstrate
that either survival or molting were affected during long-term exposure to
k yg/liter (Figures 37 and 39)-
Propanil appears to be considerably less toxic to aquatic organisms
than DEF.
freshwater
concentration for
of 4,800 yg/liter
(Sanders, 1970).
We are unaware of any toxicological data on marine species or
fishes, but Crosby and Tucker (1966) have reported a 50% effective
immobilization of the freshwater crustacean Daphnia magna
The 96-hr LC50 for Gammarus fasciatus is 16,000 yg/1 iter
These observations are in accord with our finding on the
acute toxicity of propanil to Dungeness crabs. The 96-hr LC50 for technical
propanil to first instar zoeae is 7»300 yg/liter and to adult crabs exceeds
88
-------
A Control
• Acetone Control
A 10 ^g/liter
O 100 t
0 1,000 ••
O 10,000 «
10
20
Days
30
40
Figure 51- Effect of continuous exposure to 2,4-D on survival of juvenile
Dungeness crabs. Data are from the first juvenile chronic
experiment which was initiated with newly metamorphosed first
instar crabs.
26,000 yg/liter (Table 19). As evidenced by reduced survival, toxic concen-
trations of propanil for zoeae, juveniles and adults in chronic exposure
experiments were 800, 800 and 4,000 yg/liter, respectively (Figures 35, 49
and 57)- For the same stages, no toxic manifestations of any kind were
observed at concentrations of 80, 80 and 400 yg/liter.
The effect of trifluralin on aquatic organisms has now been rather
extensively studied (Cope, 1965; Liu and Lee, 1975; Macek et al., 1969;
Parka and Worth, 1965; Sanders, 1969 and 1970; Sanders and Cope, 1966, and
1968; and Walsh, 1972). The reported toxicities range from 14 to 50,000
yg/liter in acute toxicity tests of the dissolved herbicide. Teleost fish
rank among the most sensitive species studied to date. The 96-hr TL50s for
Sal mo gai rdneri and Lepom i s machrochirus, each tested over a range of three
temperatures, range from 42 to 210 yg/liter and 47 to 190 yg/liter,
respectively, the toxicity being greatest at the higher temperatures (Macek
89
-------
100
r 2,4-D
80
"8 60
40
A Control
• Acetone Control
A KD /jg/Ifter
O 100 «
0 1,000 "
O 10,000 «
Figure 52. Effect of continuous exposure to 2,4-D on molting of first
instar Dungeness crabs to the second instar. The molt
success is expressed as the percentage of the original
number of first instar crabs.
et al., 1969). For fingerling rainbow trout in 24-hr exposures the toxicity
ranged from 14 to 210 yg/liter, and for small bluegills, from 23 to 120
yg/liter (Cope, 1965)- Similar values were reported for bluegills afld fat-
head minnows, but the 96-hr LC50 for goldfish, Carassius auratus, was 585
Freshwater crustaceans and insects were
48- and 96-hr LC50s ranging from 240
,000 yg/liter for Orconectes nai s (Sanders,
1966 and 1968). Walsh (1972T~reported
that the concentration of trifluralin resulting in a 50% decrease in growth
of four species of marine phytoplankton was 2,500 yg/liter. The only marine
animal for which toxicity data is available is the marine mussel, Myt i1 us
eduli s, (Liu and Lee, 1975)• For this species 96 yg/liter reduced larval
yg/liter (Parka and Worth, 1965)
less sensitive to the herbicide;
yg/1iter for Daphn ia pulex to 50,
1969 and 1970; Sanders and Cope,
90
-------
100
80
60
i 40
^
20
2,4-D
A Control
• Acetone Control
A 1,000 yug/lit«f
O 10,000 ••
10
20
30
40
Days
50
60
70
80
Figure 53. Effect of continuous exposure to 2,4-D on survival of juvenile Dungeness crabs. Data are
from the second juvenile chronic experiment which was initiated with second and third
instar crabs.
-------
soo
80
60
1 40
>
CO
20
o—o
TRIFLURALIN
A Control
• Acetone Control
A I /ig/liter
O 10 "
0 100
10
20 30 40 50 60 70 80 90
Days
Figure $k. Effect of continuous exposure to trifluralin on survival of adult Dungeness crabs.
-------
UD
100
80
60
i 40
•5
20
2,4-D
A Control
• Acetone Control
A 1,000 /ig/liter
O 10,000
II
0 10 20 30 40 50 60 70 80 90
Days
Figure 55. Effect of continuous exposure to 2,^-D on survival of adult Dungeness crabs.
-------
100
80
60
40
20
DEF
A Control
• Acetone Control
A 2 /ig/liter
O 20 "
0 200
O 2,000
10
20 30
40 50 60 70 80
Days
90
Figure 56- Effect of continuous exposure to DEF on survival of adult Dungeness crabs.
-------
vn
100
80
60
g
1 40
>
L_
co
20
PROPANIL
A Control
• Acetone Control
A 40 /ig/liter
O 400 «
0 4,000 «
-0 0 0-0
10
20 30 40 50 60 70 80 90
Days
Figure 57- Effect of continuous exposure to propanil on survival of adult Dungeness crabs.
-------
growth and 100 and 240 yg/liter affected attachment of adults to glass and
was lethal to 50% of adults, respectively.
Our studies with crabs. Cancer magister, indicate that the larvae of this
species has a sensitivity to trifluralin comparable to that reported for
freshwater fish and the marine mussel Mytilus. In long-term tests, larvae
were rapidly killed upon exposure to 150 yg/liter of trifluralin with 50% of
the deaths occurring in about 5 days (Figures 30 and 34). Larval survival
and molting were not affected by exposure to 15 yg/liter for up to 69 days.
