Un ted States
Environmental Protection
Agency
EPA-600/^-3l-0^1
May 1981
<>EPA Research and
Development
Acephate, Aldicarb, Carbophenothion, DEF,
EPN, Ethoprop, Methyl Parathion, and
Phorate: Their Acute and Chronic Toxicity,
Bioconcentration Potential, and Persistence
as Related to Marine Environments
Prepared for
Office of Pesticides
and Toxic Substances
Prepared by
Environmental Research
Laboratory
Gulf Breeze FL 32561
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EPA-600A-81 -Oh)
May 1331
ACEPHATE, ALDICARB, CARBOPHENOTHION, DEF, EPN, ETHOPROP, METHYL
PARATHION, AND PHORATE: THEIR ACUTE AND CHRONIC TOXICITY,
BIOCONCENTRATION POTENTIAL, AMD PERSISTENCE AS RELATED TO MARINE
ENVIRONMENTS
by
Experimental Environments Branch
Environmental Research Laboratory
U.S. Environmental Protection Agency
Gulf 3reeze, Florida 32561
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
GULF BREEZE, FLORIDA 32561
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DISCLAIMER
This report has been reviewed by the Gulf Breeze, Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the views and
policies of the U.S. Environmental Protection Agency, nor does mention of
trade names or commerical products constitute endorsement or recommendation
for use.
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FOREWORD
Protection of our nation's estuarine and coastal areas from damage caused by
toxic organic pollutants requires that regulations restricting the introduction
of such chemicals into the environment be formulated on a sound scientific basis.
As a research laboratory of the U.S. Environmental Protection Agency (EPA), the
Environmental Research Laboratory, Gulf Breeze (ERL,G3) conducts toxicological
studies of the effects and persistence of pesticides and other chemicals on a
single species and on communities of marine animals and plants.
During the last 20 years, this laboratory has developed a defined testing
protocol for determining the toxicity and persistence of a particular chemical to
marine organisms. A request from EPA's Office of Pesticides and Toxic Substances
(OPTS) for the evaluation of eight pesticides gave our scientific staff the
opportunity to utilize each bioassay procedure of our testing protocol, namely:
(1) acute static toxicity tests; (2) acute flow-through toxicity tests; (3)
embryo-juveni1e tests; (4) life-cycle tests; (5) benthic community tests;
(6) bioconcentration tests; and (7) persistence tests.
Our report on these investigations provides a reference document on the
toxicity, bioconcentration potential, and persistence of the eight subject
pesticides: acephate, aldicarb, carbophenothion, DEF, EPN, ethoprop, methyl
oarathion, and phorate. Further, it demonstrates the marine toxicological and
analytical capabilities of the Experimental Environments Branch.
Jack I. Lowe
Chief
Experimental Environments Branch
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ABSTRACT
The toxicity, bioconcentration, and persistence of the pesticides ace-
phate, aldicarb, carbophenothion, DEF, EPN, ethoprop, methyl parathion, and
phorate were determined for estuarine environments. Static acute toxicity
tests were conducted to determine the 96-h EC50 values for algae, 48-h EC50
values for oyster larvae, and 96-h LC50 values for at least two crustacean
and fish species. Flow-through acute toxicity tests, based on measured con-
centrations, were conducted to determine the 96-h LC50 values of the pesti-
cides for at least two crustacean and fish species. In addition, Maximum
Acceptable Toxicant Concentrations (MATC) were determined in life-cycle tox-
icity tests with mysid shrimp (f'ysidopsis bahia) and sheepshead minnows
(C.yprinodon variegatus), or in partial TTfe-cycle tests with grass shrimp
(Palaemonetes puqio). MATCs were estimated from embryo-juveni1e toxicity
tests with sheepshead minnows. Persistence studies on carbophenothion, DEF,
EPN, methyl parathion, and phorate investigated processes in marine systems
that contribute to those pesticides' disappearance. The relative importance
of biological and nonbiological processes (including biodegradation, photo-
lysis, hydrolysis, sediment/water partitioning, and volatility) were exam-
ined. Bioconcentration factors for fish or mollusks exposed to carbopheno-
thion, EPN, ethoprop, and phorate were determined at steady state or after
_>28-day exposures.
Acute and chronic toxicities of the pesticides differed markedly. The
crustaceans (Penaeus duorarum or M. bahia) were the most acutely sensitive
of the 7 to 10 species tested; frequently, the alga (Skeletonema costatum) or
the eastern oyster (Crassostrea virqinica) were least sensitive. The lowest
96-h LC50 in yg/i for each pesticide was: acephate, 3,800; aldicarb, 12;
carbophenothion, 0.47; DEF, 4.6; EPN, 0.29; ethoprop, 6.3; methyl parathion,
0.77; and phorate, 0.11. Of the 14 chronic toxicity tests, £. puqio was the
most sensitive to carbophenothion, and M. bahia was the most sensitive species
for the other pesticides. The MATC in yg/£ for the species most sensitive
to each pesticide was: acephate, 588 to 1,400; aldicarb, 1.0 to 1.5; carbo-
phenothion, 0.22 to 0.36; DEF, <0.34; EPri, 0.44 to 3.4; ethoprop, 0.36 to 0.62;
methyl parathion, 0.11 to 0.34; and phorate, 0.09 to 0.21.
Persistence and bioconcentration tests provide further insight needed in
hazard assessment, bioconcentration factors were less than 100 in organisms
exposed to ethoprop and phorate, and less than 10,000 for carbophenothion
and EP'l. Persistence studies in sediment/water systems that contained natural
populations of microorganisms demonstrated the following half-lives (in days):
0.5, phorate; 1.0, methyl parathion; 2.2, DEF; 2.3, EPN; and 6.5, carbopheno-
thion. Persistence of these chemicals was most frequently diminished by bio-
logical processes in water or sediment, or both.
Evaluation of the relative hazards of chemicals to aquatic environments
requires that information on toxicity, accumulation potential, and expected
environmental concentrations be compared. Data on acute and chronic toxicity
and accumulation potentials of these eight pesticides and on their persistence
will be useful for hazard assessment. Additionally, methods used will be useful
for future generation of data to assess other chemicals.
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CONTENTS
Foreword i i 1
Abstract iv
Fi gures
Tables
Acknowledgement
1. Introduction 1
2. Materials and Methods 3
Test organisms 3
Test pesticides 3
Test water 3
Environmental Research Laboratory, Gulf Breeze (ERL,GB) 3
Bionomics Marine Research Laboratory (BURL) 4
Acute toxicity tests 4
Static algal tests 4
Static oyster larval tests 5
Static copepod test 6
Static invertebrate and fish tests 6
Flow-through macrocrustacean and fish tests 6
Chronic toxicity tests 8
Hysid shrimp entire life-cycle test 8
Acephate test 8
Aldicarb test 9
Ethoprop test 9
EPN, phorate, carbophenothion, and DEF tests 10
Methyl parathion test 10
Grass shrimp partial life-cycle toxicity test 11
Sheepshead ninnow embryo/juvenile tests 12
Aldicarb and ethoprop tests 13
Carbophenothion and phorate tests (ERL.GB) 14
Sheepshead minnow entire life-cycle test 16
Benthic animal community test 17
Bioconcentration tests 18
Carbophenothion 18
r p f-| 1,g
Persistence 19
Log P. 19
Persistence test 19
Pesticide analytical methods 20
Water 21
Tissue 21
Phorate and carbophenothion 21
EPN and DEF 21
V
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Methyl parathion 21
Sediment 22
Analytical equipment and quality assurance 22
Instrumentation 22
Qua!ity control 22
Acetylcholinesterase (AChE) 22
Statistical methods 23
Algal test 23
Oyster embryo test.. 23
Acute toxicity test 23
Mysid shrimp entire life-cycle tests 23
Acephate, aldicarb, and ethoprop 23
Carbophenothion, DEF, EPN, methyl parathion and
phorate 23
Grass shrimp Dartial life-cycle tests 2^
Carbophenothion ?&
Sheepshead minnow embryo/juvenile and entire life-
cycle test 24
Bioconcentration tests 25
Persistence tests and Log P 25
3. Results and Discussion..... . 26
Acephate..... 26
Results 26
Acute static toxicity tests 26
Algae 26
Invertebrates 26
Fishes 26
Acute flow-through toxicity tests 26
Invertebrates 26
Fishes 26
Mysid shrimp entire life-cycle test 26
Discussion 27
Aldicarb 28
Results 28
Acute static toxicity tests 28
Algae 28
Invertebrates 28
Fishes 29
Acute flow-through toxicity test 29
Invertebrates 29
Fishes 29
Mysid shrimp entire life-cycle test 29
Sheepshead minnow embryo/juveni le test 29
Discussion 30
Carbophenothion 31
Results 31
Acute static toxicity tests 31
Algae 31
I nvertebrates 31
Fishes 31
Acute flow-through toxicity tests 31
Invertebrates 31
Fishes 31
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Mysid shrimp entire life-cycle test 32
Grass shrimp entire life-cycle test 32
Sheepshead minnow embryo/juvenile test 34
Renthic animal community test 35
Bioconcentration 35
Persistence 36
Discussion 36
OEF 37
Results 37
Acute static toxicity tests 37
Algae 37
Invertebrates 37
Fishes 37
Acute flow-through toxicity tests 38
Invertebrates 38
Fishes 38
Mysid shrimp entire life-cycle test 38
Persistence 38
Discussion 39
EPN -40
Results 40
Acute static toxicity tests 40
Algae 4Q
Invertebrates 40
Fishes 41
Acute flow-through toxicity tests 41
Invertebrates 41
Fishes 41
Mysid shrimp entire life-cycle tests 41
Sheepshead minnow entire life-cycle tests 41
Survival and signs of poisoning in parental fish 41
Growth of parental fish 42
Reproduction 42
Survival, growth, and signs of poisoning in progeny 43
MATC and application factor 44
Bioconcentration 44
Acetylcholinesterase activity 44
Bioconcentration tests 45
Persistence 45
Discussion 46
Ethoprop 47
Results 47
Acute static toxicity tests 47
Algae.. 47
Invertebrates 47
Fishes 4y
Acute flow-through toxicity tests 47
Invertebrates ' 47
Fishes 47
Mysid shri-np entire life-cycle test 48
Sheepshead minnow embryo/juvenile test 48.
Discussion 48
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Methyl oarathion 49
Results 49
Acute static toxicity tests 49
Algae 49
Invertebrates 49
Fishes 50
Acute flow-through toxicity tests 50
Invertebrates 50
Fishes 50
Mysid shrimp entire life-cycle test 50
Persistence 51
Discussion . 51
Phorate 53
Results 53
Acute static toxicity tests 53
Algae 53
Invertebrates.... 53
Fishes 53
Acute flow-through toxicity tests 53
Invertebrates.. 53
Fishes 53
Mysid shrimp entire life-cycle test 54
Sheepshead minnow embryo/juvenile tests 54
Persistence 55
Discussion 55
References 57
Appendices 126
A-l. Description of acephate used in tests 126
A-2. Description of aldicarb used in tests 127
A-3. Description of carboDhenothion used in tests 128
A-4. Description of DEF used in tests 129
A-5. Description of ethoprop used in tests 130
A-6. Description of EPN used in tests 131
A-7. Description of methyl parathion used in tests 132
A-3. Description of phorate used in tests * 133
B. Chemical methods for the analyses of acephate (Orthene ),
aldicarb (Tenik ), carbophenothion (Trithion*), ethoprop
(Mocap ) and methyl parathion in seawater and tissue
samples 135
C-l. Acute (96-h) toxicity of eight oesticides to the marine
diatom (Skeletonema costatum) in static tests 158
C-2a. Number of normal eastern oyster (Crassostrea virginica)
embryos per milliliter counted following 48 h cf exposure to
acephate to static, unaerated seawater 152
C-2b- Toxicity of acephate to embryos of eastern oysters (Crassostrea
virqinica) exposed for 48 h in static, unaerated seawater 153
C-3. Test concentrations and mortality of pink shrimp (Penaeus
duorarum) exposed to acephate in static acute toxicity tests
using 20 animals per concentration 154
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C-4. Test concentrations and mortality of sheepshead minnows
(Cyprinodon variegatus) exoosed to acephate in static acute
toxicity test using 20 fish per concentration 165
C-5. Test concentrations and mortality of spot (Leiostomus xanthurus)
exposed to acephate in static acute toxicity tests using 30
animals per concentration 166
C-6. Test concentrations and mortality of mysiri shrimp (Mysidopsis
bahia) exposed to acephate in flowing, natural seawater of
29+1 °/oo salinity and 22+l°C 167
C-7. Test concentrations and mortality of pink shrimp (Penaeus
duorarun) exposed to acephate ig flowing, natural seawater
of 28-30 °/oo salinity and 22+1 C 168
C-8. Ranges of dissolved oxygen and pH measured in seawater from acute
static and flow-through toxicity tests 169
C-9. Test concentrations and mortality of pinfish (Lanodon rhonboides)
exposed to acephafce in flowing, natural seawater of 28-30 °/oo
salinity and 22+1 C 171
C-10. Test concentrations and percentage of sheepshead minnows
(C.yprinodon variegatus) exhibiting complete loss of eauilib-
rium and lack of escape response after exposure to acephate
in flowing, natural seawater 172
D-la. Number of normal eastern oyster (Crassostrea virginica) embryos
per milliliter counted following 48 h of exposure to aldicarb
in static, unaerated seawater 173
D-lb. Toxicity of aldicarb to embryos of eastern oyster (Crassostrea
virginica) exposed for 48 h in static, unaerated seawater 174
D-2. Test concentrations and mortality of mysid shrimp (Mysidopsis
bahia) exposed to aldicarb in static acute toxicity tests
using natural seawater adjusted to 20 °/oo; teriDerature,
26+1 °C 175
0-3. Test concentrations and mortality of postlarval shrimo (Penaeus
sty1irostris) exposed to aldicarb in static acute toxicity
tests 176
C-4. Test concentrations and mortality of sheepshead minnows
(Cyprinodon variegatus) exposed to aldicarb in flowing
natural seawater 177
D-5. Test concentrations and mortality of spot (Leiostomus xanthurus)
exposed to aldicarb in static acute toxicity tests using ten
D-6. Test concentration and mortality of mysid shrimp (Mysidopsis
bahia) exposed to aldicarb in a flowing seawater test 179
D-7. Test concentrations and mortality of pink shrimp (Penaeus
duorarum) exposed to aldicarb in a flowing seawater test 180
D-8. "est concentration and mortality of sheepshead minnows
(C.yprinodon variegatus) exposed to aldicarb in a flowing
seawater test 181
0-9. Test concentrations and mortaltiy of pinfish (Lagodon
rhomboides) exposed to aldicarb in a flowing seawater test... 182
E-la. Number of normal eastern oyster (Crassostrea virginica) embryos
per milliliter gounted following 48-h exposure to carbooheno-
thion (Trithion") in static, unaerated seawater 183
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E-lb. Toxicity of carbophenothion to embryos of eastern oysters
(Crassostrea virqinica) exposed for 48 h in static, unaerated
sea water 184
E-2. Test concentrations and mortality of mysid shrimp (Hysidopsis
bahia) exposed to carbophenothion in static acute toxicity tests
using 20 animals per concentration 185
i-3. Test concentrations and mortality of postlarval penaeid shrimp
(Penaeus stylirostris) exposed to carbophenothion in static
acute toxicity test using 20 animals per concentration 186
E-4. Test concentrations and mortality of sheepshead minnows
(Cyprinodon variegatus) exposed to carbophenothion in static
acute toxicity tests using 20 animals per concentration 187
E-5. Test concentrations and mortality of spot (Leiostonus xanthurus)
exposed to carbophenothion in static acute toxicity tests
using 20 animals per concentration 188
E-6. Test concentrations and mortality of mysid shrimp (Mysidopsis
bahia) exposed to carbophenothion in a flowing seawater test. 189
E-7. Test concentrations and mortality of pink shrimp (Penaeus
duorarum) exposed to carbophenothion in a flowing seawater
test 190
E-8. Test concentrations and mortality of grass shrimp (Palaemonetes
puqio) larvae (1- to 7-day old) exposed to carbophenothion in a
flowing seawater test.. 191
E-9. Test concentrations and mortality of grass shrimp (Palaemonetes
puqio) exposed to carbophenothion in a flowing seawater test. 192
E-10. Test concentrations and mortality of wild-stock grass shrimD
(Palaemonetes puqio) exposed to carbophenothion in a flowing
seawater test 193
E-ll. Test concentrations and mortality of sheepshead minnows
(Cyprinodon variegatus) exposed to carbophenothion in a flow-
ing seawater test 194
E-12. Test concentrations and mortality of Atlantic silversides
(Menidia menidia) exposed to carbophenothion in a flowing
seawater test 195
E-13. Test concentrations and mortality of spot (Leiostonus xanthurus)
exposed to carboohenothion in a flowing seawater test 196
E-H. Test concentrations and mortality of pinfish (Lacodon
rhomboides) exposed to carboohenothion in a flowing seawater
test . 197
E-15. Mean salinity and dissolved oxygen during a 249-day partial
life-cycle toxicity test of carbophenothion with grass shrimp
(Pal aemonetes puqio) 198
r-la. Number of normal eastern oyster (Crassostrea virqinica)
embryos oer milliliter counted following 48 h of exposure
to DEF in static, unaerated seawater 199
F-lb. Toxicity of DEF to embryos of eastern oysters (Crassostrea
virqinica) exposed for 48 h in static, unaerated seawater... 200
F-2. "Test concentrations and mortality of mysid shrimp (Mysidopsis
bahia) exposed to DEF in static acute toxicity tests using
20 animals per concentration .... 201
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F-3. Test concentrations and mortality of postlarval shrimp (Penaeus
st.yl irostris) exposed to DEF in static acute toxicity tests
using 20 animals per concentration 202
F-4. Test concentrations and mortality of sheepshead minnows
(Cyprinodon variegatus) exposed to DEF in static acute
toxicity tests using 20 animals Der concentration 203
F-5. Test concentrations and mortality of spot (Leiostomus
xanthurus) exposed to DEF in static acute toxicity tests
using 20 animals per concentration 204
F-6. Test concentrations and mortality of mysid shrimp (Hysidopsis
bahia) exposed to DEF in a flowing seawater test 205
F-7. Test concentrations and mortality of pink shrimp (Penaeus
duorarum) exposed to DEF in a flowing seawater test 206
F-8. Test concentration and mortality of wild-stock grass shrimp
(Palaemonetes pugio) exposed to DEF in flowing seawater tests 207
F-9. Test concentrations and mortality of pinfish (Lagodon
rhomboides) exposed to DEF in flowing seawater test 2C8
F-10. Test concentrations and mortality of spot (Leiostomus xanthurus)
exposed to DEF in flowing seawater tests. 209
F-11. Test concentrations and mortality of sheepshead minnow
(Cyprinodon variegatus) exposed to DEF in a flowing seawater
test 210
G-la. Number of normal eastern oyster (Crassostrea virginica) embryo
per milliliter counted following 48 h of exposure to EPM in
static, unaerated seawater 211
G-lb. Toxicity of EPM to embryos of eastern oysters (Crassostrea
virqinica) exposed for 48 hours in static, unaerated seawater 212
G-2. Test concentrations and mortality of mysid shrimp (Hysidopsis
bahia) exposed to EPM in static acute toxicity tests using
20 animals per concentration 213
3-3. Test concentrations and mortality of postlarval penaeid shrimp
(Penaeus stylirostris) exoosed to EPN in static acute toxicit
tests using 40 or 60 animals oer concentration 214
H-6. Test concentrations and mortality of nysid shrimp (Hysidopsis
bahia) exposed to ethoprop in a flowing seawater test 232
H-7. Test concentration and mortality of pink shrimp (Penaeus
duorarum) exposed to ethoprop in a flowing seawater test 233
H-8. Test concentrations and mortality of sheepshead minnows
(Cyprinodon variegatus) exposed to ethoprop in a fowing sea-
water test 234
H-9. Test concentrations and mortality of pinfish (Lagodon
rhomboides) exposed to ethoprop in a ¦Mowing seawater test.... 235
I-la. Number of normal eastern oyster (Crassostrea virginica) embryos
per milliliter counted following 48 h of exposure to methyl
parathion in static unaerated seawater 236
I-1b. Toxicity of methyl parathion to embryos of eastern oysters
(Crassostrea virginica) exposed for 48 h in static, unaerated
seawater 237
1-2. Test concentrations and mortality of mysid shrimp (Mysidopsis
bahia) exposed to methyl parathion in static acute toxicity
tests jsing 20 animals per concentration 238
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1-3. Test concentrations and mortality of postlarval penaeid shrimp
(Penaeus stylirostris) exposed to methyl parathion in static
acute toxicity test using 20 animals per concentration 239
1-4. Test concentrations and mortality of calanoid copepod (Acartia
tonsa) exposed for 96 h to methyl parathion in static,
unaerated seawater 240
1-5. Test concentrations and mortality of sheepshead minnows
(Cyprinodon variegatus) exposed to methyl parathion in static
acute toxicity tests using 20 animals per concentration 241
1-6. Test concentrations and mortality of spot (Leiostomus xanthurus)
exposed to methyl parathion in static acute toxicity tests
using 20 animals per concentration 242
1-7. Test concentrations and mortality of mysid shrimp (Hysidonsis
bahia) exposed to methyl parathion in a flowing seawater test. 243
I-B. Test concentrations and mortality of pink shrimp (Penaeus
duorarum) exposed to methyl parathion in a flowing seawater
tests 244
1-9. Test concentrations and mortality of spot (Leiostomus xanthurus)
exposed to methyl parathion in flowing, natural seawater 245
J-la. Number of normal eastern oyster (Crassostrea vi rginica) embryos
per milliliter counted following 48 h of. exposure to phorate
in static, unaerated seawater 246
J—1b. Toxicity of phorate to embryos of eastern oysters (Crassostrea
virginica) exposed for 48 h in static, unaerated seawater 247
J-2. Test concentrations and mortality of postlarval penaeid shrimp
(Penaeus stylirostris) exposed to phorate in static acute
toxicity tests using 20 animals per concentration 2^8
J-3. Test concentrations and mortality of mysid shrimp (Mysidopsis
bahia) exposed to phorate in static acute toxicity tests using
20 animals per concentration 249
J-7. Test concentrations and mortality of mysid shrimp (Mysidopsis
bahia) exposed to phorate in a flowing seawater test 253
J-3. Test concentrations and mortality of sheepshead minnows
(Cyprinodon variegatus) exoosed to phorate in flowing sea-
water test 254
J-9. Test concentrations and mortality of spot (Leiostomus xanthurus)
exposed to phorate in flowing seawater test 255
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FIGURES
Number Page
1. Effects of carbophenothion on spawning response of grass
shrimp (Palaemonetes pugio) in a partial life-cycle tox-
icity test 63
2. A. longitudinal, horizontal section of a normal sheepshead
minnow from control group. B. similar section from fish
exposed to 2.8 ug carbophenothion per liter from the zygote
stage for 28 days 6^
3. Uptake and depuration of carbophenothion in whole-body
tissues of spot (Leiostomus xanthurus) in a flowing sea-
water bioassay 65
4. Uptake and depuration of EPN in whole-body tissues of pin
fish (Lagodon rhonboides) in a flowing seawater bioassay... 66
5. The effect of measured concentrations of phorate on mor-
tality of sheepshead minnows (C.yprinodon variegatus) in a
28-day embryo/juveni le toxicity test at 30°C. 67
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TABLES
Number Page
1-a. Scientists or laboratory responsible for conducting acute
static toxicity tests 68
1-b. Scientist or laboratory responsible for conducting acute flow-
through toxicity tests 69
1-c. Scientist or laboratory responsible for designated tests and
sections of this document 70
2. Composition of mixes added to algal growth media 71
3. Profile of sediment used in persistence studies 72
4-a. Acute toxicity of acephate, aldicarb, carbophenothion, and DEF
to estuarine organisms in static tests 73
4-b. Acute toxicity of EPTJ, ethoprop, methyl parathion, and p'norate
to estuarine organisms in static tests 74
5-a. Acute toxicity of acephate, aldicarb, carbophenothion, and DEF
to estuarine organisms in flowing seawater tests 75
5-b. Acute toxicity of EPN, ethoprop, methyl parathion, and phorate
to organisms in flowing seawater tests 76
6. Mortality (percentage killed) of mysid shrimp (Mysidopsis bahia)
during a chronic (28-day) exposure to acephate 77
7. Number of offspring produced per female of mysid shrimp
(Mysidopsis bahia) exposed to acephate in a chronic (28-day)
exposure in natural, flowing seawater 78
8. Results of chronic toxicity tests with acephate, aldicarb, car-
bophenothion, DEF, EPN, ethoprop, methyl parathion, and phor-
ate 79
9. Mortality (percentage killed) of mysid shrimp (Mysidopsis bahia)
during a chronic (28-day) exposure to aldicarb 80
10. Number of offspring produced per female of mysid shrimp
(Mysidopsis bahia) exposed to aldicarb in a chronic (28-day)
exposure in natural, flowing seawater 81
11. Survival of embryo and juvenile sheepshead minnows
(Cyorinodon variegatus) exposed to various concentrations
of aldicarb in seawater at 29+l°C 82
12. Standard lengths and survival of juvenile sheepshead minnows
(Cyprinodon variegatus) exposed to various concentrations
of aldicarb for 28 days 83
13. Mortality (percentage killed) of mysid shrimp (M.ysidopsis
bahia) during a chronic (28-day) exposure to carbopheno-
thion 84
14. 'lumber of offspring per female of mysid shrinp (Mysidopsi s
bahia) exposed to carbophenothion in a chronic (28-day) .
exposure in natural, flowing seawater 85
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Number Paqe
15. Survival of Fj generation of mysids (Hysidoosis bahia) con-
tinuously exposed to carbophenothion 86
16. Growth (head-tail length) of parental generation nysid shrimp
(Mysidopsis bahia) exposed to carbophenothion in a chronic
(28-day) exposure in natural, flowing seawater 87
17. Survival of adult grass shrimp (Palaemonetes pugio) continuously
exposed to carbophenothion for 249 days in a partial life-
cycle test 88
18. Effect of carbophenothion of fecundity of grass shrimp
(Pal aemonetes pugio) 89
19. Survival of F, generation grass shrimp (palaemonetes pugio)
larvae reared under conditions of continued exposure to
carbophenothion 90
20. Effect of carbophenothion on weight (g) of grass shrimp
(Palaemonetes pugio) exposed in a partial life-cycle test 91
21. Concentrations of carbophenothion (ug/g) whole body, wet weight
in parental generation grass shrimp (Palaemonetes pugio), and
bioconcentration factors (concentration measured in tissue
divided by the average concentration in exposure water)
at termination of a 249-day partial life-cycle toxicity test.. 92
22. Survival of embryonic and juvenile sheepshead minnows
(Cyprinodon varieqatus) exposed to various concentrations of
carbophenothion in seawater at 30°C 93
23. Standard length, tissue concentrations, and bioconcentration
factors (concentration measured in whole body, wet weight
divided by average measured water concentration) for juvenile
sheepshead minnows (Cyprinodon variegatus) continuously
exposed to various concentrations of carbophenothion in sea-
water at 30°C 94
24. Animals collected from control aquaria and aquaria exposed to
carbophenothion in eight-week benthic community study 95
25. Total number of individuals and species (in parentheses), by
phylum, collected in an eight-week benthic community test
with carbophenothion 97
26. Octanol/water partition coefficients (Log P) of methyl
parathion, phorate, EPN, carbcohenothion, and DEF, as
determined by reversed phase high pressure liquid chroma-
tography 98
27. Results of persistence studies with carbophenothion 99
28. Mortality (percentage killed) of mysid shrimp (."ysicopsis
bahia) during a chronic (28-day) exposure to DEF 100
29. Number of offspring per female mysid shrimp (Mysidopsis
bahia) exposed to DEF in a 23-day life-cycle toxicity test.... 101
30. Results of persistence studies with DEF.... 102
31. Mortality (percentage killed) of mysid shrimp (Mysidopsis
bahia) during a chronic (28-day) exposure to EDN 103
32. flumber of offspring oer female mysid shrimp (Mysidopsis
bahia) exposed to EPN in a chronic (28-day) exposure in
natural flowing seawater 104
xv
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Number Pane
33. Percentage survival of sheepshead minnows (Cyprinodon
variegatus) continuously exposed to EPN for 265 days
in an entire life-cycle toxicity test 105
34. Effect of EPN on average standard length (mm) of sheepshead
minnows (Cyprinodon variegatus) exposed for an entire
life-cycle 106
35. Egg production by sheepshead minnows (Cyprinodon variegatus)
continuously exposed to measured concentrations of EPN in
flowing seawater at 30°C 107
36. Average dissolved oxygen concentrations (mg/i) during a
265-day toxicity test in which sheepshead minnows
(Cyprinodon variegatus) were continuously exposed to
measured concentrations of EPN at 30°C 108
37. Survival of embryos, fry, and juvenile sheepshead minnows
(Cyprinodon variegatus) and standard lengths of surviving
fish continuously exposed to measured concentrations of
EPN for 28 days.... . 109
38. Concentrations of EPN (pg/g, whole body, wet weight) measured
in selected life stages of the sheepshead minnow (Cypri nodon
variegatus) in a 265-day life-cycle toxicity test 110
39. Bioconcentration factors (concentration measured in tissue
divided by the average concentration measured in exposure
water) for EPN in selected li^e-stages of the sheepshead minnow
(Cyprinodon variegatus) in a 255-day life-cycle toxicity test.. Ill
40. Acetylcholinesterase activity, expressed as micromoles of
acetylcholine hydrolyzed/hour/mg brain tissue x 100, in
brains from sheepshead minnows (Cyprinodon variegatus) that
were continuously exposed to EPN 112
41. Results of persistence studies with EPN 113
42. Mortality (percentage killed) of mysids (Mysidopsis bahia) .
during a chronic (28-day) exposure to ethoprop 114
43. Number of offspring produced per ^enale of nysid shrimp
(Mysidopsis bahia) exposed to ethoprop in a chronic
(28-day) exposure in natural, flowing seawater 115
44. Survival of embryo and juvenile sheepshead minnows (Cypri nodon
variegatus) exposed to various concentrations of ethoprop in
seawater at 30^1°C 116
45. Standard lengths, tissue concentrations, and bioconcentration
factors of ethoprop in juvenile sheeoshead minnows
.(Cyprinodon variegatus) exposed to various concentrations
of ethoprop for 28 days 117
46. Mortality (percentage killed) of mysid shrimp (Mysidopsis
bahia) during a chronic (24-day) exposure to methyl
parathion 118
47. Number of offspring per female mysid shrimp (Mysidopsis
bahia) exoosed to methyl parathion in a chronic (24-day)
exDosure in natural, flowing seawater.. 119
48. Results of persistence studies with methyl parthion 120
49. Mortality (percentage killed) of mysid shrimp (Mysidopsis ¦
bahia) during a chronic (28-day) exposure to Dhorate 121
50. Number of offspring per female mysid shrimp (Mysidopsis
bahi a)' exposed to phorate in a chronic (28-day) exposure
in natural flowing seawater 122
xvi
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Number Pape
51. Survival of embryonic and juvenile sheepshead minnows
(Cyprinodon variegatus) exposed to various concentrations
of phorate in seawater for 28 days at 30°C 123
52. Standard -¦ lengths , tissue concentrations, and bi oconcentrati on
factors of phorate in juvenile sheeoshead minnows
(Cyprinodon variegatus) exposed to various concentrations
of phorate for 28 days 124
53. Results of persistence studies with phorate 125
xv i i
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ACKNOWLEDGMENT
Material in this report was prepared by the Experimental Environments
Branch of the Environmental Research Laboratory, Gulf Breeze (ERL,GB) and a
contractor, Bionomics Marine Research Laboratory, EG&G International, Inc.,
Pensacola, Florida. Scientists and laboratories responsible for designated
tests are identified in Tables 1-a, 1-b, and 1-c (p. 68-70). The contribution
of many individuals who assisted in the preparation of the report is apore-
ciated. Special thanks is extended to Or. Nelson Cooley, editorial advisor;
Betty L. Jackson, technical editor; and Dorothy Delk, typist. Dr. Jerry L.
Oglesby performed statistical analyses on the benthic community test,
bioconcentration tests, and chronic toxicity tests conducted at EP.L,GB.
xvi i i
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INTRODUCTION
Use of theRpesticides acephate, EPN, methyl parathion, phorate,
aldicarb (Temik ), carbophenothion (Trithion ), DEF, and ethoprop ¦
(Hocap ) has increased, especially in the southern states, because of
cancellations in the registration or restrictions in the labeling of
certain chlorinated insecticides and herbicides. (Mention of commer-
cial products does not constitute endorsement by the U.S. Environmental
Protection Agency.) Due to this increased usage, the Office of Pesti-
cide Programs (OPP) of the U.S. Environmental Protection Agency (EPA)
requested that the Agency's Environmental Research Laboratory, Gulf
Breeze (ERL,GB), Florida conduct laboratory experiments to determine
the toxicity and persistence of these pesticides in the marine environ-
ment. Tests were conducted either at ERL,G3 or under contract at EG&G
Bionomics Marine Research Laboratory (BMRL), Pensacola, Florida.
This report includes the results of testing with the above eight
pesticides. Acute static and flow-through toxicity tests were con-
ducted initially on the alga (Skeletonema costatum), oyster larvae
(Crassostrea virginica), mysid shrimp (Hysidopsis bahia), penaeid
shrimp (Penaeus duorarum or P_. styl i rostris), grass shrimo
(Palaemonetes puqio), cooepod (Acartia tonsa), Atlantic silversides
(Menidia menidia), pinfish (Lagodon rhomboides), sheepshead minnows
(Cyprinodon variegatus), and spot (Leiostomus xanthurus). The species
tested were selected on the basis of seasonal availability, ease of
culturing, and sensitivity to pesticides. Chronic toxicity tests in-
cluded mysid shrimp and sheeoshead minnow entire life-cycle, grass
shrimp partial life-cycle, and sheepshead minnow embryo/juvenile tests.
Specific tests were selected on the basis of relative acute sensitiv-
ities of fish and invertebrates and oersistence of the pesticide.
Generally, if the LC50s differed by more than 100X, the most sensitive
taxonomic group was tested. If sensitivities were similar, both mysid
and fish tests were conducted. Tissues from animals exposed in tox-
icity tests or in bioconcentration tests were analyzed for those pes-
ticides whose partition coefficients indicated that they would De
accumulable.
Since the environmental fate of a pollutant determines its avail-
ability to aquatic organisms, persistence studies have been included to
complement the toxicity data on DEF, EPN, methyl parathion, carbopheno-
thion, and phorate. These studies included the determination of the
^-octanol/water partition coefficient (Log P) and the half-life or rate
of disaopearance of the pesticide under estuarine conditions. The Log
P value has become the cornerstone for predicting the fate of organic
chemicals from physical properties through the use of structure-
activity correlations, because it is a notential predictor of bioac-
cumulation (Veith et a!., 1979b), solubility (Chiou et al., 1977),
sorption (Karickhoff et al., 1979), and oersistence (Yonezawa and
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Urushigawa, 1979). Half-lives of pesticides in aquatic systems,
particularly in estuaries, are not available and are necessary for
regulatory agencies to identify situations potentially detrimental to
man and the environment.
The fate of methyl parathion has been extensively studied by us
because we consider it to be a representative organophosphate
insecticide and use it to calibrate the natural processes affecting the
pesticide in a variety of microcos.ns under develODment at Gulf Breeze
(Bourquin et al., 1979). These microcosms represent an alternative
method to field studies for validating routine laboratory studies and
serve to identify potential pesticide oersistence or to isolate nat-
ural processes that contribute to pesticide dissipation (Pritchard et
al., 1979).
2
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METHODS AND MATERIALS
TEST ORGANISMS
All test organisms, except the oyster (Crassostrea virqinica),
post-larval penaeid shrimp (Penaeus stylirostris), and Atlantic
silversides (Menidia menidia), used in toxicity tests described herein
were either cultured at ERL,GB or BMRL, or collected from estuarine
areas adjacent to these laboratories. The alga (Skeletonena costatum)
was cultured at ERL.GB. Mysid shrimD (M.ysidopsis bahia) and sheeps-
head minnows (Cyprinodon varieqatus) used in acephate studies were
cultured at BMRL. Those used in the carbophenothion, EPN, methyl para-
thion, or phorate studies were cultured at ERL,GB. Adult oysters were
collected from Ocean Springs, Mississippi, and conditioned at BMRL in
flowing, unfiltered seawater. P_. st.yl irostris postlarvae were pur-
chased from Ralston Purina's Marine Research Center, Crystal River,
Florida; Atlantic silversides embryos were shipped from Wadmalaw
Island, South Carolina. Tables 1-a, 1-b, and 1-c describe the species
used, the chemicals tested, and the researcher responsible for each
test at ERL,GB, as well as those tests conducted at BMRL.
All foods given to test animals during culture, holding, and test-
ing at ERL.GB were analyzed chemically for chlorinated hydrocarbon con-
tent and contained less than 0.1 yg/g (micrograms per gram, wet
weight). Mysid shrimp were fed living Artemia sp. nauplii. P_. puqio
were fed living Artemi a sp. nauplii and a dry, flake food (nauplii as
larvae and both foods as juveniles and adults). P_. styl i rostri s were
fed Artemia nauplii and a pelleted experimental food formulated and
supplied by Ralston Purina. Pink shrimD (Penaeus duorarum) were fed
grouper filets, and fish were fed either dry flake food, frozen adult
Artemi a, or live Artemi a nauDlii.
TEST PESTICIDES
Descriptions of the pesticides tested at ERL,GB and BMRL are con-
tained in Appendix A. Concentrations of each pesticide are reported
here as micrograms (ug) of pesticide per liter (a) of seawater (parts
per billion) or ug of pesticide per gram (g) of tissue, wet weight
(parts per mi 11 ion).
TEST WATER
ERL.GB
All seawater used for culture and testing at ERL ,GB (except for
3
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algal studies) was pumped by -316 stainless steel pumps from Santa Rosa
Sound, Florida, through a coarse sand filter and oolypropylene filters
(10 to 20 micrometers [un] pore size) into the laboratory. Seawater
was then adjusted to the appropriate test temperature in reservoirs
located near the test apparatus, -rom the reservoirs, seawater flowed
by gravity to the various test systems. For algal studies, Rila Salt
Mix (Rila Products, Teaneck, NJ) with nutrients added was the arti-
ficial sea salt medium.
BMRL
All water used for holding, acclimation, and testing was natural
seawater pumped from Big Lagoon, Florida. The pump intake was located
about 30 meters (m) from shore, at a depth of aoproximatsly 3 rn.
Test water was pumped by a #316 stainless steel pump through hard
polyvinylchloride (PVC) pipe, a fiberglass, sand filled filter, and 10
ym polypropylene-core filters into an elevated fiberglass reservoir.
Water was aerated continuously and vigorously in the reservoir and
flowed by gravity through PVC pipes into the laboratory and the test
systems. The salinity of water in tests was that of the incoming
water.
ACUTE TOXICITY TESTS
Static Algal Tests
The chain-forming marine and estuarine diatom (Skeletonema
costatum) was exposed to the eight pesticides in static tests as
described by Walsh and Alexander (1980). Stock solutions of the pesti-
cides in nanograde acetone we^e diluted with acetone to concentrations
used in the tests. Since the pesticides were not equally toxic, con-
centrations differed for each, but working solutions were adjusted so
that when 0.1 nu was added to 26 m£ of test medium, desired exposure
concentrations were obtained.
Since toxicity of the pesticides to algae was not
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calculate the EC50 by nonlinear least squares analysis according to the
expression
Y " A " 1 + px X-p2
where
Y = estimated population density over the range of X,
X = range of concentrations,
A = mean maximum biomass,
P-i = downward sloDe, and
?2 ~ inflection point of downward slope.