Older stages were less sensitive to the herbicide. The survival of juvenile
crabs exposed to 150 yg/liter of trifluralin was not significantly less than
controls in exposures up to 80 days (Figure 44)- Adults were not affected by
exposure to 100 yg/liter, the highest concentration tested against this
stage, in 85 days (Figure 5*0 • In addition there was no adverse effect on
egg hatching or prezoeal development in our tests at the highest trifluralin
concentration tested, 330 yg/liter (Figure 26).
An assessment of the toxicity of 2,4-D to aquatic organisms is compli-
cated by the fact that this herbicide may be employed as a variety of esters
and salts as well as the free acid. Hughes and Davis (1963), in studies with
the bluegill sunfish (Lepomis machrochirus), found that the 48-hr toxicity of
various formulations ranged from 800 to 840,000 yg/liter (expressed as acid
equivalents). The alkanolamine and dimethylamine salts were the least toxic,
and the esters were the most toxic. An emulsified formulation of 2,4~D acid
was also relatively toxic at 8,000 yg/liter. Sanders (1970) found that the
relative toxicity of 2,4-D formulations could vary appreciably for different
species of freshwater crustaceans. For example, Daphnia magna 48-hr TL50s
for the propylene glycol butyl ether ester, the dimethylamine salt, the
butoxyethanol ester and the free acid were 100, 4,000, 5,600 and >100,000
yg/1iter,respectively, but Gammarus fasciatus 48-hr Tl_50s were 2,600,
100,000, 5,900 and 3,200 yg/liter for the same series. Species differences
were also noted for a series of salmonids (Meehan et al., 1974). The latter
authors also found that the propylene glycol butyl ether ester was more toxic
than the isooctyl ester,and the pure acid was the least toxic of the formu-
lations tested.
Values for the aquatic toxicity of 2,4-D acid reported in the literature
range from approximately 1,000 to 3,000 yg/liter (Butler, 1965 for the marine
shrimp fenaeus aztecus; Meehan et al., 1974 for pink salmon fry; Sanders,
1970 for Gammarus fasciatus) to over 100,000 yg/liter (Crosby and Tucker,
1966 and Sanders, 1970 for Daphnia magna; Liu and Lee, 1975 for Myt i lus
edulis larvae and adults). It is evident from our studies that the*Dungeness
crab exhibits a comparable sensitivity to the acid form of this herbicide.
The larvae, which was the most sensitive stage tested, experienced reduced
survival during extended exposure to 10,000 yg/liter, but not during exposure
to 1,000 yg/liter. However, molting appeared to be delayed at the latter
concentration. Egg hatching and prezoeal development was not affected at
concentrations up to about 33,000 yg/liter. The threshold of effects on
juvenile crabs appeared to occur at about 10,000 yg/liter,and adults were not
affected in long'-term tests at 10,000 yg/liter or in acute tests at 100,000
yg/1iter.
96
-------
THE INSECTICIDES CARBOFURAN, CHLORDANE,AND MALATHION
Unlike the results previously observed with captan, methoxychlor, and
the four herbicides 2,4-D, DEF, propanil and trifluralin, exposure of eggs to
the insecticide carbofuran did not result in an acceleration of egg hatching
(Figure 58). At insecticide concentrations ranging from 1 to 330 yg/liter,
hatching success during the 24-hr test period ranged from 2k to 50% averaging
38 ± 9%. Furthermore, no trend in hatching success was noted with increasing
carbofuran concentrations. The effects of carbofuran on zoeal development
and on motility of developed zoeae followed the usual pattern, however
motility being affected at lower concentrations of the insecticide than those
that affected the development from prezoeae to zoeae (Figure 58). The 50%
100
80
60
L
40
20
0
tr — ^
^* ^^**^^» ^
J^^^^v^
ccx *
• » » »
V \
I . \ \
u , . O
4 \ \ o
. A\
\ \^
// , ^^. A *^--TA. A
I
Carbofuran Concentration (/ig/liter)
Figure 58. Percent hatch of Dungeness crab eggs ( O O), percent
development of hatched crab larvae through the prezoeal to the
first zoeal stage (A A)> and percent of developed first
stage zoeae which are motile (^ ^)as a function of
carbofuran concentration during a 2^-hr exposure period.
97
-------
effective concentration for zoeal motility was approximately 3-3 yg/liter,
while that for zoeal development was approximately 10 yg/liter.
The results of chlordane treatment on egg hatching, zoeal development,
and zoeal motility were the most unusual. Egg hatching was unaffected or
even slightly depressed at the lower test concentrations, 1 to 10 yg/liter,
but appeared to be accelerated at higher insecticide concentrations, 33 to
330 yg/liter (Figure 59) • The most unusual response was the relationship
between zoeal development and zoeal motility. Unlike any of the other pesti-
cides, development of prezoeae into the first zoeal stage was inhibited at a
lower concentration, approximately 2 to 3 yg/liter, than the motility of
those zoeae that developed, approximately 33 yg/liter (Figure 59)•
loo > -0 -Ct.^
H//
10 100
Chlordane Concentration (/ig/lrter)
Figure 59- Percent hatch of Dungeness crab eggs (Q LO)» percent
development of hatched crab larvae through the prezoeal to the
first zoeal stage (A —A)> and percent of developed first
stage zoeae which are motile (^ ._^)as a function of
chlordane concentration during a 2k hr-exposure period.
98
-------
The pattern of effects of malathion on these early developmental stages
was like that seen with captan, methoxychlor, DEF and propanil. All concen-
trations of the organophosphate from 0.33 to 100 yg/liter resulted in
accelerated egg hatching compared to the controls; the average hatch success
of the insecticide-treated eggs being about 70% (Figure 60). The success of
development of prezoeae into first stage zoeae was 90% or higher even at the
highest pesticide treatment, 100 lag/liter, but zoeal motility was 50%
affected at only 11 to 12 yg/liter of malathion (Figure 60).
100
80
60
4O
20
-^ A
i
!
fcA,«L..*t..L~> f***m
10
*,>*~nH*n tiut/lto^A
100
Figure 60. Percent hatch of Dungeness crab eggs ( Q O)> percent
development of hatched crab larvae through the prezoeal to the
first zoeal stage (A A)> and percent of developed first
stage zoeae which are moti le ( <)> <0)as a function of
malathion concentration during a 24-hr exposure period.
99
-------
The acute toxicity of all three insecticides for zoeae -was similar
(Table 20). The 96-hr LC50 for carbofuran was 2.5 yg/liter. Based on the
sublethal criterion, inhibition of swimming, the 5®% effective concentration
was 1.5 yg/liter of the same insecticide. The LC50 for chlordane exceeded
the highest concentration tested, 10 yg/liter, but the EC50 was 1.3 yg/liter,
a value similar to that for carbofuran. Comparable data for malathion were
1.2 and 0.4 yg/liter, respectively.
TABLE 20. ACUTE TOXICITY OF INSECTICIDES TO FIRST INSTAR
ZOEAE AND ADULT CRABS IN 96-HR TESTS. DATA ARE
EXPRESSED IN yg/1iter
Carbofuran
Chlordane
Malathion
EC50
1-5
1.3
0.4
Zoeae
LC50
2.5
>10.
1 .2
Adult
EC50 LC50
190.
220.
1,330.
The toxicity of each of the insecticides to first instar zoeae was also
evaluated in chronic exposure experiments. At the highest carbofuran
exposure, 5 yg/liter, 50% mortality occurred within about 4 days and complete
mortality within 7 days (Figure 61). No lethal effect was seen at 0.5
yg/liter; larval survival still exceeded 80% at this concentration even after
50 days of exposure. The latter concentration may have resulted in some
delay of molting of the zoeal stages, however. By the time of the third
zoeal molt this group molted approximately three days later than the controls
(Figure 62).
The results with malathion were similar to those observed with carbo-
furan. At the highest exposure, 2 yg/liter, 50% mortality occurred in from
4 to 5 days, and mortality was complete by day 6 (Figure 63)- Survival of
zoeae exposed to the next lower concentration, 0.2 yg/liter, was as high as
that of the controls until the end of the experiment on day 69- At the
latter concentration, malathion had no effect on molting during the first
three molts. However, during the fourth molt zoeae at this treatment molted
about 3 days later than controls (Figure 64).
As in the egg tests, chlordane produced unusual results in the long-term
zoeal experiment. At the highest concentration, 15 yg/liter, the survivor-
ship curve is almost identical with those of the highest concentrations of
carbofuran and malathion, i.e. 50% mortality occurred on about day 4, and
100% mortality by day 6 (Figure 65). Compared with controls, however, con-
siderable mortality occurred even at 10% and 1% of this highest chlordane
concentration. At an exposure of 1.5 yg/liter, 50% mortality occurred
between day 8 and day 9, and 100% mortality on day 20. At only 0.15 yg/liter
chlordane, 50% mortality was noted on day 37 compared with survival of more
100
-------
CARBOFURAN
Control
Acttoiw Control
0.05 /tg/IHtr
O 0.5
5
Figure 61.
Effect of continuous exposure to carbofuran on survival of Dungeness crab zoeae.
from the second zoeal chronic experiment conducted during the spring of 1971*-
Data are
-------
100
£80
o
^60
X
O)
140
20-
0
II
CARBCRJRAN
in
10
20
30
50
60
Days
Figure 62. Effect of continuous exposure to carbofuran on molting of Dungeness crab zoeae beginning
with tfie first zoea1 molt. The molt success of each stage is the percentage of the
original number of zoeae. Roman numerals indicate the zoeal instar at each stage in the
zoeal sequence. Data are from the second zoeal chronic experiment conducted during the
spring of
-------
MALATHION
Control
Acetone Control
0.002 /ig/liter
0.02
0.2
2
Figure 63-
Days
Effect of continuous exposure to malathion on survival of Dungeness crab zoeae.
from the second zoeal chronic experiment conducted during the spring of
Data are
-------
o
-tr-
KX>
80
~o
§60
40
20
0
II
MALATHON
A Cortrd
AMtaw Contra!
A QOOe/ig/ltar
o oce •
0 O2 •
IV
10
20
30
40
50
GO
Days
Figure 64. Effect of continuous exposure to malathion on molting of Dungeness crab zoeae beginning
with the first zoeal molt. The molt success of each stage is the percentage of the
original number of zoeae. Roman numerals indicate the zoeal instar at each stage in the
zoeal sequence. Data are from the second zoeal chronic experiment conducted during the
spring of 1974.
-------
O
U1
CHLORDANE
A Control
Acetone Control
A 0.015 /eg/liter
O 0.15
0 1.5
O 15
Days
Figure 65• Effect of continuous exposure to chlordane on survival of Dungeness crab zoeae.
from the second zoeal chronic experiment conducted during the spring of
Data are
-------
than 90% of the control larvae. Larval molting was also apparently affected
at this concentration (Figure 66). Survival of crabs exposed to 0.015
yg/liter was not different than that of control animals at any time during
the chronic tests. Furthermore, there was no evidence that chlordane at
this concentration influenced the time of molting compared with controls
(Figure 66).