Static Oyster Larval Tests
Methods for the 48-h oyster larvae toxicity tests were based on
those of Woelke (1972) and American Society for Testing and Materials
Committee E-35 on Pesticides (1978).
For the larval eastern oyster (Crassostrea virginica) studies,
individual sexually mature females, held in glass chambers that con-
tained 1 z of filtered (5 pm) seawater, were induced to spawn by
increasing the water temperature from 29 to 35°c- Viable sperm
excised from the gonad of a sexually mature male oyster were added to
each chamber. Fertilization occurred upon, release of the eggs into the
spawning chambers and was confirmed microscopically. Fertilization
success was 91%. Population density of the embryos was determined by a
Sedgwick-Rafte^ count of 1:10 dilution (1 mi embryo suspension to 10 mi
seawater) of water from the spawning chamber.
Concentrations for definitive tests were based on the results of
48-h range-finding tests. All concentrations and the control were in
triplicate. Test containers were 1 I glass jars, each of which con-
tained 900 m£ of filtered (5 ym) natural seawater. Test concentrations
were prepared by adding appropriate weighed amounts of the pesticide to
each test container. If a solvent/carrier was required, the pesticide
was dissolved in reagent grade triethylene glycol (TEG) and the appro-
priate amounts added to the test containers. A solvent control was
also maintained to which was added the maximum volume of TEG.
Each test container was inoculated with an estimated 30,000.
embryos within 1 h after fertilization, then maintained at 25 + 1°C
in a light- and temperature-controlled environmental chamber.
After 48-h exposure, the larvae from each container were collected
and preserved seDarately in a constant volume of filtered seawater with
neutralized formalin. The number of normally developed 48-h larvae
was determined by a Sedgwick-Rafter count from each triplicate test
concentration container and each control container.
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Percentage reduction of normal embryos was determined as:
Number of normal 48-h control embryos minust the number
Percentage _ of normal 48-h embryos in each test concentration ^
reduction ~ Number of normal 43-h control embryos
Static Copepod Test
A static, 96-h toxicity test was conducted with the copepod
(Acartia tonsa) exposed to methyl parathion. The test was conducted
in 50 x 90 mm glass crystallizing dishes, each containing 100 nu of
test solution and 10 adult test animals. Test concentrations and the
control were in triplicate, resulting in 30 animals per treatment.
5a 1inity was 22 °/oo, and temperature was maintained at 22 + 1°C.
Based on results of a range-finding test, copepods were tested at
nominal concentrations of 13 to 100 yg/£. Test concentrations were
prepared by pipetting appropriate weighed amounts of methyl parathion,
dissolved in TEG, into each container. A solvent control was also
maintained, to which.was added the maximum volume of TEG (0.1 mi) that
had been added to the treatment test containers. The seawater control
received neither test material nor TEG.
Static Invertebrate and Fish Tests
Test methods used for static 96-h toxicity tests with mysid
shrimp, penaeid shrimp, and fish were generally those of The Committee
on Methods for Toxicity Tests with Aquatic Organisms (1975). The
pesticides tested and species exposed are shown in Table 1-a. Stock
solutions of each pesticide, except acephate, were prepared by dissolving
appropriate quantities of each in TEG. Acephate was dissolved in distilled
water. By using Hamilton microliter syringes, stocks were added directly to
filtered seawater in jars or culture dishes. Solutions were stirred with ten
vigorous swirls with a glass rod and allowed to equilibrate in an incubator or
water bath for 30 minutes before addition o^ test animals. Twenty animals were
exposed in each concentration of insecticide and in each of two control groups.
One control contained seawater and TEG carrier at the same concentration as
those used in the test solution chambers; the second control, seawater only.
Juvenile M, bahia less than 24-h-old and st.ylirostris postlarvae were fed
48-h-old Artemia larvae during the tests to limit cannibalism or starvation.
Mortality was recorded daily, and dead animals were removed when discovered.
Test temperature for all studies was 25 + 1°C; test salinity, 20 °/oo.
Dissolved oxygen values and pH, where monitored, are in Appendix C-3.
Flow-through Hacrocrustacean and Fish Tests
Methods for all 96-h flow-through tests were those described by
the Committee on Methods for Toxicity Tests with Aquatic Organisms
6
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(1975). Five concentrations and a seawater control were used for
acephate, aldicarb, carbophenothion, ethoprop, methyl parathion, and
phorate. At least four concentrations and a seawater control were
used for EPN and DEF. A TEG solvent control and a seawater control
were used in studies on carbophenothion, EPN, methyl parathion, and
phorate. Selection of concentrations for each test were generally
based on the results of 96-h static tests. Pesticides studied, the
species tested, and the laboratory and researcher conducting each
study are listed in Table 1-b. Dissolved oxygen values and pH, where
monitored, are in Appendix C-l.
The acephate tests were conducted in an open diluter constructed
to deliver 1.0 i/cycle/test aquarium at a dilution rate of 60%.
Each test aquarium contained 42 i of control seawater or test solu-
tion; the average number of cycles was approximately 6 per h,
providing 3 volume additions every 24 h. The stock solution, prepared
with deionized water, was delivered to the chemical mixing chamber by
a 15-mA glass "dioping bird" to produce 5 test concentrations.
To begin the acephate test, the diluter cycled for about 24 h to
equilibrate the system. Then, 10 fish were placed in each dupli-
cate test aquarium, i.e., 20 fish per treatment. Salinity, tempera-
ture, dissolved oxygen (DO) concentrations, and pH were measured
periodically during each test. A 450-iu water sample was taken on Day
1 and 4 from each duplicate aquarium, and samples for each day were
combined in amber-glass bottles. Samples were preserved, with 15 m of
chloroform {CHC13) for later chemical analysis.
Aldicarb tests were conducted in a closed diluter designed to de-
liver at a dilution rate of 60%. The test species and test aquaria
volumes were: M. bahia, 8.5 i\ P_. duorarum, 8.5 i\ C. variegatus,
53 i\ and U rhomboides, 52 1. At least eight volume additions per
day were provided for the crustacean tests; at least two for the f^sh
test. Triethylene glycol was used as a solvent.
Flow-through acute toxicity tests with carbophenothion were con-
ducted by BMRL on the following species: M. bahi a, C. vari egatus, and
L. rhomboides. An open diluter (1 z/cycle/42 1 test aquarium) was
used for the mysid test; closed, 1-*, diluters were used for the fish
studies. Aquaria volume for the C. varieqatus test was 53 I; that for
the pinfish study was 52 1.
All flow-through acute toxicity studies with ethoprop were con-
ducted by BMRL, using closed diluters. At least 2 volume additions
were provided daily to each 52- or 53-* test aquarium. Solvent for
all tests was TEG.
All remaining flow-through penaeid shrimp and fish tests were
7
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conducted at ERL,GB with the pesticides EPN, carbophenothion, DEF,
methyl parathion, and phorate. Appropriate quantities of the
pesticides, dissolved in TEG, were metered by syringe punDs at 10 ml
day into the filtered seawater that entered each aquarium from glass
siphons calibrated to deliver 30 i/h. One control aquarium received
the same quantities of seawater and solvent but no insecticide; a
second received only seawater. Twenty animals per aquarium were
exposed to each concentration for 96 h. Mortality was recorded daily,
and dead animals were removed when discovered. Fishes and pink shrimp
that survived the EPN test were rinsed with acetone and analyzed for
whole-body residues.
Flow-through toxicity tests with mysid shrimp, because of the
shrimp's small Size, required several modifications in the above pro-
cedures (Nimmo et al., 1977). Tests with acephate, aldicarb,
carbophenothion, ethoprop, and methyl parathion used the di^uters
previously described. Tests with DEF, EPN, and phorate used syn'nge
pumps and calibrated siphons described earlier for fish and shrimp
tests with these pesticides. TEF, EPN, phorate, and methyl parathion
were dissolved in TEG or acetone; therefore, a solvent control was
provided. Juvenile mysids were placed in test containers that
consisted of glass Petri dishes to which a 150-mm-high nylon screen
collar (210 to 315 ym mesh opening) was attached with silicone
sealant. Test containers were placed in each aquarium, resulting in
20 mysids per treatment. Self-starting siphons in the aquaria caused
water depth to fluctuate, ensuring an exchange of water in containers.
Mysids were counted by gently lifting containers from the aquaria,
draining water through the nylon screen to the depth of the Petri
dish, and placing them on a back-lighted table. Mysids were fed 48-h-
old Artemia nauplii.
When dissolved oxygen concentrations in water of static and
flow-through tests were less than 50% saturation—a violation in the
procedures in the method stipulated by The Committee on Methods for
Toxicity Tests with Aquatic Organisms (1975)--data were presented in
Tables 4, 5, and 3, but were appropriately footnoted. Several LC50
values in Tables 4, 5, and 3 were from tests in which species recom-
mended in the method were used, but control mortality was excessive.
Penaeus stylirostris was not a species recommended by the Committee.
CHRONIC TOXICITY TESTS
Mysid Shrimp Entire Life-cycle Test
Acephate test (BMRL)
The acephate entire life-cycle test was conducted in an open dil-
uter constructed to deliver 0.5 2/cycle/test aquarium at a dilution
rate of 60%. The stock solution (1.932 g technical acephate/z
3
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deionized water) was delivered to the chemical mixing chamber by a
13.2-nu, glass "dipping bird" to produce five nominal test
concentrations that ranged from 1,300 to 10,000 ug/i. A seawater
control was also maintained; there was no solvent control.
Aldicarb Test (BMRL)
The aldicarb test was conducted in a closed diluter constructed
to deliver 0.5 Ji/cycle/test aquarium at a dilution rate of 60%. The
stock solution, prepared with reagent grade TEG (19.5 mg technical
aldicarb/100 mfc TEG), was delivered to the chemical mixing chamber by
a 50-mfl. glass syringe to produce five nominal test concentrations
that ranged from 0.6 to 5.0 ug/a. A seawater control and a solvent
control were maintained.
EthoDrop Test (BMRL)
The ethoprop test was conducted in a closed diluter constructed
to deliver 0.5 Ji/cycl e/test aquariun at a dilution rate of 60%. The
stock solution, prepared with reagent grade TEG (10.0 mg technical
ethoprop/100 ma, TEG), was delivered to the chemical mixing chamber by
a 50-mi, glass syringe to produce five nominal test concentrations that
ranged from 0.3 to 2.0 ug/A- A seawater control and a solvent control
were maintained.
In all aforementioned tests, the diluter cycled for about 24 h
to allow the system to equilibrate before adding test animals. The
test aquaria contained aporoximately 8 £ of control seawater or test
solution; the average number of cycles was approximately 6/h,
providing >9 volume additions every 24 h.
To begin the test, five mysids, 34- to 48-h-old, were placed in
each test container and the containers were placed in the aquaria.
The containers were glass Petri dishes to which a 15-cm-high nylon
screen collar (Nitex HC-315 um mesh opening) was attached with
silicone sealant. Two test containers were placed in each of two
sections of one aquarium of all treatments, providing duplicates (a
total of 20 mysids). Recorded during the test were number of dead
nysids, time to formation of brood pouches, and number of offspring.
Mysids were fed 48-h-old Artemia nauplii ad 1ibiturn, daily throughout
the test. After hatching occurred, a maximum of 10 juvenile progeny
were isolated within each duplicate test container for continued
exposure and observation. To observe mysids in the test containers,
each container was lifted gently from the aquarium, the water allowed
to drain through the screen to the top level of the Petri dish, the
container then placed on a back-lighted table, and the number of
living animals counted. Duration of these tests was 28 days.
Salinity, temperature, 00 concentrations, and pH were measured
periodically during each test. A 450-nu water sample was taken from
each duplicate for chemical analysis on Days 1, 7, 14, 21, and 28 of
9
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each test. The two samples from each treatment were combined in an
amber-glass bottle and preserved with 15 mi of chloroform (CHCl^)
for later chemical analysis.
EPN, Phorate, Carbophenothion, and DEF Tests (ERL,GB)
Test methods for the mysid EPN, phorate, carbophenthion, and DEF
entire life-cycle tests were those of Nimmo et al. (1977). These
tests employed syringe pumps and calibrated siphons. EPN, phorate,
and DEF dissolved in a TEG carrier were metered by syringe pumps into
seawater at 10 mi/day. Carbophenothion dissolved in TEG was metered
into seawater at 5 m£/day.
The seawater, delivered by calibrated siphons, was mixed with the
dissolved toxicant and entered each experimental or control aquarium
at the rate of: 24 1/h for the EPN and phorate tests, 25 £/h for the
carbophenothion test, and 30 %Ih for the DEF test. Two controls were
provided for each test (one control with and one control without TEG
carrier). TEG carrier concentrations were: 0.017 mi/i for EPN and
phorate, 0.008 nu/i for carbophenothion, and 0.014 mi/i for the DEF
test. Nominal toxicant concentrations selected were: 0.075, 0.1, 0.3
and 3.0 gg/i. for EPN; 0.03, 0.08, 0.16, and 0.32 ggji for phorate;
0.18, 0.375, 0.75, 1.5, 3.0, and. 6.0 yg/£ for carbophenothion; and
0.5, 1.0, 2.0, and 4.0 yg/z for DEF.
The number of water additions per day for each test was: 12 for
carbophenothion, EPN, and phorate tests; >14 for the DEF test; and 8
for the methyl parathion test.
Methyl Parathion Test (ERL,GB)
The methyl parathion test employed a 1-i diluter similar to that
of Mount and Brungs (1967) that delivered, an intermittent flow of test
water to the test chambers. Methyl parathion was dissolved in three
parts TEG, and one part deionized water mixed with seawater, and
delivered to the test chambers about every four minutes. Two controls
were provided for the methyl parathion test (one with and one without
the TEG carrier at 0.016 ml!i)• Nominal toxicant concentrations
selected for this test were: 0.095, 0.19, 0.375, and 0.75 ug methyl
parathion/* seawater.
In each test (EPN, phorate, carbophenothion, DEF, and methyl
parathion) as each test chamber achieved its maximum volume, a self-
starting siphon drained the water to a volume of 1 I. Fluctuating
levels of seawater ensured an exchange of water within each aquarium
and test container. Toxicant-laden test water was introduced into the
test aquaria 24 h in advance of test animal exposure.
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To begin each test, forty 24- to 48-h-old juvenile mysids (5 per
test container) were placed in each concentration of toxicant and the
two controls. The test containers were 100-nm glass Petri dishes to
which a 15-cm-hiah nylon screen collar ('litex HE-210 um mesh) was
attached with silicone sealant. Mysids were fed 43-h-old Artemia
nau pii i ad libitum daily throughout the test. Duration of the tests
was 28 days, except for methyl parathion which was 24 days. During
this time, juveniles matured, became sexually active, and females
produced multiple broods.
The total number of live mysids (by sex) and the number of
progeny produced in each test container were recorded daily; dead
mysids and progeny produced were removed daily. A bottom-lighted
table was used to make daily observations of the test animals. Test
containers were cleaned or replaced, as required, to ensure a proper
exchange of seawater through the nylon mesh collars throughout the
test.
One-liter water samples taken weekly from each test chamber and
controls were analyzed for pesticide content.
Grass Shrimp Partial Life-cycle Toxicity Test
Test methods used for the grass shrimp carbophenothion partial
life-cycle test were modified from those of Tyler-Schroeder (1978a,
1979). Laboratory-reared juvenile shrimp (12 to 19 itfi rostrum-telson
length) were used in the partial life-cycle test. Ovigerous females
were collected from a natural population near ERL,GB. Larvae were
hatched and reared to the onset of sexual maturity (approximately 1.5
months) in a flow-through system, using filtered, ambient seawater
from Santa Rosa Sound. 3hotoperiod during this time was 4 to 6 h and
was maintained, using 15-watt incandescent bulbs (Tyler-Schroeder,
1978b).
All exposures were conducted in a flow-through system, using a
modified Mount and Brungs diluter (Mount and Brungs, 1967) to deliver
0.5 i/cycle/tank, with a dilution ratio of 50%. The number of cycles
averaged 513/day. The exposure aquaria contained approximately 57 i
and there were 4.5 volume additions every 24 h. The 3 test aquaria
were '40.6 cm wide x 15.2 cm deep x 91.4 cm long. Egg trays used in
the reproductive portion of the test were 29.8 cm wide x 13.3 en deep
x 55.9 cm long and nested on two strips of glass glued to the inside
of the test aquaria. Stock solutions of carbophenothion were prepared
in triethylene glycol (TEG) to give nominal concentrations of 0.125,
0.25, 0.50, 1 .0, 2.0, and 4.0 ug carbophenothion/*, water. There was
also a seawater and a carrier control. The highest test concentration
was essentially the same as the 96-h LC50.
Grass shrimp were exposed to carbophenothion in a partial life-
cycle to determine its effect on all- aspects of development. One
hundred laboratory-reared juveniles (12- to 19-mm rostrum to telson
11
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length) were randomly -distributed to each test aquarium. The parental
generation was observed daily, dead shrimp removed, and population
counts were made on Days 1, 33, and 249 (test termination).
The light regime for the first two weeks was 3-h, provided by 15-
watt incandescent bulbs. After this period, to allow for uptake of
chemical by the shrimp, spawning was induced by increasing photoperiod
to 10-h, provided by 100-watt incandescent bulbs. The light regime
was increased further by a 47-minute increment at 2-week intervals
until a regime of 14 h 1ight:10 h dark was reached.
. To determine the. effects of carbophenothion on reproduction, the
number of spawning females per concentration was recorded daily. The
first 10 mothers were stripped of their eggs to determine individual
production. The next ten ovigerous females were placed in a hatch-
ing apparatus (Tyler-Schroeder, 1979) that emptied into individual
containers. These containers were constructed of 1-z heavy duty
beakers with a 4 cm diameter bole approximately 1 cm from the bottom
and covered with 363 um Nitex screen, similar to those described by
Buchanan et al. (1975). A self-starting siphon in each egg tray
provided the fluctuating water levels which ensured an exchange of
exposure medium in each beaker.
Larvae that hatched into beakers were exposed throughout their
development to the juvenile .stage to the same concentration as their
parents. They were counted and fed fresh Artemia nauplii daily. As
larval survival was seemingly affected by test conditions other than
toxicant concentration (i.e., wide salinity fluctuations, low dis-
solved oxygen, and presence of hydrogen sulfide), multiple replicates
of larval survival were run in each concentration. Ultimately, sur-
vival data from only the first 10 days of exposure were used.
After 35 days of exposure during larval development, the rostrum-
telson lengths of the postlarval shrimp were measured. Postlarval
exposure to carbophenothion was continued past Day 35 to test termina-
tion. 3ecause of differences in hatch times, postlarvae at test
termination varied in age from 45 to 78 days. The age was noted for
each group and used in the statistical analysis.
At test termination, shrimp from parental and generation
populations were measured individually (rostrum, to telson length).
All shrimp from each exposure concentration were sacrificed and
weighed together, then frozen and placed in glass tubes for residue
analysis.
Sheepshead Minnow Embryo/Juvenile Tests
Twenty-eight day embryo-juvenile toxicity tests were conducted
with the insecticides aldicarb, carbophenothion, ethoprop, and phorata
12
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to determine effects on embryonic development, hatching success, sur-
vival, and growth of hatched sheepshead minnows (Cyprinodon
variegatus).
Aldicarb and Ethoprop Tests (BMRL)
Sheepshead minnow embryos for the aldicarb and ethoprop embryo-
juvenile tests were obtained from fish collected from Big Lagoon, a
Gulf of Mexico estuary adjacent to BMRL. Eggs were obtained from
females whose egg production was enhanced by injections of human
chorionic gonadotrophic hormone on two consecutive days. The eggs
were fertilized by the addition of a sperm suspension made from
macerated testes excised from adult male fish.
Both tests were conducted in an intermittent-flow system by using
a proportional diluter (Mount and Brungs, 1967) constructed to deliver
1 Jt/cycle/test aquarium at a dilution rate of 60%. ror each test, a
stock solution of the pesticide was delivered into a mixing chamber
where it was diluted, then siphoned down into the chemical cells. A
Teflon solenoid valve, controlled by a float switch, regulated sea-
water flow into the water cells. Mixing of uncontaminated dilution
seawater with test solution in the chemical cells produced the five
concentrations of test solution that were distributed to the
appropriate test chambers.
Five concentrations, a control, and a solvent control were all
duplicated. Selection of concentrations was based on the results of
96-h flow-through tests.
Light- was provided by two 3.7-m fluorescent bulbs suspended 53
centimeters (cm) above the test containers, providing approximately
1,300 lux incident to the water surface. Photoperiod was 12 h.
To begin each test, the diluter cycled for about 4 days to permit
system equilibration. Eggs and sperm were obtained as previously
described, and within 4 h after visual confirmation of fertilization,
two groups of 50 embryos each per treatment were placed randomly in
incubator cups (Pyrex tubing 51 mm in diameter and 75 mm in length
with 315 urn square mesh nylon screen attached to one end with silicone
sealant). The incubator cups were placed into embryo cup holding
chambers equiDped with automatic siphons which allowed the holding
chambers to fill and empty with each diluter cycle. Embryos were
removed from each cup by pipette daily, counted, and the cups washed
with seawater to clean the screens. This procedure was repeated until
all living embryos had hatched. Embryo mortality and time to hatch
were recorded. After hatch, 40 juveniles from each replicate were
impartially selected and placed in glass chambers (14 cm wide x 20.5
cm high x 26 cm long with 480 um square mesh ny'on screen over one
end). Juveniles were maintained in the growth chambers until the end
of the test and fed live brine shrimp (Artemia salina) nauplii daily.
Survival was monitored daily for 28 days posthatch and any changes in
13
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physical appearance of the fish or changes in behavior were recorded.
Growth of juveniles (standard length) was determined photographically
at the end of the exposure. All surviving fish were collected, pooled
according to treatment, wrapped in aluminum foil, placed in a glass
jar, and frozen for later chemical analysis.
Diluter function was checked daily by observation and weekly by
measurement of toxicant concentration. Water samples were collected
every 7 days, starting on Day 1; 450-mz samples were taken fron both
replicates and combined for each treatment.
Salinity and temperature were measured daily, except as noted.
The pH and DO concentrations were measured in one duplicate set of
test containers daily, except as noted.
Test chambers contained approximately 50 l of control seawater or
test solution and there were >2 volume additions every 24 h. The
stock solution 0.62 g aldicarb/z of TEG or 7.0 g ethoprop/z TEG was
delivered to the chemical mixing chamber by a 50-rru glass syringe to
produce the five nominal test concentrations that ranged from 17 to
134 ug aldicarb/i or 26 to 200 ug ethoprop/i. Another syringe
containing only TEG provided the test with a solvent control.
Carbophenothion and Phorate Tests (ERL,GB)
The exposure apparatus used for both studies was a modified Mount
and Brungs (1967) diluter similar to that used by Schimmel et al.
(1974). Seawater used in these studies was filtered (2.20 ym) t heated
to 30 + 2°C and aerated before delivery to the diluter.
Adult sheepshead minnows were collected near ERL.GB and accli-
mated to 30°C at ambient salinity for 5 days prior to hormonal
enhancement of egg production. Female sheepshead minnows (10 for the
carbophenothion test, 12 for the phorate test) were injected intra-
peritoneal ly with 50 I.U. of human chorionic gonadotrophs hormone on
two consecutive days, then manually stripped of eggs on the second day
following the final injection. Eggs were fertilized in approximately
100 m£ of 30°C filtered, seawater with sperm from macerated testes
from 9 male fish and held at 30°C for one hour before distribution.
Chemica1 analysis of excess eggs revealed no detectable chlorinated
insecticides or polychlorinated biphenyls (<0.05 ug/g carbophenothion;
<0.02 yg/g phorate).
In the carbophenothion test, the diluter cycled an average of 273
times per day, delivering 500 mi of exposure water to each of two 3.2
£ aquaria per concentration per cycle. A carbophenothion stock
solution was prepared in triethylene glycol to give nominal exposure
concentrations of 0.38, 0.75, 1.5, 3.0, 6.0, and 12.0 yg o^
carbophenothion/i of seawater. Average measured concentrations and
standard deviations in weekly composite water samoles were: non-
detectable (control = <0.05 yg/i), 0.36 _+ 0.053, 0.59 _+ 0.015,
14
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1.3 + 0.082, 2.8 + 0.34, 5.4 + 0.33, and 10.8 ug/i +_ 1.3 (n = 4 in all
cases except 10.8 yg/£ where n - 3). Triethylene glycol was delivered
to each toxicant concentration and the control at a concentration of
8.0 mg/«, of seawater.
In the phorate test, the diluter cycled an average of 243 times
per day, delivering 500 mz of exposure water to each of two 3.2 t
aquaria per concentration per cycle. A stock solution was prepared in
TEG to give nominal concentrations of 0.094, 0.19, 0.38, 0.75, 1.5,
and 3.0 yg of phorate/z of water. Average measured concentrations
and standard deviations in weekly composite water samples were: non-
detectable (control = <0.02), 0.16 + 0.04, 0.24 + 0.06, 0.41 + 0.18,
0.77 + 0.29, 1.2 + 0.60, and 2.4 yg/z + 0.49 (n = 4, except control,
where n = 5, and 2.4 yg/Z, where n = 2j. Solvent was delivered to
each toxicant concentration and the control at a concentration of 8.0
mg of TEG/z of seawater.
In each test, one hour after fertilization, 20 embryos we^e
distributed randomly in egg cups (Petri dishes with a 9 cm collar of
450 um nylon mesh). Distribution in the test system was: two egg
cups per aquarium, two aquaria per concentration, and six toxicant
concentrations and a control with carrier, or a total of 28 egg cups.
To ensure exchange of water in each egg cup, self-starting siphons
produced 5 cm fluctuations of water levels in aquaria approximately 60
times/day in the carbophenothion study; 18 times/day in the ohorate
study. Dissolved oxygen, measured weekly by the modified Winkler
method of Strickland and Parsons (1968), remained above 50% saturation
in all aquaria throughout each test. Salinity averaged 18.9 °/oo in
each test (range 8 to 28 °/oo in the carbophenothion test, 6.5 to
28 °/oo in the phorate. test). Aquaria were partially submerged in a
recirculating water bath that maintained the exposure temperature at
30 _+ 2°C- Photoperiod was 12 h. After hatching, fish were fed live
Artemia salina nauplii that contained no detectable (<0.05 ug/g)
carbophenothion and no detectable (<0.02 ug/g) phorate, chlorinated
insecticides, or PCBs.
Embryos and hatched fish were observed daily for mortality and
signs of poisoning. On Day 28, fish in each egg cup were ohotographed
for measurement of standard length. Fish from each treatment in the
carbophenothion test were preserved for pathological examination and
the renaining fish from 0.59, 1.3, and 2.8 ygii were sacrificed by
immersion in hot water, towel-dried, weighed, and frozen for later
chemical analysis. Fish in the phorate study were not preserved for
pathological examination, but were analyzed for phorate content.
Ten fish each from control, 0.36, 0.59, 1.3, and 2.8 yg carbo-
phenothion/z and four fish 'rom 5.4 ygfi were fixed in Davidson's fix-
ative prior to being embedded, sectioned, and stained by the methods
of Couch et al. (1979). Samples of fish were coded so the path-
ologist had no a priori knowledge of the sample's exposure history.
15
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Therefore, samples were classified as effect or no-effect groups
solely on histopathological findings.
Sheepshead Minnow Entire Life-cycle Test
A sheepshead minnow entire life-cycle toxicity test lasting 265
days was conducted with the insecticide EPN. Seawater was gravity-
fed -to a constant-head box, where it was heated to 30 + 2 C and
aerated. Ambient salinity water (average 24.9 °/oo, range = 6.5 to
32.5 °/oo) was then delivered intermittently to the fourteen 70-£
exposure aquaria by a 2-£ diluter similar to that of Schimmel et al.
(1974). Water flow from the diluter was divided so that 1 £ was
delivered to each of two duplicate aquaria in each of the seven
treatments. The exposure tenperature• was maintained at 30 +_ 2°c in
an upper and a lower water bath; each bath contained one aquarium for
each of the seven treatments. During each cycle of the diluter, 12 of
the 14 aquaria received 1 £ of seawater containing EPN and 6.7 g£ of
the carrier solvent TEG. The two control aquaria received the same
volume of seawater and TEG. The photoperiod was 12 h. Nominal EPN
concentrations were: 0.31, 0.62, 1.2, 2.5, 5.0, and 10.0 yg/£.
Average measured EPN concentrations and standard deviations of weekly
water samples in yg/£ were: nondetectable (control = <0.05); 0.25 +
0.09; 0.50 + 0.13; 0.88 + 0.22-, 2.2 + 0.38; 4.1 + 0.61; and 7.9 vq/i +
1.32 (n = 38 except in 4.1 and 7.9 ug/£, where n = 37 and in 0.25
pg/£, where n = 39).
The test began with embryos spawned from adult sheepshead minnows
that were collected near EPA's Gulf Breeze Environmental Research
Laboratory, maintained for 48 days at the same temperature and photo-
period used in the toxicity test, and injected with human chorionic
gonadotrophin to enhance egg production, as in the embryo/juvenile
fish study. Eggs from 17 females were incubated with macerated testes
from eight males for approximately 1 h in approximately 100.ru of
30°c filter seawater. Chemical analyses of excess eggs revealed no
detect- able EPN (<0.25 ug/g), organochlorine pesticides (<0.02 ug/g),
or polychlorinated biphenyls (<0.25 ug/g)•
Twenty fertile eggs were placed in each of three egg cups (9-cm
1.D. glass Petri dish bottoms to which 10-cm-high 450 gm nylon mesh
tubes were attached) per exposure aquarium. Egg cups were olaced in
trays within the aquaria that received water from the diluter; the
water then flowed to. the aquaria via self-starting siphons that caused
the water level within the trays to fluctuate approximately 5 cm and
vary the water volume in the tray from approximately 2.75 to 6.67 i.
Embryos and hatched fish were observed daily for mortality and
signs of poisoning. On Day 28, fish in each egg cup were photographed
for measurement of length and some fish from each egg cup were removed
and combined within duplicate treatments for analysis of E-'N content.
After replacing the self-starting siphon with a stand-pipe, ten fish
16
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from each of the three egg cups per duplicate aquarium were released
into the tray in the aquarium. The trays were removed on exposure Day
49 and the fish were released into their aquaria. All fish were also
photographed on Days 60, 88, 118, and 140.
We investigated the effect of EPN on sheepshead minnow reproduc-
tion in three spawning trials between exposure Days 150 and 231.
After fish attained reproductive size (>_26 mm S.L.), groups of three
females and two males were placed in glass spawning chambers in each
aquarium (345 x 190 x 230 mm high and a water depth of approximately
150 mm) (Hansen et al., 1977). The ends and bottom of the chambers
were covered with 6.4 mnr mesh stainless steel screen that allowed
eggs to fall onto a removable tray constructed of Plexiglas and 450
un nylon mesh. Trials one and three lasted 10 days; trial two, 14
days. Spawning groups were monitored in each of the 14 aquaria in all
spawning trials, except the third, where all spawning groups were not
available. Eggs were removed daily, counted, and examined micro-
scopically to determine percentage fertility. Adult fish from the
first and third spawning trials were sacrificed for analysis of brain
acetylcholinesterase activity and analysis of EPN content.
Where adequate numbers of eggs were obtained, we investigated the
effect of EPN on progeny of adults that had been continuously exposed
for 222 to 231 days by placing 20 fertile eggs in each of two egg cups
contained in trays located in the same aquarium in which the eggs were
spawned. Whenever eggs were not produced in one of the duplicates,
but were available from the other, eggs were transferred between dup-
licates. Extra eggs from the spawning trials were combined within
exposure concentration and frozen until analyzed for EPN. Embryos and
hatched fish were observed daily for death and signs of poisoning.
Fish were fed live brine shrimp nauplii. After a 28-day exposure,
surviving fish were photographed for length measurement, combined
within treatments, and frozen for later analysis of EPN content.
Benthic Animal Community Test
The effect of carbophenothion on colonization by macrobenthic
organisms was determined by comparing numbers and species of animals
that grew from planktonic larvae in contaminated aquaria for 3 weeks
and 3 days (March 8 - May 7, 1979) with those of animals that grew in
uncontaminated aquaria. Larvae entered the aquaria from the natural
component of plankton in flowing unfiltered seawater. We used 4 plex-
iglass apparatuses (shown in Tagatz et al., 1979), each consisting of.
a central constant-head box and 3 aquaria (40 cm long, 10 cm wide, and
12 cm high). Aquaria were filled to a depth of 5 cm with clean silica
sand (size range of 96% of the particles was 250-710 ym) dredged from
Santa Rosa Sound, Florida. Water levels in aquaria were maintained at
8 cm (3 cm above substratum).
-------
Seawater with its constituent plankton was pumped from Santa Rosa
Sound to a splitter box, where adjacent glass tubes supplied water
at a rate of 2 1/min to the 4 constant-head boxes that supplied water
to the aquaria. Flow to each aquarium was maintained at 200 mfc/min by
adjusting the height of a 3-mm diameter hole in the wall of the con-
stant-head box. Water flowed from each aquarium through a notched
end-opening; large predators escaped through these openings before
their numbers could drastically affect community structure.
Technical grade carbophenothion dissolved in triethylene glycol
(TEG) was metered by syringe pump into and mixed with the seawater
that entered the center of the constant-head box of each contaminated
apparatus. The same amount of TEG was metered into the control
apparatus. Desired concentrations of carbophenothion in seawater were
0.01, 0.1, and 1.0 pgy%
Samples of water were taken from constant-head boxes once a week
for carbophenothion analyses. At the end of exposure, meats of the
clam (Mulinia lateralis) were obtained rrom individuals in control,
0.01 ug/4, and 0.1 ug/I aquaria for carbophenothion analyses (pooled
sample of 5 g for each treatment).
After 8 weeks and 3 days, animals were collected by siphoning the
contents of the aquaria into a 1-mm mesh sieve, then preserved and
identified.
Bioconcentration Tests
Carbophenothion
Juvenile spot (Leiostoinus xanthurus, 24 to 36 mm standard length,
Y = 32.4 mm) were exposed to carbophenothion for 15 days followed by
a 4-day depuration period. The exposure apparatus was that of
Schimmel et al. (1977). A stock solution of carbophenothion dissolved
in 95% TEG: 5% acetone was prepared at the beginning of the test;
calculated deliveries were 5.0 and 50 gg carbophenothion/i seawater.
Each exposure concentration was duplicated, two control aquaria being
provided, one with and one without TEG. We selected these
concentrations because: (1) acute toxicity studies indicated that
they were well below the carbophenothion lethal threshold for spot;
and (2) they were quantifiable by gas chromatographic analyses.
Fifty-five juvenile spot were placed in each experimental and
control aquarium at the beginning of the test. Three fish from each
control, 5.0 and 50 ug/2 aquarium were sampled for bioconcentration at
Days 1, 2, 4, 8, 11, and 15. The chemical exposure was then termi-
nated and remaining fish were held in carbophenothion-free seawater
and sampled as they deourated on Days 1, 2, and 4. Seawater tempera-
ture averaged 24.6 Z (range = 24-25 C); seawater salinity averaged
23 °/oo (range = 16 to 30 °/oc).
18
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EPN
Juvenile pinfish (44 to 77 mm standard length, Y = 59 mm) were
exposed to EPN for 26 days, followed by an 3-day depuration period.
The exposure apparatus was that used in the carbophenothion test
(Schimmel et al., 1977). A stock solution of EPN dissolved in TEG was
prepared at the beginning of the test; calculated deliveries were 0.25
and 2.5 ug EPN/A seawater. Each exposure concentration was
duplicated, two control aquaria being provided, one with and one
without TEG. We selected these water concentrations, using the same
criteria as those in the carboohenothion test.
Thirty juvenile pinfish were placed in each experimental and con-
trol aquarium at the beginning of the test. Two fish from each con-
trol, 0.25, and 2.5 yg/£ aquarium were sampled for bioconcentration at
Days 1, 2, 4, 8, 15, 18, and 22. The chemical exposure was terminated
after 26 days, and remaining fish were held in EPN-free seawater and
sampled as they depurated on Days 1, 2, 4, and 3. Tissues were
analyzed on a whole-body, wet-weight basis. Seawater temperature
averaged 25°C (range = 23 to 28°C); seawater salinity averaged
25.7 °/oo (range = 22 to 31 °/oo).
PERSISTENCE
Log P
The octanol/water partition coefficient was calculated using
retention tine on a high pressure liquid chronatograph (HPLC) (Veith,
1979a). The instrument was a Waters Associates High Pressure Liquid
Chromatograph with a Model 440 Absorbence Detector operated at 254 nm.
The column was a Waters Associates Micro Bondapak C18 with the
theoretical plates in excess of 4000. The solvent system was 20%
water in methanol (v/v) at a flow rate of 1.0 nu/min. Column calibra-
tion standards were benzene (Rt 42 mm); biphenyl (Rt 65 mm); p.p'-DDE
(Rt 168 mm); and hexachlorobenzene (Rt 224 nm). A plot of Log Rt for
these standards versus Log P (n-octanol/water) was linear with r
for the correlation equal to 0.98. Using the equation for this
relationship, the retention times for methyl parathion, phorate, EPN,
DEF, and carboohenothion were determined by HPLC and converted to
Log P.
Persistence Test
The persistence of EPN, methyl parathion, phorate, DEF, and
carbophenothion was studied in the laboratory to resolve those
processes in marine environments that contribute most to the
pesticide's disappearance. The relative importance of nonbio-
logical and biological orocesses was investigated by eliminating or
amending the sediment and water components into three types of
19
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systems: one with water only (Seawater), a second with water and
sterilized sediment (Sterile), and a third with water and unsterilized
sediment (Untreated). Water was filtered (20 um) natural seawater
from Santa Rosa Sound; sediment (described in Table 3) was collected
from a salt marsh adjacent to Range Point, Santa Rosa Island, Escambia
County, Florida.
The experimental apparatus consisted of 150 mil Corex centri-
fuge bottles fitted with No. 6 Neoorene stoppers. A typical system
contained 10 g (wet weight) of sediment and 100 m£ of seawater (27
°/oo). Some of these systems were sterilized by addition of 5 n£ of
formalin to identify non-biological processes affecting pesticide
persistence. A series of systems we^e prepared that contained only
100 mi of seav/ater to better resolve the role of sediments in
pesticide persistence. Systems were fortified with 10 ug of pesticide
delivered in 10 of acetone, stoppered, and aerated at 50 nu/min.
Air exiting each system was passed through a column of Amberlite XAD-4
resin to trap vaporized pesticide. Systems Were incubated at 25°C
with 12-h photoperiod under G.E. White Fluorescent lights.
Duplicate unsterilized sediment and water systems were ana"!yzed
on Day 0 and 5; systems containing water or sterilized water and sedi-
ment were analyzed on Day 0 and 7. Contents of the bottles were cen-
trifuged at 1,600 G and the water ohase decanted into a separatory
funnel. Water and sediment were extracted and analyzed by procedures
in the analytical methods section. Pesticide was eluted from the
resin with acetone and quantitated by procedures described in the
analytical methods section.
In a separate experiment, the effect of sunlight on the persis-
tence of methyl parathion was investigated. We added 300 mg of methyl
parathion in acetone to a amber bottle and purged the acetone with
a gentle stream of nitrogen. Three Jl of seawater were added to the
bottle and it was agitated vigorously on a shaker at 25°C over-
night; 100 ma. aliquots were added to 250 ma Erlenmeyer flasks fitted
with ground glass stoppers. Half of the flasks were covered with
aluminum foil, and the other half were not covered. The systems were
exposed outdoors to direct sunlight and sampled on Days 0, 3, and 7.
Temperatures in the flasks varied from a high of 40°c during the day
to a low of 22°C at night. Duplicate samples were analyzed each
sampling day by procedures described in the analytical methods
section.
PESTICIDE ANALYTICAL METHODS (3MRL)
The following methods apply only to toxicity testing conducted by
BMRL, Pensacola on acephate, aldicarb, and ethoprop. These methods
and the instrument parameters are detailed in Appendix 5.