The relative toxicity of malathion and carbofuran to adult crabs in
96-hr acute tests was reversed from that noted with first instar zoeae
(Table 20). The 96-hr LC50 for malathion and adult crabs was 1,330 yg/liter,
but for carbofuran was only 190 yg/liter. For chlordane the 96-hr LC50
with adult crabs was 220 yg/liter. Sublethal responses were difficult to
distinguish in adult crabs and, therefore, EC50 values are not reported.
Adult crabs exposed chronically to carbofuran were greatly affected by
an exposure concentration of 250 yg/liter. Of the crabs in this group, 50%
died by day k and 90% by day 10. As was observed in tests with the zoeae
exposed chronically to carbofuran, exposure of the adults to a concentration
of the insecticide 10 times less, 25 yg/liter in this instance, had no dis-
cernible effect on survival (Figure 67). Crabs exposed chronically to 100
yg/liter chlordane experienced 50% mortality by day 8, but the mortality rate
subsequently declined as one animal survived until day kS (Figure 68). High
mortality was also noted in the group of crabs exposed to only 10 yg/liter
chlordane; 50% mortality in this group occurred on day 23, but one animal
survived until termination of the test on day 90. Crabs exposed to 0.1 and
1 yg/liter chlordane survived as well as the controls. Mortality of crabs
exposed chronically to 1,500 yg/liter malathion was 50% by day 3 and 100% by
day 12 (Figure 69). At the next lowest concentration, 150 vg/liter
malathion, no effect of the pesticide exposure was noted on survival.
The acute toxicity of the organophosphate insecticide malathion to
Dungeness crab first instar zoeae is comparable to that reported for fresh-
water crustaceans and stonefly naiads, but about 30 to 70 times greater
than that reported for three adult marine crustaceans. The 48-hr EC50s for
immobilization of Simocephalus serrulatus and Daphnia pulex were found to be
3-5 and 1.8 yg/liter, respect ively (Sanders and Cope, 1966). Sanders and
Cope (1968) reported that the 96-hr LC50s for two species of stonefly
naiads were 1.1 and 2.8 yg/liter and for a third species, Pteronarces
californica, was 10 yg/liter. We found that the 96-hr LC50 for malathion to
first instar zoeae was 1.2 yg/liter. In contrast, Eisler (1969) reported
that 96-hr LC50 concentrations of malathion for the sand shrimp, Cranqon
septemspinosa, grass shrimp, Palaemonetes vulgaris and the hermit crao,
Pagurus long!carpus were 33> 82 and 83 yg/1i ter, respectively. The lesser
sensitivity of these latter three species of marine Crustacea may be related
to their greater size or age compared to the crab zoeae. Although Eisler
(1969) did not examine the toxicity of malathion to larval stages of these
three species, it is likely that they would have been substantially more
sensitive to the insecticide. We found that the crab first instar zoeae
were more than 1100 times as sensitive to malathion as the adult crabs
(Table 20).
106
-------
CHUORQANE
A OonM
Aotlont Control
Figure 66. Effect of continuous exposure to chlordane on molting of Dungeness crab zoeae beginning
with the first zoeal molt. The molt success of each stage is the percentage of the
original number of zoeae. Roman numerals indicate the zoeal instar at each stage in the
zoeal sequence. Data are from the second zoeal chronic experiment conducted during the
spring of 197**-
-------
O
CO
100
80
60
i 40
k_
CO
20
10
CARBOFURAN
A Control
• Acetone Control
A 025 yug/liter
O 2.5 M
0 25 M
O 250 ••
-O-O-
20
30 40 50
Days
60
70
8
90
Figure 67. Effect of continuous exposure to carbofuran on survival of adult Dungeness crabs.
-------
100
80
60
I 40
20
0
CHUORDANE
A Control
• Acetone Control
A O.I /ig/liter
O I "
0 10 •
O 100
0
10
20 30
40 50
Days
60 70 80 90
Figure 68. Effect of continuous exposure to chlordane on survival of adult Dungeness crabs.
-------
MALATHION
A Control
• Acetone Control
A 1.5 yag/liter
O 15
0 150
O 1,500
Figure 69- Effect of continuous exposure to malathion on survival of adult Dungeness crabs.
-------
Our data, those of Sanders and Cope (1966) and Eisler (1969) for fresh-
water and marine Crustacea, and of Sanders and Cope (1968) for insects, indi-
cate that the toxicity of malathion for arthropods is substantially higher
than for freshwater and marine fishes and marine mol1 uses. Macek and
McAllister (1970) found that the 96-hr LC50s for 12 species of freshwater
fishes ranged from a low of 101 yg/liter for coho salmon to 12,900 yg/liter
for the black bullhead, Ictalurus melas. Marine fishes may be slightly more
sensitive to the insecticide. Eisler (1970) found that the 96-hr LCSOs for
seven species of marine fishes ranged from a low of 27 yg/liter for the blue-
head, Thalassoma bifasciatum, to 3,250 yg/liter for the Northern puffer,
Sphaeroides maculatus. The most sensitive fish tested were fingerlings of
the striped bass, Morone saxati1i s for which the 96-hr LC50 was reported to
be 14 yg/liter (Korn and Earnest, 1974). Molluscs are the least sensitive to
malathion. Davis and Hidu (1969) found that the 48-hr LC50 for egg develop-
ment of Crassostrea virginica was 9,070 yg/liter and the 14-day LC50 for
larval development of the same species was 2,660 yg/liter. Clams, Merceneria
merceneria, exposed to the insecticide were not killed at 37,000 yg/1i ter
(Eisler and Weinstein, 1967), but altered metal profiles were noted in the
tissues at only 37 yg/liter.
The acute toxicity of the cyclodiene insecticide chlordane to Dungeness
crabs is similar to that reported for a multitude of other aquatic species
representing several phyla. The 96-hr EC50 concentration for inhibition of
swimming of first stage zoeae was 1.3 yg/liter of chlordane (Table 20). The
chlordane 96-hr LC50 for this stage was not precisely determined since con-
centrations higher than 10 yg/liter were not employed, but would be expected
to fall only slightly above 10 yg/liter (Table 20). With adult crabs, the
96-hr LC50 for this insecticide was 220 yg/liter (Table 20). Henderson
et al. (1959), Katz (1961), Korn and Earnest (1974) and Macek et al. (1969)
have found that the 96-hr LC50s for 10 species of fish are in the range of
11.8 yg/liter for striped bass, Morone saxat i1i s, to 190 yg/liter for the
guppy, Lebistes reticulatus. The 96-hr EC50s for immobilization of the
freshwater crustaceans, Simocephalus serrulatus and Daphnia pulex, are 20 and
29 yg/liter, respectively (Sanders and Cope, 1966), and for naiads of the
stonefly Pteronarces californica, the 96-hr LC50 is 15 yg/liter (Sanders and
Cope, 1968)^The growth of young oysters, Crassostrea virginica, was ad-
versely affected by 10 yg/liter chlordane (Butler et al.,1960).
We are unaware of studies of the acute toxicity of the carbamate insec-
ticide carbofuran to aquatic species for which comparisons to our work can
be made. Our results indicate, however, that the toxicity of this compound
to the Dungeness crab is similar to that reported for another carbamate
insecticide, Sevin. Buchanan et al. (1970) have reported that the 96-hr
LC50 for the first instar zoeae exposed to Sevin at 10°C was 10 yg/liter and
that the 48-hr LC50 at 17°C was 5 yg/liter. We have found that the carbo-
furan 96-hr LC50 for the first instar zoeae at 13°C is 2.5 yg/liter (Table
20). Buchanan et al. (1970) also determined the 96-hr LCSOs of Sevin to
adult female Dungeness crabs at 11 and 18°C, and these values were approxi-
mately 280 and 180 yg/liter, respectively. In our work with adult crabs, the
96-hr LC50 for carbofuran at 13°C was 190 yg/liter (Table 20).
Several studies have been concerned with the effects of a variety of
111
-------
insecticides on the larval development of crabs. These investigations,
covering four major families of insecticides, the organochlorines, the
organophosphates, the cyclodienes and the carbamates, are summarized in
Table 21. Although there is substantial overlap in the toxicities of repre-
sentatives of the four families, there is some indication that the toxicities
can be ordered as follows: organochlorines = organophosphates> cyclodienes =
carbamates. For all pesticides and all species of crabs, the range of con-
centrations, representing the lowest concentrations at which adverse effects
on the larvae have been observed, is from 0.01 to 5-0 yg/liter. Our obser-
vation with methoxychlor falls within the range of toxicities reported for
other organochlorine insecticides and other species. Our data for chlordane
and carbofuran represent the highest toxicities reported for the cyclodiene
and carbamate insecticides,respectively, and our data for malathion provide
the only indication of the toxicity of the organophosphates to larval crabs.
As a result of the high toxicities of chlordane, carbofuran and
malathion to larval C_. mag i ster, each of these insecticides should be con-
sidered as potentially serious contaminents of marine waters. However, with
the possible exception of chlordane, these compounds are not thought to be
particularly persistent in aquatic ecosystems (Liu and Lee, 1975; Sanborn,
197^) a factor which would minimize the hazard of their use near marine
envi ronments.
SUMMARY OF PESTICIDE TOLERANCE
The relative sensitivity of the life history stages of C_. mag i ster
employed in this study for each of nine pesticides is summarized in Table 22.
The data given are the highest no-effect concentrations derived from the
particular test except in the case of zoeae. The data given for zoeae are
the highest concentrations at which no adverse effect was seen on survival
during chronic exposures. In some instances, to be discussed below, these
concentrations were found to have an effect on the molting of larvae.
Although the range of toxicity varied by more than five orders of magnitude,
from the highly toxic methoxychlor to the relatively non-toxic 2,4-D acid,
a uniformity of response with respect to the relative sensitivity of various
life history stages is evident. The stage exhibiting the highest sensitivity
to pesticides was the zoeal stage. Data were not obtained for the long-term
toxicity of chlordane, malathion and carbofuran to juvenile crabs, but for
the other six pesticides, the tolerance of juvenile stages was some five to
ten times greater on the average than the zoeae. Propanil represented an
extreme case in which juvenile stages did not appear to be more resisfant
than zoeal stages. At the other extreme, juvenile stages exposed to
trifluralin, captan and 2,4-D acid were more than ten times as resistant as
zoeal stages.