20
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PESTICIDE ANALYTICAL METHODS (ERL ,GB)
The following analytical methods apDly only to toxicity and per-
sistence testing conducted by ERL,GB on EPN', methyl parathion, phor-
ate, DEF, and carbophenothion.
Water
One liter of seawater was extracted twice with 100 m£ cf petro-
leum ether. The combined extracts were collected in a Kuderna-Danish
concentrator and evaporated on a steam table with a Snyder column to
no less than 20 nu. For further concentration, samples were trans-
ferred to a nitrogen evaporator with a water bath maintained at 35°c
and then analyzed.
Tissue
Phorate and Carbophenothion
One to ten grams of tissue were weighed into 150 mm x 25 mm .
screw cap test tubes and extracted four times with 5 nu portions of
acetonitrile, using a Will ems Model PT 10-ST polytron (Brinkman
Instruments, Westbury, M.Y.). The test tube was centrifuged after
each extraction and the combined extracts were diluted with 75 ml of
2% (w/v) aqueous sodium sulfate and extracted four times with 5 ru
portions of hexane. The hexane extracts were combined in a 25 mn
concentrator tube and evaporated with a gentle stream of nitrogen to
1.0 nu. The concentrate was transferred quantitatively to a Sep-9ak
silica cartridge (Waters Associates, Mil ford, Mass.) with two 0.5 nu
portions of hexane. Phorate or carbophenothion was eluted from the
cartridge with 15 mi of 1% (v/v) diethyl ether in hexane and analyzed.
Tissue concentrations were calculated on a wet weight basis.
EPri and DEF
Tissues were extracted similar to phorate except that the hexane
concentrate was transferred to a 9 mm Chromoflex column (Kontes Glass
Company, Vineland, N.J.) containing 3.0 gm of florisil topped with 2.0
gm anhydrous sodium sulfate. (The column had been previously washed
with 10 nu of hexane.) Following the transfer of the extract, the
column was eluted with 20 mi of %% diethyl ether in hexane to remove
PCBs and chlorinated pesticides. EPN or DEF was eluted with 25 mi of
10% isopropanol in isooctane and then analyzed.
Methyl Parathion
Tissues were extracted similarly to phorate, except that methanol
was substituted for acetonitrile, and the aqueous sodium sulfate/meth-
anol mixture was extracted with petroleum ether instead of hexane.
Methyl parathion was eluted from the Chromoflex column described for
21
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EPM tissues with 20 mil of 50% (v/v) diethyl ether in hexane and then
analyzed.
Sediment
Ten grans of sediment (wet weight) were extracted four tines with
25 nl of acetonitrile for 30 seconds with a Branson Sonifier Model
350. The combined extract was diluted with 1 i of seawater and
extracted according to the water procedure previously described. The
concentrated extract was then cleaned, using the method previously
described for tissue samples and then analyzed. Sediment concentra-
tions were calculated on a dry weight basis.
ANALYTICAL EQUIPMENT AND QUALITY ASSURANCE
Instrumentation
Analyses were performed with Hewlett-Packard Model 5730A gas
chromatographs, using either a Model 1879A dual ni trogen-p'nosphorus
flame ionization detector or a Nickel-63 linear electron capture
detector. Glass colunns (182 cm by 2 mm I.D.) were packed with either
2% SP 2100 on 100/120 mesh Supelcoport or 5% QF-1 on 80/100 nesh Gas
Chrom Q. Compounds were quantitated by peak area, using a Hewlett-
Packard Model 3352B Laboratory Data System.
Quality Control
The lower limit of detection for EPM, methyl parathion, DEF,
carbophenothion, and phorate in seawater was 0.02 ug/i. and in tissues
and sediment, 0.02 pg/g, unless otherwise noted. Recoveries from
fortified samples of seawater, tissues, and sediment were greater than
35?. Each set of samples analyzed included a fortified sample to
evaluate recoveries for that day. Analyses were not corrected for
percentage recovery of fortifications. The chemicals used in these
studies are described in Appendix A and were oositively identified by
mass spectrometry, using a Finnigan Model 400C EI/CI gas chromatograph
mass spectrometer.
ACETYLCHOLINESTERASE (AChE)
Acetylcholinesterase activity in brains of adult sheepshead
minnows continuously exposed to EPN was assayed by the pH-stat method
of Coppage (1971). For each AChE determination, nine fish brains were
divided into pooled samples of three brains each. Percentage AChE
inhibition was determined by comparison with mean nomal activity o^
control fish on each day of sampling.
22
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STATISTICAL METHODS
Algal Test
Nonlinear regression procedures (Rahner and Oglesby, in press)
were used to analyze the algal inhibition data. From the resulting
equations and parameter estimates, an approximate EC50 and 95%
confidence limits were established.
Oyster Embryo Test
Test concentrations from the oyster study were converted to
logarithms and the corresponding percentage reduction of normal
embryos was converted to probits to estimate the concentration that
would in 48 h reduce the number of normal embryos by 50% as compared
to the number of control embryos (48-h EC50) (Finney, 1971). The
estimated 95% confidence limits for the 48-h EC50 were then obtained.
To determine whether normal development of embryos in the solvent
control differed from that of the control, data were analyzed by
Student's t-test (Steel and Torrie, 1960). Differences, were
considered significant at a = 0.05.
Acute Toxicity Test
Mortality data from acute toxicity tests were analyzed by the
probit analysis method of Finney (1971), moving average, or binomial
test to estimate concentrations of insecticide in water that killed
50% of the test animals (LC50) and the 95% confidence intervals for
these concentrations. Abbot's correction was used in all cases where
control mortality was observed (Finney, 1971).
Mysid Shrimp Entire Life-cycle Tests
Acephate, Aldicarb, and Ethoprop
At the termination of the acephate tests by 3VRL, the differences
among the percentage mortalities of control and exposed rnysids were
determined for adults for each 7-day period by analysis of variance
(AN0VA), after an arcsin transformation (Neter and Wasserman, 1974).
Statistical comparison between the control and each concentration was
made by Williams' (1971) method. Significant differences in number of
offspring produced in different treatments were determined by AN0VA
and Williams' method. Differences were considered significant at a =
0.05.
Carboohenothion, DEF, EPN, Methyl Parathion, and Phorate
One way AN0VA and various post hoc tests were used to determine
i^ mortality or reorocluction were significantly affected (a = 0.05) in
mysid entire life-cycle tests conducted at ERL,G8. In the DEF and
carbophenothion studies, AN0VA with Duncan's procedure (Steel and
23
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Torrie, 1960) was used to detect significant differences in survival
and reproductive success. In the EPN study, ANOVA and Student-
Newman-Keul's (SNK) test (Steel and Torrie, 1960) were used to detect
significant differences in mortality while ANOVA and Dunnett's test
(Steel and Torrie, 1960) were used to detect significant differences
from control in reproductive success (mean number of progeny per
female). In the methyl parathion study, significant differences in
survival and reproductive success were detected by ANOVA and the SNK
test. In the phorate study, ANOVA and SNK post hoc tests were used to
detect significant differences in survival, and ANOVA with Duncan's
multiple range test were used to detect significant differences in
reproduction. .
Grass Shrimp Partial Life-cycle Tests
Carbophenothion
The adult mortality data were analyzed at Day 33 and Day 249
(end of test) of the exposure. An LC50 was generated, by either the
probit, binomial, or moving average method.
Spawning success was examined by comparing the total number of
shrimp that had spawned in each test concentration on four different
days during the test (Day 21, 84, 147, and 191), using a t-test with a
Bonferroni adjustment for the number of test run (Meter and Wasserman,
1974).
Lengths and weights of parent and generation shrimp were
analyzed by one-way ANOVA followed by Duncan's test to determine dif-
ferences between means.
Possible effect of carbophenothion on fecundity (egg production
and hatching efficiency) was determined by one-way ANOVA, using the
length of the female producing the eggs or larvae as a single
covariate. The length of ^emale shrimp is related to the number of
eggs produced, and, therefore, to the number of larvae to hatch
(Jensen, 1958). A post hoc test was performed on adjusted neans with
an adjustment to the a level for the number of tests performed.
Sheepshead Minnow Embryo/Juveni1e and Entire Life-cycle Test
At termination of the embryo/juvenile toxicity tests, the percen-
tage survival of embryos to hatching, juvenile survival, overall sur-
vival, and average lengths were calculated. Except for phorate, all
survival data were analyzed using analysis of variance (ANOVA).
Aldicarb and ethoDrop survival percentages we^e transformed using
arcsins and differences between treatments were determined using
Williams' test. Differences in survival between treatments in the
carbophenothion test were determined using Duncan's test. Chi-square
24
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tests (Meter and Wassernan, 1974) were used to analyze phorate survival
data. Analysis of variance arid Duncan's test (earbophenothion) or
Williams' test (aldicarb and ethoprop) were used to determine differ-
ences in growth. Analysis of covariance and Student-Newman Keul's
test (phorate) or Duncan's test (Neter and Wasserman, 1974) (earbo-
phenothion) were used to determine differences in growth. The covar-
iate was the number of fish in a particular exposure chamber summed
over the number of days over which length data were compared (fry-
feeding-days, FFD).
At termination of the EPN entire life-cycle toxicity test, per-
centage survival of embryos and fish, average' lengths, percentage fer-
tility, average number of eggs spawned, and AChE levels were calcu-
lated. Survival data for embryos and 28-day-old fish from the
parental and progeny generations and reproductive data were analyzed
by ANOVA; other survival data were analyzed by chi-square tests.
Growth data were analyzed by analysis of covariance using the
covariate FFD and Duncan's test. AChE activity of fish exoosed to EPN
was compared with that of control fish by Student's t-test.
All tests were performed, using a level of significance of 0.05.
Bioconcentration Tests
The statistical model of Banner and Oglesby (1979) was used to
determine steady-state bioconcentration factors (BCFs = steady-state
concentration in whole animals, wet weight, divided by concentration
measured in exposure water) and to describe uptake and depuration of
earbophenothion in spot and EPN in pinfish.
Persistence Tests and Log ?
Log P was estimated according to the method of Veith et al.
(1979b). A linear calibration of the logarithm of retention time
(relative to methanol) with the logarithm of the partition coefficient
(Log P) was determined using benzene, biphenyl- p,p'-DDE, and hexa-
chlorobenzene. The correlation coefficient (r ) was 0.98.
Persistence tests were run in duplicate, with the average values
being reported.
25
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RESULTS AND DISCUSSION
ACEPHATE
Results
Acute Static Toxicity Tests
Alqae--The 96-h EC50 for Skeletonema costatum exposed to acephate
was >50,000 yg/z (Table 4-a and Appendix C-l); the no effect
concentration was not determined.
Invertebrates--The toxicity of acephate to embryos of eastern
oysters (CrassosTrea Virginia) is shown in Table 4-a, Appendix C-2a,
and Appendix C-2b. The 48-h EC50 was 150,000 (8,000 to 300,000} ug/z.
The 96-h LC50 for juvenile pink shrimp (Penaeus duorarum) was >10,000
ug/ji (Table 4-a and Appendix C-3).
Fishes--The static acute toxicity expressed as the 96-h LC50s of
acephate to sheepshead minnows (Cyprinodon variegatus.) was >3,200,000
ug/i and for spot (Leiostomus xanthurus) was >100,000 gg/£ (Table 4-a,
Appendix C-4, and Appendix C-5X
Acute Flow-through Toxicity Tests
Invertebrates--The 96-h LC50 of acephate to mysid shrimp
(Mysidopsis bahiaT based on measured concentrations was 7,300 (300 to
190,000) ug/z (Table 5-a and Appendix C-6). The 96-h LC50 of acephate
to pink shrinp, based on nominal concentrations, was 3,800 (2,000 to
7,300) yg/z (Table 5-a and Appendix C-7). Ranges of DO and pH Treasured
in seawater used in these tests are given in Appendix C-3.
Fishes--The 96-h LC50 of acephate to pinfish (Lagodon rhomboides),
based on nominal concentrations, was 85,000 (7,800 to 933,000) ug/z
(Table 5-a and Appendix C-9). Sheepshead minnows were affected by
acephate and in some concentrations, completely lost equilibrium, but
only one fish died in the highest concentration tested (1,100,000
ug/l). The 96-h EC50, based on loss of equilibrium was about 910,000
ug/z (Table 5-a and Appendix C-10). Ranges of dissolved oxygen and pH
in seawater from these tests are shown in Appendix C-8.
iMysid Shrimp Entire Life-cycle Test
Mysid shrimp exposed to acephate in the 28-day test were adversely
affected. At Day 7, mortality was significantly greater in an average
26
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measured concentration greater than 4,200 ugM than in the control
(Table 6). At Day 14 and throughout the remainder of the exposure,
mortality was significantly greater in a concentration of 1,400 ug/£
than in the control. After 28 days, mortality of mysids exposed to a
concentration of 580 ug/£ was not significantly greater than mortality
of the control mysids (Table 6).
There appeared to be a difference in time to brood pouch formation
between mysids exposed to 4,200 ug/z and the control. Brood pouches
were first observed on Day 17 in control mysids, but on Day 19 in
mysids exposed to 4,200 ug/A. Time to brood pouch formation in mysids
exposed to concentrations <2,000 ug/£ was similar to that of the
control (Table 5).
The average number of offspring produced per female mysid exposed
to measured concentrations of 2,000 and 4,200 ug/ji was significantly
less than that produced by control mysids: 4.9 for mysids exposed to
2,000 ug/fc, 3.4 for nysids exposed to 4,200 ugh and 8.4 for the con-
trol (Table 7).
Survival of progeny of acephate-exposed mysids was not affected;
none died in any treatment.
The maximum acceptable toxicant concentration (MATC, highest con-
centration that produced no significant effect and the lowest concen-
tration in which significant effects were observed) of acephate, based
on measured concentrations, was >580 <1,400 ug/I and the application
factor limits (MATC divided by the 96-h LC50 in a flow-through test)
were 0.079 to 0.19.
Discussion
The acute toxicity of acephate is the lowest of the eight pesti-
cides tested, and is characterized either by high EC50 or LC50 values
or by no observed effect at concentrations tested (Table 4-a, 5-a, and
8). In static tests, acute intoxication in fish is characterized by
signs of cholinergic poisoning (erratic swimming, loss of equilibrium,
no reaction to gentle prodding, scoliosis, and rapid ventilation).
Other investigators have also noted that acephate is relatively
non-toxic. KlaverkanD et al. (1975) in acute lethality studies on
Salmo gairdneri with acephate at 15 C obtained 24-h LC50s of 395,000
ug/2. and 1,050,000 ug/£. Miyaska and Kobayashi (1974) reported the
48-h TLM to "young carp" to be 30,000 yg/ji and to "shrimp," 299,000
pg/2.. Duangsawaski (1977) found that aceDhate directly inhibited
cholinesterase activity in adult rainbow trout [S. gairdneri) and noted
a decreased heart rate and increased ventilation rate, which indicate
that the cardiovascular and respiratory systems of fish are important
sites for acephate toxicity. Lyons et al'. (1976) reported the 24-h
LC50 of Orthene (acephate) to frog (Rana clamitans) larvae exposed
in static bioassays to be 6,433,000 ug/i.
11
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After a helicopter spraying of acephate on a Maine spruce-fir
forest, Rabeni and Stanley (1979) studied stream biota from two
ecosystems, one within and one outside the sprayed a^ea. Post-spray
residues of acephate peaked (140 ug/Jt) within an hour after
application, and disappeared at about 40% per day. No mortalities of
stream biota were detected. However, acephate caused several
perturbations to the stream ecosystem; drift of benthos temporarily
increased; invertebrate standing crop remained unchanged; no dead fish
were observed; brain acetylcholinesterase activity was depressed in
suckers, but not in salmon or trout; brook trout altered their diet but
their growth was unchanged. Evidence from this study indicate that
acephate caused less impact on a forest stream ecosystem than other
spruce budworm insecticides.
USD I (1979) described intensive. management of coniferous forests
where pesticides are used against spruce budworm epidemics, noting that
potential contaminant problems exist for salmonid populations of the
western and northeastern states. Orthene (acephate) was considered
the most acceptable of five insecticides studied, but is not
extensively used due to limited availability and high cost.
Available data indicate that acephate may be the least toxic of
the insecticides tested. Acute lethality is not the manifestation of
concern. LC50s and EC50s were high for all species in both static and
flow-through tests. In the chronic toxicity test, there was little
difference between concentrations that produced chronic effects on
mysid shrimp and the 96-h LC50 to this invertebrate.
ALDICARB
Results
Acute Static Toxicity Tests
A1qae—The 96-h EC50 (the calculated concentration that would
inhibit growth by 50* in relation to control cultures) for Skeletonema
costatum was greater than the highest concentration tested, 50,000 yg/e
(Table 4-a; Appendix C-l).
Invertebrates--The amount of aldicarb necessary to cause a 50%
reduction in the number of normal oyster embryos in 43 h (48-h EC50)
was 8,800 ug/£ (Table 4-a;-Appendix Q-la and Appendix D-lb). "he
percentage "eduction of embryos that developed normally to the
straight-hinged, veliger stage after 48 h was from 9% in 1000 ugh to
71% in 32,000 ug/A. Aldicarb was most toxic to the two crustacean
species tested ("able 4-a). Mysid shrimp (Mysidopsis sahia) showed
sensitivity at 13 uQ/l (10-15) (Appendix D-2), whereas white shrimo
postlarvae (Penaeus stylirostris) had a 96-h LC50 of 72 ugh (65-82)
(Appendix D-Tf^
28
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Fishes—The 96-h LC50 of aldicarb to sheepshead minnows
(Cyprinodon variegatus) was 168 yg/J. and to spot (Leiostomus xanthurus)
202 yg/2 (Table 4-a; Appendix D-4 and Appendix D-5).
Acute Flow-through Toxicity Test
Invertebrates—The 96-h LC50 of aldicarb to mysid shrimp
(Mysidopsis bahiaT, based on measured concentrations, was 16 ugh
(13-20) (Table 5-a and Appendix D-6). The 96-h LC50 of aldicarb to
pink shrimp (Penaeus duorarun) based on measured concentrations, was 12
ug/2. (7.5-18) (Table 5-a; Appendix D-7). Ranges of DO and pH measured
in seawater used in these tests are given in Appendix C-8.
Fi shes—The 96-h LC50 of aldicarb for sheepshead minnows
(Cyprinodon variegatus) was 41 yg/2. (55-72) (Table 5-a and Appendix
D-8) and for pinfish (Lagodon rhomboides), 80 ug/i (43-150) (Table 5-a
and Appendix D-9). Ranges of DO and pH measured in seawater used in
these tests are in Appendix C-8.
Mysid Shrimp Entire Life-cycle Test
Mysid shrimp exposed to aldicarb in the 28-day test were adversely
affected by concentrations significantly less than the 96-h LC50
(Tables 5-a and 9). Significant mortality of parental generation
shrimp occurred after 14 days of exposure to measured test
concentrations of 1.5 and 2.1 yg/fc. The highest test concentration,
2.1 yg/i, had an average 40% mortality by the end of test. There
appeared to be a meaningful difference in time to brood pouch formation
between mysids exposed to 2.1 yg/£ and the controls (Table 9). Brood
pouches were first observed on Day 16 in the two controls and on Day 20
in mysids exposed to 2.1 yg/£. No significant difference existed
between the average number of offspring of mysids exposed to test
concentration of 1.5 yg/z and the average number of offspring produced
by control mysids. No offspring were produced by mysids exposed to 2.1
yg/Jl (Table 10). Survival of juveniles (Fi generation) in a post-
hatch exposure was not significantly affected after 4 to 8 days in
either controls or any exposure concentration. The MATC (maximum
acceptable toxicant concentration) based on measured concentrations was
1.0 to 1.5 yg/H, and the application factor limits were 0.063-0.094.
Sheepshead Minnow Embryo/Juveni1e Test
Hatching success of sheepshead minnows was not affected by
exposure to <38 yg aldicarb/*.. Percentage survival from fertilization
through embryonic development to hatching ranged from 91% to 98% in all
exposure aquaria. Embryonic abnormalities or hatching delays were not
observed.
A 28-day posthatch exposure to _<88 ug aldica^b/'z did not
significantly affect juvenile survival (Table 11), based on counts o^
29
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living fish on Days 7, 14, 22, and 28. The majority of the mortality
in all test concentrations occurred by Day 14 posthatch. No physical
abnormalities were observed among juvenile fish in any treatment.
Growth (mean standard length) of juvenile fish was affected at the
highest exposure concentration, 88 ug/2. (Table 12). Chemical analyses
of fish that survived each test exposure did not yield any residues of
aldicarb. The estimated MATC for embryos and juveniles of sheeoshead
minnows exposed to aldicarb was >50 <88 yg!%. The 96-h LC50 (44 ug/
Table 5-a) was lower than the MATC limits. No obvious explanation for
a lower LC50 value is evident. Bionomics Marine Research Laboratory
conducted both tests, and the measured aldicarb concentrations in water
from each test were approximately 50% of nominal and other critical
test conditions (i.e., DO, temperature, etc.) were similar.
An attempt was made by personnel at Bionomics Marine Research
Laboratory to conduct a life-cycle toxicity test with aldicarb and
the grass shrimp, Palaemonetes pugio. Poor water quality and rapid
growth of bacterial slime and algae in test tanks contributed to high
mortality in the solvent control and several of the test concentra-
tions. For these reasons, data from the test are not included here.
Discussion
Acute toxicity testing of aldicarb, in either static or flow-
through 96-h exposures, showed it to be more acutely toxic to
invertebrates than fish. The range of toxicity varied from 12 ugA to
72 ugh for marine invertebrates; and from 41 pg/ji to 202 yg/JZ. for
marine fish (Tables 4-a and 5-a). Freshwater fish have similar or less
susceptibility to aldicarb than estuarine species. Several
investigators found the toxicity to bluegill (Lepomis macrochirus) to
range from 76 to 100 pg/Ji and that of rainbow trout to range from 560
to 880 ygIl (Midwest Research Institute, EPA-540/1-75-013, 1975). A
saltwater phytoplankter (Skeletonema costatun) and oyster larvae
(Crassostrea virqinica) were much less sensitive than either estuarine
fish or invertebrate species tested (Table 4-a).
Longer-term life-cycle exposures of estuarine fish and inverte-
brate species to aldicarb once again revealed that invertebrate species
are more sensitive. Results of a life-cycle toxicity test with nysid
shrinp (Mysidopsis bahia) showed long-term effects to occur at signifi-
cantly lower concentrations (1.5 and 2.1 ug/z) than that at which acute
toxicity occurred (16 pg/1). Mortality of animals exposed to aldicarb
concentrations 2 1-5 ugh was significantly greater than that of con-
trols, but the difference did not become significant until Day 14 of
the exposure. At the highest concentration tested, 2.1 ug/I, there
were also effects on reproduction (no release of young), as well as
reduced survival.
30
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CARBOPHENOTHION
Results
Acute Static Toxicity Tests
A1gae—The 96-h EC50 for the marine diatom (Skeletonema costatun)
exposed to carbophenothion was the lowest among the eight pesticides
tested (Table 4-a, Appendix 'C-l). The 96-h EC50 and 95% confidence
interval was 109 (100-115) yg carbophenothion/fc. The no-effect
concentration was 10 yg/i..
Invertebrates—The 48-h EG50 and 95% confidence intervals for
embryos of the eastern oyster (Crassostrea virqinica) exposed to
carbophenothion were 99 (96-102) ygfi (Table 4-a, Appendix E-la, and
Appendix E-lb). Stock solutions chemically analyzed from this test
were 109% of nominal.
The 96-h LC50s and 95% confidence intervals for Mysidopsis bahia
and Penaeus stylirostris are shown in Table 4-a, Appendix E-2, and
Appendix E-3. These results were 13 (10-17) yg/i, for M. bahia and 4.3
(3.8-5.1) m9/i for P_. styl irostris.
Fishes--Sheepshead minnows (Cyprinodon variegatus) and spot
(Leiostomus xanthurus) had strikingly different static toxicity test
results when exposed to carbophenothion (Table 4-a, Appendix E-4 and
Appendix E-5). The 96-h LC50s and 95% confidence intervals were 17
(14-21) ug/i for £. variegatus and 500 (390-630) yg/i for L_.
xanthurus. Thus £. variegatus was 29 times more sensitive; a very
uncharacteristic difference as spot is typically more sensitive.
Acute Flow-through Toxicity Tests
Invertebrates--The 96-h LC50 values and 95% confidence intervals
for the invertebrate species are listed in Table 5-a, and Appendices
E-6 to E-10. Pink shrimp (Penaeus duorarum) was the most sensitive
species (96-h LC50 = 0.47 ug/£, 0.36-0.56) followed by M. bahia (3.0,
2.4-3.7) and wild Palaemonetes pugio (4.6, 3.8-6.2). Three acute
studies were conducted on separate groups of P_. pugio. Two groups
consisted of animals that were hatched in the laboratory; the third
was obtained from field collections. The LC50 values from three
groups were not appreciably different, ranging from 2.0 to 4.6 uq/l.
Fi shes—Four fish species were exposed to carbophenothion in
separate f 1 ow-t'nrough toxicity tests. Results of these studies (Table
5-a and Appendices E-ll to E-14) show a wide range in species
sensitivity. Sheepshead minnows were the most sensitive with a 96-h
LC50 of 2.8 (0.85-9.6) ug/i\ Menidia menidia (5.7-12) and pinfish
(Lagodon rhomboides,.4.6-12) nad identical LC50 values of 7.7 ug/Jl,
while spot (Leiostomus xanthurus) was relatively insensitive (96-h
LC50 >206 ugfx).
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Mysid Shrimp Entire Life-cycle Test
Effects of carbophenothion on nysid shrimp (Hysidopsis bahia) in
a 28-day life-cycle toxicity test were observed at concentrations rel-
atively close to the measured 96-'n LC50 of 3.0 yg/£ (Table 13). Mor-
tality of adult mysids exposed to 0.3 and 4.1 ug/£ was significantly
greater than nortality of controls. Mortality in the 0.30 pg/£ con-
centration was DrobaDly related to bacterial and fungal populations
enhanced by the presence of triethylene glycol (TEG) as a solvent car-
rier and not to the presence of carbophenothion. This is further
supported by the lack of significant nortality in the next three
higher concentrations. Mortalities in 4.1 yg/z were probably carbo-
phenothion-related because onset of poisoning was early and paralleled
effects seen in progeny.
Growth of parental mysid shrimp was significantly diminished in
concentrations of carbophenothion 1.2 pg/l and greater (Table 16). To
obtain more consistent measurements from photographs, lengths were
taken from the bottom of the cornea of the eyestalk to the statocyst
at the base of the telson. Therefore, lengths may appear shorter than
is normal for adult mysid shrimp.
Results of the reproductive portion of the test suggest effects
at 4.1 yg/£ (Table 14). The total number of offspring, the number of
offspring per female, and the number of days until onset of spawning,
while not statistically different, appeared impacted in this concen-
tration but not in any others.
Survival of juveniles for 10 days post release was significantly
reduced in 4.1 yg/j, (Table 15). Behavioral aberrations, including
abnormal and erratic swimming, were noted among juveniles reared in
1.9 and 4.1 yg/z.
The maximum acceptable toxicant concentration (MATC) of carbo-
phenothion for this mysid shrimp was >0.48 <1.2 ug/£, based on
statistically significant reduction in lengths. The measured 96-h
LC50 for this animal is 3.0 ug carbophenothion/£. The application
factor limits then are >0.16 <0.4.
Grass Shrimp Entire Life-cycle Test
Survival of adult shrimp in 2.9 yg/£, the highest concentration,
was significantly less than controls (Table 17). Survival of shrimp
on Day 33 was 24% in 2.9 yg/£ and 91 to 98% in all other treatments.
Survival at the end of the test was 45 to 75% for all treatments,
except 2.9 ug/£, where none remained.
The spawning of grass shrimp was significantly affected by
carbophenothion (Table 18 and Figure 1). Ovarian maturation and onset
of spawning, as measured by deposition of eggs on pleopods, first
occurred on Cay 32 in 2.9 yg/£-exposed shrimp. In contrast, it was
32
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first observed on Days 16 and 18 in controls and Days 14 to 13 in all
other treatments. The total number of spawning females was signifi-
cantly reduced compared to controls at concentrations of 0.36 ug/£ and
above.
Carbophenothion did not significantly affect the number of eggs
produced/female or the number of larvae to hatch in any exposure
concentration (Table 18). While egg counts were not made in the
highest concentration due to insufficient availability, it did not
appear that egg numbers were diminished. This is supported by the fact
that the number of hatched larvae was not affected in this concentra-
tion.
Repeated attempts to rear larvae under conditions of continuing
toxicant exposure were only partially successful due to salinity fluc-
tuations and other test complications (Appendix E-15). Therefore,
effects on larval survival were only noted for the first 10 days of
larval development.
Carbophenothion significantly diminished survival of larvae reared
in 2.9 ug/z, as compared to those reared in the controls and all other
test concentrations. Survival of larvae reared in 2.9 ug/i averaged
5%, while in all other treatments it averaged 82 to 90% on Day 10
(Table 19). Larvae hatched from five ovigerous females exposed to 2.9
ug/i were reared separately. Complete mortality occurred within 3 to 8
days in larvae hatched from three of the females and only 5% and 26% of
the larvae from the other two females survived to Day 10. All larvae
hatched in 2.9 yg/i were hyperactive, swam in a chaotic manner, and
their response to light was abnormal. Although larvae hatched in 1.3
ugh also demonstrated a disrupted response to light, survival was not
impaired.
Growth of shrimp was not significantly affected by carbophenothion
(Table 20). Weight measurements of all survivors of the 249-day
exposure did not reveal any differences between control and exposed
shrimp.
The average bioconcentration factor of carbophenothion in shrimp
tissue was low: 420X that measured in water (Table 21). Carbopheno-
thion was only detected in tissues of shrimp exposed to _>0.36 ug/£ in
water. It was these concentrations in which reproductive effects,
i.e., impaired spawning response, were noticed (Figure 1, Table 18).
The maximum acceptable toxicant concentration (MATC) for grass
shrimp, based on impaired reproduction, was >0.22 ygji <0.36 ugh and
the 96-h LC50 was 2.9 (2.5 to 3.3) The application factor limits
then are >0.076 <0.12.
33
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Sheepshead Minnow Embryo/Juvenile Test
Continuous exposure of sheepshead minnow embryos to carbopheno-
thion caused no significant difference (a = 0.05) in the percentage
that hatched (Table 22). Survival of embryos to hatching ranged from
85 to 95% per treatment.
Carboohenothion was lethal to hatched fish at concentrations of
11 and 5.4 yg/a (Table 22). All fish exposed to 11 ug/Ji died by Day 18
post-fertilization and 96% of those exposed to 5.4 ugh died within 28
days. Although survival of embryos or hatched fish was not signif-
icantly affected (a = 0.05) by exposure to 2.8 ug carbophenothion/z,
the cumulative effect was significant with only 79% survival through 28
days (Table 22).
The occurrence, frequency, and severity of signs of carbopheno-
thion poisoning were dependent upon the exposure concentration. Fish
exposed to 0.36 and 0.59 yg/i appeared unaffected. Although occasional
abnormal lateral flexure of the body was observed in fish exoosed to
1.3 yg/£ during the first week after hatching, these fish appeared
unaffected during the remainder of the experiment. Signs of poisoning
observed in fish exposed to 2.8 yg/A were the occasional occurrence of
darkened areas on the posterior one-third of the body, lethargy, abnor-
mal lateral flexure of the body, and an abnormally thickened appearance
when the fish were observed from a dorsal or ventral view. Signs of
poisoning observed in fish exposed to 5.4 and 11 gg/i were similar to
those observed in the 2.8 ugti exposure, except they did not exhibit
abnormal discoloration. However, muscular tetany was frequently
observed in these fish when the egg cups were placed on a lighted stage
for observation.
Although more food was available per fish in 2.8 yg carbopheno-
thion/2 because of significant mortality, these fish were significantly
shorter in standard length (a = 0.05) than those in lower concentra-
tions and in the control (Table 23). Fish exposed to 5.4 yg/£ averaged
9.7 mm standard length, but were excluded from the lengtn analysis
because there were so few survivors and there was much more available
food per fish than in other treatments.
The results of this study imply that the estimated MATC for
carbophenothion is between 1.3 and 2.8 ug/fc.
Carbophenothion accumulated in the tissues of fish surviving the
28-day experiment (Table 23). In the three concentrations for which
.residues were determined, the 3CF averaged 950 (range 780 to 1,200).
Pathological findings suggest a threshold of effect between 1.3
and 2.8 ug carbophenothion/i. Ove^ 60% of those fish examined from 2.8
and 5.4 yg/z had vertebral dysplasia consisting of aonormal thickening
(5 to 15X normal) of vertebral walls (Fig. 2), particularly in the
34
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caudal area and occasionally in the trunk region. Foci or clusters of
osteoblast-1ike cells were associated with the dysplasia, and osteo-
cytes were included within the thickened bone. Fish exposed to carbo-
phenot'nion concentrations <1.3 ug/jl were pathologically indistinguish-
able from control animals. This vertebral dysplasia is similar to, but
less pronounced than, the dysplasia reported by Couch et al. (1979) for
sheepshead minnows exposed to 5.5 ug/A of the herbicide trifluralin.
Benthic Aninal Conmunity Test
Concentrations (nominal) of 0.01, 0.1, and 1.0 ug carbopheno-
thion/*. had no significant adverse effects on the total numbers of
individuals and species of macrobenthic animals colonizing sand in
aquaria (Table 24). A total of 1,600 animals representing 41 species
of 7 phyla were collected from control aquaria and from those exposed
for 8 weeks, 3 days to carbophenothion. Numerically dominant phyla
were Molluscs, Arthropoda, and Annelida (Table 25). Averages and
ranges of measured concentrations of carbophenothion were: Control =
nondetectable (limit of detection was 0.05 ug/z); 0.01 ygfl nominal =
nondetectable; 0.1 yg/i nominal = 0.1 (0.06-0.25) ug/£; and 1.0 pgfl
nominal = 0.9 (0.52- 1.3) ug/fc. Seawater salinity averaged 20 °/oo
(5.5- 31.5 °/oo) and temperature 19°C (15.0-23.0°C).
After eight weeks and three days exposure, the clam (Mulinia
1 ateralis) bioconcentrated carbophenothion to 5,100 times that measured
in exposure water.
Bioconcentration Test
Spot exposed to 2.9 uG/£ (5.0 ug/£, nominal concentration) carbo-
phenothion bioconcentrated the insecticide rapidly, reaching 90% of
steady state in approximately 3 days (Figure 3). Uptake data for the
36 ug/£ (50 yg/*., nominal concentration) were not illustrated because
of excessive mortality. Mortality was not expected in this test
because the acute toxicity of carbophenothion to spot was not great
(96-h LC50 >206 ugfTable 5-a). At steady state, the concentration
of carbophenothion in whole spot exposed to 2.9 ygfi was 1.8 ug/g, a
bioconcentration factor of 620. In comparison, spot surviving a 96-h
carbophenothion exposure bioconcentrated an average of 140 X. Reasons
for this difference are unclear; however, the elevated concentrations
of carbophenothion in the water of the acute study (>135 ug/fc) may have
altered the rate of carbophenothion uptake.
After four days in carbophenothion-free seawater, only 10% of the
steady-state concentration (0.18 ug/g) was measured in spot tissues; no
fish were available beyond Day 4.
Persistence
The Log P for carboohenothion is 4.9 (Table 26). If this value is
35
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applied to the Log P/Loa BCF regression curve of Veith (1979a), the BCF
for fathead minnows (Pimephales promelas) is approximately 2900X. In a
28-day BCF study at steady state, spot (Leiostomus xanthurus) concen-
trated carbophenothion 620X.
Persistence tests on carbophenothion show it to be the most per-
sistent of the pesticides tested ("""able 27). Untreated and sterile
sediment/water systems retained 57% after 5 days and 61% after 7 days,
respectively, suggesting that biological processes are not responsible
for the degradation of carbophenothion. This is contrary to.studies
with the other pesticides. Carbophenothion was not detected in the
resin traps of any sediment/water systems, but in the seawater systems
all of the pesticide that was accounted for (41%) was present in the
resin trap. Presumably, hydrolysis in the seawater systems removed
over half of the pesticide and sorption to sediments in the sediment/
water system interferred with this degradative process and resulted in
increased persistence. The observations that carbophenothion was pur-
geable and adsorbed to sediment predict a low potential 'or persis-
tence in the water of aquatic systems. .'The sediment compartment re-
mains the key factor in the ecological implications of carbophenothion
in the aquatic environment.
Pi scusssion
The acute toxicity of carbophenothion to species of marine organ-
isms we tested is similar to that reported for freshwater species. The
96-h LC50 of the insecticide to the glass shrimp (Palaemonetes
kadiakensis) was 1.2 ug/jI (Sanders 1972); for various ages of the estu-
arine shrimp, P. puqio, the LC50 was between 2.0 and 4.6 yg/z (Table
5-a). Sanders X1969) reported that the 96-h LC50 for the scud (Gammarus
lacustris) was 5.2 ug/£. The LC50 or EC50 values we report for inver-
tebrate species range from 99 yg/2. for the larvae of the eastern oyster
to 4.3 ug/i for Penaeus stylirostris in static tests (Table £-a).
The BCF from the bioconcentration test was one-fourth of that cal-
culated for a freshwater fish. Our steady-state BCF in the spot study
was 620, that calculated for fathead minnows was 2900 (Veith, 1979b).
Persistence tests indicate tnat carbophenothion degradation is not
biologically mediated; hydrolysis may be responsible for some degrada-
tion of the compound. As in many compounds with chemical and physical
properties similar to carbophenothion, the sediment compartment is
likely to be the key to the fate of this insecticide in the acuatic
envi ronment.
36
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Carbophenothion has been found at concentrations from 0.103 to
0.109 yg/g in juvenile estuarine fishes from Texas and Maryland (Butler
and Schultzmann, 1978). Residues probably are associated with agricul-
tural activities in those areas. Our bioconcentration and persistence
data indicate that carbophenothion may not remain in yvater or tissues
for long periods of time; we assume that the fish were recently
exposed.
In summary, carbophenothion is acutely toxic to three invertebrate
and fish species at concentrations <20 yg/z. Chronic toxicity tests on
Mysidopsis bahia, Palaemonetes pugio, and Cyprinodon variegatus demon-
strate that concentrations ranging from 0.36 to 2.8 yg/z oroduce
adverse effects. Bioconcentration of carbophenothion by spot achieved
90% steady state in three days and depurated to 10% after four days;
the steady-state BCF was 620. Persistence studies indicated minimal
biological degradation, with the sediment compartment serving as a
sink.
DEF
Results
Acute Static Toxicity Tests
A1gae—The 96-h EC50 and 95% confidence interval for marine dia-
toms (Skeletonema costatum) exposed to DEF were 366 (315-391) yg/z
(Table 4-a, Appendix C-l); the no-effect concentration was 200 yg/z.
Invertebrates--The 43-h EC50 for embryos of the eastern oyster
(Crassostrea virqinica) exposed to DEF was 197 (120-323) yg/z (Table
4-a, and Appendix F-la and Appendix F-lb).
Two crustaceans exposed to DEF were mysid shrimp (Mysidopsis
bahia) and postlarval white shrimp (Penaeus stylirostrisT^ Results of
these acute static tests are shown in Table 4-a., Appendix F-2, and
Appendix F-3: the 96-h LC50s and 95% confidence intervals were 5.8
(4.4-6.8) yg/z for M. bahi a, and 25 (16-49) yg/z for P_. sty 1 i rostri s.
Mysids were roughly 4X more sensitive to DEF than were postlarval
penaeid shrimp.