Adult crabs were also substantially more tolerant of the nine pesticides
than the zoeal stages. At one extreme, adults were only five times more
tolerant of propanil and DEF than the zoeal stages, and at the other extreme,
adults were 750 times more tolerant of malathion than the zoeae. Aftersummariz-
ing the data for all nine pesticides, it would appear to be a reasonable
approximation that adult stages averaged from 10 to 100 times more resistant
112
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TABLE 21. TOXIC CONCENTRATIONS OF VARIOUS INSECTICIDES TO LARVAL CRABS
Insect ic ide
Species
Toxic
concentrat ion
(ug/1iter)
Reference
Organochlor i ne
Mi rex
Mi rex
Methoxychlor
Endri n
DDT
M i rex
Organophosphate
Malathion
Cycled iene
Chlordane
Dieldr in
D ieldr in
Carbamate
Carbofuran
Sevin
Rhi thropanopeus harrisii
Menippe mercenaria
Cancer magi ster
Cancer magister
Cancer magister
Callinectes sapidus
Cancer magister
Cancer magister
Leptodius floridanus
Panopeus herbst i i
Cancer magister
Cancer magister
0.01
0.01
0.05
0.25
0.50
1 .0
0.2
0.15
1 .0
5-0
0.5
3-2
Bookhout et al . , 1972
Bookhout et al . , 1972
This study
Poole and Will is, 1970
Poole and Will is, 1970
Bookhout and Costlow, 1975
This study
This study
Epifanio, 1971
Epifanio, 1971
This study
Buchanan et al . , 1 970
Lowest concentration at which adverse affects seen during long-term exposures
-------
TABLE 22. RELATIVE SENSITIVITY OF VARIOUS LIFE HISTORY STAGES OF C. MAGISTER TO NINE PESTICIDES. DATA
ARE EXPRESSED IN yg/LITER
Life history stage
Pest ic ide
Methoxychlor
Chlordane
Malathion
Carbofuran
DEF
Tr if lural in
Captan
Propan i 1
2,4-D acid
Egg *
>10
>330
>100
>330
>100
>330
>10,000
>33,000
100,000
Prezoeal +
0.033
<|
33
1
33
>330
>1 0,000
>33,000
33,000
zoeal 4
0.005
0.015
0.2
0-5
4
15
20
80
1000
j uveni 1 e 4
0.04
15
>150
>200
80
>10,000
adult *
0.4
1
150
25
20
>100
>200
400
>1 0,000
* Highest concentration at which no decrease in egg hatchability was observed during a 24-hr
observation period.
+ Highest concentration at which no decrease in percentage of prezoeae molting to zoeae was observed
during a 24-hr observation period.
4 Highest concentration at which no adverse effect on survival was seen during exposure periods of at
least 69 days.
-------
to pesticides than the zoeal stages.
The toxicity data for the egg and prezoeal stages are not as readily
compared to those for the zoeae because these stages were tested during
extremely short periods of time, and because the criteria of effect were
different. The prezoeal stage is of extremely short duration, from 10 to 60
min at the temperatures tested (Buchanan and Millemann, 1969), and represents
the brief period in the life history between the hatching of the crab eggs
and the first zoeal stage. The criterion of effect employed for prezoeae was
failure to molt to the zoeal stage, which, considering the 24-hr observation
period, could be considered a lethal effect. By this criterion, the toler-
ance of the prezoeal stage relative to that of the zoeae ranged from twice
for carbofuran to >k\2 and >500 for propanil and captan, respectively (Table
22.)
In all probability, the greater apparent tolerance of prezoeae is
attributable to the very short duration of this stage, and, therefore, the
short duration of pesticide exposure, relative to that of the first through
fifth zoeae. If the periods of exposure were the same, the prezoeae would
likely be about as sensitive to pesticides as first stage zoeae. This is
indicated by the ratios obtained for each pesticide of the 24-hr LCSOs for
zoeae to the "no effect" levels for prezoeae. These ratios range from <0.04
for malathion to 27-9 for methoxychlor. Since the actual exposure period for
prezoeae is undoubtedly much less than 2k hr, these ratios are probably con-
servatively low. Therefore, where only short-term contamination of marine
waters by pesticides is expected, effects on the prezoeae may be as important
or more important than effects on zoeae, especially for some compounds.
Nevertheless, it is evident that under conditions of chronic pesticide expo-
sure, the prezoeal stage should not be considered as a sensitive stage in the
life history, since at concentrations below those that affect prezoeae, the
zoeal stage is still likely to be affected (Table 22).
The eggs, as judged by egg hatchabi1ity, appear to be even less affected
by pesticide exposures on the average than the prezoeal stage (Table 22).
With the exception of 2,4-D acid, egg hatchabi1ity was not adversely affected
by any. of the pesticides examined at the highest concentrations - - concen-
trations which greatly exceeded those that affected zoeae in the chronic
exposure tests. In the egg hatching tests, the highest concentrations of DEF
and trifluralin were 100 and 330 yg/liter, and exceeded the no-effect con-
centrations in the zoeal tests by 25 and 22 times, respectively. Further-
more, no-effect concentrations for these two pesticides and eggs could have
been substantially higher. At the other extreme, the highest concentration
of chlordane in the egg tests, 330 yg/liter, a concentration which had no
effect, was 22,000 times greater than the highest no-effect level of
chlordane in the zoeal tests. Only with 2,4-D acid was an adverse effect
observed on egg hatching, but the no-effect level for this response was
100,000 yg/liter, a concentration exceeding the no-effect level against first
stage zoeae by 100 times. These data suggest that eggs are not a particu-
larly sensitive stage of the life history of crabs with respect to pesticide
exposure. This observation, however, should not be considered conclusive
until much more extensive tests with eggs are conducted. The experiments
reported here involved exposure of eggs only just prior to hatching and for a
115
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very short period of time. Exposures of eggs to pesticides earlier in their
developmental sequence and for longer periods of time might result in differ-
ent conclusions.
The objective of the studies described in this report was to determine
for the Dungeness crab, Cancer magister, the maximum concentration of each
pesticide that could be tolerated by the most sensitive stage in the life
history of the crab. Since the time required vfor this species to complete
the life cycle is about 3 to k years (Butler, 1961), it was not feasible to
estimate the maximum acceptable toxicant concentration as recommended by
Mount and Stephen (1967)• Our approach, therefore, was to expose distinct
stages in the life history of the crab to uniform concentrations of each
pesticide for periods of up to two to three months and determine for each
such stage the maximum tolerable concentration of each compound by using
survival and appropriate sublethal criteria to judge effects.