Fishes--Sheepshead minnow (Cyprinodon variegatus) and spot
(Leiostomus xanthurus) acute static DEF toxicity test results (Table
4-a, Appendix F-4, and Appendix F-5) expressed as the 96-h LC50 and 95%
confidence interval were 440 (410-473) yg/z and 158 (135-320) yg/z,
respectively. The DEF toxicity to j_. xanthurus was approximately 3X
greater than that for C_. variegatus.
37
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Acute F1ow-through Toxicity Tests
In vertebrates--The 96-h LC50 of DEF to mysid shrimp, based on
measured concentrations, was 4.6 (4.2-4.9) ugh (Table 5-a, Appendix
F-6). The 96-h LC50 of DEF to pink shrimp (Penaeus duorarum) based on
measured concentrations was 14 (10-18) ugII (Table 5-a and Appendix
F-7); that for grass shrimp (Palaemonetes pugio) v/as 22 u
-------
importance of biological processes in the disappearance of the
pesticide. While none of the systems mentioned above displayed any .
detectable pesticide loss in the air, the DEF seawater systems
contained 12% of the original pesticide in the resin trap after 7 days.
Similar to previous studies with EPN, only 38% of DEF was recovered
from the water of the seawater systems after 7 days, with the remaining
50% loss attributed to hydrolysis in the water compartment. As in EPN
studies, sorption of DEF to sediments in the sterile systems reduced
hydrolysis either because the pesticide was not susceptible to
hydrolytic processes in a sorbed state or because biological processes
were responsible for hydrolysis in the water.
Pi scussi on
The .acute toxicity of DEF, a systemic herbicide widely used to
defoliate cotton and for the control of undesiraDle plant species, to
aquatic organisms is most pronounced in crustaceans. The static and
flowing acute results for M. bahia (Tables 4-a and 5-a) are not
statistically different. Also, our Penaeus sp. static and flowing 96-h
LC50s (Tables 4-a and 5-a) are both within 2X of an earlier 48-h LC50
by Lowe (personal communication: 1980, Jack I. Lowe, U.S. Environ-
mental Protection Agency, Environmental Research Laboratory, Gulf
Breeze, Florida). He determined that the 96-h EC50 (the concentration
of DEF causing a 50% decrease in shell growth) in eastern oysters
(Crassostrea virqinica) was 100 pg/z. The 48-h LC50 for brown shrimp
(Penaeus aztecus) was 28 pg/£; and the 48-h LC50 for spot (Leiostonus
xanthurus) was 240 yg/z. Sanders (1969) determined the acute static
toxicity of DEF to freshwater scuds (Gammarus lacustris). The 96-'n
LC50 and 95% confidence interval was 100 (78-150) pg/£.
Our toxicity data indicate that fishes are less sensitive to DEF
than crustaceans. The static 96-h LC50s for C. varieqatus and L_.
xanthurus were 440 (410-473) uq/i and 158 (135-320) ug/£. Flow-
through acute test results for L_. rhonboides, L. xanthurus, and C.
varieqatus were 130 (100-170) ug/£, 290 (240-379) pg/£, and >440 ug/t,
respecti vely.
Fabacher et al. (1976) reported a pronounced increase in the
toxicity of DEF by methyl parathion in mosquitofish (Gambusia affinis).
The 24-h LC50 of DEF alone to G_. affinis is approximately 300 pg/i. No
mortality occurred within 24 h in J3. affinis treated with 500 ug DEF/i.
In £. affinis treated with 5,000 ug methyl parathion/2 alone, mortality
was 3%. However, mortality was 89% in fish treated with 500 ug DEF/z
combined with 5,000 pg methyl parathion/i.
Caldwell et al. (1979) conducted toxicity tests with DEF on sev-
eral life stages of the Dungeness crab (Cancer magister). The percent-
age of hatched crab prezoeae successfully molting to the first zoeal
stage during the 24-h test period was affected at 100 ug DEF/iu Motil-
ity of developing first stage zoeae declined. At 10 pg/2. 80% were
39
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motile, and motility decreased to below 40% at 33 and 100 ug/z after
24-h exposure. Zoeae exposed to the high concentration (5.9 ug/*)
molted one day later than controls through the first three instars.
In long-term toxicity tests, zoeae survival exceeded 30% in controls
and in each concentration (0.04, 0.95, and 6.9 ug/z) of DEF until Day
50. Then survival decreased in all cultures until the test was
terminated on Day 69. Chronically exposed larvae were killed at
concentrations similar to those in acute toxicity tests. The lethal
threshold for DEF is approximately 6.9 ug/z. Survival of juvenile
c^abs was reduced at 270 ug DEF/z, but not at lower concentrations.
Adult crabs chronically exposed to DEF exhibited reduced survival at
380 and 2600 ugh compared to controls. Survival of adult crabs was
significantly reduced after 50 days at 380 ug/z and 32 days at 2,600
ug/z. The no-effect concentration for developing C. magister exposed
to DEF was estimated to lie between 0.95 and 6.9 ug/z.
In our life-cycle toxicity test with mysids, the lower value for
the MATC limits was not determined for M. bahia exposed to DEF since a
no-effect concentration was not observed. Therefore, the MATC for
reproductive effects is less than the lowest concentration, 0.34 ug/z.
The application factor is less than 0.074 (based on a chronic MATC of
<0.34 ug/Z and a measured acute flow-through 96-h LC50 of 4.6 ug/z),
which indicates a chronic exposure hazard at least an order of
magnitude greater than the acute toxicity.
Evaluation of toxicity and *ate data is necessary to predict the
hazard of DEF to marine organisms. Persistence tests indicate that
DEF degrades rapidly in the presence of estuarine sediments. Appli-
cation of DEF near coastal areas or watersheds could affect estuarine
crustaceans, even at exposure concentrations below 1 ug/z.
EPN
Results
Acute Static Toxicity Tests
A1gae—The 96-h EC50 for Skeletonema costatum exposed to EPN was
340 ug/z (Table 4-b and Appendix C-l), the second lowest of the eight
insecticides tested.
Invertebrates—The 43-h EC50 of 2,200 ug/z to eastern oysters
(Crassostrea virqinica) (Table 4-b, Appendix G-la and Appendix 3-lb)
was over 15 tines greater than that for sheepshead minnows (Cyprinodon
variegatus), the next "least sensitive species. Reduction in numDers
of oyster embryos that developed normally to the straight-hinged,
veliger stage after 48 h ranged from 4% in 320 ug/z to 76% in 5,600
ug/z (Appendix G-lb).
Acute toxicity of E^N to crustaceans (Mysidopsis bahia and
Penaeus stylirostris) is shown in Table 4-b, Appendix 3-2, and
40
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Appendix G-3. The 96-h LC50 and 95% confidence intervals for K. bahia
were 13 yg/z (in to 13); for P^. styl irostris, 4.6 yg/ii (2.4-9.0).
Fishes—The 96-h LC50 values and 95% confidence intervals for the
two fish species exposed to EPN differed greatly (Table 4-b, Appendix
G-4, and Appendix 6-5). Spot (Leiostomus xanthurus) were more
sensitive, LC50 = 37 yg/z (33 to 40), than sheepshead minnows, 140
yg/z (59 to 500).
Acute Flow-through Toxicity Tests
Invertebrates—The crustaceans, M. bahi a and P_. duorarum, were
particularly sensitive to EPN in flow-through tests (Table 5-b,
Appendix G-6, Appendix G-7). The 96-h LC50 and 95% confidence limits
for M. bahi a were 3.4 yg/2 (2.5 to 5.8 yg/z), a value nearly one-
fourth that measured in.static tests (Table 4-b). P_. duorarum were
more sensitive to EPN (96-h LC50 = 0.29 yg/z).
Fi shes--the 96-h LC50 values for the three fish species exposed
to EPN are listed in Table 5-b, Appendix G-8, Appendix G-9, and
Appendix G-10. Pinfish (U rhomboides) was the most sensitive fish
species with LC50 and 95% confidence limits being 18 yg/z (15 to 24
ug/z); spot, 26 yg/z (19 to 34 yg/z), and sheepshead minnows, 190 yg/z
(150 to 260 yg/z).
Mysid Shrimp Entire Life-cycle Tests
The lowest concentration of EPN that produced no adverse effect
on survival or fecundity was 0.44 yg/z (Tables 31 and 32).
A concentration of 4.1 yg EPN/z adversely affected both survival and
number of young produced per female (Table 32). Approximately 63% of
the deaths in 4.1 yg/z occurred during the final 14 days of the test
and may have been a result of the increased stress associated with
reproduction and accumulated stress of the toxicant. The number of
young produced was less in 4.1 yg/'z than in the control aquaria; and
in most chambers, no young were produced. Of nine females in 4.1
ug EPN/z, only two produced young during the test. This could have
been a result of toxicant-induced sterility in either male or female
mysids or of disruption of their sexual behavior.
The MATC for EPN is >0.44 <3.4 yg/z; the 96-h LC50 in a flow-
through test was 3.4 yg/z. Therefore, the application factor was 0.13
to 1.0 of the 96-h LC50. Because the limits on the MATC were about an
order of magnitude apart, the estimated "safe" concentration is poorly
defined.
Sheepshead Minnow Entire Life-cycle Tests
Survival and signs of poisoning in parental fish--Survival of
embryos to hatching and of hatched fish to Day 28 in the six EPN
-------
concentrations was not significantly different from that of controls,
but significantly fewer fish survived from Day 28 to Day 140 in
7.9 uQ/A (Table 33). Survival of embryos to hatching averaged 38%
(range, 33 to 94% per concentration) and survival of fish from hatching
through Day 23 averaged 97% (range, 96 to 99% per concentration).
Survival from Day 28 to 140 was significantly less in fish exposed to
7.9 ug/i than in controls. Overall survival of embryos to Day 140,
calculated from data on embryo, juvenile, and adult survival, appeared
to be concentration-dependent. Overal1.survival averaged 87% (range,
82 to 90%) for control fish and fish exposed to 0.25, 0.50, and 0.88
pg/l. Survival decreased from 73 to 67% as concentration increased
from 2.2 to 7.9 ug/£. Survival of fish from Day 140 to the end of the
test (Day 265) could not be analyzed because of mortality from exces-
sive handling or mortality associated with spawning trials.
External signs of poisoning observed in fish exposed to 2.2 ug/i
through Day 28 were lethargy and edema. Fish exposed to 4.1 and 7.9
ug/l exhibited lethargy, edema, and abnormal lateral flexure of the
body.
Signs of poisoning observed in juvenile and adult fish after
release from the egg cups were: occasional blackening of the poste-
rior third of the body of fish exposed to 0.25, 0.50, and 0.88 yg/£;
blackening plus reduced feeding, abnormal lateral flexure of the body,
and abnormal muscular tetany in fish exposed to 2.2, 4.1, and 7.9
Fish exposed to 4.1 and 7.9 yq/i were lethargic. Fish exposed
to 2.2, 4.1, and 7.9 ug/£ frequently experienced muscular tetany when
food was first placed in the aquaria. Generally, the frequency of
occurrence of these signs seemed dependent on the exposure concentra-
tion.
Growth of parental fish—Chronic exposure of sheepshead minnows
to EPN affected their growth (Table 34). Average standard lengths of
juvenile control fish at Day 28 were greater than lengths of fish in
each concentration tested. Thereafter, fish exposed to 7.9 ug/i were
always significantly shorter than control fish. Their lengths aver-
aged 2.6 to 3.9 ttti less than lengths of control fish between Days 60
and 140, (11 to 16% shorter than controls).
We believe that the significant decrease in lengths of fish
exposed to 7.9 ug/I may be biologically significant, but that signif-
icant decreases in lengths of fish in lower concentrations observed at
Day 28 and, in one instance, at Day 60 are probably not biologically
significant. Differences in lengths in these -ower concentrations at
Day 28 and 60 are within measurement error of about + 1 mm and are not
consistent throughout this experiment; thus, we doubt their biological
significance.
Reproduction--Egg production was extremely variable among the
three different spawning trials and between duplicates within the same
42
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trials (Table 35). Although control fish averaged 7.9 eggs per female
per day and fish in 7.9 ug EPN/2, averaged only 0.5 eggs per female per'"
day, the variability was so large that significant differences (a =
0.05) we"-e not observed.
Percentage fertility averaged >_97.9% in all concentrations,
except 7.9 ug/i, in which fertility averaged 93.8% and was signif-
icantly different from all other concentrations. However, statistical
analyses of fertility data revealed no differences between controls
and fish exposed during individual spawning trials.
When our experiment is compared with published accounts of the
effects of toxicants on sheepshead minnow reproduction in which data
are presented by spawning groups (Parrish et a 1 ., 1977; Parrish et
al., 1978; Goodman et al., 1979), this EPN test is the only experiment
in which there were control groups that did not spawn. The average
number of eggs oer female per day, for a single spawning group (3
females and 2 males) in control or control-with-carrier aquaria or
both, in these published studies ranged from four in a life-cycle
toxicity test with chlordane (Parrish et al., 1978) and 12 to 39 in a
life-cycle toxicity test with methoxychlor (Parrish et al., 1977).
Two difficulties were encountered in this experiment that prob-
ably contributed to the large variability in the reproduction data.
First, it was unusually difficult to determine the sex of the fish
when placing them in the spawning chambers. Sheepshead minnows are
sexually dichromatic, but differences in coloration are sometimes
subtle. At present, we do not know whether the lack of expression of
sexual dichromatism was due to genetics of the brood stock or to some
factor in our exposure system. Although we attempted to place 3
females and 2 males in each spawning group, the ratio was frequently
wrong when determined by dissection after trials I and III. The sex
of fish from spawning trial II was not determined by dissection
because we were reasonably confident of the sex »-atio and we needed to
keep the fish alive for subsequent experiments. Oata analyses were
based on the average number of eggs per female per day, but there may
have been differences in egg production attributable to the male/-
female ratio. Secondly, we had difficulty in maintaining DO levels
during the reproductive portion of the experiment (Table 36). Average
DO concentrations were 2.5 mgM in trial I, 3.6 nig/4 in trial II, and
3.3 mg/£ in trial III.
Survival, growth, and signs of poisoning in progeny—There was no
significant (a = 0.05) toxicant-related effect on survival of progeny
from fish continuously exposed to average measured concentrations of
0.25, 0.50, and 0.88 ug EPN/ji. Survival of embryos to hatching aver-
aged 38% (range, 81 to 92% per concentration); and of the fish that
hatched, an average of 96% (range, 93 to 100% per concentration)
survived through Day 23 (Table 37).
The average standard length of surviving progeny was unaffected
by EPN exposure through 28 days (Table 37). Average standard lengths
43
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of progeny ranged from 3.6 mm in 1.25 gg/i to 9.0 mm in 0.31 yg/Z;
'control fish averaged 3.9 mm.
No signs of poisoning were observed in progeny exposed to 0.25
and 0.50 gg EPN/£. Progeny exposed to 0.38 yg/z infrequently appeared
to be affected; signs of poisoning were those described for parental
fish.
MATC and application factor--The MATC for sheepshead minnows
continuously exposed to EPN at 30°C, lies between 4.1 and 7.9 ggh
based on: (1) significantly reduced survival from Day 28 to 140; (2)
reduced length of fish; and (3) reduced fertility of eggs from fish
exposed to 7.9 gg EPN/z of water. However, Cripe (Appendix G-ll)
determined the critical swimming speed of adult fish during this
exposure and found that the threshold level of effect on swimming
performance lies between 0.88 and 2.2 gg/z.
The application factor for sheepshead minnows, obtained oy divid-
ing the MATC limits based on reduced survival, growth, and egg fertil-
ity by the 96-h LC50 (190 yg/z at 25°C; Table 5-b), lies between
0.022 and 0.042. Although we are unaware of any MATC being based on
critical swimming speed, if the threshold levels reported by Cripe
(Appendix G-ll) were used to calculate the application factor, it
would lie between 0.0046 and 0.012.
Bioconcentration--EPN was bioconcentrated by all life stages of
the sheepshead minnow (Table 38). Concentrations in fish or their
embryos generally increased with increasing exposure concentration.
In embryos and 28-day juveniles, tissue concentrations were generally
less for the same concentration of exposure than in adult fish.
Concentrations in males and in females varied considerably, but seemed
to be similar.
Bioconcentration factors (3CFs) appeared to be greater in adult
fish than in juveniles or embryos (Table 39), averaging 1,000 for
juvenile fish, 5,800 for adult males, 5,300 for adult females, 1,300
for embryos from exposed fish, and 650 for juvenile progeny from
exposed parents. Bioconcentration factors in each of the three groups
of adult fish appeared to increase with increasing concentration of
exposure. 3ioconcentration factors averaged 2,100 for adult fish
exposed to the three lowest EPN concentrations and 7,700 for adult
fish exposed to the three highest EPN concentrations. This increase
in bioconcentration factor with increase in concentration of exposure
is unusual. This anomaly may occur because fish in the highest con-
centrations were adversely affected by EPN, which may alter jsual
uptake and depuration rates in adult sheepshead minnows.
Acetylcholinesterase activity—Acetylcholinesterase (AChE) inhi-
bition of parental fish from spawning grouDs I and III was directly
dependent on the exposure concentration (TaDle 40). Average AChE
activity throughout the study ranged from 38.0% inhibition in fish
44
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exposed to 0.25 yg/2. to 85.5% inhibition in fish exposed to 7.9 ug/£,
with good correlation in extent of inhibition between the two spawning
groups.
Bioconcentration Tests
Pinfish exposed to 2.4 yg/i bioconcentrated E?N raoidly, reaching
an apparent steady state in approximately two days (Figure 4). Uptake
and depuration data for the 0.17 yg/a (0.25 ugh, nominal concentra-
tion) exposure were not illustrated, because residues in these tissues
were usually below the level of detectabi1ity (0.02 yg/g). At steady
state, the concentration of EPN in whole pinfish exposed to 2.4 ug/z
was approximately 1.7 ug/g, giving a bioconcentration factor of 7C0.
In comparison, pinfish that survived a. 96-h exposure bioconcentrated
EPN an average of 740X. Additional species and their mean EPN bic-
concentraf:on factors were pinK shrimp, <43 (none detectable), and
spot, 260X.
After a faur-day post-exposure period, only one pinfish of four
sampled had detectable (0.04 yg/g) EPN in its tissues; after eight
days, no EPN was detected in any fish samoled.
Persistence
The Log P for EPN is 4.0 (Table 26). If this value is applied to
the Log P/Log BCF regression curve for *athead minnows (Pimephales
promelas) of Veith (1979a), the BCF is approximately 5Q0X. The BCF
observed for pinfish exposed to EPN was 700X, which correlates well
with the predicted value and demonstrates a relatively low DOtential
for bioaccumulation of EPN in the tissues of aquatic organisms. In
contrast, in a life-cycle test, sheepshead minnows bioconcentrated EPN
650 to 5,800X the water concentration, depending on life-stage.
In the results of persistence tests with EPN (Table 41), the
untreated systems retained much greater amounts of oesticide after
five days than did similar methyl parathion systems. Like methyl
parathion systems, used here as a reference, sterile EPN systems
showed less disappearance than untreated systems, with 79% of the
starting concentration of the pesticide being recovered after seven
days. More EPN disappeared from seawater systems than did methyl
parathion after seven days, which indicates that EPN is more sus-
ceptible to hydrolysis in the water column than is methyl parathion.
This observation is supported by the diminished disaopearance of.EPN
when sorbed to sediments in the sterile systems. The untreated sys-
tems displayed the greater disappearance of EPN, due to the combina-
tion of hydrolysis in the water column and biological processes in tne
sediment compartments. VolatiHty was not a significant factor in
these studies since EPN was not detected in the resin traps. Studies
with natural sunlight were not conducted for EPN.
45
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Discussion
The 96-h LC50s were similar for static and flow-through toxicity
tests with EPN in which the same species were exposed (^able 4-b and
5-b). Differences between these values for the species tested (V.
bahia, C. varieqatus, and L. xanthurus) ranged from a factor of 1.3 to
OT~
Our 96-h EPN acute toxicity test results are comparable to those
for freshwater species. Sanders (1972) reported 96-h LC50 values of
6.8 yg/z for scud (Gammarus fasciatus) and 0.56 yg/z for glass shrimp
(Palaemonetes kadiakensis). Pickering et al. (1962) reported the 96-h
LC50 values of EPN for several freshwater fishes: bluegill (Lepomis
macrochirus), 100 yg/Z; fathead minnows (Pimephales pronelas), 255
ug/Z; goldfish (Carassius auratus), 450 yg/ji; and guppies (Lebistes
reticulatus), 32 yg/z. Korn and Earnest (1974) exposed juvenile
striped bass (Morone saxatilis) to EPN in flowing seawater and
calculated a 96-h LC50 of 60 yg/z. Our LC50s for spot (25.6 yg/z,
flow-through tests; 36.6 yg/z, static tests) and pinfish (18.3 yg/z,
flow-through tests) indicate that these species are more sensitive
than freshwater fishes, whereas sensitivity of sheepshead minnows to
EPN (LC50 = 189 yg/z, flow-through tests; 144 yg/z, static tests) was
similar to that of fathead minnows and bluegills.
Pink shrimp (P_. duorarum) were especially sensitive to EPN (LC50 =
0.29 yg/z), and there was even a 20% mortality among shrimp exposed for
96 h to a nondetectable concentration. Further,' since no'EPN was de-
tected in tissues of shrimp exposed to concentrations as great as 0.42
yg/z, it is conceivable that pink shrimp in estuarine.habitats could
suffer significant mortality if exposed for more than 96 h to EPN
concentrations below our chemical detection limits, and the identity of
the toxicant that caused the mortality could not be determined by
residue analysis.
The MATC for EPN in chronic studies with mysid shrimp lies be-
tween 0.44 and 3.4 yg/z; that for sheepshead minnows, between 4.1 and
7.9 yg/z (Table 8). The application factor for sheepshead minnows,
obtained by dividing the MATC limits by the 96-h LC50 of 190 yg/z
(Table 5-b), is between 0.022 to 0.042; that for mysids is between 0.13
and 1.0. If the application factor for sheepshead minnows can be
applied to more sensitive fishes, chronic effects may be expected at
concentrations less than 1 yg/z. Based on the mysid MATC, therefore,
it is evident that a concentration of 0.44 pg EPN/z in estuarine waters
could cause acute effects to sensitive species, such as P_. duorarum
(96-h LC50 = 0.29 yg/Z; Table 5-b), and .may cause chronic effects to
M. bahia and to sensitive fishes.
Information obtained on the persistence of EPN in laboratory
studies demonstrates the importance of fate data in attempting to pre-
dict the probability that EPN will remain in tne marine environment
for durations sufficient to oroduce acute effects, chronic effects, or
45
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result in significant bioaccumulation. Hydrolysis and biologically
mediated degradation did occur in our tests. This information coupled
with data on expected extent and duration of inputs from freshwater
agricultural run-off may aid in predicting the likelihood of EPN
reaching estuaries. If these predictions, or monitoring data, demon-
strate that EPN would be present intermittently, then this insecti-
cide's hazard relative to its chronic toxicity or bioaccumulation
potential seems minimal. Chronic tests with M. bahia and C_.
variegatus demonstrated that effects occurred during the later stages
of exposure; thus, chronic effects nay be unlikely if EPN occurs
intermittently. Although EPN is bioconcentrated, it is also rapidly
depurated; thus, significant accumulation is unlikely unless EPN is
present continuously. Our studies suggest that the acute toxicity of
EPN to estuarine invertebrates is oossibly the greatest hazard because
death can occur at concentrations that cannot be detected.
ETHOPROP
Results
Acute Static Toxicity Tests
A1gae—The 96-h EC50 for Skeletonema costatum exposed to ethoprop
was 8,400 ug/A (Table 4-b and Appendix C-lJ^ In these tests nany
elongated cells were evident, possibly a result of inhibited cell
division.
Invertebrates—The toxicity of ethoprop to embryos of eastern
oysters (Crassostrea virginica) is shown in Table 4-b, Appendix H-la,
and H-lb. The 48-h EC50 was 16,000 yg/£-. The 96-h LC50 values and
95% confidence intervals for the crustaceans were 23 (19 to 27) yg/i
for Mysidopsis bahia, and 6.4 (5.5 to 7.4) yg/i for Penaeus
stylirostris (Table 4-b; Appendix H-2 and H-3).
Fishes--The 96-h LC50 and 95% confidence intervals of ethoprop
for sheepshead minnows (Cyprinodon variegatus) was 740 (585 to 1,070)
yg/£; for spot (Leiostomus xanthurus) 33(0 to 48) ygIt. (Table 4-b,
Appendix H-4, and Appendix H-5.)
Acute Flow-through Toxicity Tests
Invertebrates--Based on measured concentrations, the 96-h LC50
and 95% confidence intervals of ethoprop for mysid shrimp, was 7.5
(6.4 to 9.2) ug/4 (Table 5-b and Appendix H-6) and for pink shr.imp
(Penaeus duorarum) 13 (4.7 to 37) ygji (Table 5-b and Appendix H-7).
Fishes--The 96-h LC50 and 95% confidence intervals of ethoprop .
for the sheepshead minnow was 180 (85 to 390) yg/n based on measured
concentrations (Table 5-b and Appendix H-8) and for pinfish (Lagodon
rhomboides) was 5.3 (4.8 to 3.4) yg/Jl (Table 5-b and Appendi x H-9).
47
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Ranges of 00 and pH measured in seawater used in most acute tests
are in Appendix C-8.
Mysid Shrimp Entire Life-cycle Test
Mysid shrimp exposed to ethoprop in a 28-day test were adversely
affected. From Day 21, to the end of the exposure, mortality was
significantly greater in mysids exposed to the two highest concentra-
tions (0.62 and 1.4 ug/A; Table 42). Time to the formation of brood
pouches appeared not to be affected by ethoprop at the concentrations
tested (Table 42). The number of offspring produced per female was
significantly reduced in the highest (1.4 u9/i) concentration (Table
43).
There was no significant mortality of mysids during a oost-
hatch exposure of four to eight days. Survival ranged from 94 to 100%
in each treatment.
The MATC of ethoprop, based on measured concentrations, was >0.36
<0.62 ugh and the application factor limits (MATC divided by the 96-h
LC50 in a flow-through test) were 0.048-0.083.
Sheepshead Minnow Embryo/Juvenile Test
Sheepshead minnows exposed to ethoprop during their embryonic to
juvenile stages were adversely affected. Exposure to 98 ug/Ji produced
no significant effect on time-to-hatch or hatching success compared
with those in control aquaria, nor were abnormalities in embryonic
development observed (Table 44). Exposure to 21 ygh significantly
increased mortality of juvenile sheepshead minnow. By post-hatch, Day
7, approximately 33% of all fish that died during the entire 28-day
test had died; and by Day 14, 95% of all deaths had occurred. No
physical abnormalities were observed among juvenile fish in any
treatment.
The growth of surviving juveniles was not significantly affected
in concentrations <2\ u9!l (Table 45). Few fish survived concentra-
tions >35 ug/l, and they were not measured.
Ethoprop was detected in the tissues of sheepshead minnows that
survived the 23-day exposure (Table 45). The tissue concentrations
"¦anged f^om 0.1 to 0.48 pg/g; bioconcentration factors from 4 to 17.
The estimated MATC of ethoprop for embryos and juveniles of
sheepshead minnows was >12<21 ug/I. The 96-h LC50 for sheepshead
minnows exposed to ethoprop was 180 ug/i, (Table 5-b); therefore, the
application factor limits were 0.067-0.12.
Pi scussion
The results of toxicity tests with ethoprop indicate that estuar-
ine crustaceans and fishes are sensitive at concentrations >6.3
48
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£740 pg/Z, and that oysters and a phytoplankter are probably less
sensitive. Like phorate, the acute toxicity values of ethoprop to the
majority of the fishes tested are similar to those of the crustaceans
(Tables 4-b and 5-b). A comparison of the ethoprop static and flow-
through tests on the sane species indicates that the static results
significantly underestimate ethoprop toxicity to mysid shrimp and
sheepshead minnows. This may be due to a number of factors, including
loss of the chemical in the exposure water by adsorption on glass, up-
take by the test organism, and metabolism or alteration of the parent
compound to a less toxic substance. Since no persistence studies have
been conducted, no definitive explanation can be made.
Search of the literature produced very little additional data on
the toxicity of ethoprop to aquatic organisms.. The only information
found was static acute toxicity data on the freshwater fish, rainbow
trout (Salmo qairdneri), and the cladoceran (Daphnia magna) (U.S. EPA,
1975). The 96-h LC50 of concentrated (92%) ethoprop was 1,150 ug/I
and that of 10$ granular material to Daphnia magna was 690,000 yg/z.
Reasons for the wide disparity in sensitivity to ethoprop between the.
freshwater and estuarine species are unclear. However, data collected
from flow-through tests with freshwater species may permit explanation
of these differences.
Chronic toxicity tests with the mysid shrimp and sheepshead min-
now both demonstrated that the highest concentration that produced no
significant deleterious effect in the long-term studies was not vastly
different from concentrations that are lethal in acute tests (Tables
5-b, 42, and 43). It is interesting to note that although LC50s
differed by a factor of 25, the range of the application factors over-
lapped (0.048 to 0.083 for mysids, 0.067 to 0.12 for sheepshead min-
nows). The time required to elicit lethal responses in each test
differed. In the mysid test, significant differences in mortality
were not observed until after 3 weeks exposure, whereas in the
sheepshead minnow test, 83% of all total deaths occurred by post-hatch
Day 7 and 95% by oost-hatch Day 14. In both tests, however, mortality
was the factor that determined the MATC.
METHYL PARATHION
Results
Acute Static Toxicity Tests
A1qae--The 96-h EC50 and 95% confidence limits for Skeletonema
costatum exposed to methyl parathion were 5,300 ug/z, (4,300 to 5,700
yg/z) [Table ^-b, and Appendix C-l). The no-effect concentration was
1,000 ug/z.
Invertebrates--The calculated 48-h EC50 and 95% confidence limits
for embryos of eastern oysters (Crassostrea virqinica) exposed to
nethyl parathion was 12,000 (1 ,000 to 160,000 ug/z) (Table 4-b,
Aopendix I-la and I-1b). Reduction in numbers of embryos that
49
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developed normally to the straight-hinged, veliger stage after 48 h
ranged from 25% in 3,200 ug/£ to 71% in 56,000 ug/£.
Acute toxicity values of methyl parathion to three species of
crustaceans are shown in Table 4-b, Appendix 1-2, Appendix 1-3, and
Appendix 1-4. The 96-h LC50's and 95% confidence intervals were
0.98 pg/£ (0.81 to 1.20) for mysid shrimp (Mysidopsis bahia), 1.4 pg/£
(1.3 to 1.6) for white shrimp (Penaeus sty!irostris), and 28 gg/z (16
to 49) for a copepod (Acartia tonsa). Mortality ranged.from 0% in
0.32 ugIi to 90% in 1.8 gg/i for mysid shrimp, from 33% in 0.32 ggh
(equal to control mortality) to 100% in 5.6 ug/£ for white shrimp, and
from 20% in 13 gg/£ to 100% in 1C0 yg/£ for the copepod.
Fishes—The 96-h LC50s and 95% confidence intervals for two
species of fishes were 12,000 pg/£ (10,000 to 14,000) for sheepshead
minnows, Cyprinodon variegatus, and 93 pg/£ (56 to 320) for spot,
Leiostomus xanthurus, (Table 4-b, Appendix 1-5, and Appendix 1-6).
Mortality ranged from 0% in 10,000 gg/2 to 100% in 14,000 gg/£ for
sheepshead minnows, and from 0% in 56 gg/£ to 100% in 320 ug/£ for
spot.
Acute Flow-through Toxicity Tests
Invertebrates—The 96-h LC50's and 95% confidence intervals for
two invertebrates tested were 0.77 gg/£ (0.65 to 0.98) for mysid
shrimp and 1.2 ugIl (0.91 to 1.4) for pink shrimp (Penaeus duorarum)
(Table 5-b, Appendix 1-7, and Appendix 1-8). Methyl parathion was not
detected in pink shrimp that survived the tests.
Fishes-- The 96-h LC50 for spot and 95% confidence limits were
59 ug/£ (45 to 74) (Table 5-b). Mortality ranged from 10% in 25 pg/£
to 100% in 250 ug/£ (Appendix 1-9).
Ganges of DO and pH measured in seawater used in .most acuts tests
are shown in Appendix C-8.
Mysid Shrimp Entire Life-cycle Test
Results of exposure of M. bahia to methyl parathion in a life-
cycle test show that survival (Table 46) and reproductive success
(number of orogeny produced per female) (Table 47) are both reduced in
0.37 gg/£. A low survival in the TEG carrier control group was at-
tributed to excessive TEG (>50
-------
Persistence
'he Log P for methyl parathion is 2.4 (Table 26). If this value
is applied to the Log P/Log BCF regression curve for fathead minnows
(Veith, 1979a), the estimated BCF for nethyl parathion is 22X. We
conducted no long-term study to verify this result.
The results of persistence studies with methyl parathion are
reported in Table 48. Only 3% renained in untreated sedinent/water
systems after 5 days. In contrast, EPN and methyl parathion remained
in the sterilized sedinent/water systems after 7-days. From these
data, the implications a^e that biological processes are most impor-
tant in the disappearance of methyl parathion. Examination of the
fortified seawater .systems indicate a less significant role for
biological and nonbiological processes present in the water colunn,
which substantiates that the biological component of the sediments is
primarily responsible for the disappearance of nethyl parathion in the
untreated systems. Volatility of the pesticide was not a factor in
the disappearance of methyl parathion in any of these systems since
the resin tubes fails to reveal the presence of any pesticide. In the
studies with natural sunlight (Sunlight), thermal factors did not
affect disappearance since the foil-covered systems (Dark) did not
differ from the seawater systems. Although natural sunlight, compared
to artificial light, did enhance this pesticide's disanpearance, bio-
logical processes in the sediments remain the major contributor.
Di scussi on
Methyl parathion was not very toxic to the alga, S. costatum
(95-h EC50 = 5,300 yg/z). We know of no other studies of the toxicity
of nethyl parathion to algae or other aquatic plants with which to
compare our test (Table 4-b).
Invertebrates, particularly crustaceans, were more sensitive to
methyl parathion than we're fish. In our tests, the mysid shrimp were
particularly susceptible. There was little difference in the results
of acute and chronic exposures of mysids to methyl parathion or
between the 96-h LC50s derived from static (0.98 yg/i) and flow-
through (0.77 ug/i) tests. Two species of aenaeid shrimp were almost
as sensitive as mysid shrimp. The 96-h LC50 for P. sty!irostris
(static test) was 1.4 yg/z; that for duorarun Tfl ow-through test)
1.2 yg/z. The copepod (A. tonsa) was the least sensitive (28 yg/z
static test) crustacean tested. Methyl oarathion had relatively
little effect on embryos of the oyster (C_. virginica) (static test,
48-h EC50 = 12,000 yg/z), representing the least sensitive to methyl
parathion of all the species we tested.
Other studies on the toxicity of this compound to aquatic inver-
tebrates appear to be limited to crustaceans. The range of acute tox-
icity of methyl parathion reported for other saltwater and freshwater
crustaceans was from 2 to 40 yg/z. In addition, concentrations lower
-------
than I yg/£ adversely affected the behavior of grass shrimp. Static
48-h LCBOs for freshwater crayfish (Procanbarus clarki) (Muncy ana
Oliver, 1963) were 40 ug/£ and for two populations of _P. acutus, 2.4
and 3.4 ug/A (Albaugh, 1972). Naqvi and Ferguson (1970), using 24—h
static bioassays, found that the LC50s for four populations of the
freshwater shrimp (Palaemonetes kadiakensis) ranged from 2.5 to 23.3
ug/z. Saltwater crustaceans are equally sensitive to methyl para-
thion. In static tests, 96-h LC50s ranged from 2 to 7 gg/z for sand
shrimp (Crangon septemspinosa), grass shrimp (Palaemonetes vulgaris),
and hermit crao (Pagurus longicarpus) (Eisler, 1959).
Farr (1977) determined that 0.1 ug methyl parathion/£ impairs the
ability of grass shrimp (Palaemonetes pugio) to escape predation by
the gulf kiltifish (Fundulus grandisT- Even lower concentrations
(0.024 ug/jz.) altered the relative proportions of prey (grass shrimp
and sheepshead minnows) in the diet of this predator (Farr, 1973).
Available data also indicate a wide range of toxicity of methyl
parathion to fish. Our 96-h LC50s for soot (static, 93 ug/2; flow-
through, 59 ug/O are the lowest concentrations of this toxicant in
water that have been reported to kill fish. Acute toxicity to the
sheepshead minnow and to other saltwater and freshwater fish ranged
from 1,900 ug/Z to 75,800 ug/£. The 96-h LC50s for 19 species of
freshwater fish in static tests ranged from 1,900 ug/i for the blue-
gill (Lepomis macrochirus) to 15,200 ugji for the banded ki'llifish
(Fundulus diaphanus) (Pickering et al., 1962; Macek and McAllister,
1970; Rehwoldt et al., 1977). Sensitivity (96-h LC50 values) of 7
species of saltwater fish in static tests ranged from 5,200 ug/z for
the striped mullet (Muqil cephalus) to 75,800 ugh for the northern
puffer (Sphaeroides naculatus) (Eisler, 1970).
The relationship of brain AC'nE inhibition to death of sheepshead
minnows after exDosures up to 72 h to methyl parathion was determined
by Coppage (1972),. The chemical reduced brain AChE activity to less
than 17.7% of normal when 40 to 50% of the fish were killed.
Pathological changes also occur in rish following methyl para-
thion poisoning. McCann and Jasper (1972) observed hemorrhaging in
bluegill exposed less than 6 h to concentrations that "-anged from
3,900 to 6,900 ug/*-' Anees (1978) found that 1,050 ug/z caused
intestinal and liver lesions in the freshwater -ish (Channa.ounctatus)
in 96 h.
Data on the toxicity, oioaccumulat'1 on, and fate of methyl para-
thion must be compared if the hazard of this insecticide to the marine
environment is to be assessed. Because crustaceans are particularly
sensitive to acute and chronic effects of methyl parath;on, they are
more likely to be imoacted than algae, other invertebrates, or fishes
if this insecticide enters the marine environment. The results of our
laboratory persistence tests and those using more complex laboratory
systems (Pritchard et al., 1979) demonstrate that biological and
52
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chemical hydrolysis act to rapidly degrade methyl parathion. If this
occurs at the same rate in estuaries and input is intermittent, it
would seem unlikely that methyl parathion would persist for sufficient
durtions for chronic exposures or bioaccumulation to occur.
PHORATE
Results
Acute Static Toxicity Tests
A1gae—The 96-h EC50 and 95% confidence limits for Skeletonema
costatum exposed to phorate were 1,300 uq/i (1,000 to 1,400 ug/*,)
(Table 4-b and Appendix C-l). Growth of this alga in 100 ng/z did not
differ from growth of control cultures.
Invertebrates--The 48-h EC50 of phorate to eastern oysters
(Crassostrea virqinica) was 900 ug/i (400 to 1,900 yg/i)'(Table 4-b,
Appendix J-la, and Appendix J-lb).
In static tests, the 96-h LC50 was 0.27 ugh (0.18 to 0.32 yg/z)
for white shrimp postlarvae (Penaeus stylirostris) and 1.9 ug/£ (1.0 to
3.2 ug/i) for mysid shrimp (Mysidopsis bahia) (Table 4-b, Appendix J-2,
and Appendix J-3).
Fishes—The 96-h LC50s of phorate to two fishes were similar, 4.0
pg/£ for C_. varieqatus and 5.0 pg/£ for L. xanthurus (Table 4-b,
Appendix J-4, and Appendix J-5). Survival decreased only slightly
between Day 1 and Day 4 for each species during each test.
Acute Flow-through Toxicity Tests
Invertebrates--The 96-h LC50s of phorate, based on rlow-through
tests with measured concentrations in the exposure water, to pink
shrimp (Penaeus duorarum) and mysid shrimp were less than 1 yg/£ (Table
5-b, Appendix J-6, and Appendix J-7); 0.33 (0.27 to 0.43) yg/;i for
mysid shrimp, and 0.11 (0.08 to 0.16) yg/z for pink shrimp. The LC50
for mysid shrimp in this flow-through test was only one-fifth of the
static LC50 for that species and the 96-h LC50 for juvenile pink shrimp
was two-fifths of the LC50 for postlarval white shrimp in a static
test. These data suggest that chemical, physical, or biological
factors are reducing the actual concentration of phorate in static
tests, thus increasing the LC50.
Fishes--Sheepshead minnows were slightly more sensitive to phorate
in flow-through acute tests than were spot (Table 5-b, Appendix J-3,
and Appendix J-9). The 96-h LC50s based on chemically measured water
concentrations were 1.3 (0.97 to 1.7) yg/ji for sheepshead minnows and
3.9 (3.1 to 5.6) yg/i for spot. The LC50s for fishes were lower in
flow-through than in static acute toxicity tests.
53
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Ranges of DO and pH mesured. in seawater used in most acute tests
are shown in Appendix C-3.
Mysid Shrimp Entire Life-cycle Test
In an entire life-cycle toxicity test, mysid shrimp were exposed
to phorate for 28 days. Survival, the most sensitive neasure of
effect, was significantly reduced in an average -measured concentration
of 0.21 yg/z (Table 49), a concentration only slightly less than the
96-h LC50. Twenty-seven of the 30 mysids that died in this concentra-
tion did so in the first 8 days of exposure. There were no signifi-
cant effects on reproduction at the concentrations tested (Table 50).
Visual observations indicated that growth was probably not affected.
We observed no statistically significant effects on survival or repro-
duction at an average measured concentration of 0.09 yg/z. The MATC
limits for phorate are between 0.09-0.21 yg/z. The application factor
is >0.29 <.68 of the 96-h LC50 from a' flow-through test reported in
Table 5-b." Therefore, the effect of phorate on mysid shrimp is acute,
rather than chronic in nature.
Sheepshead Minnow Embryo-Juvenile Tests
There was no significant difference (a = 0.01) between survival of
control embryos and embryos exposed to phorate ("""able 51). Survival of
embryos to hatching ranged from 88.8% to 98.8% per treatment.
Phorate was toxic to juvenile sheepshead minnows at a measured
concentration of .0.41 yg/z, when the fish were continuously exposed for
four weeks post-fertilization (Table 52 and Figure 5). All fish
exposed to 2.4'yg/A died within 96 h after hatching; 96% of .the fish
exposed to 1.2 yg/z and 42.9% of those 1n 0.77 ygh died during the
test. Mortality was significantly different between control fish and
those exposed to 2.4, 1.2, and 0.77 yg/z (ct = 0.01) and between control
fish and those exposed to 0.41 yg/z (a = 0.05). Mortality of fish
exposed to 0.^1 yg/z for 24 days was 2.5%, but by Day 28, had increased
to 11.4% with some survivors visibly affected, indicating that further
exposure could have increased mortality.
Time of. occurrence and extent of signs of poisoning were dependent
on duration of exposure. Initially, affected fish were lethargic with
some developing lateral flexure of the body; thereafter, the body
became thickened when viewed from above, fish failed to feed, lost
equilibrium, and died. Within 24 h after hatching, all fish exposed to
2.4 yg/z were lethargic and over 50% nad visible flexure. Twenty-fou1"
hours later, over half the fish exposed to 2.4 yg/z had died and those
in 1.2 yg/z were lethargic and some flexed. Deatns began in the 1.2
ug/l exposure concentration 4 days after hatching and body thickening
became apparent in survivors 3 days later. Lethargy and flexure were
observed in/ish exposed to 0.77 yg/z four days after hatching;
noticeable thickening occurred 3 days ^ter, and deaths commenced 12
days after edema was first observed. The fish in 0.41 yg/z were
54
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lethargic 5 days after hatching; flexure occurred intermittently,
thickening was apparent 12 days after becoming lethargic, ana some
mortality occurred by the end of test. No signs of poisoning were
observed in fish exposed to 0.16 or 0.24 ug/i.
The three remaining juvenile fish in 1.2 ug/l were significantly
larger (a = 0.01) than fish in all other aquaria (Table 52). This
probably resulted from the greater availability of food and space per
juvenile in the egg cups. When the number of fry-feeding days was used
as a covariate, the adjusted lengths of these fish were net
significantly different (a = 0.01) from controls.
Phorate did not accumulate to any great extent in the tissues of
sheepshead minnows. The BCF was 90 for juveniles that contained
detectable phorate concentrations (Table 52).
\
Persistence
The Log P for phorate is 3.2 (Table 26). If this value is applied
to the Log P/Log BCF regression curve for fathead minnows (Veith,
1979a), the estimated BCF of phorate is 100X. Although long-term BCF
studies were not conducted to verify this result, juvenile sheepshead
minnows after 28 days of exposure bioconcentrated phorate 90X.
The results of persistence studies with phorate (Table 53) demon-
strate that phorate is less persistent than our reference pesticide,
methyl parathion. Vie cannot determine the relative significance of
biological and non-biological processes in the sediment/water systems
because the pesticide was not detectable after five days in untreated
systems or seven days in sterile systems. Volatility of phorate from
sediment/water systems was not evident since analysis of ^esin tubes
failed to reveal any pesticide. However, data from the seawater sys-
tems demonstrate that some disaDpearance of pesticide was the result of
removal with exiting air. Since the purging rate is competitive with
other processes affecting the disapoearance of phorate in the seawater
systems, the failure to account for the remaining pesticide in the wa-
ter from the systems indicates that hydrolytic processes in the water
column account ror much of the phorate disapoearance in these systems.
The exact significance of sediments in the disappearance of phorate is
not evident from these data, although the oresence of sediment in-
hibited volatilization in both untreated and sterile systems.
Discussion
The results of acute toxicity tests with phorate inriicat
-------
communication: 1980, Jack I. Lowe, U.S. Environmental Protection
Agency, Environmental Research Laboratory, Gulf Breeze, Florida). Lowe
found that the nominal concentration of ohorate required to immobilize
or kill 50% of the brown shrimp (Penaeus aztecus) in 48 h was 0.46 ug/i
and to reduce shell growth of the eastern oyster by 50% in 96 h was
estimated to be 640 yg/£. He also determined that the 43-h LC50 based
on nominal concentrations for spot was 3.2 pg/£; for longnose killifish
(Fundulus simi lis) 0.36 gg/i. Coppage (1972) exposed sheepshead
minnows to 9 pg/z for 48 h and observed 40 to 60% kill.
Chronic toxicity tests demonstrated that the highest concentration
that caused no significant effect in entire life-cycle tests with mysid
shrimp and in an embryo-juvenile test with sheepshead ninnows differed
only slightly from the 96-h LC50 for these species (Tables 5-b and 8}.
The only significant effect of phorate on mysids in the 23-day test was
that survival was adversely affected during the first eight days. In
contrast, sheepshead minnows died throughout the 28-day embryo/juvenile
test, indicating that the lower limit on the MATC (0.24 to 0.41 ug/z)
.nay not be a "safe" concentration of phorate. However, embryo-juveni 1 e
tests for other fishes and chemicals have been shown to be good predic-
tors of MATC's in entire life-cycle toxicity tests (McKim, 1977; Macek,
1977).
Examination of data on the toxicity, bioaccumulation, and persis-
tence of phorate is necessary to determine the hazard of phorate to
marine environments. Persistence tests with phorate indicate that this
insecticide disappears from these laboratory systems more rapidly than
EPN or methyl parathion. If this relationship is also true for
estuaries, this could limit the expected environmental concentration,
chronic effects, and bioaccumulation, especially if concentrations in
freshwater run-off are low and intermittent. Even if concentrations in
estuaries are low and occur intermittently, the hazard of phorate
should not be minimized because intermittent exposure to concentrations
less than 1 pg/2 would be expected to impact sensitive invertebrates
and fish.
56
-------
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58
-------
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59
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60
-------
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-------
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62
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• SW CONTROL
° TEG CONTROL
¦ 0.06 uq/l
¦ 0 22
* 0 36
a 0 69
o l 31
» 2 94
j€~:
143 155 167 179 191
DAY 0F EXPOSURE
Figure 1. Effects of carbophenothion on spawning response of grass shrimp
(Palaemonetes pugio) in a partial life-cycle toxicity test. The
number of new ovigerous females in each exposure chamber was
recorded daily during the test. Data were analyzed for differences
in onset and duration of spawning and total number of females to
spawn per concentration.
63
-------
Figure 2. A. Longitudinal, horizontal section of a normal sheepshead
minnow from control group. Note normal thin wall of centa of
vertebra (VW) and vestigial or inconspicuous notochord (N).
B. Similar section from fish exposed to 2.8 pg carbophenotMon
per liter from the zygote stage for 28 days. Note thickened wall
of vertebra (VW) and prominent notochord (N).
64
-------
10.0
o>
N
CT>
5
CL
.0
0.1
Detection Limit
UJ
0.0
CD
cr
<
o
UPTAKE
_l
±
15 18
-I — DEPURATION'
21
TIME (days)
Figure 3. Uptake and depuration of carbophencthion in whole-body tissues of
SDot (Leiastomus xanthurus) in a flowing seawater bioassay.
Exposure concentration averaged 2.S yg carbop'nenothion per liter
seawater.
65
-------
lOOOOr
- IOOO
100
10
/r
I
I
I
Detection Limit
14
UPTAKE-
T
28
35
DEPURATION
42
TIME (days)
Figure 4. Uptake and depuration of EPN in whole-body tissues of pinfish
(Laqodon rhomboides) in a flowing seawater bioassay. Exposure
concentration averaged 2.4 ug EPN per liter seawater.
66
-------
20r
_i
<
t-
cr
c
2
id
>
h-
<
1
60
70
30
SO
ICO
20
Tl'vIE (days)
sheepshead frnnnws7cypr°^ °f
area. gg per llter 1s within the shaded
67
-------
Table 1-a. Scientist or laboratory responsible for conducting acute static toxicity tests.*
Species Acepliate Aldicarb Carbophenothi on DFF EPN Ethoprop Methyl Phorate
" parathi on
CD
OO
Skelet.qnema cost.at.ujn GFU GFW
marine diatom
f.rassost.rea vir<)inica BURL RMRI.
eastern oyster
Acartia tonsa
marine copepod
Mysidopsis bahid - PUR
mysid shrimp
PenrUM^s sty 1 i rostri s - PIJR
white shrimp
Penaeus duorarum PUR
pink shrimp
Cypr inodon variepatus PWI'. Pl/P
sheepshead minnow
Leiostonuis xant.hurus PWR PWR
spot.
GFW
PWR
GEW (UH
GEW
RI1RL RMRL BMRL
PWR
PUR
PUR PWR
PUR PWB
PWR
PUIS
(JEW
RMRL
RMRI
PWR
PUR
GFW
RMRL
PUB
PUR
PWR
PWR
PWR Pl/R
PWR PUR
PWR
PUR
PUR
PWB
PWR
PUR
*GFU is an abbreviation for Herald F. llalsh, U.S. EPA, KRI , Gulf Rreeze, l-lorida; RMRI , Rionomics Marino Research
Laboratory, FG&G Internationa 1, Inc., Pensacola, Florida (Patrick R. Parrish, Pirector; P. Thomas Hcit.nullor,
Terry A. Ilollister, Trank I. Saksa, and G. Scott Ward); PUR, Patrick W. Rorthwick (Kinberly J. Rutler and
Richard A. Zern), U.S. EPA, f.RL, Gulf Rreo^e, florida.
-------
lable 1-b. Scientist or laboratory responsible for conducting acute flow-throuqh toxicity tests.*
Species Acephate Aldicarb Carhophenothion OFF EPfl Ethoprop riot hy 1 Phorate
_ parathion
M.ysidopsis bahia PURL RMRL BURL DUN Dl/N BMIIL DUN SCS
I'enaeus duo r a rum BURL RMRL SCS SCS SCS BMRL SCS SCS
Palaemonctes pugio - - SCS SCS - -
'lrass shrimp
Cypri nodon varieijatiis RMRL RMRL RMRL SCS SCS BMRL - SCS
Moniriia menidia - - SCS -
Atlantic silverside
I anodon rhomboidos RMRI RMRL RMRL SCS SCS BMRL
pinfish
Leiostoimis xanthurus - - SCS SCS SCS - BMRL SCS
*RMRI is defined in I able 1-a. SCS is an abbreviation for Steven C. Schimntel (James M. Patrick, dr.), U.S. t- P A,
tT.L, C.ulf Rreeze, Florida; DUN, Del Wayne flimno (Timothy L. Ilamaker, Charnell A. Sommers, and Ro^er Hood), U.S.
EPA,-URL, 'inIf Breeze, Florida.
-------
Table lc. Scientist or laboratory responsible for designated tests and sections of this document.1
Species Acephate Aldicarb Carbophenothion DEF FPN Ethoprop Methyl Phorate
. . __ ^ parathion
Finhryo/juveni le
£yplan(lqn varie.gaf.us - RMRL DJII - - RHRL - DJII
Fntire lifo-cycle
Mysi clops is bahia RMRI . " BMHI. DRT DUN. DUN RMRI OWN DUN
Palaemonet.es pugjo - - DRT . - - • -
Cyprinodon variegatus - RMRL - DJU -
Renthic community - - MET - - - -
Rioconcentration
Lagodon rhomboides - - - - SC S -
Leiostomus xanthurus - - SCS -
Persistence - - RLG RLG RLG - RLG RLG
Contributor PWH DRT DJH PWB SCS SCS MFT DJII
*Rf1RL and Pl/R are defined in Table la. SCS and DWN are defined in Table lb. DJII- is an abbreviation for David J. Hansen
(Larry R. Goodman, Peggy K. Iligdon, Charles S. Manning, and Edward Matthews); DR[, Dana Reth Tyler (J.V. l/heat and
R.D. link); MET, Marlin F.. Taqatv (Joel M. Ivey); RL(i, Richard L. Garnas (Jerrold Forester, Johnnie Knight, and Jaines
C. Moore); U.S. EPA, ERL, Gulf Rreeze, Florida.
-------
Table 2. Composition of mixes added to algal growth nedia.
Nutrient Mixes Amount/Liter
Metal Mix3
FeCl3.6H20 0.048 g
MnC^-^O 0.144 g
ZnS04.7H20 0.045 g
CuS04.5H?0 0.157 mg
CoC12-6H2O 0.404 mg
H3B03 0.140 g
Na2EDTAb 1.0 g
Glass-distilled or deionized water 1 n
Vitamin Mixc
Thiamin hydrochloride 50 mg
Biotin 0.01 ng
B«2 0.01 mg
Glass-distilled or deionized water 100 m£
Mi nor Salt Mi x^
K3P04 3.0 g
NaN03 50.0 g
Na2Sin3.9H2n 20.0 g
Glass-distilled or deionized water 1 1
j^Add 15.0 m1/1 of test solution.
•Added to stock culture nediun for all pesticides except
aldicarb.
^Add 0.5 mi/i of test solution.
Add 1.0 m1/1 of test solution.
71
-------
Table 3-. Profile of sediment used in oersistence studies.
Sediment profile3 Percentage
Particle si ze^ ¦ (mm)^
2.0 - 0.05
18.1
0.05 - 0.005
9.5
0.005 - 0.002
15.3
<0.002
57.1
Organic matterc
40.0
Moisture content^
89.6
aRange Point saltnarsh on Santa Rosa Island, near Pensacola
, Beach, Florida.
Settling Rate-Soil Hydrometer Method (ASTM D422-63, 1964).
"•Total organic matter on ignition at 500°C for 4 h.
Twenty-five g sample of sediment dried on a steam table to
constant weight.
72
-------
Table 4-a.
Acute toxicity of acephate, aldicarb, carbophenothion, and OFF to estuarine organisms in static tests.
Values are 96-h'IC50s, except where indicated. Nominal concentrations are in ug/2 and 95% confidence
intervals are in parentheses.
Species Acephate Aldicarb Carbophenothion DC F
Skeletonema costatum3 >50,000 >50,000 109 366
diatom " " (100-115) (315-391)
("rassostroa virqinica'1 150,000 8,800 99 197
easternoyster "larvae (8,000-300,000) (1 ,400-56,000) (96-102) (120-323)
Mysidopsis hdhia - 13 13 5.8
mysid shrimp (10-15) (10-17) (4.4-6.8)
24-h-old
Penaous st.yl irost.risc - 72 4.3 25
white shrimp (65-82) (3.8-5.1) (16-49)
post larvae
Penaeus duorarum >10,000 - - -
pink shrimp
field collected
Cyprinodon variegatus >3,200,000 168 17 440
slieepshead minnows (102-320) (14-21) (410-473)
28-day-old
l.eiostomus xanthurus >100,000 202 500 158
spot. (116-293) (390-630) (135-320)
f%-h IC50 values.
48-h FC.50 values.
rP. siyli rostris post-larval stages were used iri these tests; control mortality exceeded 10%,
-------
Table 4-h. Acute toxicity of EPN, ethopro, methyl parathion, and phorale to estuarinc organisms in static tests.
Vd 1 iios arc 96-h LCSOs, except,-where indicated. Nominal concentrations are in nn/e. and 95% confidence
intervals are in parentheses.
Species TPN Fthoprop Methyl Phorat.e
parathion
Skoletonena costatunid 340 8,4(10 5,300 1,300
diatom "" (330-350) (7,600-9,000) (4,300-5,700) (1,100-1,400)
Orassostrea virqinira^1 ?,?00 16,000 1?,000 900
"eastern oyster larvae (600-8,100) (7,000-38,000) (1 ,000-160,000) (400-1,900)
Ac art, i a tonsa - - ?8 ¦
copepod (16-49)
Mysidopsis bahia 13 23 0.98 1.9
Piysid shrimp (10-18) (19-27) (0.81-1.?) (1.0-3.2)
?4-h-old
Pendens sty I irostri_sC 4.6 6.4 1 .4 0.?7
white shrimp (2.4-9.0) (5.5-7.4) (1.3-1.6) (0.18-0.3?)
post larvae
Cyprinodon varieqatus 140 740 12,000 4.0
sheepshead minnows (59-500) (585-1,070) (10,000-14,000) (3.5-4.5)
28-day-old
Leiost.oi'ius xanthurns 37 33 93 5.0
spot (33-40) (0-48) (56-320) , (4.2-5.6)
^96-h IT.50 values.
48-h t C50 values.
^P. stylirosfris post.-larval st.aqes were used in these tests; control mortality exceeded 10%.
-------
Table 5-a. Acute toxicity of acephate, aldicarb, carbophenothion, and l)EF to estuarine organisms in flowing seawater
tests. Values are 96-h LC50s, except, where indicated. Measured concentrations are in jig/s. and 95% confidence
intervals are in parentheses.
Species
Acephate
A1di carb
Carbophenothi on
DFF
•-J
CJ1
Hysidopsis bahia
mysid shrimp
I'enaeus duoraruni
pink shririp
Palaenonetes pugio
qrass shrimp
wiIdstock
larvae, 1-7 day
lab-reared, 4()-day
^Lnorion variegat.us
sheepshend minnow
Men id id nenidi a
Atlantic silvers ides
I a'jndnn rhomhoidns
pi nfish
Leiostonins xanthunis
spot
7,30(1
(300-190,000)
3,noo
(2, 000-7,300)
910,000*
8!i ,000
(/,800-933 ,000)
16
(13-20)
1?
(7.5-1R)
41
(r>6-/2)
HO
(43-160)
3.0
(2.4-3.7)
0.47
(0.36-0.66)
4.6
(3.8-6.?)
2.0
(] .R-2.6)
2.9
(2.!)-3.3)
2.8
(0.R5-9.6)
7.7
(!>./-1 ?.)
7.7
(4.6-12)
>206
4.6
(4.2-4.9)
14
(10-1H)
22
(19-23)
>440
290
(240-3/9)
130
(100-170)
196-h rCSO (nominal concentrations) based on loss of equilibrium.
-------
Table 5-b. Acute toxicity of FPN, ethoprop, methyl parat.hion, and phorate to estuarine organisms in flowing seawater
tests. Values are 96-h LC50s; measured concentrations are in u
-------
Table 6. Mortality (Dercentage killed) of mysid shrimp (Hysidopsis bahia)
during a chronic (28-day) exposure to acephate. The nysids were •
24- to 48-h-old at the beginning of the test. Twenty mysids were
placed in each treatment (10 per replicate). Salinity was fron
26-31 °/oo and temperature, 22 1°C-
Measured concentration (uq/&)
Day
Control
580
520
1,400 ¦
2,
000
4,
200
A
B
A
B
A
B
A
B
A
B
A
' B
1
0
0
0
o.
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
3
0
.0
0
0
0
0
0
0
0
o .
0
0
4
0
0
0
0
0
0
0
0
. 0
0
0
0
5
0
0
0
0
0
0
0
0
0
0
0
0
6
0
0
0
0
0
0
0
0
3
0
0.
o.
7a
0
0
0
0
0
0
0
10
C
0
10b
20
n
0
0
0
0.
0
0
0
10
10
0
0
10
20
9
0
0
0
0
0
0
10
10
0
0
10
20
10
0
0
0
0
0
0
10
10
0
o ¦
10
20
11
0
0
0
0
0
0
10
10
0
0
10
20
12
0
0
0
0
0
0
10
10
0
0
10
20
13
0
0
0
0
0
0
10h
10b
10h
l0
0,
iob
°h
l0b
10K
20.
14a
0
0
0
0
0
0
10
30
15
0
0
0
0
0
0
10
10
10
20 •
10
30
16
0
0
0
0
0
0
10
20
10
. 20
10
30
17
oc
Cc
0C
or
0
0
10
20„
10
20
10
30
13
0
0
0
0C
0C
oc
10C
20c
10C
20c
20
40
19
0
0
0
0
0
0
10 .
20
10
20
20
40c
20
0
0
0
0
0
0
1 o.
20.
ldh
20k
20^
20 s
4°,
01 3
C 1
0
0
0
0
0
0
10b
30b
I0b
?0b
50
22
0
0
0
0
0
0
10
30
10
20
20
60
23
0
0
0
0
0
0
10
30
10
20
30
60
24
0
0
0
0
0
0
10
40
10
20
30
60
25
0
0 .
0
0
10
0
10
40
10
20
40
60
26
0
0
0
0
10
0
10
40
10
20
40
60
27,
0
0
0
0
20
0
10,
4°,
10,
20.
^0
6CL
23
0
0
0
0
20
0
10°
40
10°
20°
40°
6 Or
Average 0
0
10
25
b
15b
501b
aStatistical analysis performed.
^Significantly greater (a = 0.05) than the control.
cFornation of brood pouches first observed.
-------
Table 7. Number of offspring produced per female of nysid shrimp
(Mysidopsis bahia) exposed to acephate in a chronic (28-
day) exposure in natural, flowing seawater.
Measured
Replicates
Total
Females with
Offspri ng
concent rati on
A -.••••
B
offspri ng
brood pouches
per
(ug/f)
female
Control, NDa
8.8
8.0
76
9
3.4
580
8.8
5.3
60
8
7.5
520
7.3
6.0
46
• 7
6.6
1,400
7.0
5.3
51
8
6.4
2,000
4:8
5.0
49
10
4.9b
4,200
3.5
3.3
17
5
3.4b
j^ND = nondetectable (<400 mq/i).
Significantly less {a = 0.05) than the control as determined by ANOVA and
Wi 11 iams 1-test (Williams, 1971).
78
-------
Table 8. Results of chronic toxicity tests with acephate, aldicarb, carbophenothion,
Pfc'F, HPN, ethoprop, methyl parathion, and phorate. The maximum acceptable
toxicant concentration (MATH) range is the highest concentration (yg/i)
that produced no significant effect and the lowest concentration in which
significant effects were observed. The application factor (AF) is the MATC
range divided by the 9f>-h LC50 value from a flowing toxicity test.
Pesticide
Mysidop:
MATC
sis hahiaa
AF
Palaemonetes
MATC
puqio^
AF
Cynrinodon varienatusc
riATC AF
Acephate
5H0-1,400
0.079-0.19
Aldicarb
•1.0-1.5
0.063-0.094
-
-
50-88
-
Carbophenothion
0.48-1.2
0.16-0.40
0.??-0.36 f
).76-0.12
1.3-28
0.46-1.0
PEF
<0.34
<0.074
-
-
-
-
CPN
0.44-3.4
0.13-1.0
-
-
4.1-7.9a
0.022-0.042a
Ethoprop
0.S6-0.fi?
0.048-0.083
-
-
12-21
0.067-0.12
flothyl parathion
0.11-0.37
0.14-0.48
-
-
-
-
Phorate
0.09-0.?!
0.?9-f!.68
-
—
0.24-0.41
0.18-0.32
^MATC and AF derived from entire life-cycle toxicity test.
riATC and AF derived from partial life-cycle toxicity test.
criATr. and AF derived from ombryo/juverii 1 e toxicity test, unless noted.
-------
TaMe 0. Mortality (percentage killed) of mysid shrimp (flysidopsis bahia) during a chronic (28-day) expo-
sure to aldicarb. The mysids were 24- to R-h-olcl al beginning of test. Twenty mysids were
placed in each treatment (10 per replicate). Salinity was fron 26-31 °/oo and temperature 22_+l°C.
Carrier
0
.30
0.
50
1.0
1.
5
2
1
Day
Control
control
A
R
A
B
A
. R
A
R
A
C
1
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
.0
3
0
0
n
0
0
0
0
0
0
0
0
0
4
0
0
0
o -
o -
0
0
0
0
0
10
0
R
0
0
0
0
0
0
0
0
0
0
10
0
6
. 0
0
0
0
0
0
0
0
0
0
10
0
7a
0
0
n
n
0
0
0
0
0
o
10
0
8
0
0
0
0
0
0
0
0
0
. 0
10
0
0
(1
0
0
0
0
0
0
0
0
0
10
0
10
0
0
. 0
0
0
0
0
0
0
0
10
0 '
11
0
0
0
0
0
0
0
0
0.
0
20
10
12
0
0
0
0
0
0
0
0
10
0
20
10
13
0
0
0
0
0
0
0
10
1 o,
10b
n
c: c:
' 20h
20 b
1 o,
10b
I /I ^
0
0
n
0
0
0
0
10
0
0
0
0
0
0
0
10
10
10
20
10
If.
oc
oc
0
0
0
0
0
10
10
10
20
10.
1 /
0
0
oc
0C
oc
oc
oc
10c
10
10
20
10
18
0
0
0
10
0
10
0
10
10c
10
20
20
0
0
10
10
0
10
0
20
.10
20°
20
20
20
0
0
10
10
10
10
0
20
10K
10b
30
30 b
20 c
20 b
¦ZOh ,
21
0
0
10
10
10
10
0
20
20 '
OO
(. c.
0
0
10
20
10
20
0
20
10
30
20
20 "
23
0
0
10
20
10
20
0
20
10
30.
20
30
24
0
0
10
20
10
20
0
20
. 10
30
20
30
2b
0
0
10
20
10
20
0
20
20
40
20
30
2f>
10
0
10
20
10
20
0
20
30
40
20
40
27
2f!d
10
II!
0
0
10
10
20
20
10
25.
20
20
0
0
20
20
30
30
40
1
| OjO
40.
40h
Averages
10
0
1
.
15
10
35
40
^Analysis of variance (AtlOVA) performed.
Significantly greater (f1 _ .(lb) than the control as determined by ANOVA and Wil Nans' test '(Williams, 1071).
c f o t ma Lion of brood pouches first observed.
-------
Tab! e 1Q.. Number of of^snring produced per *emale of mysid shrimp (^ys'doosis
bahi a) exoosed to aldicarb in a chronic (28-day) exposure "'n
natural, flowing seawater.
Measured Replicate Replicate Total Females with Offspring per
concentration A 3 offspring brood pouches fema"!e
(ng/i)
Control
24
24
4
6.0
Carrier control
28
28
4.
7.0
0.38
26
22
48
7
6.3
0.50
16,
40
56
9
6.2
1.0
38
29
67
9
7.4
1.5
10
9
19
4
4.8
2.1
0
0
0
3
0.0a
Significantly less (a = 0.05) than the control as determined by ANOVA and
Williams' test (Williams, 1971).
-------
Table 11. Survival of embryo and juvenile sheepshead minnows (Cyprinodon
variegatus) exposed to various concentrations of aldicarb in
seawater at 29+l°C. Juvenile fish were exposed for 28 days
posthatch.
Exposure concentration (uq/Jt) Survival , %
Nominal
Measured
Embryo
Juveni1es
Control
\T)a
"92
86
Carrier control
ND
KG
00
95
17
8.7
• 95
86
29
14
96
71
48
28
92
79
80
50
94
70
.130
88-
91 \
64
aND = nondetectable {<0.2 ugfi).
32
-------
Table 12. Standard lengths and survival of juvenile sheepshead minnows (Cyprinodon
varieqatus) exposed to various concentrations of aldicarb for 28 days.
One hundred embryos were placed in each treatment at the beginning of
the test.
Exposure concentration (yq/O
Nominal Measured
Number of
surviving fish
Average
length (mm)
Control
NDa
69
11.5
Carrier control
ND
76
12.5
17
8.7
69
13.0
29
14
57
12.0
48
28
63
11.0
80
50
56
11.0
130
88
51
9.5b
j*ND = nondetectable (<0.2 ug/i)
F1sh significantly smaller (a = 0.05) than the controls as determined by ANOVA
and Williams' test (Williams, 1971).
83
-------
Table 13. Mortality (percentage killed) of inysid shrimp (H.ysidopsis bahia) during a chronic (28-day)
exposure to carbophenothion. The mysids v/ere 24-h-old at the beginning of the test. Forty
mysids were placed in each treatment. Salinity averaged 20.7 °/oo (17-23 °/oo); temperature,
25.2°C (23.5 - 26.2°C).
Day
Control
Carrier Controla
0.18
0.30
Measured Concentration (pg/fc)
0.48
1.2
1.9
4.1
1
2.5
0
7.5
0
2.5
7.5
0
2.5
2
2.5
0
7.5
2.5
5.0
7.5
0
2.5
3
5.0 ,
2.9
7.5
2.5
7.5
7.5
0
/ .5
4
5.0
2.9
12.5
2.5
7.5
7.5
0
17.5
5
5.0
2.0
12.5
5.0
7.5
7.5
0
20.0
6
5.0
5.7
12.5
7.5
7.5
7.5
0-
20
/
5.0
5.7
12.5
7.5
7.5
7.5
0
20
a
5.0
5.7
12.5
7.5
7.5
7.5
0
22.5
9
7.5
5.7
12.5
7.5
. 7.5
7.5
0
22.5
10
10
5.7
12.5
7.5
7.5
7.5
0
27.5
li
10
11.4
12.5
10
7.5
10
0
27.5
12
10
14.3
12.5
10
7.5
10
5
27.5
13
10
14.3
12.5
10
10
10
7.5
27.5
14
10
14.3
12.5
12.5
10
12.5
7.5
30
15
10
14.3
15
- 17.5
10
12.5
7.5
30
16
20
14.3
15
22.5
12.5
15
15
35
17
20
17.1
20
22.5
15
17.5
17.5
35
18
20
17.1
20
25
15
17.5
17.5
37.5
19
20
20
20
35
15
17.5
22.5
3/.5
20
20
22.9
20
40
15
20
22.5
37.5
21
20
22.9
20
40
15
22.5
25
37.5
22
20
22.9
20
40
15
27.5
25
37.5
23
20
22.9
20
42.5
15
27.5
27.5
37.5
24
20
25.7
20
42.5
15
27.5
27.5
37.5
25
20
25.7
20
42.5
15
27.5
27.5
42.5
26
20
25.7
20
42.5
15
32.5
27.5
42.5
2/
25
28 .6
2?.5
42.5
15
32.5
27.5
42.5
28
30
28.6
22.5
42.5n
17.5
35
27.5
47.51
aN = 35
'^Si (jni f i cant ly different, t.han controls (u = 0.05) at the end of test.
-------
Table 14. Number of offspring per f enal e of nysid shriirp (Hysidopsis bahia) exposed
to carbophenothion in a chronic (28-day) exposure in natural, flowing sea-
water.
Measured concentration Total Offspring Onset of spawning
(yg/z) offsDring per female (day of test)
Control 104 34.2 19
Carri er control 78 25.5 19
0.18 102 35.6 ¦ 20
0.30 110 47.5 18
0.48 121 42.6 20
1.2 112 47.9 20
1.9 84 31.5 20
4.1 52 16.8 22
85
-------
Table 15. Survival of F.^ generation of mysids (Mysidopsis bahia)
continuously exposed to carbophenothion. Values given
are percentage survival 10 days Dosthatch.
Measured concentration
(yg/O
No. of trials
% survival
Range
Control
6
90
(80-100)
Carrier control
d
93.5
(30-100)
G.ie
8
87.7
(50-100)
0.30
5
. 93.1
(80-100)
0.48
6
89.2
(75-100)
1-2
7
75.7
(¦20-100)
1.9
5
77.3a
(60-100)
4.1
1
33a'b
-
aJuvenile swimming behavior erratic, i.e., swimming in backward
somersaults, . frequent change of direction, very ^apid swimming.
Different from controls {a = 0.05).
86
-------
Table 16. Growth (head-tail length) of parental generation mysid shrimp
(Mysidopsis bahia) exposed to carbophenothion in a chronic
(28-day) exposure in natura1, flowing seawater.
Measured
concentration
(ug/O
"lumber of
shrimp measured
Average
length (mm)
Range
(mn)
Control
26
5.9
5.0-6.5
Carrier control
2^
5.9 '
5.0-6.2
0.18
31
5.7
4.5-6.2
0.30
23
5.7.
. 5.0-6.5
0.48
31
5.6
5.0-6.0
1.?
23
5.4a
4.8-6.0
1.9
22
5.2 a
4.5-5.8
4.1
5.1a
4.0-5.8
aSiqnificantly different fron controls (a = 0.05).
87
-------
Table 17. Survival of adult grass shrimp (Palaenonetes puqio) continuously
exposed to carbophenothior for 249 days in a partial life-cycle
test. One hundred shrimp were'placed in each treatment.
Exposure concentration (uq/£)
Survival, %
Nominal
Heasured
Day 33
Day 249
Control
'ID a
96
75
Carrier control
no
97
63
n .125
0.06
94
79
0.25
0.22
98
64
0.50
' 0.36
91
52
1.0
0.69
97
i5b ¦
2.0
1.3
92
52
4.0
2.°
24b
0b
aND = nondetectab 1 e (<0.2 vq!l).
'"'Significantly different from controls (a = O.OB).
88
-------
Toll I e 18. Fffect of carhopherioth ion on fecundity of grass shricip (Palaemonetes pugio). The three
components of fecundity examined included total number of ovigerous females, average egg
production and hatching success in each tost concentration. The number of eggs produced
per female was determined for at least 10 shrimp per test concentration. Using a.separate,
group of shrimp, hatching success was determined for each test concentration. Since egg
production is proportional to length of female, rostrum-telson length of each female was
used as a covariate in all statistical analyses.
Measured Total No. Average Average Average Average
concentration ovigerous egg production female length No. larvae female length
(ug/iO females (eggs/9) (mm) hatched/? (mm)
Control
45
154 .5
26 .8
125.1
27.1
Carrier control
40
154.6
27 .4
115.1
20.2
0-06
46
13? .9
26.6
118.1
28.0
0.22
50
157.4
26.6
94.7
26.6
0.3 6
3od
154.1
26.')
142.8
30.9
0.69
o0a
163.3
27.1
164.4
29.7
1.3
32*
156.n
27.1
120.2
29.4
2.9
fia>h
c
_
125
28.5
^Significantly less than the controls by Day 147.
Significantly less than the controls by Day 84.
cFgg production not determined tor this concentration.
-------
Table 19. Survival of F-, generation grass shrinp (Palaenonetes
puqio) 1arvae'reared under conditions of continued exposure
to carbophenothion. Each larval rearing trial was run with
larvae produced by a single fenale.
Concentrati on
(ug/O
• -lumber of larval
rearing trials
Average survival
from hatchina to
Day 10,"%
Range
Control
10
90
46-100 '
Carrier control
16
82
50-100
0.06
14
83
36-1C0
0.22
12
83
63-97
0.36
13
82
£3-100
0.69
14
85
67-100
1.3
7
84
70-97
2.9
5
5a
0-26a
aSigniflcantlv different from survival of controls (a = 0.05).
90
-------
Table 20. Effect of carbophenothion on weight (gl of' grass shrimp
(Palaenonetes pugio) exposed in a partial life-cycle test.
Weight determinations were made for all survivors in each
test concentration at the end of the test (Day 2^9).
Measured
concentration
(ug/i)
Average weight3
"(g)
N
Control
0.1771
75
Carrier control
0.1342
68
0.06
0.1555
79
0.22
0.1619
64
0.36
0.1510
52
0.69
0.1530
45
1.3
0.1222
52
2.9
dNo significant difference (a = 0.05) between control and exposed
animals.
-------
Table 21. Concentrations of carbophenothion (pg/g) whole body,--wet weight
in aarental generation grass shrimp (Palaenonetes nugio), and bio-
concentration 'actors (concentration neasure^1 in tissue divided
by the average concentration"in exposure water) at termination
of a 249-day partial .life-cycle toxicity test.
Measured water
concentration (ug/i)
Measured tissue
concentration (ug/g)
Bioconcentrat ion
factor
Control
MDa
Carrier control
nn
-
0.06
ND
-
0.22
r.'D
-
. .0.3*
0.14
390
0.69
0.36
520
1.3
0.47
360
X = 420
aNP = nondetectable (<0.02 ug/g).
92
-------
Table 22. Survival of enbryonic and juvenile sheepshead ninnows (C.yprinodon
varieoatus) exposed to various concentrations cf carbophenothion
in seawater at 30°C.
Exposure
concentration (uq/£)
Survi val
<5)
Nomi nal
Measured
F.nbryos
Juveni1es
Roth stages
Control
NDa
. ¦ 95
96
91
0.38
0.36
92
96
89
0.75
0.59
89 .
97
86
1.5-
1.3
86
96
82
3.0
2.8
85
92
79b
6.0
5.4
86
4C
4C
12.
11.
86 .
,0C
0C
®MD = nondete.ctable (<0.05 ug/O •
bSignificantly different frcn control and 0.36 pg/£ (n = 0.05).
cSignificantly different fron control and all other concentrations (a = 0.05).
93
-------
Table 23. Standard lengths, tissue concentrations, and r>
-------
Table 24. Animals collected fror^ control aquaria and aquaria exposed to carbcphenothion in
eight-week benthic cormunity study.
Exposure concentration (uq/£)
Nominal Control 0.01 0.1 1.0
Taxa Measured (ND)a (ND) (0.1) (0.9) Total
Annelida
Pectinariidae
Cistenides gouldi i
Nerelrlae
Neathes succinea
Mereis pelaqica
Laeonereis cul ve>"i
Peri nereis f1oridana
CaDitel1idae
Capitel1 ides jonesi
Capitel1 a capitata
Caoitonastus aciculatus
Medionastus californi ensi s
Spionidae
Nerine aqi1i s
PoVydora 1 i gni
Polydora websteri
Po'jydora social i s
Streblospi o benedi cti
Spiophanes bonbyx
Sthenelais articulata
Sabel1idae
Sabe:•a melarostigna
Phyl1cdocidae
Ne^elphyl1 a fraci1is
Eteone heteropcda
Eumicia sangui nea
Opheli idae
Apnardia agi1 is
Oroi niidae
Haplcsco:3p1os robustus
Map!oscclop"!os f "-ag1 1 is
41
25
2
0
0
n
o
0
8
5
1
5
4
0
1
0
0
.38
19
a
0
0
2
1
2
1
<1
9
0
2
3
1
0
2
40
a
4
0
1
n
o
n
o
o
o
p
34
20
5
1
0
D
0
1
0
1
1
0
3
2
0
0
n
o
153
72
15
4
7
2
2
1
14
16
2
10
1
C
0
35
-------
Table 24 continued.
axa
Nominal Control
Measured (ND)a
Fxposure concentration (uq/z)
n.oi
C no)
n.i
(0.1)
1.0
(0.9)
Tota'
Gv/eni i dae
Owenia fusifomis
Total Annelids
^'enertea
0*~der Heteronenertea
Crder Hop!onenertea
Oder ^aleonenertea .