We have shown for each pesticide that the zoeal stages during chronic
exposures represented the most sensitive stages for the species (Table 22).
In addition, we have found in the majority of cases that survival was as
sensitive a criterion of effect for this stage as molt inhibition, the
sublethal effect employed. For methoxychlor, chlordane, trifluralin,
propanil and DEF, the highest concentration that had no effect on survival
also had no apparent effect on larval molting. In the case of methoxychlor,
the threshold concentration for effects on survival and molting appeared to
be approximately the same. Larvae exposed to 0.5 yg/liter were rapidly
killed (Figure 14); those surviving the time of the first molt also experi-
enced a significant delay in molting (Figure 15). Larvae exposed to 0.05
yg/liter of methoxychlor exhibited a slightly decreased survival compared to
controls and also appeared to molt slightly later than the control zoeae.
Those exposed to 0.005 yg/liter experienced neither a decrease in survival
nor a delay in molting.
For larvae exposed chronically to captan and 2,4-D, and possibly also
for those exposed to malathion and carbofuran, molting was a slightly more
sensitive response to the pesticide exposure than survival. The survival of
larvae was not affected during 69 days of exposure to 20 yg/liter of captan
(Figure 6), but this concentration resulted in a delay in molting of about
3 to k days during the third and fourth zoeal molts (Figure 7)- Even at 2
yg/liter, captan delayed the time of the third molt, but this delay was no
longer apparent by the time of the fourth molt and may represent an anomolous
event. A concentration of 10,000 yg/liter of 2,4-D acid significantly
affected survival of developing crab larvae (Figure 36) and also substan-
tially delayed their molting (Figure 41). This herbicide had no effect on
survival of larvae at 1,000 yg/liter, but molting was still delayed. Larvae
exposed to this concentration of 2,4-D molted about 2 to 3 days later than
control larvae during each molt, indicating that the effect on molting
occurred at the time of the first molt; i.e., the duration of second, third
and fourth zoeal stages was not actually longer than these stages in the
control larvae. Since a lower concentration of 2,4-D was not employed in
these tests, the threshold concentration for the effect on molting could not
be ascertained, but is presumed to fall only moderately below 1,000 yg/liter.
116
-------
The data for carbofuran and malathion are equivocal and, as for methoxy-
chlor, the threshold of effects on survival and molt delay may be very simi-
lar.^ The highest no-effect concentration of carbofuran for survival was 0.5
yg/liter (Figure 6l), but at this concentration the larvae appeared to be
molting slightly later than the control larvae (Figure 62). A similar
relationship was observed for the insecticide malathion at an exposure con-
centration of 0.2 yg/liter. For this pesticide, a delay in molting was not
at all apparent until the time of the fourth molt, and the results are only
suggestive of a delay even at that time (Figure 6k).
No other sublethal effects were systematically examined in larval crabs
during the chronic exposure experiments. However, we did not, by casual
observation, detect any effects on swimming activity, positioning behavior
or size. The only other sublethal effects studied were those relating to
osmotic and ionic regulation in adult crabs and the size of molted juvenile
crabs exposed to methoxychlor. In the former experiments, crabs were
exposed for up to 14 days to 10 yg/liter of the pesticide, a concentration
which should have resulted in 50% mortality upon continuous exposures for 30
to 60 days (Figure 19). Under these exposure conditions, an effect was noted
on salinity tolerance (Table 17), gill NaKMgATPase activity (Table 18) and
blood/urine Mg++ regulation (Figure 25), but not on regulation of Na+ or K+
ions or on blood osmotic pressure (Figures 22 to 24). Juvenile crabs exposed
to 4.0 yg/liter of methoxychlor were smaller than controls following the molt
to fourth instar, but this effect also occurred at a concentration of the
insecticide that was eventually lethal (Figure !?)• These studies with older
crabs also suggest that sublethal effects are only marginally apparent except
at very nearly lethal concentrations. In addition, these sublethal effects
on adult and juvenile crabs occurred at concentrations substantially higher
than those causing impaired survival of larvae during chronic exposure.
We believe, therefore, that it is reasonable to conclude that the
maximum acceptable toxicant concentrations for the nine pesticides studied
against the Dungeness crab, Cancer magister, are equivalent to the highest
concentrations which do not affect larval survival during chronic exposures
for methoxychlor, chlordane, trifluralin, propanil, and DEF, and are the
highest concentrations which do not affect larval molting for the pesticides
malathion, carbofuran, captan, and 2,4-D acid. These concentrations are
listed in Table 23- Although we believe that these represent "safe" concen-
trations for long-term exposures of £. magister to each pesticide, individu-
ally, it should be reemphasized that they are not strictly equivalent to the
maximum acceptable toxicant concentrations as defined by Mount and Stephen
(1967). These workers defined the maximum acceptable toxicant concentration
as that concentration which during continuous exposure throughout an entire
life cycle does not affect any aspect of the biology of the organism. It is
necessary to reemphasize that we have not been able to extend our tests for
the full 3-to 4-year period necessary to achieve this resu1tsand we cannot
be certain that the concentrations given in Table 22 would have no effect on
reproductive processes of the organism. We believe, however, that they may
be applied as criteria for the protection of this species with reasonable
assurance.