P1atyhelni nthes
Mol1usca
'¦lul i ni a 1 ateral i s
Ens i s ninor
Polinices dun 1icatus
Cytopl eura costata
M u s c u1n s 1aterali s
Total Molljsca
Arthropoda
Balanus inprovi sus
Anoplodactylus 1entus
¦ Phithropanopeus harrisi i
Coronhiun acherusicjn
Family Caprel1idae
Cxyurostyli s snitni
Tcta^ Arthropoda
Coelenterata
A-'otasia pal • ida
Chorriata
f'ol qui a "anhattensi s
Total all phyla^
Individuals
Speci es
n
(97)
i
n
178
51
n
o
o
(229)
78
9
0
0
c
(88)
0
(93)
193
43
n
t.
o'
n
C?38)
79
7
n
l
1
n
(83)
2
..n
(58)
n
0
n
2
114
C
0 ¦
(142)
. 102
7
1
0
0
(111)
i 7p ^
\ ' r i
0.
n
20C
44
r?A6)
112
n
n
0
0
n
(11?)
2
(326)
2
2
685
165
¦>
(855)
371
L J
[ pqg }
419
425
29
315
18
4^1
21
16CO
41
3f!D = nondetectabl e.
96
-------
Table 25. Total
nunber of individuals and species (in oarentheses) , by phylum,
col 1ected
i n a n
eight-week bentnic
community
test with
carbophenothion
•
Exposure
concentrati on
(uq/a)
Nominal
Control
0.01
fl.l
1.0
Phylun
Measured
(ND)a
(ND)
(0.1)
(0.9)
Total
Mol1usca
229
233
142
246
355
(2)
(3)
'3^
(4)
(5)
Arthropoda
38
33
111
112
399
(3)
;*)
;4)
(1)
(6)
Annelida
97
93
53
73
326
(13)
(17)
(9)
(14)
(24)
Coelenterata
i
2
2
4
9
(1)
(1)
V i
(1) •
(1)
Nernertea
3
2
0
0
5
(3)
(2)
(0)
fo\
(3)
PIatyhelni nthes
0
1
2
1
4
(0)
(1)
:d
'l11
\ J- /
(D
Chordata
1
X
0
0
2
(1)
(1)
(0)
(0)
(1)
Total all phyla
Indi viduals
419-
425
315
ddl
1,600
Species
(23)
(29)
(18)
(21)
(41)
a'!D = nondetectable.
97
-------
Table 25. Octanol/water partition coefficients (Log P) of methyl
parathion, phorate, EPM, carbophenothion, and QEF, as
determined by reversed phase high pressure liquid chroma-
tography.
Chemical Log P
Benzene3
2.1
Methyl parathionb
2.4
Phoratec
3.2
Biphenyl a
3.8
EPNd
4.0
Carbophenothi one
4.9
DEFf
5.7
£,£-DDEa
5.7
Hexachlorobenzene3
•6.2
^Reference chemical for calibration.
b0_,_0-dimethyl -0-£-ni tropheny! phosphorothioate.
^0_,0-diethyl -S-(ethyl ,thio) nethyl phosphorodithioate.
0_,ethyl -£-£-ni troDhenyl phosphorothi oate.
p-chl orophenyl thi o)methyl ]0,_0-di ethyl phosphorodi thi oate.
S_,_S,S-tri butyl phosphorctrithioate.
98.
-------
Table 27. Results of persistence studies with carbophenothion.
Numbers represent micrograms (ug) of the pesticide detected
in the respective compartment. The totals are the per-
centages of the combined micrograms of pesticide detected
in a system when compared to the micrograms originally added
to the system. MD means no pesticide detected.
System Compartment Time
Dav 0
Day 5
Day
Untreated5
water (ug)
1.9
ND
sediment (yg)
3.8
5.7
total (%)
87%
57%
Sterile^
water (ug)
ND
sediment (ug)
6.1
total [%}.
61%
Seawater0
water (ug)
' 9.5
MD
resin (ug)
rm
£.1
total (%)
95%
.<11%
aTen grams of sediment (wet weight) and 100 rM of 20 pm filtered seawater
of 27 parts per thousand salinity fortified with 10 ug of carbophenothion
in 10 vi of acetone so that at Day 0, water concentration equivalent to
49 yg/'i. and sediment concentration eouivalent to 3800 ug/kg (dry weight).
Systems aerated at midpoint of water column at 50 mz ner minute flow rate,
with pesticide in exiting air purged through XAD-4 resin trap.
Sane as untreated systems except that 5 nz of formalin was added 24 hours
before Day 0. Sterility was confirmed by plating aliquots frcm the water
column en IB parts per thousand Zobell's media throughout the test.
cSame as untreated systems, exceot that sediment was not added.
99
-------
Table 28. Mortality (percentage killed) of nysid shrirp (Mysidopsis hahia) during a
chronic (28-day) exposure to DEP. The rrys'ds were 24-h-old at the
beginning of the test. Forty mysids were placed in each treatnent unless
otherwise noted. Salinity was f^om 24-32 /oo and tenperature,
22-27°C.
Day
Control
Carrier control
Measured concentration
(uQ/l
(M = 36)
0.34
0.58
(N = 39)
1.7
3.3
1
0
0
0
0
0
5
2
0
0
0
0
0
5
3
• n
0
0
0
0
5
. 4
2.8
0.
0
n
5.0
57.5
K
2.8
0
0
0
7.5
82.5
5
2.8
0
0
0
7.5
82.5
7
5.6
0
0
7.7
17.5
92.5
p
5.6
0
0
7.7
20.0
97.5
9
• 5.6
0
0
7.7
20.0
100.0
10
5.6
0
0
7.7
20.0
100.0
11
. 5.6
. 0
0
10.2
45.0
100.0
12
5.6
n
0
in.2
45.0
100.0
13
8.3
o
0
12.8
45.0
100.0
K
8.3
0
0
17.9
47.5
100.c
15
8.3
0
0
17.9
50.0
100.0
15
11.1
2.5
0
17.9
55.0
100.0
17
14.9
2.5
o
20.5 .
55.0
100.0
18
14.9
5.0
n
23.1
55.0
100.0
19
14.9
¦ 5.0
0
28.2
'57.5
100.0
20
14.9
10.0
0
30.1
57.5
100.0
21
19.4
12.5
7.5
35.9
60.0
100.0
22
22.2
12.5
7.5
*1.0
62.5
100.0
23
22.2
12.5
7.5
48.7
65.0
10C.0
24
25.0
17.5
7.5
51.3
72.5 •
100.0
25
25.0
17.5
10.0
51.3
75.5
100.0
26
30.1
17.5
in .n
51.3
77.5
100.0
27
30.1
17.5
12.5
56.4
80.0
10C.0
23
30.1
22.5
15.0
58.9a
80.0a
100.0
aSinnificantly different £ron controls [a = 0.05).
100
-------
Table 29. Number of of'sDring per female mysid shrimp (Hysidop-sis bahia)
exposed to DEF in a 28-day life-cycle toxicity test.
Concentration (wq/JZ.)
Nominal
Measured
Total
offspri ng
Offspri ng
per female
(range)
Control ND
Carrier control ND
Q.5 0.34
1.0 0.63
2.0 1.7
362
413
254
88
0
22.6 (13.7-42.0)
20.5 (5.3-28.0)
10.8
6.6*
(7.0-16.0)
(0.0-10.0)
®ND = nondetectable (<0.02 ug/z).'
bSignificantly different from controls at (<
.05)
-------
Table 30. Results of persistence studies with HEF. Numbers represent
micrograms (ug) of -the pesticide detected in the respective
compartment. The tota">s are the percentages of the combined
micrograms of pesticide detected in a system,when compared to
the micrograms originally added to the system. ND means no
pesticide detected.
System Compartment Time
Day 0
Da.y 5
Day 7
Untreated3
water (ug)
7.5
ND
sediment (ug)
3.1
1.9
total (7-.)
106%
19%
Steri le*3
water (yg)
sediment (yg)
total (%)
ND
7.7
77%
Seawater^
water (yg)
9.B
3.8 '
resin (ug)
:!D
1.2
total (%)
98%
50%
aTen grams of sediment (wet weight) and 100 mi of 20 ym filtered seawater
with 27 Darts per thousand salinity fortified with 10 yg o' CEF in 10 y£
acetone so that at Day 0, water concentration equivalent to. 75 yg/£ and
sediment concentration equivalent to 3100 yg/kg (dry weight). Systems .
aerated at midpoint a* water column at 50 m£ per minute flow rate, with
pesticide in exiting air purged through XAD-4 resin trap.
Same as untreated systems except that 5 r*z of formalin was added 24 hours
before Day 0. Sterility was confirmed by plating aliquots from the-water
column on 15 .parts per thousand Zobe'l's media throughout the test.
cSame as untreated systems, except that sediment was not added'.
102
-------
Table 31. Mortality (percentage killed), of mysid shrimp ''-lysidopsis bahia) during a
chronic (28-day) exposure tc EPM. The mysids were 24-h-old at the begin-
ning of the test. corty mysids were placed in each treatment. Salinity
was fron 24-32 °/oo and temperature, 21-24°'.
Hay
Control
Carrier control
0
Measured concentrat*on
06 0.03 0.44
(uq/u.)
4.1
1
0
0
0
0
n
0
2
2.5
0
0
2.5
o
0
3
2.5
2.5
0
2.5
0
2.5
4
2.5
2.5
0
2.5
0
5.0
5
2.5
2.5
0
2.5
0
5.0
6
2.5
2.5
0
2.5
o
5.0
7
2.5
2.5
0
2.5
0
c; n
8
2.5
2.5
0
2.5
0
5.0
9
2.5
2.5
0
2.5
0
5.0
in
2.5
2.5
0
2.5
0
5.0
H
2.5
2.5
0
2.5
0
7.5
12
2.5
n t;
u • -•
c
5.0
-.0 '
10.0
13
2.5
2.5
0
5.0
5.0
15:0
14
2.5
2.5
0
5.0
5.0
20.0
15
2.5
2.5
0
5.0
7.5
20.0
16
2.5
2.5
0
5.0-
7.5
20.0
17
2.5
2.5
0
5.0
7.5
22.5
13
2.5
2.5
0
5.0
7.5
25.5-
19
2.5
2.5
0
5.0
7.5
30.0
20
2.5
2.5
0
5.0
7.5
35.0
21
2.5
2.5
0
5.0
7.5
37.5
22
5.0
2.5
c
5.0
7.5
47.5
23
5.0
2.5
0
7.5
7.5
57.5
24
7.5
2.5
0
7.5
7.5'
65 .0
25
7.5
2.5
0
7.5
7.5
55.0
25
7.5
2.5
0
7.5
7.5
65.0
27
7.5
2.5
0
7.5
7 .5
55.0
23
7.5
2.5
0
7.5
7.5
57.5a
aSignificantly different fron controls (a = 0.05) at the end of test.
103
-------
Table 32. Number of offspring per fenale ~ysid shrimp (Mysidoosis bahia)
exposed to EPM in a chronic (28-day) exposure "in natural, flow-
.i ng seawater.
Concentration f
yq/2)
Total
Offspring
• lomi nal
Measured
. offspring
per
fema1e
(range)
Control
NDa
' 306
15.1
(10.0-21.0)
Carrier control
fin
219
14.6
(6.0-31.5)
0.075
n oc
u • JO
426
18.5
(10.0-32.5)
0.10
0.08
296
14.8
(9,0-23.0)
0.30
0.44
301
15.9
(9.5-25.5)
3.0
4.1
-J
t
"3 C,-
5 (1.0-6.0)
!ND = iondetecta.ile (<0.O2 yg/z).
¦Significantly different from controls (a = 0.05)
1C4
-------
Table. 33. Percentage survival of sheepshead minnows (C.yprinotion variegatus)
continuously exposed to EPN for 265 days in an entire 1 ife-cycle
toxicity test. The exposure began with 118 to 121 embryos per treat-
ment. The survival data are divided into: (1) percentage of embryos -
that hatched; (2) percentage of hatched fish that survived as juveniles
until the 23th day of the test; and (3) percentage of adult fish that
survived from '"'ay- 28, when the total number of fish was reduced to 60
per treatment, until Day 140. Since thei^e were no significant differences,
duplicates were combined.
Measured Percentage Survival
concentration Embryos to Hatching Hatching to Day 28 Day 23 to 140 Tot a Ta
(vg/i)
Control , MD*5
90.0
99.1
96.8
86.3
0.25
90.0
99.2
100.0
89.3
0.50
88.8
96.1
96.6
82.4
0.88
94.1
97.4
98.3
90.1
2.2
. 83.4
94.9.
93.3
73.8
4.1
89.2
95.9
85.7
73.3
7.9
83.3
96.7
83.0C
66.9
aP»*oduct of survival percentages for embryos to hatching, hatching to Hay 28, and
Day 28 to 140. Statistical analyses were not performed on data in the "~ctal"
column.
Carrier control, "ID = nondetectable (<0.05 pg/z).
cSicnificantly different from survival of controls, (Chi-square test, a =0.05).
105
I
-------
Table 34. Effect of CPN on average standard length (imi) of sheepshead minnows (Cyprinodon
variegatus) exposed for an entire life-cycle.
Measured
concentration (iig/£)
--
Day of exposure
28 d
60a
88 J
118
140
ND; Controll>
10.6
19.7
24.6
29.4
32.4
0.25
10.0C
19.6
24.5
29.7
32.0
0.50
9.9C
19.3
24.2
29.0
32.1
0.88
10.0C
19.5
24.0
28.1
32.2
2.2
9.7C
18.5C
23.6
28.1
31.8
4.1
9.6C
18.8
23.4
28.5
31.6
/ .9
9. 7C
17. lc
20.7C
2b.9C
28 .8C
a Location was significant in ANOVA (a <0.05) but there was no significant location x
concentration interaction; therefore, duplicates were combined for these analyses.
Control with carrier; NO = nondetectable (<0.05 ug/n).
c Standard lengths differed from those of control fish; Analysis of Variance and
Duncan's Multiple range test (a = 0.05).
-------
Table 35. Fgg production by sheepshead minnows (Cyprinodon variegatus) continuously exposed to measured concen-
trations of KPN in flowing seawater at 30°C. Spawning groups usually consisted of two males and
three females from a giver* concentration. Percentage fertility is in parentheses.
Exposure
concentration
(hQ/*)
l)up I i cate
Trial" T
Average number eggs/female/day
rb j -rrrflr
Trial TP
Trial HI
Duplicate
Afl Trial s
X
O
Nnc
upper
12. 8
(99.0)
'13.2
(98.7)
13.8
(96.9)
13.3
(98.2)
7.9
(98.1)
ND
lower
7.5
(96.0)
0.0
(100rt)
0.0e
-
2.5
(98.0)
0.25
upper
18.2f
(100)
. 7.8
(99.4)
5.0
(98.0)
-10.3
(99.1)
13.1
(97.9)
0.25
1 ower
17.6
(98.9)
0.4
(94.1)
29.8°
(97.3)
15.9
(96.8)
0.50
upper
23.0°
(99.8)
20.1
(99.8)
9.1
(99.3)
1/.4
(99.6)
11.8
(99.6)
0.50
1 ower
17.5
(99.0)
1.0
(100)
0.0
(I00d)
6.2
(99.7)
0.88
upper
16.2
(99.0)
2.4
(100)
17.6
(99.4)
12.1
(99.5)
8.6
(99.4)
0.88
lower
15.2
(99.3)
0.0
-
0.0e
-
5.0
(99.3)
?.?
upper -
12.3
(100)
f). 0
-
0.0
-
4.1
(100)
3.8
(99.2)
2.2
lower
10.7
(98.4)
0.0
-
0.0
-
3.6
(98.4)
4.1
upper
9.0°
(97.2)
0.0
-
0.0e
-
3.0
(97.2)
2.6
(98.6)
4.1
lower-
3.9
(100)
0.0
-
2.0
(100)
7.9
upper
0.1
(100)
0.5
(100)
0.1
(100)
0.2
(1 oo)
0.5
(93.8)
7.9
1 ower
1.7
(/ 5.0)
0.0
-
0.9
(75.0J'h)
Spawning data for If) days were analyzed.
Spawning data for 14 days were analyzed.
^Carrier control, ND - nondetectable (<0.05 pg/Jt).
'Although less than 0.1 eggs/female/day were produced all were fertile.
^Two feinales and three males.
One female and four males.
^Significantly different from all other treatments (a = 0.05).
Significant (a = 0.05) duplicate "^concentration interaction.
-------
Table .16. Average dissolved oxygen concentrations (mg/£) curing a 265-day toxicity test
in which sheepshead ninnows (Cyprinodon vari egatus) were continuously exposed
to measured concentrations of EPN at 3n°Ti Dissolved oxygen content was
determined weekly in one duplicate exposure aquarium in each exposure
concentration.
Day Portion of test N Mean Standard
deviation
0-28 Parental errbryo/ 28 4.9a 0.64
juvenile survival
29-149 Juvenile and adult 119 3.5 0.76
survival
150-161 Spawning trial I 14 ?.5a 0.80
192-209 Spawning trial II 21 3.6 0.8*
216-231 Spawning trial III 14 3.2 0.87
150-231 All spawning trials 84 3.5 0.97
222-259 Progeny survival 35 3.3 0.86
0-265 Entire test 266 3.6 0.94
aSignificantly different from other portions of the test; a = 0.05.
1C8
-------
Table 37. Survival of embryos, fry, and juvenile sheepshead minnows
(Cyprinodon varienatus) and standard lengths of surviving
fish continuously exposed to measured concentrations of EP'I
for 28 days. Exposure began with 80 embryos per duplicate
aquarium. Embryos were spawned by adult fish that had
been continuously exoosed in the respective concentrations
for greater than 222 days. Since no significant difference
(a = 0.05) existed between treatments, duplicates have been
combi ned.
Exposure
concentrati on
Survival (%)
Average
standard
length (mm
(uG/O
Embryos Fry and Both stages
juveniles
NDa
cn 9
>J « L.
100.0
98.7
92.7
75.n
90.0
88.9
85.0
8.9
0.25
9.0
0 .50
8.8
0.88
8.6
^Carrier control, MP = nondetectable (<0.05
81 embryos.
c 120 embryos.
109
-------
Table 38. Concentrations of EPN (pg/g, whole body, wet weight) measured in selected life stages of
the sheepshead minnow (Cyprinodon variegatus) in a 265-day life-cycle toxicity test.
Life stage, Hays exposed
Average measured exposure concentration, (pg/&)
Control,
nit
0.25
0.50
0.88
2.2
4.1
7.9
Juveniles, 20
N0a
ND
NO
0.40
2.3
6.4
8.0
Adult males, 161
ND
0.31
1.0
1.8
44.
3.9
67.
Adult females, 161
ND
0.21
0.64
2.9
15.
38.
81.
Adult fish, 235
Nl)
0.39
1.0
4.3
5.4
21.
46.
Lmbryos, 146 to 200
no
NO
0.60
1.9
1.5
Juvenile progeny, 28
Nil
ND
0.29
0.63
aND - nondetectable (<0.05 ug/«. water, <0.25 ug/g tissues).
-------
Table 39. Rioconcentratiori factors (concent rat ion measured in tissue divided by the average concen-
tration measured in exposure water) for EPN in selected life-stages of the sheepshead
minnow (Cyprinodon variegatus) in a ?6!>-day life-cycle toxicity test. EPN was not
detected in control fish or embryos.
Life stage, Days exposed
Exposure
concentrati on
(u9/4)
0.25
0.50
0.88
2.2
4.1
7.9
Jtiveniles, Day 2B
NDd
m
450
1,000
1,600
1,000
Adult males, Day 161
1 ,200
2,000
2,000
20,000
950
8,500
Adult females, Day 161
840
1,300
3,300
6,800
9,300
10,000
Adult fi sh, Day 235
1,600
2,000
4 ,900
2,500
5,100
5,800
Kmbryos, Days 146 to ?()()
MD
1 ,200
2,200
fiHO
Juvenile progeny, Day 28
ND
580
720
aND - nondetectable EPfl in tissues (<0.?5 pg/g); therefore, no hi oconcent.rati on factor could be cal-
cuIated.
-------
Table 4D. Acetylcholinesterase activity, expressed as micromoles of acetyl-
choline hydrolyzed/hour/mg brain tissue x 100, in brains from
sheepshead minnows (Cyprinodon variegatus) that were continuously
exposed to EPN. Each assay sample consisted of pooled brains
from three fish; three samples were assayed to obtain each datum.
Measured Day 161 Day 235
concentration (yg/z)
AChE
% Inhibition
AChE
% Inhibition
NDa
111
205
0.25
117 b
34
118 b
42
0.50
81b
54
81b
60
0.88
51b
71
LH
00
cr
72
2.2
38b
78
35 b
83
4.1
32b
82
36 b
82
7.9
24 b
86
30'b
85
^Carrier control, ND = nondetectable (<0.05 yg/z).
Significantly less than that of controls (a =0.05).
112
-------
Table 4-1. Results of persistence studies with EP"!. Numbers represent
micrograms (ug) of the pesticide detected in the respective
compartment. The totals are the percentages of the combined
micrograms of pesticide detected in a system when compared to
the micrograms originally added to the system. NO means .no
pesticide detected.
System
Compartment
Time
Untreated3
Steri 1 e^
Seawaterc
water (ug)
sediment (uq)
total {%)
water (yg)
sediment (ug)
total '%)
water (ug)
resin (ug)
total (?)
Day 0 Hay 5 Pay 7
6.2
2.1
83%
9.7
no
97%
NP
2.8
28%
MD
7.9
79%
3.9
m
29%
aTen grams of sediment (wet weight) and 100 m£ of 20 un Altered seawater
with 27 parts per thousand salinity fortified with 10 ug of EP?i in 10 u£ of
acetone so that at Day 0, water concentration was equivalent to 62 ug/£ and
sediment concentration equivalent to 2100 pg/kg (dry weight). Systems
aerated at midooint of water column at 50 n£ per minute flow rate, with
pesticide in exiting air purged through XAD-4 resin trap.
Same as untreated systems except that 5 nu of formalin was added ?A hours
before Day 0. Sterility was confirmed by plating aliquots from the water
column on 15 parts per thousand Zobell's media throughout the test.
cSame as untreated systems, except that sediment was not added.
113
-------
'anle 42. Mortality (percentage killed) of mysids (Mysidopsis bahia) during a
chronic (28-day) exposure to ethoprop. The mysids -were ?A- to 48-h
old at the beginning of the test. Twenty Tiysids were placed in each
ethoprop treatment (ten per replicate); ten mysids in the control and
solvent control treatments. Salinity was from 26 to 31 °/oo and
temperature, 22 + 1°C-
Measured concentration (ug/i)
Carrier 0.15 0.27 0.36 0.62 1.4
Day Control control _A _B _A _J5 __A _B _A _J3 _A 3
1
n
'J
0
0
0 .
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
o
•J
¦ 0
3
0
0
0
0
0
0
0
0
0
0
0
0
4
0
0
0
0
0
0
0
0
0
0
0
0
5
0
0
0
0
0
0
0
0
0
0
0
0
6
0
0
0
0
0
0
0
c
10
0
0
0
7a
0
0
0
0
0
0
10
0
20
0
0
0
8
0
0
0
0
0
0
10
0
20
c
0
0
Q
c
0
0
0
0
0
10
0
20
0
0
0
10
0
0
0
0
0
0
10
0
20
0
0
0
11
0
0
0
.0
0
0
10
0
20
0
0
0
12
0
0
0
0
0
0
10
0
20
n
0
0
13
0
0
0
0
0
0
10
0
20
0
0"
:o
14 a
0
0
. 0
0
0
0
10
0
20
0
0
10
15
0
0,
0
0
0
0
10
0
20
0
0
10
15
0.
ob
o
w L.
0°
0.
0°
10h
0,
20
0
0
10
17
ob
0
0b
0
cb
0
10b
o3
20h
0
oK
10
18
0
0
0
0
0
0
10
0
20
0,
ob
* n
i ^
19 '
0
0
0
0
G
0
10
0
20
nD
0
10
20
0
0
0
0
0
0
10
0
20
0
10
10
21a
0
0
0
0
0
0
10
0
20c
10C
20 c
20
22
0
0
0
0
0
0
10
0
20
10
30
20
23
0
Q
0 .
0
0
3
10
0
20
10
30 ¦
20
24
0
' 6
10
0
0
0
10
0
20
20
30
30
25
0
0
10
10
10
0
10
0
20
20
30
40
26
0
0
10
10
10
0
10
0
20
20
40
40
27
0
10
10
10
10
0
10
0
20
20
50
40
28a
0
10
10
10
10
0
10
0
9QC
23c
50L
<10
Aver-
o -
10
10
5
5
20
r-
V
¦4
5C
age:
^Statistical analysis performed.
-orT.ation of brood touches first observed.
cSignificantly greater (a = 0.05) than the control.
114
-------
Table 43. Nunber of offsoring produced per female of nysid shrimp
(Mysidopsis bahia) exposed to ethoprop in a chronic (28-day)
exposure in natural, flowing seawater.
Measured Number of offspring
concentration Females with Offspring
(uq/O Rep A Rep B Total brood pouches per female
Control, NDa
38
-
38
6
6.3
Carrier control
38
-
38
6
6.3
0.15
41
35
76
11
6.9
0.27
30
36
66
11
6.0
0.36
26
11
37
6
6.2
0.62
22
16
38
7
5.4
1.4
11
5
16
4
4.0
|*ND = nondetectable (<0.2 ug/i).
^Significantly less (a = 0.05) than the control.
-------
Table 44. Survival of enbryo and juvenile sheeoshead minnows (Cypri nodon
varieqatus) exposed to various concentrations of ethoprop in
seawater at 30+1°0. Juvenile fish were exposed for 28 days
postnatch.
Exnosure concentration (yq/z}"
Survi val, %
Noninal
Measured
Embryo
Juverr' 1 es
Control
NDa
95
74
Carrier control
NO
96
78
26
12
94
68
43
21
39
45 b
72
35
95
ll3
120
54
91
*5b
200
98
90
2.5b
j*ND = nondetectable (<0.5 uqfi).
Significantly different from control (a = 0.05).
116
-------
Table 45. Standard lengths, tissue concentrations, and bioconcentraticn factors
of ethoprop in juvenile sheepshead minnows (Cyprinodon varieqatus)
exposed to various concentrations of ethoprop for 23 days.
Concentration (pg/fc) Concentration Average Bioconcentration
Nominal Measured in fish length factor
/
Control
NDa
fID
12
-
Carrier control
ND
NO
14
-
26
12
0.20
12
17
43
21
0.10
13
5
72
35
n.36
_b
10
12H
54
n.as
_b
9
200
98
0.42
_b
4
^MD = nondetectable (<0.05 yg/z water, <0.09 pg/g tissue).
Length not measured because of snail number of surviving fish.
117
-------
Table 46. Mortality (sercentage killed) of mysid shrimp (Mysidopsis bahia)
during a chronic (24-day) exposure of methyl parathion. The nysids
were 24-h-old at the beginning of the test. Forty nysids were
placed in each treatment unless otherwise noted. Salinity was from
12-25 °/oo and temperature, 2<*-270C.
Measured concentration (uq/a)
Day Control Carrier control O.OR 0.11 0.37 0.59
(N = 35) {<< = 35)
1
0
0
0
0
0
0
2
0
0
O
0
0
0
3
n
0
0
0
0
12.5
4
0
0
0
0
2.5
17.5
5
o
0
2.5
2.5
2.5
20.0
6
0
0
2.5
2.5
2.5
30.0
7
0
0
5.0
2.5
5.0
35.0
8
Q
0
7.5
2.5
10.0
37.5
o
3.0
3.0
7.5
10.0
17.5
55.0
10
3.0
11.0
10.0
15.0
27.5
55.0
11
3.0
17.0
• 10.0
15.0
37.5
80.0
12
3.0
17.0
10.0
15.0
42.5
82.5
13
3.0
20.0
10.0
25.0
50.0
82.5
14
3.0
20.0
10.0
25.0
60.0
85.0
15
3.0
23.0
10.0
25.0
62.5
92.5
16 .
3.0
23.0
10.0
30.0
75.0
97.5
17
3.0
23.0
10.0
32.5
77.5
97.5
18
3.0
29.0
15.0
37.5
80.0
97.5
19
6.0
34.0
15.0
37.5
80.0
100.0
20
6.0
34.0
15.0
40.0
82.5
100.0
21
6.0
34.0
15.0
42.5
85.0
100.0
22
9.0
34.0
15.0
42.5
85.0
100.0
23
9.0
37 .0
15.0
42.5
87.5
100.0
24
9.0
43.0
15.0
50.0
37.5a
100.0
aSiqn1ficantly different from controls (a = 0.05) at the end of test.
118
-------
Table 47. Number of offspring per female mysid shrimp (Mysidopsis bahia)
exposed to methyl parathion in a chronic (24-day) exposure
in natural, flowing seawater.
Concentrati on
Noninal
(ug/i)
M.easu red
Total
offspring
Offspri ng
per female
(range)
Control
NDa
133
8.3 (5.0-15.7)
Carrier control riD
53
5.9 (3.5-11.0)
0.095
0.05
206
11.4 (3.3-20.3)
0.18
C.ll
139
11.6 (2.0-15.0)
0.38
0.37
0b
nb
0.75
0.59
0b
0b
j*ND = nondetectable; CO.n? in seawater.
Significantly different fron controls at a = 0.05.
119
-------
Table 48. Results of persistence studies with methyl parathion.
Numbers represent micrograms (ug) of the pesticide detected
in the respective compartment. The totals are the per-
centages of the combined micrograms of pesticide detected
in a system when compared to the micrograms o^ininally added
to the system. f'D means no pesticide detected.
System
Compartment
Time
Day 0
nay 5
Pay 7
Untreated3
water
(ug)
8.8
0.11
'¦:n
sediment (yg)
n.p
0.15
t
total
(%)
97%
3%
Sterileb
water
(uq)
5.5
sediment (ug)
3.9
total
(?>}
1 p/le/
Seawater0
water
(yg;
0.0
7 C
! 4
res i n
(yg)
f ID
ftp
total
(*)
99"
75%
Hay 0
Hay 3
Cay 6
Sunli nht^
1 i ght
(yg)
8.8
6.6
3.8
total
100%
75%
43%
dark (
ug)
S .8
7.5
6.5
total
100%
85%
73%
aTen grams of sediment (wet weight) and 100 ri of filtered seawater (20 urn)
with 27 oarts Der thousand salinity were fortified with 10 ug metnyl
oa^athion in 10 of acetone, so that at Oav 0, water concentration of
8R 'jg/£ and sediment concentration of 900 ug/kg (dry weight). Systems were
aerated at midpoint of water co'umn at SO mz cer minute flow rate; pesticide
K in exiting air was sorben by a XAP-4 resin trap.
"As in untreated systems except that 5 mz of formalin was added 2^ Hours
before Pay 0. Sten 1ity was confirmed ny c'ating al'iquots from the water
column on 15 parts oer thousand Zobell's media throughout the study.
^Same as untreated systems, except that sediment was not addon.
"Systems were 250 mz Pyrex E^ienmeyer flasks fitted with ground class
stoppers. A homogeneous so-ution in filtered (20 urn) seawater of 27 parts
per thousand was prepared by adding 350 ug of methyl ra^athion in acetone to
a 4_£ amber bottle, purging the acetone with a stream of nitrogen, and add-
ing 2500 mi seawater. The bottle was shaken overnight and 100 mi alinuot was
added to each flask. The flasks were stopoered and half were covered with
foi1. The systems were exposed outdoors to direct sun'ight, where tempera-
tures in the flasks varied from a high of 46°C during the day to a low of
20°C at night.
120
-------
Table . Mortality (percentage killed) of nysid shririp ('lysidopsis bahia)
during a chronic (28-day) exposure to ohorate. The Tiysids were
24-to 48-h-old at t^e beginning of the test. Forty nysids were
placed in each treatnent unless otherwise noted. Salinity was frcn
20-26 °/oo and temperature, 26-29°C.
Measured
concentration
(ug/i)
Day
Control
Carrier control
0.02
0.06
0.09
0.21
(N = 35)
(M- = 39)
N = 38
1
0
0
0
0
0
n
2
0
0
0
0
0
c
3
0
0
0
0
0
0
4
0
2.5
3.0
0
0
0
5
. 0
2.5
3.0
0
0
0
5
2.5-
2.5
3.0
5.0
5.0
24.0
7
2.5
2.5
9.0
5.0
5.0
24.0
3
5.0
2.5
9.0
5.0
20.0 •
66.0
9
5.0
2.5
9.0.
5.0
20.0 .
66.0
10
7.5
10.0
9.0
5.0
20.0
74.0
11
7.5
10.0
9.0
5.0
20.0
74.0
12
7.5
12.5
9.0
5.0
20.0
74.0
13
10.0
15.0
9.0
7.5
. 20.0
74.0
14
12.5
15.0
14.0.
10.5
26-0
74.0
15
12.5
15.0
14.0
12.5
26.0
74.0
16
12.5
15.0
14.0
12.5
26.0
74.0
17
15.0
15.0
¦20.0
12.5
26.0
76.0
18
15.0
15.0
20.0
12.5
23.0
76.0
19
17.5
15.0
20.0
12.5
28.0
76.0
20
17.5
15.0
20.0
12.5
31.0
79.0
21
17.5
20.0
26.0
12.5
31.0
a 2.0
22
17.5
25.0
26.0
12 .5
33.0
82.0
23
20.0
27 .5
26.0
15.0
38.. 0
82.0
2*
30.0
30.0
29.0
20.0
41.0
82.0
25
30.0 '
32.5
34.0
22.5
46.0
82.0
26
37.5
32.5
37.0
35.0
51.0
82.0
27
37.5
32.5
43.0
35.0
53.0
82.0
28
37.5
40.0
43.0
37. d
53.0
82.0a
aSignificantly different f^om controls (a = 0.05) at the end of test.
121
I
-------
¦Table 50. Number of offspring per renale mysid shrimp (f-'ysidopsis bahia)
exposed to phorate in a chronic (28-day) exposure in natural ,
flowing seawater.
Concentrati on
Momi nal
(ug/*)
Measured
Total
offspring
Offspring
per female
(range)
Control
NDa
292
16.8 (3.0-25.5)
Carrier control ND
234
12.5 (6.0-21.0)
0.03
0.02
155
9.2 (2.0-13.7)
0.08
0.06
.91
6.8 (2.0-12.0)
0.16
0.09
141
12.8 (2.0-23.0)
0.32
0.21
19
8.2 (2.5-14.0)
aND = Nondetectable (<0.02 ug./£)«
122
-------
Table 51. Survival of embryonic and juvenile sheepsheac! minnows (Cyprinodon
variegatus) exposed.to various concentrations of phorate seawatsr
for 28 days at 30°C.
Exposure concentration (pg/JL) Survival {%)
N'onina1 Measured Embryos Juveniles 3oth stages
Control
NDa
97.5
98.7
96.2
0.09
0.16
92.5
97.3
90.0
0.19
0.24
95.0
97 A
92.5
0.38
0.41
98.8
R8.fic
87.5
0.75
0.77
96.2
57. lb
55.0b
1.5
1.2
97.5
3.8b
3.8b
3.0
2.5
88.8
0b
0b
^ND = nondetectable (<0.02 yg/£).
bSignificantly different from control (a = O.ni).
cSignificantly different from control (a = 0.05).
123
-------
Table 52. Standard lengths, tissue concentrations, and bioconcentrat ion factors
of phorate in juvenile sheepshead minnows (Cyprinodon variegatus)
exposed to various concentrations of phorate for 28 days.
Concentrati on
(pq/i)
Concentration
Average
Bioconcentration
Nomi nal
Measured
in fish
(pg/g)
1ength
(mm)
factor
Control
NDa
MD
9.5
n .094
0.16
ND
9.3
-
0.19
0.24
ND
9.3 .
-
0.38
0.41 .
r:p
8.9
-
0.75
0.77
C .069
a.»
90
1.5
1.2
-
.10.7
-
aMD = nondetec
table (<0.02 yg/fc water, <0.02 yg/g
tissue).
124
-------
Table 53. Results of persistence studies with phorate. Numbers repre-
sent nicrcgrans (yg) of the pesticide detected, in the respective
compartment. The totals are the percentages of the combined
rnicrograns of pesticide detected in a system when comoared to
the micrograms originally added to the system. *.'D -"eans no
pesticide detected.
System
Compartment
T i me •
Pav 0
Day 5
Day 7'
Untreated3
water (ug}
sediment (ug)
total (%)
6.5
1.9
84%
MD
ND
Sterileb
water (ug)
sediment (ug)
total (%)
W
MD .
Seawaterc
water (ug)
resin (ug)
total (*)
9.7
nc
97%
riD
3.1
31%
aTen g^ans of sediment (wet weight) and 100 m of filtered seawater
(20 ym) of 27 parts per thousand salinity fortified with 10 ug of Dhorate
in 10 ui of acetone, so that at Day 0, water concentration of 65 yg!i and
sediment concentration of 1,900 uQ/kg (dry weight). Systems were aerated at
midpoint of water column at 50 nu per minute flow rate; pesticide in exiting
air was sorbed by a XAD-4 resin trap.
Same as untreated systems except that 5 n£ of formalin was added ?A h
p^ior to Day 0. Sterility was confirmed by plating aliquots fron the water
column on 15 parts per thousand Zobell's media throughout the study.
cSame as untreated systems, except that sediment was not added.
125
-------
Appendix A-l. Description of acephate used in tests.
Acephate (Qrthene). The technical material, as supplied by the
manufacturer was labeled "Chevron, ORTHO, ORTHENE, Analytical Standard, S X
985; Chevron Chemical Company, Ortho Division/San Francisco, CA 94119,
Richmond, CA 94804" and "Orthene, t 169.5." Amount: = 20 a.
EPA/FDA fio. ' 481 •
Lab Code: 73-31
Name: acenhate; Orthen^; Ortho 12,420
Systematic Name: 0,S-Dinethyl acetylphosphoramidothioate
Empirical Formula: C^HigNO^PS M.W. 183.17 ¦ State: Solid
Structure:
Use: Insecticide
Stability: Slightly hygroscopic, sensitive to heat, relatively stable to
"Toxicity: Acute Oral LP.^q (rats) female 866 mg/kq; ^.ale 945 tig/kg.
Mass spectra1 analysis was not run on this pesticide.
0
P-NHCOCH
ch3o
II
hydrolysi s
1 oc.
-------
Appendix A-2. Description of aldicarb used in tests.
Aldicarb (Temik). The technical material was labeled "Aldicarb,
Union Carbide, 5/2/78, Purity: 100%, Lot 4-CHA-32-2, amount: 1 gm."
The primary standard was obtained from the Pesticide Reference
Standards Section of EPA and had the following characteristics:
FDA No. 262
Name: aldicarb; Temil^, UC-21149
Systematic flame: 2-'1ethyl-2-(methylthio)propionaldehyde 0-(methyl-
carbonyl) oxime
Empirical Formula: ^i^O^S 190.26. State: Solid
Structure:
CH3 0
ch3-s-c-ch=n-o-c-mh-ch3
ch3
Stability: Decomposes on prolonged heating; oxidized by air to the
sulfoxide and sulfone
Toxicity: Acute Ora 1 lc50 ' rats) 1 mg/kg
Purity: 99.8% + 0.1%
Mass spectral analysis was not run on this pesticide.
127
-------
Appendix A-3. Description of carboDhenothion used in tests.