117
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TABLE 23. ESTIMATED MAXIMUM ACCEPTABLE TOXICANT CONCENTRATIONS (MATC)*
FOR CONTINUOUS EXPOSURE OF CRABS TO EACH OF NINE PESTICIDES
Pesticide
Estimated maximum
acceptable toxicant
concentration (MATC)
for continuous
exposure
(yg/liter)
Stage and criteria
employed for
estimation of MATC
Methoxychlor
Chlordane
Malath
ion
Carbofuran
Captan
DEF
Tr i f lural in
Propan
2,4-D
il
acid
0.005
0.015
0.02
0.05
2
4
15
80
1000
zoeal
zoeal
zoeal
zoeal
zoeal
zoeal
zoeal
zoeal
zoeal
surv
i
val
survival
mol
mol
mol
t
t
t
i
i
i
survi
ng
ng
ng
val
survival
surv
mol
t
i
i
val
ng
(Figure 14)
(Figure 65)
(Figure
(Figure
(Figure
(Figure
(Figure
(Figure
(Figure
64)
62)
7)
37)
34)
35)
41)
Maximum acceptable toxicant concentration (MATC) is defined as the
highest concentration of each pesticide tested individually at which no
lethal or sublethal effects were observed in any acute or chronic tests
in this study. It does not represent the highest no effect concentra-
tion for continuous exposure throughout an entire 3~to 4-year life
history cycle and, therefore, may not adequately define the "safe,
level for reproductive processes other than larval development and egg
hatching.
118
-------
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124
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PUBLICATIONS
Armstrong, D. A., D. V. Buchanan, and R. S. Caldwell. 1976. A Mycosis
caused by Lagenidium sp. in Laboratory-Reared Larvae of the Dungeness
Crab, Cancer magister, and Possible Chemical Treatments. J. Invert.
Pa t ho 1" 2TT329-336.
Armstrong, D. A., D. V. Buchanan, M. H. Mallon, R. S. Caldwell, and R. E.
Millemann. 1976. Toxicity of the Insecticide Methoxychlor to the
Dungeness Crab, Cancer magister. Mar. Biol. 38:239-252.
Buchanan, D. V., M. J. Myers, and R. S. Caldwell. 1975- Improved Flowing
Water Apparatus for the Culture of Brachyuran Crab Larvae. J. Fish.
Res. Bd. Canada 32:1880-1883.
Caldwell, R. S. 197^- Osmotic and Ionic Regulation in Decapod Crustacea
Exposed to Methoxychlor. pp 197-223- In: Pollution and Physiology of
Marine Organisms (Vernberg, F. J. and W. B. Vernberg, Eds.). Academic
Press, N.Y.
Caldwell,-R. S., D. A. Armstrong, D. V. Buchanan, M. H. Mallon, and R. E.
Millemann. 1977- Toxicity of the Fungicide Captan to the Dungeness
Crab, Cancer magister Dana. Mar. Biol. (Submitted).
Caldwell, R. S., D. V. Buchanan, D. A. Armstrong, M. H. Mallon and R. E.
Millemann. 1977- Toxicity of the Herbicides 2,^-D, DEF, Propanil and
Trifluralin to the Dungeness Crab, Cancer magister. (in preparation).
Caldwell, R. S., D. V. Buchanan, D. A. Armstrong, M. H. Mallon, and R. E.
Millemann. 1977- Toxicity of the Insecticides Carbofuran, Chlordane
and Malathion to the Dungeness Crab, Cancer magister. (in preparation)
125
U.S. GOVERNMENT PRINTING OFFICE: 1978 — 740-263/1525 Region No. 4
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
FPA-600/3-77-131
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
5. REPORT DATE
Biological Effects of Pesticides on the
Dungeness Crab
September 1. 1977
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Richard S. Caldwel1
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Oregon State University
Department of Fisheries & Wildlife
Marine Science Center
Newport, Oregon 97365
10. PROGRAM ELEMENT NO.
IEA615
11. CONTRACT/GRANT NO.
68-01-0188
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory - Gulf Breeze
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/600/4
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The toxicity of nine pesticides to various life history stages of the Dungeness
crab, Cancer magister, was examined to establish the most sensitive life stage of the
crab, and the highest concentration of each pesticide having no discernible effect on
that most sensitive stage during prolonged exposures. The compounds tested were the
insecticides carbofuran, chlordane, malathion and methoxychlor; the herbicides 2,k~D,
DEF, propanil and trifluralin; and the fungicide captan.
For each pesticide the zoeal stages were found to be the most sensitive in long-
term tests, approximately 5 to 10 times and 10 to 100 times more sensitive than
juvenile and adult crabs, respectively, and were also affected at lower concentrations
than those that affected egg hatching and prezoeal development. The maximum acceptable
toxicant concentrations for continuous exposures of C_. mag i ster zoeae to each of the
nine pesticides are: methoxychlor, 0.005 yg/liter; chlordane, 0.015 yg/liter;
malathion, 0.02 yg/liter; carbofuran, 0.05 yg/liter; captan, 2 yg/liter; DEF, 4 yg/liter
trifluralin, 15 yg/liter; propanil, 80 yg/liter; and 2,4-D, 1000 yg/liter.
The toxicity of each of these pesticides to crabs is compared with literature
reports of their toxicity to other aquatic species.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Tox ici ty
Invertebrates
Bioassay
Mar i ne An imals
Pest ic ides
Crustaceans
zoeae
1arvae
captan
herbicide
fung icide
carbofuran
chlordane
ma lathion
Cancer mag i ste
methoxychlor
i nsect i cide
2,4-D
propan i1
DEF
tri f1uralin
18. DISTRIBUTION STATEMENT
release to publi c
19. SECURITY CLASS (This Report)
unclass i f ied
20. SECURITY CLASS (This page)
unclass i fi ed
21. NO. OF PAGES
125
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
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