P
Carbophenothion (Trithion ). The technical material was frcn 2
stock supply and was labeled "Carbophenothion, Chen Services, 12/77,
9/6/78, 6/6/79; Purity: 94.6%; amount: 10 gm."
The primary standard.was obtained from the Pesticide Reference
Standards Section of EPA and had the following characteristics:
FDA N. 147
Lab Code: 70-66
D
Name: Carbophenothion; Trithion
Systematic Name: S-[(p-Chlorophenylthio)nethy1] 0,0-diethy1
phosphorodithioate
Empirical Formula: -n^l6^2P^3 342.87 State: Liquid
Structure:
CI
S-CH2-S-P(0C2H5)2
Stability: Relatively stable in water
Toxicity: Acute Oral LR^ (rats) 10 - 30 mg/kg. Absorbed 'through
the skin.
Manufacturer's Assay: 95%
The nass spectra of the technical and analytical material were identical,
with the molecular ion visible at ''/E 343.
CAR90PHENOTHI0N
ANALYTICAL STANDARD
M.W 142 BT
100-
80-
jj 60**
® 40-
u.
O
* 20H
r 10
1
Jit
f 1 j ' 1
111
20 40 ' SO 80 ' 100 * 120 * 140 ' 6C ' 180 200 ' 220 240 ' 260 ' 280 ' 100 320 340
M/E
. O
-0
360
128
-------
Appendix A-4. Description of DEF used in tests.
OEF. The technical material was from a stock .simply and was
labeled "DEF, Lot: 7710549Chen Services, Date: 3/25/78, Purity: 95%,
amount: 10 grn."
The primary standard was obtained from the Pesticide Reference
Standards Section of EPA and had the following characteristics:
FDA No. 61
Lab Code: 58-33
Name: DE@
Systematic Name: S,S,S-Tributyl phosphorotrithioate
Enpi»-ical formula: C^H^OPS-j ?1.W. 31-"-.50 State: Liquid
Structure: (C^HgS^ ?=0
Stability: Hydrolyzed by strong acid and alkali.
Toxicity: Acute O^al LD^g (rats) 325 ma/kg. Absorbed through the
Manufacturer's Assay: Technical; approximately
The mass spectra of the technical and analytical material were identical,
with the nolecular ion visible at M/E 314.
DEF
ANALYTICAL STANDARD
MW 31450
skin.
lOO-i
UJ
CL
<
CD
U.
° 40-
20-
-------
Appendix A-5. Description of Ethoprop used in tests.
Ethoprop (Mocap^). The technical material was supplied by
Bionomics Research Laboratory and was not labeled.
The primary standard was obtained from the Pesticide Reference
Standards Section of EPA and had the following characteristics:
FDA No. 350
Lab Code: 68-106
Name: Moca^); Propho^
Systematic Name: 0-Ethyl S,S,-dipropyl phosphorodithioate
Empirical Formula: CgHjg OpPSj, M.W 242.34 State: Liquid
Structure:
0 S-C-jH-,
j /
c2h5-o-p.
Stability: Hydrolyzed by alkali
S_C3H7
Toxicity: Acute 0ra: LD^q (rats) 30-56 mg/kg. Absorbed through
the skin.
Manufacturer's Assay: 95%
Mass spectral analysis was not run on this pesticide.
130
-------
Appendix A-6. Description of EP"! used in tests.
EPN. The technical materia1, was obtained from a stock supply and was
labeled "F.PN, Supplier: Chern Servi ces, Date: 3/25/7B, Purity: 97.2%,
amount: 10 gn."
The primary standard was obtained *rom the Pesticide Reference Standard
Section of EPA and had the following characteristics:
FDA No. 51
Mane: EPF!
Systematic Name: O-Ethyl O-p-nitroohenyl pheny'phosphonothioate
Emphirical Formula: C^H^NO^PS 323.31 State: Solid
Structure:
p.0^"yN02
oc2H5
Stability: Unstable in alkali
Toxicity: Acute Oral LO^q (rats) 9-45 mg/kg. Absorbed throuqh the skin.
The mass spectra of the technical and analytical material were identica
with the molecular ion visible at M/F. 323.
EPN
TECHNICAL MATERIAL
MW 12131
OO-
BO
1
40'
¦5 H
o
3
2C 40 SO 30 ,00 120 140 160 leo 2O0 220 240 2&C 280 300 320 340
M/E
131
-------
Appendix A-7. Description of methyl parathion used in tests.
Methyl Parathion. The technical naterial was obtained rrom a stock supply that
was labeled "Methyl Parathion, HGF: Chem Services, Date: 12/9/77, Purity: 99%,
amount: 10 gm."
The primary standard was obtained from the Pesticide Reference Standards
Section of EPA and had the following characteristics:
FDA No. 88
Name: Methyl Parathion
Systematic Name: 0,0-Dimethyl 0,p-nitrophenyl phosphc>~othioate
Emphirical Fornula: CgH^NO^PS M.W. 253.21 State: Solid
Structure:
S
f
op(och3;2
Stability: Hydrolized in alkali.
Toxicity: Acute Oral LD^q (rats) 9-25 rng/kq. Absorbed through the skin.
The mass spectra of the technical and analytical material were identical,
with the molecular ion visible at M/E 26.1.
METHYL PARATHION
TECHNICAL MATERIAL
M.W. 263 21
lOOl
80-
*
< _
u 60'
o.
UJ
in
2 40
20'
20
li, Li 4^
-t
40
60
llrll -,U . nlh—, ill, ill.—,-L-,—,—.—,—4—,—,—,1 I ' I—I Ml ! l-c
00 160 lio i4o i6o 160 260 zio zk> 260 2*0
M/E
10
o
H
132
-------
Appendix A-3. Description of phorate used in tests.
Phorate (Thionet"). The technical mat en a 1 was fron a stock supply and was
labeled "Phorate (Thionet), Lot: SPS-11548, Anerican Cyanamid, Date: 12/77,
Durity: 39.5%, amount: 5 nm."
The prinary standard was obtained from the Pesticide Reference Standards Section
of EPA and had the following characteristics:
FDA Mo. 160
Lab Code: 70-14
D
'lame: phorate; Thionet
Systematic Pane: 0-0-Diethyl S-(ethvlthio)methyl phosphorodithioate
Empirical Formula: -7^7 260.38 State: Liquid
Structure:
S
t
(C2H50)2P-S-CH2-S-C2H5
Stability: Hydrolyzed by alkali
Toxicity: Acute Oral LO^q (fats) 1-2 ng/kq. Absorbed through the skin.
The mass spectra of the technical and analytical material were identical,
with the molecular ion visible at M/'E 260.
PHORATE
TECHNICAL MATERIAL
M W 260 38
100
x
4
01
<
OD
80
60-
£ 40'
20-
J—J-
20
40
60
"1—
80
M*
100
r ' "j 1—1 "1—1 1 , "|—r
120 140 i£o 180 2<
200
—, ,—| 7 1
220 240 260 280
M/E
133
-------
APPENDIX B
CMEHICAL METHODS FOR THE ANALYSES OF ACEPHAIE (CRTHENE ),
ALDICAPE (TEMIKR), CARROPHENOTHICN (TRITHIOHR), ETHOPROP
(WCAPR), AND METHYL PA.RATHION IN SEAWATER AMD TISSUE SAMPLES
ER&G International, Inc., R">ononics ODerations
Diononics Marine Research Laboratory
Route 6, Box 100?
Pensacoia, Florida 3?507
In Partial f01fillnent of:
Contract Number 68-01-497^
U.S. Environmental Protection Agency
Ecological Effects Branch
Office of Pesticide Programs
l/ashington, O.C. 20460
134
-------
1
The chemical methods describee in this report were used for
the quantification of acephate (Orthene®) aldicarb iTemik®) ,
carbophenothion (Trithion©) , ethoprop (Mocap'3) , snd methyl
parathion in seawater and tissue samples from toxicity tests con-
ducted at Bionomics- Marine Research Laboratory (BMRI.) in Pensaccla,
Florida. This report should be referred to in conjunction with the
following reports submitted to the U.S. Environmental Protection
Agency: BP-79-2-16, BP-79-2-17, 3P-79-2-18, BP-79-2-19, and BP-79-2-22.
All cata related to th^se studies are stored at BM?LL.
All chemicals used in preparing the seawater and tissue samples
for chemical analysis were pesticide grade.
CHEMICAL METHODS
Water samples for acephate (Crther.ei
Seawater samples (900-railliliters [:?.£•]) were placed in amber
glass bottles with Teflon®-lined caps. Each sample was preserved
with 15 of chloroform and stored in the dark at anbient temperature
until analyzed.
A 25-rai subsample was removed from the sample bottle and added
to a 0.5-liter (i) jar. One hundred grans (g) of anhydrous sodium
sulfate was added to the jar with stirring to prevent cakinc. The
sodium sulfate had been washed with ethyl acetate - ar.c oven dried
at 275 decrees .Celsius (°C) to eliminate interfering components.
Then 100 ml of ethyl acetate was quickly added and blended v;tn the
sodium sulfate at high speed for twe minutes with a top-drive blender.
The solids were allowed to settle and the extract was decanted
through a glass funnel plugged with glass wool ar.d containing approxi-
mately 50 of g sodium sulfate. The extraction ana decanting were
135
-------
2
repeated twice with 50-..x,£ portions of ethyl acetate. The sodium
sulfate was then washed with a 25-mi porticn of ethyl acetate and
then the combined solvent fractions were collected in a round bcttoir.
flask and evaporated to less than 1 rat on a rotary evaporator with
a 40-45°C water bath.
The sample was then transferred to a 15-ni. graduated centrifuge
tube with methyl-isobuty1-ketone and diluted to a known volume.
Finally, the sample was transferred to a glass vial, capped with a
Teflon-faced rubber septun and stored at 3°C until analysed.
In order to verify the recovery of acephate from seawater,
quality control standards were produced by fortifying seawater with
known masses of acephate dissolved in acetone. The samples were
prepared for analysis by the preceding methodology, Analytical
standards of acephate i nme thy 1 - i sobu ty 1- ke tone were prepared bv
serial dilution of the acephata-in-acetor.e stock solution.
The samples were analyzed by using the following .instrument
conditions:
Instrument: Perkin-Elrr.er Model Sigma-2 gas chromatograph .
equipped with a rubidium bead nitrogen-
phosphorus detector.
Column: 3-m x-2 nun ID PyrexS packed with 10% carbovrax 20 M
on 80/130 mesh Supelccport
Temperatures (°C): Injector: 225
Oven: 2 00
Detector: 250
Gas flows: Carrier: nitrogen, 20 mVninutes (ir.in)
Reactar.t: hydrogen, 5 m£/nun
Supcort: air, .100 ni/min
Retention time: 2.2 min
Chart spead: 1 centimeter (cm)/mm
Amplifier range: 1 millivolt irav)
Attenuation: 1
-------
3
Bead adjust: 400
Response: Approximately half-chart deflection with'60 ng acephate
The results of the recovery of acephate from seawater are sum-
marized in the following table.
Sample and
reDlicate
Acephate mass
added (ir.q)
Acephate .T.ass Percentage
recovered (mg) recovery
Blank A
0.0
<0 .01
N/A
3
0.0
<0.01
N/A
C
0.0
<0.007
N/A
D
0.0
<2.008
N/A
2
0.0
<0.005
N/A
Low range A
0.05
0 . 018
36
B
0.05
0.030
SO
C
0 .03
0.040
80 '
D
0.05
0 .0 25
50
E
0.05
<0.007
Mic range A
0 .50
0 . 22
44
B
0.50
0.17
34
C
0.50
0 .19
38
D
0 .50
0.11
22
E
0 .50
0.32
64
High ranga A
5.0
2.4
48
3
5.0
2 . 4
48
C
5.0
3.2
64
D
5.0
4 .0
80
E
5.0
3 .1
62
Mean percentage
low rar.ce E was
recovery ± standard
not included in the
deviation;
calculation
52+17.
of the mean.
137
-------
The analytical results cf the samples were calculated by coir-
paring the peak height at retention time 2.2 minutes with the peak '
heights of external acephate standards. A calibration curve was
calculated using the parabolic curve fit with the sum of the squares
of the errors minimized JAdams, 1976) for the peak heights (mn)
versus mass (ng) of acephate. The results vers corrected for a
52?. recovery. The minimum detectable concentr atior. of acephate in
seawater was 0.4 mg/x based on a 25~n£ water sample concentrated to
5.0 nf.
Hater samples fcr aldicarb (Teir.ik)
Seawater samples (900-nI) were placed in amber glass bottles
with Teflon-lined caps. Each sample was preserved with 15 n»i of
chloroform and stored in the dark at ambient temperature until
analyzed.
Samples were extracted twice in a 1-2. separatory funnel with
50 mi portions of dichloronethane. The organic phase was passed
through glass wool to break up any emulsions. The pooled organic
phase plus 2 m£ toluene were evaporated almost to dryness on a rotar
evaporator in a water bath at 4o-50°C.
The samples were then quantitatively transferred to a 15-xii
graduated centrifuge tube with toluene and diluted to a known volume
The sample was -hen transferred to a clear glass vial and capped wit
a Teflon.-sided septum. The sample was stored in a.refrigerator
at 8CC prior to analysis.
In order to verify the recovery of aldicarb frcir. seawater,
quality control standards were prccucsd by fortifying seawater with
known masses of aldicarb dissolved in acetone. The samples wore the
prepared for analysis by the preceding method. Analytical standards
138
-------
of aldicarb in toluene were produced by serial dilution of stock
aldicarb solutions ir. toluene. A calibration curve was calculated
using the parabolic curve fit with the sum of the squares of the
errors minimized (Adams, 1976) for the peak heights (m\) versus
rr.ass (ng) of aldicarb.
The samples were analyzed by using the following instrument
conditions:
Instrument: Perkin-Elmer Model Sigma 2 gas chromatcgraph
equipped with a rubidi—n bead nitrogen/
phosphorus detector
Column: 3-m x-2 nun ID Pyrex packed with 10% carbowax 20 M
on 80/100 mesh Supelcoport
Temperatures (°C): Injector: 225
Oven: 1-4 0
Detector: 250
Gas flows: Carrier: nitrogen, 50 miL/rain
Heactant: hydroqer., 5 mi/rrSn
Support: air, 100 ni/xdn
Retention time: 1.1 min
Chart speed: 1 c.^/ir,in
Amplifier range.: 1 mv
Attenuation: 1
3ead adjust: 370
Response: Approximately half-chart deflection with 1.9 ng
aldicarb
The results of the recovery of aldicarb from seawater are
su.Tjnarized in the following table.
139
-------
Sample and
replicate
Aldicarb nass
added (uq)
Aldicarb riass
recovered 'jg)
Percentage
recoverv
Blank A
0.0
<.02
N/A
B
0.0
<.02
'¦ / A
n
0.0
<.02
N/A
D
0.0
<.02
N/A
E
0.0
<. 02
*;/a
Low range A
3.2
2.6
si
B
3.2
2.4
75
C
3.2
2.0
62
D
3.2
2.0
62
E
3.2
1.7
53
Mid range A
32
40
120
B
32
25
78
C
32
13
56
D
32
20
62
E
32
23
72
High range A
320
210
66
B
320
210
6 6
C
320
270
34
D
320
270
84
S
320
240
75
Mean percentage
recovery i standard
deviation: 70+10.
Mid range A was
not included in the
calculation of the
;ne a n .
140
-------
7
The analytical results cf the samples were calculated by com-
parison of the peak height with the retention tine of 1.1 minutes
with the peak heights of aldicarb external standards. The results
were corrected for a 70£ recovery. The minimum detectable concen-
tration of aldicarb in seav.'ater was 0.2 uq/i based on a 900-mi
seawater sample being concentrated to 5.0 mi.
Water samples for ethocrop JMocap), carbopnenothion
ITnthion) , and metr.yl parathion
Seawater samples (900-m£) were placed in amber glass bottles
with Teflon-lined caps. Each sample was preserved with 15 n2 cf
chloroform and stored in the dark at ambient temperature.
Seawater samples were extracted twice with 50 —ra£ aliquots of
dichloronethane. The pooled organic phase was passed through a
20-mm ID chromatography colunn containing 30 Tim of anhydrous sodium
sulfate which had been dried overnight at 275°C. The sodium sulfate
was then washed with an additional 30 mi of dichloronethane.
The organic phase was collected in a 50 0-mi. Kudarna-Dar.ish
evaporator equipped with a 10-rr.i collector. A porcelain boiling chip
was added to the evaporator which was then fitted with a three-ball
Snyder column. The solvent was concentrated to approximately 3 mi on
a steam bath. The evaporator was cooled and then rinsed with 2 mi
hexane .
The collector tube was fitted with a Micro-Snyder column,
placed into an 30°C Xontes tube heater and the solvent was then con-
centrated to less than 0.5 mi.
The sample was quantitatively transferred to a 15 xt-graduated
centrifuge tube and diluted to a known volume with haxane. The
141
-------
3
sample was transferred to a clear glass vial capped with a Teflons-
faced rubber septum and stored at 3°C until analyzed.
The samples were analyzed by using the following instrument
conditions:
Instrument: Perkin-Elrr.er Model Sigma 2 gas chromatcgraph
equipped with a rubidium bead nitroger./
phosphorus detector
Column: 2-m x - 2 mm Pyrax packed with 3% OV-1 on 100/12C
mesh Supelcoport
Temperatures (aC): Injector: 225
Detector: 250
Oven tenperature prograir.:
Initial Final Post
Oven temp. (°c; 14 5 2 00 20 5
Time (mm) 2.0 3.3 1.0
Rate (°C/min) 40 13
Gas flows: Carrier: nitrogen, 60 mi/nin
Reacrant: hydrogen, 3 mji/min
Support: air, 100 mi/nin
Amplifier range: 1 mv
Detector setting: 520
Ethoprop Kethyl parathion Carbophenothion
Retention time (mia): 2.6 4.0 7.2
Attenuation : 8 128 4
Response (half-
chart deflection): 0.44 ng 4.5 ng 0.35 ng
In order to quantitate the recovery of ethoprop, carbophenothion,
and methyl parathion from seawater, quality control samples were pro-
duced by fortifying seawater with known masses of each pesticide
dissolved in acetone. The samples were prepared for analysis by the
preceding method.
142
-------
9
Analytical standards were prepared by dilution of the stock
solutions with hexane. A calibration curve was produced by cal-
culating the parabolic curve fit of the sum. of the squares of the
errors minimized for the peak heights (wan) versus i,ass (ng) of the
pesticides (Adams, 1376).
The analytical results of the sair.ples were calculated by com-
parison of the peak heights found at 2.5, 4.0, and 7.2 rr.inutes for
ethoprop, methyl carathion, and carbophenothion, respectively.
Sample and Ethoprop nass Ethoprop mass Percentage
replicate added (ng) recovered (uq) recovery
N/A
N/A
N/A
N/A
N/A
Low range A 0.073 0.059 81
B 0.073 0.047 64
C 0.073 0.038 52
C 0.073 0.044 60
E 3.073 0.043 60
Mid range A 0.73 0.50 63
3 0.73 0.56 77
C 0.73 0.52 71
D 0.73 0.64 83
E 0.73 C . 59 SI
(continued;
3lank A 0.0 <0.01
B 0.0 <0.01
C 0.0 <0.01
D 0.0 O'.Ol
E 0.0 <0.01
143
-------
10
continued
Sample and Ethoprop mass Ethcprop mass Percentage
replicate added (ug) recovered (ug) recovery
High range A 7.3 -5.0 68
B 7.3 6.3 86
C 7.3 6.0 82
D 7.3 4.3 55
E 7.3 5.0 '68.
Mean percentage recovery i standard deviation: ' 71±i]
Sample and'
replicate
Blank A
B
C
D
E
Methyl oarathion
mass added (ug)
0 .:
0.0
0.0
0.0
0.0
Methyl parathion
mass recovered tug)
<0.1
<0.1
<0.1
<0.1
Percentage
recovery
N/A
"/A
N'/A
N/A
N/A
Low range
B
C
D
E
1-5
1.5
1. 5
1.5
1.5
1 . 5
Lost
1 . 3
1.2
1.1
140
Lost
.8 7
80
Mid rar.ge A
3
C
15
15
15
11
12
10
73
(con tinusc. 5
144
-------
11
continued
Sample and Methyl parathior.
replicate mass added (yg)
Methyl parathion
mass recovered (jq)
Percentage
recovery
Mid range D 15
9 . 3
62
2 15
15
J. w V
High range A 7 6
73
96
3 76
73
96
C 76
77
130
D 76
50
66
E 76
67
38
Mean percentage recovery ± standard
deviation: 36+20.
Sample and Carbopher.othion
replicate mass added tug)
Carbophencthion
mass recovered (uc)
Percentage
recovery
Blank A 3.3
<0.33
N/A
B 0.3
<0.33
N / A
C 0.0
<0.33
N/A
D 0.3
<0.33
N/A
E 0.3
<0.03
N/A
Low range A 0.378
0 .087
110
3 0.373
0.063
86
C 0.373
0.373
94
D 0.073
0.073
94
E 0.378
0 .033
130
(continued)
145
-------
continued
Sample and
replicate
Mid range A
B
C
D
E
Carbophenothion
mass added fug)
.0 .78
0.73
:. 79
0 . 78
Carbophenothion
.•nass rscoversa fug)
0.93
0.33
0.30
0.93
0.93
Percentage
recovery
120
120
100
12 0
120
High range A
B
C
D
E
7.8
7.3
7 .3
7.8
7.8
8.7
8.7
9.0
6 . 7
8.7
110
110
12:
85
110
Mean percentage recovery ± standard deviation: 110t10.
The analytical results of ethcprop and methyl parathion were
corrected for a 71% and 36 J recovery, respectively. Carbophenothion
results were not corrected for percentage recovery.
146
-------
13
Tissue samples for acephate (Orchene! and aldi^arb (Tenik)
Acephate (Orthene) residues in ir.arine tissues were semiquanti-
tated by thin-laver chromatography (TLC! according to procedures of the
U.S. SPA (1974) for organophcschorus pesticides except: as mentioned.
Me thy1eye1ohe xa ne was not used as the developing solvent because it
was of insufficient polarity to cause acephate to migrate from the
origin. Additionally, because a high polarity migrating solvent (acetone)
was used, dimethylformamide was not used as the immobile solvent.
Tissue samples were weighed and extracted with two portions of
acetone solvent using a Brinkman Polytron® homocenizer with a sise ??-1C
rotor at 27,000 rpn. The acetone extracts were combined, centrifuges
to remove solids, transferred to a 50-mi. volumetric flask, and diluted
to a 50-mi volume with acetone.
Tissue extracts were spotted on Silica Gel (mean particle size of
250-'jm) 6C glass plates. Acephate standards or tissue extracts C 50 — u <-3
were spotted 1.2 5 cm from the bottom of the plate and developed with
50* aeetor.e-in-hexane. The solvent front traveled 15C m tc a line
near the top of the plate. Acephate was developed by spraying with
D-nitrober.zy1 pyridine, heating the plate at 110°C for 10 minutes, and
oversoraying the plate with tetraethylenepentanir.e. The R- (reference
front) value of acephate standards and acephate spotted with tissue
extract was 0.14.
Cleanup of the extract prior to TLC was not necessary because
the fats and oils present migrated further up the plate with the mi-
grating solvent used and the compound detection chemicals are known to
react only with thio- and nor. thic-organoDhosphates. Acephate was
147
-------
detectable at 0.1 jg as a pale blue spot cn a pale yellow background.
The blue color intensity ranged from pale blue with 0.1 s.c to car):
blue with 1.0 of aeephate. Spots containing in excess of 1.0 jg
of aeephate were of intense and equal dark blue color, therefore, all
tissue sample extract volumes were spotted to contain between 0.1 to
1.0 ug of aeephate.
Aeephate spots present in chrcmatocrams of tissue extract were
of larger diameter than the corresponding weight of aeephate standard,
probably because of the larger volume of tissue extract spotted or the
presence of extraneous biogenic materials which affected the migration
of aeephate. Aeephate did appear as syrrinetricai circular spots in the
chromatogram of tissue extracts and the sects were well resclvec from
other spots present on the chromatogram.
Quantitation was accomplished by comparing aeephate standards and
unknowns to estimate the amount present in the unknown sample based or.
an equivalent intensity of blue color. Next, the areas of the aeephate
standard and sample spots were measured and the ratio of sanple to
standard spot area was multiplied by the weight of aeephate (derived
by cclor intensity) tc give the total weight present in the sanple.
For example, assuming that an aeephate sample spot color intensity
corresponded to 0.5 jg of aeephate standard and that the sanple and
standard spots were 5C.Tjn' and 10 mn2, respectively. The weight of ace
phate present in, the sar.ple would be calculated as follows:
2
0.5 jc X 50 run = 2.3 vg total
10 inr^
Aeephate added to three pinfish tissues at 5.0 ppm and analysed
by TLC as described above was quantitatively recovered. Tr.e Tiinimun
detectable concentration {MDC) of aeephate was 5.0 yg/g (wet weight! of
tissue extracted.
L4S
-------
15
Aidicarb (Temik) residues in marine tissue were semiauantitated
by thin-layer chromatography. Tissue samples and' aidicarb standards
were oxidized to the sulfone and extracted according to the procedures
outlined in Pesticide Analytical Manual (FDA, 19701, Method I, Step 2,
Extraction-Oxidation. A portion of the extract.was spotted on 25C-jm
Silica Gel 6C glass plates using 2:1 hexane-acetone migrating solvent
system. The solvent front was 150 cm- and the Rf for aidicarb sulfor.e
was 0.29. Spots ware developed by sprayir.g with 0.2 per cer.t ninhydrir.
and 2 per cer.t pyridine-in-acetcna, according to the procedures i- Hand-
book of Chromatography (1972). Resulting spcts were purple in color.
Aidicarb was not detected in any of the tissue samples analyzed
by thin-layer chronatcgraphy as described abcve. Quantitative recovery
of aidicarb was demonstrated by fortifying three pinfish tissue samples
with 1.0 vq aldicarb/g tissue and analyzing the extract as described.
Although the acephate sulfone spots present in fortified samples were
larger in diameter than the equivalent weight of aidicarb sulfone stan-
dard, no streaking of the spot occurred in the chrcmatogram of fortified
sanples and the spot of aidicarb sulfcne was well-resolved from the other
spots present.
The larger spots due to aidicarb sulfone in sample chromatcgrarr.s,
compared to standards, were probably caused by the larger volume of
tissue extract spotted cr to the effect of extrar.ecus biogenic material
on the migration of aidicarb sulfone in the sample chromatocram. The
minimum detectable weight of aidicarb sulfone was 1-0 uc/g (wet weight)
of tissue analyzed.
149
-------
Tissue samples for ethoprop (Mocap), carbopher.othj.on
jTrithion), and methyl parathior.
Frozen tissue samples were prepared for gas chromatography
by extracting the sample three tirr.es with 2C-m£ portions of
dichloromethane for one minute using a Polytron PT20 homogenizes.
The sample was centrifuged between extractions and the extract
was decanted into a 100-mJ. Griffin beaker. The extract was con-
centrated over a stearr, bath to 5 cii.
The concentrate was transferred to a 1-x 30-cir, Pyrex chroma-
tographic column containing. l'O cm of activated (130CC) Florisil
;50/100 mesh) with a i-cm surface layer of anhydrous sodium sulfate.
The column was pre-rinsed with 50 rt£ of 30% ethyl ether ir. petroleum
ether and then with 35 .Til of petroleum ether beCors sanple application.
The column was eluted with a 75-mi, volume of 1:1 ethyl ether:
petroleum ether. The eluar.t was collected in-a 500-ni Kuderna-
Danish evaporator equipped with a porcelain boiling chip ar.d a- 10-.?. i
graduated collector. ' The concentrator was fitted with a three-ball
Snyder column and placed on a steam bath until the eluant concentrated
to approximately 5 ml. The sample was diluted to a known volume with
hexar.e, transferred to a glass vial capped with a Teflon-faced
rubber septum, and stored at 8°C until analysis.
Gas chromatographic analyses were, performed by using the
following instrument conditions:
Instrument: Perkir.-Elmer Model S'.gir.a 2 gas chromatograph
equipned with a rubidium bead nitrogen-
phosphorus detector.
Column: 2-ra• x 2-mn* ?yrex packed with 3% OV-1 or. 100/120
mesh Supelcopcrt
150
-------
Tenperatures (°C): Injector: 225
Detector: 250
Oven temperature program:
Initial Final
Cven tenp. (°C) T8D 250
Time Cr.in) 1.0 1.1
Rate (°C/min) 4 0
Gas flows: Carrier: nitrogen, 60 mi/rain
Reactant: hydrcgen, 5 ni/:nin
Support: air, 100 mi/mm
Chart speed: 1 cm/rain
Amplifier range: 10
3ead setting: 370
Sthoprop Methyl parathion Carbophenothion
Retention time
(min) 0.9 2.0 3.5
Attenuation: 4 16 16
Response (half-
chart deflection): C.6 ng 4.5 ng ' 7.2 rig
Calibration curves were produced by plotting peak heights (nun)
verses mass (ng) of standard injected. Analytical standards were
prepared by dilution of analytical pesticide standards with hexar.e
to yield working standards of the required concentrations.
Unexposed tissue !'»1 g) was fortified with pesticide standards-
in-acetone and analyzed by the preceding method. The analytical re-
sults of all samples were corrected for the nean percentage recoveries
shown in the following tables.
-------
Sample and
replicate
Blank A
3
D
E
low range A
S
C
D
E
Ethoprop mass
added (ug)
0.3
0.0
0.0
0.0
0.0
0.14
0.14
0.14
0.14
0 .14
Ethoprop mass
recoversc jug)
<0.03
<0.03
<0.03
<0.02
<0.03
0.12
0.12
0.17
0 .095
0.13
Percentage
recovery
N/A
M/A
N'/A
N/A
N'/A
86
56
120
6 3
93
Mid range A
B
C
D
£
High range A
3
D
E
1.4
J. . **
1 .4
1 . 4
1.4
14
14
14
1.1
1.1
1.0
0.77
0 .85
13
4
12
11
13
70
78
71
55
61
93
23
86
Mean percentage recovery t standard deviation: 82:16 .
High rar.ge 3 was net used to calculate mean percentage recovery.
152
-------
Sample and
replicate
Blank A
B
C
D
Methyl parathion
mass added (tq)
0.3
0.3
0.0
0.0
0.0
Methyl parathion
nass recovered j-jq)
<0.02
<0.02
<0.02
<0.02
<0.02
Percentage
recovery
N/A
N/A
N/A
N/A
N/A
Low range A
B
C
D
0.15
0.15
0.15
0.15
0.15
0.30
0 .14
0.13
0.1c
0 .14
200
93
37
110
93
Mic range A
3
C
0
1.5
1.5
1.5
1.5
1.5
1.0
0.97
0.53
0.92
0.93
67
5 5
57
51
52
High range A
3
C
D
E
15
15
15
15
15
13
<1
12
9 . 7
11
37
80
65
73
Mean percentage recovery t standard deviation: 77+16.
Low range A and High range 3 were not used to calculate mean
percentage recovery.
153
-------
Sample and Carbophencthion Carbophenothion Percentage
replicate mass added (ugl mass recovered (ugl recovery
Blank A 0.0 <0.02 N/A
B 0.0 <0.02 N/A
C 0.0 <0.02 N/A
D 0.0 <0.02 N/A
E 0.0 <3.02 N/A
Low range A 0.12 0.053 44
B 0.12 J .087 72
C 0.12 0.023 19
D 0.12 0.060 50
E 0.12 0.067 56
ilid range A 1.2 3.52 4 3
3 1.2 0.50 42
C 1.2 0.52 52
D 1.2 0.52 43
E 1.2 0.07 5.a
High range A 12 8.7 72
B 12 <2
C 12 7.0 53
D 12 4.7 39
E 12 6.3 52
Mean percentage recovery ± standard deviation: 52+11.
Low range C, Mid range E, and High range B were not used to
calculate the mean percentage recovery.
154
-------
The detection Lirr.i-s of ethoprcp, methyl parathion, and
carbophenothion in tissue were 0.03, 0.02, and 0.02 jq, res-
pectively, based on a sample mass of 1 gin.
155
-------
REFERENCES
Adams. 1976. Application programs. International Software
Clearinghouse. Vol 1: 15-18
U.S. Environmental Protection Agency. 1974. Manual of analytics
methods for the analysis of pesticide residues in hix-nan and
environmental samples. Environmental Toxicology Division,
Research Triangle Park, N.C. Section 123: 1-14.
U.S. Fooc and Drug Administration. 1970. Pesticide Analytical
Manual. Method I ana Method A, Temik. Vol II: Section
120-269
Zweig, Gun-er, editor. 1972. Handbook of Chromatography.
Chemical Rubber Co, Cleveland, OH. Vcl II: 117
156
-------
PREPARED BY:
Peter J. Shuba, Ph.D.
Senior Scientist
REVIEWED BY:
Peter J. Shuba, Ph.D
\
l\^o
Senior Scientist
APPROVED 3Y:
Rod Parrish
\
Director
157
-------
Appendix C-l
. Acute (96-h) toxicity
of eight
pesticides to the
marine diatom
(Skeletonema costatum)
in stati
c tests. Effect measured was specific
conductance; triplicate samples
were measured for
each concentration
and control.
96-h
Mean
Insecticide
Concent rat i on
Absorbance
Absorbance
(96-hour EC50, ug/fc) (ug/i)
(@ 525 nn)
Acephate
Trial 1: Control
0.089
0.072,0.085
0.082
(>50,000)
100
0.068
0.061 ,0.086
0.072
1,000
0.086
0.091,0.087
0.088
5 ,000
0.087
0.090,0.071
0.083
10,000
0.081
0.088,0.084
0.084
'rial 2: Control
0.083
0.073,0.100
0.085
100
0.092
0.087,0.092
0.090
1,000
0.082
0.096,0.084
0.087
5,000
0.088
0.087,0.090
0.088
10,000
0.088
0.078,0.102
0.089
Tnal 3: Control
0.080
0.077,0.073
-0.077
10,000
0.098
0.074,0.087
0.086
25,000
0.087
0.071,0.101
0.086
50,000
0.083
0.092,0.082
0.086
A1 dicarb
Trial 1: Control
0.060
0.031,0.049
0.046
(>50,000)
100
0.041
0.052,0.034
0.042
1,000
0.042
0.053,0.046
0.047
5 ,00C
0.040
0.050,0.037
0.042
10,000
0.030
0.057 ,0.041
0.043
Trial 2: Control
0.088
0.092,0.088
0.089
100
0.065
0.068,0.080
0.071
1,000
0.087
0.080,0.072
0.080
5,000
0.080
0.105,0.079
0.C88
10,000
0.081
0.090,0.087
0.086
Trial 3: Control
0.094
0.076,0.078
0.083
10,000
0.080
0.876,0.068
0.075
25,000
0.080
0.074,0.075
0.076
50,000
0.077
0.075,0.072
0.075
Carbo-
Trial 1: Control
0.123
0.121,0.103
0.115
phenothion
0.097
0.108,0.118
0.108
(109)
10
0.116
0.097 ,0.099
0.104
100
0.109
0.082,0.088
0.093
1,0C0
0.012
0.012,0.016
0.013
5,COO
0.018
0.018,0.015
0.017
10,000
0.000
0.015,0.008
0.C08
158
-------
Carbophenothion (continued)
Trial 2: Control
100
125
150
175
200
Tri aV 3 : Control
100
200
400
600
800
Trial 4: Control
100
200
400
600
800
DEF Trial 1: Control
(366) 100
200
500
700
900
Trial 2: Control
1C0
200
300
400
5C0
Trial 3: Control
10C
200
300
4CC
500
Trial 4; Control
100
20C
0.131,0.136,0.140
0.136
0.084,0.072,0.053
0.071
0.066,0.022,0.022
0.037
0.041,0.018,0.018
0.026
0.007,0.007,0.008
0.007
0.004 ,0.007 ,0.007
¦ 0.006
0.084,0.110,0.108
0.101
0.068,0.075,0.071
0.071
0.021,0.016,0.020
0.019
0.014,0.014,0.017
0.015
0.011,0.019,0.015
0.015
0.010,0.009,0.013
0.011
0.099,0.098,0.091
0.096
0.065,0.068,0.058
0.064
0.019,0.019,0.018
0.019
0.018,0.018,0.016
0.017
0.013,0.012,0.015
0.013
0.017,0.017,0.011
0.015
0.110.
0.110
0.072,0.074,0.089
0.078
0.059,0.076,0.050
0.062
0.017 ,0.006,0.014
0.012
0.014,0.020',0.015
0.016
0.010,0.016,0.016
0.014
0.123,0.118,0.095
0.112
0.108,0.094,0.108
0.103
0.115,0.106 ,0.098
0.106
0.084 ,0.087 ,0.064
0.078
0.070,0.074,0.057
0.067
0.044,0.010,0.016
0.023
0.093,0.099,0.099
0.097
0.086,0.090,0.059
0.088
0.077 ,0.074 ,0.064
0.072
0.046,0.048 ,0.055
0.050
0.047,0.038,0.033
0.039
0.038,0.031,0.022
0.030
0.142,0.138,0.121
0.134
0.114,0.112,0.128
0.113
0.108,0.104,0.089
0.100
159
-------
DEF (continued)
EPN Trial 1
(340)
Tr-: a 1 2:
"rial 3:
EthoDrop
(3,400)
Trial 1:
Trial 2:
'rial 3;
Methyl Trial 1
parathion
(5,300)
300
0.098
0.070
0.077
0.082
400
0.055
0.066
0.024
0.048
500
0.003
0.006
0.007
0.005
Control
0.106
0.120
0.098 ¦
0.108
mo
0.123
0.095
0.122
0.113
200
0.094
0.078
0.096
¦0.089
300
0.099
0. i 11
0.099
0.100
400
0.090
0.063
0.074
0.076
• 500
0.020
0.034
0.038
0.031
Control
0.151
0.170
0.151
0.157
200
0.123
0.136
0.110
0.123
300 .
0.088
0.087
0.096
0.090
400
0.027
0.009
0.021
0.019
450
O.Olfi
0.C15
0.018
0.016
500
0.014
0.016
0.014
0.015
Control
0.143
0.146
0.163
0.151
200
0.141
0.130
0.144-
0.138
300
0.130
0.120
0.133
0.129
400
0.103
• 0.103
<150
0.028
0.022
0.024
0.025
500
0.026
0.024
0.025
0.025
Control
0.122
0.107
0.09
-------
Methyl Parathion (continued)
Trial 2: Control
2,000
3,000
4,000
5,000
5,000
Trial 3: Control
2,000
3,000
4,000
5,000
5,000
Phorate Trial 1:" Control
(1,300) 1,000
2,000
3,000
4,000
5,000
Trial 2: Control
1,000 ¦
2,000
3,000
4,000
Trial 3: Control
500
1,000
2,000
0.143
0.138,0.128
0.136
0.137
0.124,0.116
0.126
0.127
0.140,0.084
0.117
0.099
0.087,0.104
0.097
0.071
0.066 ,0.065
0.067
0.036
0.031,0.032
0.033
0.146
0.143,0.149
0.146
0.152
0.173,0.183
0.169
0.130
0.146,0.168
0.148
0.128
0.128,0.136
0.131
0.098
0.116,0.110
0.108
0.068
0.063,0.076
0.069
0.104
0.112,0.094
0.103
0.063
0.054,0.074
0.064
0.086
0.034,0.020
0.027
0.020
0.024,0.017
0.020
0.020
0.018,0.018
0.019
0.018
0.016,0.014
0.016
0.146
0.147,0.152
0.148
0.091
0.078,0.091
0.087
0.051
0.041,0.046
0.046
0.011
0.010,0.015
0.012
0.010
0.009,0.012
0.010
0.140
0.156,0.136
0.144
0.131
0.116,0.120
0.122
0.109
0.108,0.107
0.108
0.031
0.021,0.025
0.027
161
-------
Appendix C-2a. Number of normal eastern oyster (Cra'ssostrea virginica)
embryos per milliliter counted following 4p h of
exposure to acephate in static, unaerated se?-water.
Salinity was 20 °/oo and tenneratare, 25^1°C. If
all enbryos in the initial 'inoculun:-had developed, the
expected count would have been 31°. anbryos per nil-li-
1 iter.
Nominal concentration
Number of
normal
enbryos
(ug/a)
RepdA
Rep 3
Rep
C Mean
SD^
Control
638
5^2
604
594
idfi-
56, one
583
526
629
579
+ 51
100,300
509
523
441
491
320,000
125
211
158
154
+^3
560,000
0
0
0
' • o
0
1,000,000
3
C
' 0
0
0
^Replicate.
Standard deviation.
162
-------
Appendix C-2b. Toxicity of acephate to embryos of eastern oysters
(Crassostrea virginica) exposed for 48 h in static,
unaerated seawater. Criterion of effect v/as reduction
of the number of normal enbryos in test concentrateons
as compared to the number of normal control embryos.
Salinity was 20 °/oo and temperature 25+l°C-
Nominal concentration Percentage reduction3
(ug/4)
Control
56,000 2
100,000 17
320,000 73
560,000 100
1,000,000 100
Mumber of normal 4^-h control embryos minus the number of
Percentage _ normal 48-h embryos in each test concentration ^ irQ
reduction ~ Number of normal ^8-h control embryos
163
-------
Appendix C-3. Test concentrations and mortality of pink shrimp 'Penaeus
duorarum) exposed to acephate in static acute toxicity tests
using 20 animals oer concentration. Natural seawater was
adjusted to 20 °/oo; temperature was 25_+l°C.
Nominal concentration Mortality (%)
(ug/a} ¦
24hQ
£8h
72h
96h
' Control
0
0
0
0
Carrier control
0
0
0
0
1,000
0
0
' 0
0
3,200
0
0
0
o
10,000
0
0
0
n
aHours.
164
-------
Appendix C-4. Test concentrations and mortality of sheepshead
minnows (Cyprinodon varieqatus) exposed to ace-
phate in static acute toxicity tests using 20 fish
per concentration. Natural seawater was adjusted
to 20 °/oo; temperature was 25+1°C-
Nominal concentration Mortality (%)
(yt)/£) ~24h* 48h 72h • 96h
Control
0
0
0
0
320,000
0
0
0
0
560,000
0
0
0
0
1,000,000
0
0
0
10
1,800,000
0
0
0
10
3,200,000
n
\>
0
0
10
aHours
.165
-------
Appendix C-5. Test concentrations and mortality of spot (Leiostomus
xanthurus) exposed to acephate in static acute toxicity
tests using 30 aninals per concentration. • "latural
seawater was adjusted to 20 °/oo; temperature was
?5+l°C.
Nominal concentration
•Mortality (")•
(ug/i)
24 h 4
¦ *8h 72 h
96h
Control
0
0 0
0
100,000
0
10 17
23
aHours.
166
-------
Appendix C-S. Test concentrations and mortality of mysid shrimp
(Mysidopsis bahia) exposed to acephate in flowing,
natural seawater of 29^1 °/oo salinity and
22^1°C. Twenty animals were tested in each
treatment.
Concentration (ug/£) Mortality {%)
Nominal Measured 24h* 48h~" 72h 96FT
Control
MDb
0
0
0
0
13,000
ND
0
0
15
. 30
22,000
3,500
0
0
15
35
36,000
7,400
0
0
10
50
60,000
15,000
0
15
40
65
100,000
32,000
0
5
30
75
^Hours.
¦londetectable (<400 yg/i).
167
-------
Appendix C-7. Test concentrations and mortality of pink shrimp (Penaeus
duorarun) exposed to acephate in flowing, natural seawater of
28-30 °/oo salinity and 22+l°C. Twenty animals were tested
in each treatment.
Concentration (uq/£) Mortality {%)
N'omi nal
Measured
2*ha
48h
72 h
96 h
Control
NDb
0
0
0
0
13,000
MD
0
0
0
0
22 ,CC0
3,500
0
0
15
70
36,00.0
7,400
0
15 .
50
55
60,000
15,000
0
40
100 •
' 100
100,000
32,000
15
75
100
100
j^Hours.
Nondetectabl e (<>100 yn/£).
168
-------
Appendix C-8. Ranges of dissolved oxygen and dH measured in seawater frcm acute
static and flow-through toxicity tests.
Insecticide Species Test Dissolved oxygen pH
(pg/2, % Saturation)
Acephate
A1dicarb
Carbophenothion
DEF
rpr!
Mysidopsis bahia
F"a
6.2-7
.4
(86-103)
8
.0-
-8.fi
Penaeus duora^un
FT
6.2-7
.4
(86-103)
8
.1-
-8.4
Cyprinodon varienatus
CT
1
2.7-7
.2
(37-99)
6
. 9-
-8.3
Lanodon rhomcides
FT
4.0-6
.2
(56-86)
7
.6-
-8.3
M. bahia
Sb
6.8-7
.4
(92-100)
M. bahia
FT
6.4-6
.8
(89-94)
8
.1-
-8.4
P. duorarun
FT
4.6-6
.7
(6£-93)
8
.3
Penaeus stylirostris
S
5.3-6
.3 ¦
(72-85)
C. varienatus
5
5.1-7
.1
(69-96)
C. varieqatus
L. rhonboides
FT
FT
5.2-6
4.6-7
.6
.0
(80-100)
(66-97)
CO 00
.3-
.0-
-8.4
-8.2
Leiostomus xanthurus
S
2.4-4
.5
(32-61)
M. bahia
S
6.7-7
2
(91-97)
M. bahia
FT
5.2-7
.7
(72-101)
8
¦8.2
P. stylirostris
S
-
-
C. varienatus
S
6.0-7
A
(81-100)
C. varienatus
FT
5.6-7
.1
(80-101)
8
.0-
¦8.3
L. rhonboides
FT
3.4-8
.1
(47-112)
«
¦ i ¦
¦8.2
L. xanthurus
S
3.4-5
8
(46-78)
M. bahia
s
6.1-8
.5
(82-116)
P. stylirostris
S
5.1-5
g
(69-30)
C. varienatus
S
6.3-7
4
f85-100)
L. xanthurus
s
3.3-4
4
(45-59)
bahia
s
6.1-8
6
(82-116)
P. st.yi irostri s
s
5.1-5
0
(69-80)
C. varieqatus
S
6.3-7
4
(85-100)
L. xanthurus
S
3 .3-4.
4
(45-59)
^-T = ow-through •
bS = static.
169
-------
Appendix C-8. (Continued)
Ethoprop M. bahia
M. bahi a
£• ^uordr'jfr,
£. st.yl i rostri s
_C. variegatus
Z. variegatus
_L. xanthurus
L_. rhomboirtes
Methyl oarathion M. bahia
P_. sty 1 i *~ostri s
Acartia tonsa
C. variegatus
xanthurus
L. xanthurus
Phorate M. bahi a
st.yl i rostris
£¦ vari egatus
L. xanthurus
s
6.1-7.1
(82-96)
-
FTa
3.8-7.0
(53-97)
8.1
FIb
5.6-7.8
(77-108)
8.0-8.2
J
S
5.2-6.7
(70-91)
FT
5.2-6.4
(82-98)
8.1-8.2
S
2.6-4.7
(35-64 V
-
FT
4.4-7.0
(61-97)
8.1
S
4.3-5.5
(58-74)
_ .
s
5.6-6.3
(76-35)
-
s
7.0-7.5
(93-101)
8.1-8.2
s
4.6-5.7
(62-77 )
-
<;
3.2-4.5
(43-611
-
FT
5.8-7.8
(78-101)
7.8-8.0
S
2.0-3.8
(32-51)
-
s
4.9-6.3
(66-85)
-
s
5.6-6.6
(76-89)
-
s
4.4-4.9
(59-681
-
aFT = flow-through.
bS = static.
-------
Appendix C-9. Test concentrations
rhonboides) exposed
water of 28-30 °/oo
aninals were tested
and mortality of pinfish (Lagodon
to acephate in flowing, natural sea-
salinity and 22+l°C. Twenty
in each treatment.
Concentration (gq/Q ; Mortality {%)
Nominal Measured 2iha ~ 48h 72h 96h
Control
NDb
0
0
0
0
65,000
6,200
0
0
0
0
110,000
39,000
0
0
0
0
180,000
49,000
0
0
0
0
300,000
21,000
0
0
5
5
500,000
120,000
0
50
85
95
^Hours.
Nondetectable (<^00 ug/£).
171
-------
Appendix C-10. Test concentrations and percentage of sheepshead -ii rrows
(Cyprinodon variegatus) exhibiting connlete loss of equilibrium
and lack of escape response after exposure to acephate in flow-
ing, natural seawater. Salinity was 30 °/co and temperature
22^1 °C. Twenty aninals were tested in each treatment.
Corcentrati on
(uQ/i)
Mortality {%)
Norn-: na 1
''easured
?Aba
48h 72h
96 h
Control
MDS
C
0 0
0 .
270,000
ion, 000
o
0 0
0
450,000
190,000
0
0 0
0
750,000
400,000
0
0 0
'•J
1 ,200,000
721,000
0
0 0 .
oc
2,100,000
1,100,000
n
0 100
100
j* Hours.
:!cndetectabl e (<40C ug/£).
cFish were jittery and lay on the bottom.
172
-------
Appendix D-la- Number of normal eastern, oyster (Crassostrea virqinica) embryos
per mil 111 iter counted following 48 h of exposure tc aldi-
carb in static, unaerated seawater. "If all embryos in the
initial inoculum had developed, the expected count would have
been 739 embryos per milliliter. Salinity was 20 °/oo and
temperature, 25+1 -C- .
Nominal concentration flunber of normal embryos
(ug/a) Rep®A Rep B Rep C Mean — SD
Control
611
563
592
588
+ 24
1,000'
565
490
552
535
+ 40
3,200
419
490
501
470
_+44
5,600
368
307
373
349
+ 36
10,000
143
184
212
• 179
+34
32,000
208
135
164 ¦
169.
+36
j^Rep 1 icate.
Standard deviation.
173
-------
Appendix 0-1 h. Toxicity of alrMc-i^b to embryos of" eas en oysters
(Oassostroa vi rqinica) exposed fo^ ¦-? '1 in staf'c,
unaerated sea water. The criterion of ..-ffect ./as the
reduction of the nunber of noma! enbryos in test con-
centrations as. compared to the nunber of normal contro1
enbrvos. Salinity was 20 °/co and tenpe^atjre,
25+1dC.
Mcnina; concentration Percentaqe reduction3
(ug/O
Control
1,000 O
3,200 20
5,600 -'"-1
10,000 70
32,000 71
Number of normal 43-n contro1 embryos minus the number o?
•'Percent^ne _ ncnal enbryos in each test concentration ^ ,qq
reduction 'Jinoer of noma; -?>-h control enorvcs A
174
-------
Appendix D-2. Test concentrations and mortality of nysid s'nrinp
(Mysidopsis bahia) exposed to aldicarb in static acute
toxicity tests using natural seawater adjusted to
20 °/oo; temperature 26+2°C. Twenty animals were •
tested in each treatment.
Nominal concentration Mortality (%)
(ug/O 24h* 48h 72h 96h~
Control
0
0 ¦
5
15
Carrier control
0
0
0
0
10.0
5
30
45
45
13.5
5
5
30
55
18
50
60
60
60
24
5°
70
•80
80
32
40
60
75
95
42
80
95
100
100
aHours.
175
-------
Appendix 0-3. Test concentrations and ncrtaVity of postlarval shrinp
(Penaeus st.yl irostris) exposed to aldican in static
acute toxicity tests. Natural seawate'" was adjusted to
20 °/oo; temperature 25+l°C.
Nominal concentration
(yg/Jt)
24h
48 h
72h
96 r
Control
3
22
30
")C
Carrier control
.20
30
. 43
48
5.6
0
1C
10
10
10
0
5
5
5
18
7
20
30
38
32
^5
33
37
43
56
¦ 34
44
50
58
100
38
58
83
85
180
50
100
100
100
aHours.
176
-------
Appendix 0-4. "est concentrations and mortality of sheepshead minnows
(Cyprinodon variegat'jsV exposed to aldicarb in flowing
natural seawater. Salinity was 28 °/oo and tenDera-
ture, 28+_l°C. Twenty animals were tested in each
treatment.
Measured concentration Mortality {%)
(ug/i) m* *8h 72h 1 96h
Control
u
o
0
0
Carrier control
0
0
0
r>
12.5
0
n
c
0
29.5
Q
0
0
0
46.5
10
25
. 25
25
58.5
40
85
35
85
115
100
100
100
100
aHours.
177
-------
Appendix D-5. Test concentrations ana mortality of spot (Leiostcrius
xanthurus) exposed to aldicarb in static acute • toxicity
tests using ten aninals per treatment.• Natural seawater
was adjusted to 20 °/oo; temperature 25_+l°C.
Mominal concentration
(yg/A)
Morta"ity
(%)
24 hd
<18 h
72h
96 h .
Control
0
0
0
0
Carrier control
n
13
10
10
100
0
in
20
20
320
60
60
60
60
560
100
100
100
100
1,000
100
100
100
10f>
aHours.
178
-------
Appendix D-6.
Test concentration and mortality of mysid shrimp
bahia) exposed to aldicarb in a flowing seawater
idopsis
Shrimo
size averaged 5 nm, total
in each treatment. Test
°/oo.
length;
temperat
twenty animals
ure was 22+1°C;
were tested
salinity 28
Concentration (
pg/O
Mortality (%)
Noni nal
Measured
24ha
48h
72h
96 h
Control
NDb
0
n
0
0
Carrier control
MP
0
0
0
0
6
2.8
n
0
0
0
11
5.4
0
0
0
0
18
9.7
0
0
0
15
30
14.0
5
5
20
£0
50
25.0
0
0
25
80
a
b.
Hours.
ID = Nondetectable (<0.2 ug/z).
179
-------
Appendix D-7. Test concentration and mortality of pink shrimp (Penaeus duorarum)
exposed to aldicarb in a flowing seawater test. Shrimp size ranged
19 to 31 mm (rostrum to telson length). Test temperature was
22+l°C; salinity ranged from 28 to 30 °/oo.
Concentration (ug/z) Mortality {%}
Nominal Measured 24ha 48h 72h 96h
Control
ND
0
0
0
o-
Carrier control
r'D
0
0
0
0
13
6.8
0
0
5
5
22
8.6
15
35
45
45
36
16
35
70
70
75
60
23
40
75
75
80
100
41
90
100
100
100
^Hours.
bND = Nondetectable (<0.2 ygfi).
130
-------
Appendix D-8. Test concentrations and mortality of sheeps'nead minnows (Cyprinodon
variegatus) exposed to aldicarb in a flowing seawater test. Fish
size ranged 10 to 15 rm, standard length'. Test temperature was
28+l°C; salinity 28 °/oo.
Conccntrati on
Nomi nal
(uq/O
Pleasured
24Tr
48h
Mortality {%)
72h
96 h
Control ND
Carrier control ND
33 12
55 29
91 46
151 68
252 120
0
0
0
0
10
40
100
0
0
0
0
25
85
100
0
0
0
0
25
85
100
0
n
o
0
25
85
.00
^Hou'-s.
ND = Nondetectable (<0.2 ug/2).
181
-------
Appendix D-9. Test concentrations and nortality of pinfish (Lagodon rhomboiqes)
exposed to aldicarb in a flowing seawater test. Fish size ranged
from 53 to 90 mm, standard length; twenty animals were tested in
each treatment. Test temperature averaged 22^1°C; salinity
ranged from 29 to 32 °/oo.
Concentration (uq/&) Mortality (%)
Nominal Measured 24ha 4Fh 72h 96h
Control
ndd
0
0
0
0
Carrier control
no
a
0
0
0
52
26
0
0
0
0
86
40
0
0
0
0
140
54
0 .
0
0
0
240
80
50
80
85
85
400
160
100
100
100
100
j^Hours.
bMD = Nondetectable (<0.2 yg/1).
182
-------
Appendix F-la. Number of normal eastern oyster (Crassostrea virginica) embryos per milliliter
counted following 4B~h exposure to carhophonothion (Trithion ) in static,
unaerated seawater. If all embryos in the initial inoculum had developed, the
expected count would have been 'IRQ embryos per milliliter. Salinity was
20 °/oo and temperature, 20+2°C.
Nominal concentration Number of normal embryos ,
(m9/0 RepaA Rep B Rep C Rep D Mean SO
Seawater control
454
409
497
383
436
+ 50
Carrier control
482
464
446
-
464
+ 18
0.3?
344
343
. 459
-
382
+67
l.n
409
312
378
-
366
+50
3.2
447
42f>
458
-
444
+ 13
10
410
45ft
486
-
451
+38
32
468
490
351
-
436
175
100
197
2fi3
182
-
214
+43
320
2
0
1
_
1
+ 1
aRepli cate
^Standard deviation
-------
Appendix E-lb. Toxicity of carbophenothion to embryos of eastern oysters
(Crassostrea virginica) exposed for h in static, unaerated
seawater. The criterion for effect was the number of normal
embryos in test concentrations as compared to the number of
normal control embryos. Salinity was 20 °/oo and tempera-
ture, 20+2°C.
Nominal concentration
(yg/O
Control
Carrier control
n .32
1.0
3.2
10.0
32.0
ino.n
320.0
Percentage reduction'
12
16
0
0
0
51
99.8
Number o* normal 4R-h control embryos minus the number of
percentage _ normal 48-h embryos in each test concentration y
^eduction ~ Number of normal control emoryos
184
-------
Appendix E-2. Test concentrations and mortality of mysid shrimp
(Mysidopsis bahia) exposed tc carbophenothion in static
acute toxicity tests using 20 animals per concentration.
Natural seawater was adjusted to 2C °/oo; temperature,
25+l°C.
Nominal concentration Mortality {%)
(ug/A) 48h 7% S6h~
Control
0 .
5
10
10
ier control
0
5
10
15b
3.2
0
0
5
10
5.6
5
15
35
35
10.0
0
10
10
50
O
oc
0
35
45
65
32.0
20
80
80
35
j^Hours.
bExcessive control mortality (>10%).
135
-------
Appendix E-3. Test concentrations and mortality of postlarval per.aeid
shrinp (Penaeus stylirostris) exposed to carbophenothion 1
static acute toxicity test using 20 aninals per concentrat
Natural seawater was adjusted to 2D °/oo; temperataure,
25+l°C.
Nominal concentration Mortality (%)
(ug/i) 24F3 58h 72h 96h
Control
5
23
33
38
Carrier control
18
35
<13
^8
0.32
10
20
20
20
0.56
10
10
10
20
1.0
25
38
40
43
1.8
20
28
40
45
3.2
28
48
55
68
5.6
33
55
68
78
10.0
55
85
95
95
^Hours.
Excessive control mortality (>10%).
136
-------
Appendix E-4. Test concencentrations and mortality of sheeoshead
minnows (Cyorinodon varieqatus) exposed to carbopheno-
thion in static acute toxicity tests using 20 aninals
per concentration. Natural seawater was adjusted to 20
°/oo; temperature 25+1°C.
Nominal concentration Mortality (%)
(yg/£) 24P 48h 72h 96h~
Control
3
0
0
0
Carrier control
0
0
0
0
10
0
0
0
10
18
5
20
50
55
32
10
85
85
90
56
100
100
100
100
100
100
100
100
100
aHours.
187
-------
Appendix E-5. "Test concencentrations and mortality of spot (Leiostonus
xanthurus) exposed to carbophenothion in static acute
toxicity tests using 20 animals per concentration.
Natural seawater was adjusted to 20 °/oo; temperature
25+l°C.
Noninal concentration Mortality {%)
(ygfi) 24h* 48h 72h 96h
Control
0
0
0
0
Carrier control
0
0
0
0
320
0
0
0
10
560
0
30
60
60
1,000
20
70
100
100
1,800
30
90
100
100
3,200
10
100
100
100
aHours.
133
-------
Appendix E-6. Test concentrations and rortaiity of mysid shrimp (f-'.ysidops"' s
bahia) exposed to carbophenotbion in a 'lowing seawater test.
Shrimp averaged 5 m, total length. Test temperatjre was 2°C;
salinity 30 /oo.
Concentration (ug/JZ.) Mortality {%)
Noninal Measured 24hd 48h 72 h 96h
Control
NDb
0
0
0
0
arrier control
NO •
0
0
0
0
1.7
1.8
0
o
5
20
2.8
2.7
0
0
n
25
4.7
3.6
0
10
15
70
7.8
6.0
0
20
90
100
l t a
Is; •
15.0
5
^5
100
100
^Hours.
NO = Mondetectable (<0.C8 uq/£).
139
-------
Appendix E-7. Test concentrations and mortality of pink shrirrp (Penaeus
duorarum) exposed to carbophenothicn in a flowing seawater test.
Shrimp size averaged 88 mr rostrum to telson length. Test temper-
ature averaged 25.5°C (25 to 26°C); salinity averaged 24.3 °/oo
(19.0 - 29 /oo).
Concentration (yg/l) Mortality [%)
Nominal Measured 24hft
-------
Appendix E-3. Test concentration and mortality of grass' shrimp (Palaemonetgs
pugio) larvae (1- to 7-day-old) exoosed to carbophenothion in a
flowing seawater test. Test temperature averaged 25.5°C. (25 to
25°C); salinity 25 0too. Forty larv-je were placed in oac!' treat-
ment .
Concentration (yig/£) Mortality (%)
Nominal
Measured
?Ah*~
" 48h
72 h
96h
Control
no13
2.5
5.0
5.C
7.5
Carrier control
ND
n
2.5
7.5
12.5
1.15
1.0
2.5
5.0
5.0
12.5
1.55
1.3
15.C
15.0
15.0
25.5
2.1
1.8
17.5
17.5
22.5
?7.5
2.8
2.1
12.5
12.5
45 .0
65.3
3.7
2.6
15.0
22.5
65.0
82.5
^Hours.
10 = 'londetectable (
-------
Appendix E-9. Tost concentrations and mortality of grass shr^'np
(Palaenonetcs pugio) exposed to carbophenothicn in a
flowing seawater test. 3hrimp size averaged 15.4 mm,
rostrum-to-telson lennth; twenty animals were tested in
each treatment. "Test teirmerature averaged 29.1°C
(29.0 to 29 . 5cC); salinity 27.4 °/oo (25.5 to
?Q °/oo).
Concentration (yQ/O Mortality (%)
1 ¦* —' _ _ a — » ' _ ¦_
"Jomi nal
Measured
2ihQ
48h
7 2 h
96 h
Control
NDb
0
0
0
0
Carrier control
MD
0
0
0
r
0.75
0.78
0
0
0
0
1.35
1.30
0
0
0
0
2.4
1.8
0
0
0
5
4.2
3.5
0
30
45
75
7.5
5.5
15
IOC
100
100
^Hours.
bND = nonaetectable (<0.02 ya/2).
192
-------
Appendix E-1Q. Test concentrations *nd mortality of wild-stock
grass shrimp (Palaemoretes pugio) exposed to carbo-
phenot'oion in a flowing seav/ater test. Shn'mp size
averaged lo.6 it, rostrun to tel son length; twenty
animals were tested in each treatment. Test tempera-
ture averaged 29.1°C (29.0-29 .5°C); salinity
averaged 27.£ °/oo (25.5 to 29.0 "/oo).
Concentration (ug/ft) Mortal 1 ty [%) .
Nominal Measured 24h(i 43h 72h 96h
Control
NDb
0
0
a
ier control
ND
0
o
0
0
0.75
0.78
0
n
n
0
1.35
1.3
0
0
0
0
2.4
1.8
n
0
0
n
a.2
3.5
n
5
25
30
7.5
5.5
0
40
85
100
^Hours.
'ID = Nondetectabl e (<0.02 yg/P.}.
-------
Aopendix E-li
Test concentrations and mortality of sheepshead
minnows (Cyprinodon variegatus) exposed to carbo-
ohenothion in a flowing seawater test.' Fish size
ranged from 10 to 15 mm, standard length. Test
temperature was 23+l°C; salinity 28 °/oo.
Concentration (ug/&)
Mortality (%)
Noninal
Measured
24h
48 h
72h
96h
Control
NDb
0
0
0
o
Carrier control
ND
¦ 0
0
0
0
1-7
.0.5
o
0
0
0
2.8
0.6
0
0
0
0
4.7
1.0
0
0
15
15
7.8
1.6
0
5
5
10
13
i.O
10
10
40
70
j^Hours.
Mc = N'ondetectable (<0.OS ug/fc).
194
-------
Appendix E-12. Test concentrations and mortality of Atlantic'
silversides (Henidia Tienidia) exposed to carho-
phenothior in a f7 owing seawater test. Fish size
averaged 11 Tin, standard length; twenty aninals
were tested in each treatnent. Test tenperatjre
averaged 24.9°C (23 to 2S°C); salinity 25.5 °/oo
(24 to 26 °/oo).
Concentration (pq/&) Mortality (%)
Nominal Measured 24 h3" 48 h 72h 96 h
Control
NDb
0
0
0
0
¦ier contro1
MD
0
0
0
• 0.
1.8
2.2
in
15
20
20
3.2
4.9
5
5
10
25
5.6
6.9
20
20
20
35
7.5
7.5
20
35
35
55
13.5
12.9
35
65
75
75
^Hours.
bND = Nondetectable (
-------
Appendix E-13. Test concentrations and mortality of soot
(Leiosto.TiLJS xanthurus) exposed to carbophenothion
in a flowing seawater test. Fish size averaged
30.7 nm, standard length; twenty anira"s -were tested
in each treatment. Test temoerature averaoed 2o.?°C
[?.S to 26.5°C); salinity 25.5' n/oo {19 to 28 °/oo).
Concentration (yg/Q Mortality (%)
Nominal. Measured 24h3 43 h 7?h 06h
Control
ND
0
J
0
0
Carrier control
riD
0
0
0
0
42
-
0
u
0
0
75
-
c
o
n
n
135
0
0
0
0
240
35
0
0
5
5
420
206
0
f>
i
35
35
^ Hours.
D = Nondetectab1e (<0.C2 yg/1).
-------
Appendix E-14
Test concentrations and nortality
of pinfisii
{Lagodon
rhonboidesj exposed to carbophenothion in a
flowinq
seawater test.
Fish size ranced
from 53 to
?0 -n,
standard length.
Twenty animals
V;prg tested
in each
treatment.
Concentration
(yq/£)
Mortality (
ro
Nominal
Measured
?A ha *8 h
12 h
96h
Control
*IDb
0 0
. o
0
Carrier control ND
3 0
0
0
13
4.9
0 0
0
0
22
5.5
0 0
0
0
36
5.2
0 0
0
30
60
6.2
0 0
5
70
100
34
0 n
100
100
^Hours.
"ID = liondetGctable (<0.08 ug/n).
197
-------
Apoendix E-15. Mean salinity and dissolved oxygen during a ?49-day partial
li£e-cycle toxicity test of carbophencthion with grass shrimp
(Palaemonetos punio). The test tank temperature averaged
25.5°C (range 22.4 - 28.3°C), whereas the larval tray tem-
perature averaged 25.0°C (range 22.2 - 30°C) for the entire
exposure.
Test day Salinity (°/oo) Dissolved oxygen (nn/z) for each treatment
Mean
Range
Control
Carri er
control
0.06
0.22
0.36
0.69
1.31
2.94
1-8
22.9
21-26
6.7
5.5
5.9
6.2
5.5
5.8
5.g
5.5
9-39
25.7
21-30
6.3 .
5.0
4.8
5.3
5.1
5.3
5.1
5.4
40-69
28.7
27-30
5.7 .
3.0
3.4
3.2
3.6
3.5
3.0
4.2
70-100
2a.fi
21-30
6.1
3.2
4.1
4.4
£.2
4.4
4.4
4.9
101-131
22.7
18-27
6.2
3.0
3.7
4.4
A.l
4.2
4.1
5.2
132-159
21.2
18-27
6.2
a.1
2.5
3.0
3.3
4.2
4.0 .
4.6
160-190
17.5
10-24
6.0
2.2
3.3
3.8
2.7
4.1
3.6
5.1
191-220
23.5
20-26
6.2
5.5
5.3
5.6
4.9
4.3
5.3
6.7
??1-2<19
23.7
22-25
7.0
6.4
6.3
6.4
6.4
5.4
6.2
6.4
198
-------
Appendix F-la. Number of normal eastern oyster (Crassostrea virginica)
enbryos per milliliter counted following 48 h of expo-
sure to DEF in static, unaerateri seawater. If all
embryos in the initial inoculum had developed, the
expected count would have been 1,059 embryos per milli-
liter. Salinity was 20 °/oo and temperature 25j+l°C.
Nominal concentration
(iig/a)
Number of
nonnal
embryos
1-
RepaA
Rep
B Rep
C
flea n
Control
7H
827
793
778
+57
Carrier control
758
812
809
793
+30
100
786
753
731
•756
+27
180
498
426
384
436
+57
320
187
231
212
210
+ 22
560
0
0
0
0
0
1,000
0
0
0
n
o
|*Repl i cate.
Standard deviation.
-------
Appendix F-lb. Toxicity of DEF to embryos of eastern oysters (Oassostrea
virninica) exposed for 48 h ,-n static, jnaerated seawate>".
The criterion for effect was the reduction of the number of
normal embryos in test concentrations as compared to the
number of normal control embryos. Salinity was 20 °/oo and
temperature, 25^1°C.
¦ ¦ - ¦ ¦ —. ».
flominaT concentration Percentage reduction3
(ug/O
Control
Carrier control
100 3
180 44
320 73
560 100
1 ,000' 100
'lumber of norma1 48-h control embryos minus the number
percentage _ of normal 48-h embryos in each test concentration ^
reduction ~ Number of normal ^8-h control embryos
200
-------
Appendix F-2. Test concentrations and mortality of mysid shrimp
(M.ysidopsis bahia) exposed to CEF in static acute
toxicity tests using 20 animals per concentration.
Natural seawater was adjusted to 20 °/oo; temperature
was' 2.5+ l°C.
Nominal concentration Mortality {%)
(ug/A) W 58h 72h 9Fh
Control
0
0
0
0
Carrier control
5
5
5
5
5-6
5
10
30
45
10.0
0
^5
90
95
18.0
5
an
95
ion
32.0
10
95
100
100
56.0
15
100
100
ICO
aHours
201
-------
Appendix c-3. Test concentrations and mortality of postlarva1 shrimp
(Penaeus stylirostris) exnosed to DEF in static acute
toxicity tests using 20 aninals oer concentration.
Natural seawater was adjusted to 20 °/oo; tenperature
26+2°C.
Nominal concentration Mortality (%)
(pg/£) 24F9 48h 72h"^ 96h
Control
8
18
25
33
Carrier control
10
13
23
28
3.2
10
10
10
20
5.6
¦) .
•*/
g
20
28
10.0
5
13
20
23
18.0
13
43
60
63
32.0
25
48
60
68
56.0
50
55
100
100
^Mours.
Excessive control mortality (>1C%).
202
-------
Appendix F-4. Test concentrations and mortality of sheepshead ninnov/s
(Cyprinodon varieqatus) exposed to DEF in static acute
toxicity tests using 20 animals per concentration.
Natural seawater was adjusted to 2D °/oo; temperature
25+l°C.
Noninal. concentration Mortality {%)
(wg/a.) "24F® 48h 72TT "96h
Control
0
0
0 .
0
Carrier control
0
0
0
c
240
. - 0
n
n
0
320
0
0
o
5
420
0
0
10
25
560
0
40
90
100
750
35
95
100
100
aHours.
203
-------
Appendix F-5. Test concentrations and nortality of spot (Leiostorus
xanthurus) exposed to DEF in static acute toxicity
tests using ?0 aninals per concentration, Natural sea-
water was adjusted to 20 °/oo; temperature ?6+l°C.
floninal concentration . Mortality (%)
(ug/Z) 48h 72h 96F"
Control'
0
0
0
n
Control carrier
3
3 '
. 3
3^
56
0
0
7
7
.75
0
n
CI
0
100
0
0
7
13
135
0
0
0
in
180
0
0
43
83
320
1°
90
100
mo
560
100
100
100
100
^Hours.'
Excessive control mortality (>10%).
2.04
-------
Appendix F-6. Test concentrations and mortality of mysid shrimp
(Mysidopsis bahia) exposed to DEF in a flowing seawater
test. Early juveniles (24 h; total length = 1 mm) were
used. Test temperature averaged 25°C (2^-26°C);
test salinity 16 °/oo (15-17 /oc).
Concentration (
uq/JL)
Mortality (%)
Moninal
Measured
24ha
48h
72h
96h
Control
NDb
0
5
10
10
Carrier control
ND
0
0
0
0
1.0
0.58
0
5
5
5
2.0
1.6
0
0
0
0 '
4.0
3.7
0
10
15
a v
8.0
5,6
10
25
45
90
j^Hours.
Nondetectable (<0.02 yg/i.).
205
-------
Appendix F-7. Test concentrations and mortality of pink shrimp
(Penaeus duorarun) exposed to DEF in a flowing seawater
test. Shrimp size averaged 84 mm, rostrum to telson
length.; twenty animals were tested in each treatment.
Test temperature averaged 25.6°C (25 to 26°C);
salinity 2D °/oo (15-26 °/oo).
Concentration (pg/ji) Mortality (%)
Nominal
Measu red
24ha
48 h
72 h
96h
Control
MDb
0
0
. 0
0
Carrier control
ND
0
0
5
5
4.2
4.5
0
0
5
5
7.5
6.9
20
20
25
' 25
13.5
9.7 •
5
5
15
45
24.0
16.0
35
50
60
70
42.0
29.0
50
75
35
90
flours.
Nondetectable {<0.02 ug/ii).
2D6
-------
Append fx F-8. Test concentration and mortality - of wild-stock grass
shrimp (Palaenonetes puqio) exposed to DEF in flowing
seawater tests. Shrinp size averaged IS.? mm, rostrum
to telson length; twenty animals were used in each
treatment. Test temperature averaged 23.1°0 <27.5-
29.0°C); salinity 21.fi °/oo (16.0 to 28 °/oo).
Concentration (pg/fc) Mortality (%)
nominal Measured 2iha AJfh 72h 96h
Control
NDb
0
0
5
f
0
Carrier cont**o^
ND
0
0
0
0
13.5
16
0
10
20
20
24
20
0 .
20
40 '
40
2^
5
-go
100
100
75
30
0
' 70
100
100
135
160
40
100
100
100
^Hours.
ND = "iondetectabie {<0.n2 wg/fc).
207
-------
Appendix F-9. Test concentration and mortality of pinfish (Lagodon
rhomhoides) exposed to DEF in flowing seawater tests. .
Fish size averaged 2,4 nnr, standard length; twenty
animals were used in each treatment. Test tenperature
averaged 24.9°C (24 .5 to 25.5°C) -y salinity
17.9 "/oo (12 to 23 °/oo).
Concentration (ug/2)
Mortality
(%)¦
Nominal
Measured 24ha
48 h
72h
96h
Control
'JDb
0
0
o
J
0
Carrier control
N'D '
n
C
0
0
75
47
Q
c
c
G
135
69
C
0
0
o
yj
180
189 '
0
0
15
30
240
240
0
0
0
40
320
343
0
0
10
60
^Hours. .
MD = Nondetectable (
-------
Appendix F-10- Test concentrations and mortality of spot (Leicstonus
xanthurus) exposed to DEF in flowing seawater tests.
Fish averaged 17 mm, standard length; twenty animals
were used in each treatment. Test temperature
averaqed 25.2°C (25 to 25°Ch salinity 18.2 °/oo
(15.5 to 23.5 °/oo).
Concentration (ug/fc) i'ortalitv (TO
Nominal Pleasured 24ha 5HT5 T?R 96 h
Control
>!Dd
0
0
0
0
arrier control
: !D
0
0
0
0
24
23 ¦
n
n
0
0
42
59
0
¦ 0
0
5
75
90
Q
0
0
10
135
162
0
0
25
85
j^Hours.
NO = MondetectabIe (<0.02 ug/fc).
209
-------
Appendix F—II- Test concentrations and mortality of sheepshead minnow
(Cyprinodon varienatus) exposed to OEF in a flowing
seawater test. Fish size averaged 3.7 rm, standard
•enqth; twenty animals were used in each treatment.
Test temperature averaged 15.40D '1^.5 to 16.5°C);
salinity 17.9 °/oo (12 to 21 °/oo).
Concentration .(
Ml/*)
Mortal ity
(%) ¦:
Norninal
Measured
24h°
48h
72h
96 h
Cont rol
N'Db
0
0
. 0
0
Carrier control
ND
0
0
0
0
135
47
0
G
0
0
320
270
0
0
0
0
420
330
0
0
G
0
750
440'
C
c
0
n
w
j^Hou^s.
1ND = Nondetectable (<0.C2 pa/t).
210
-------
Apoendix G-la. Number of normal eastern oyster (Crassostrea virgim'ca) embryos
per milliliter counted following 48 n of exposure to EPN in
static, unaerated seawater. If all embryos in the initial
inoculum had developed, the expected count would have been 739
embryos oer milliliter. Salinity was 20 °/oo and tenperature,
25+1 C.
Nominal concentration
(ug/A)
Number of
normal embryos
t
Rep^A
Rep
3 Rep
C Mean
SCL
Control
611
563
592
588
+24
Carrier control
548
587
579
571
+20
320
528
606
549
561
+40
560
517
493
558
522
+32
1 ,D00
490
cr>
CM
lT>
509
509
-19
3,200
190
106
150
148
+42
5, son
184
98
137
139 •
+43
^Repli cate.
Standard deviation.
211
-------
Appendix G-lb. Toxicity of EpN to embryos of eastern oysters {Crassostrea
virainica) exposed ror 48 h in static, unaerated sea-
water. Criterion of effect was reduction of the number of
normal embryos. Salinity was 20 °/oo and temperature
25+1°C.
Nominal concentration Percentage reduction3
(ygfi)
Control
Carrier control 3
320 4
560 11
1,000 13
3,200 75
5,600 76
dumber of normal 48-h control erhryos minus the number
aPercentaqe _ of normal 48-h embryos in each test concentration .Y
reduction ~ Number of normal 48-h control embryos
212
-------
Appendix G-2. Test concentrations and nortality o£ nysid sbrinn
(Mysidopsis hah 1 a) exposed to E?N in static acute
toxicity tests usinn 20 aninals per concentration
Natural seawater was adjusted to 20 °/oo;
temperature, 25+l°C.
Monina1 concentration
(uq/2.)
24 ha
Mortali ty
48 h
-
72n
?~6*
Control
0
5
0
10
Carrier control
0
0
0
0
3.2
0
0
10
10
5.6
0
5
5
15
m.n
fi
5
in
15
13.0
n
30
75
95
32.0
50
80
100
100
aHours.
213
-------
Appendix G-3. Test concentrations and mortality of postlarval
penaeid shrimp (Penaeus sty:irostris) exposed tc
EPN "'n static acute toxicity tests using *0 or 60
animals per concentration. Natural seawater was
adjusted to 20 °/oo; temperature, 25+l°C.
Nominal concentration
(wg/4)
Mortality
(%)
24hd
48h
72h
96h
Control
7
10
23
28
Carrier control
7
15
22
25
1.0
0
10
15
23
1.8
23
33
40
47
3.2
17
28
33
40
5.6
20
43
57
60
10.0
38
72
83
93
18.0
67
90
92
98
aHours.
214
-------
Appendix G-4. Test concentrations and mortality of sheepshead
ninnows (Cyprinodon variegatus) exposed to EPN in
static acute toxicity tests using 20 animals per
concentration, "latural seawater was adjusted to
20 °/oo; temperature, 25+l°C.
Nominal concentration Mortality '%)
(yg/jt) 24^ 48h 72h %h
Control
o
0
0
0
Carrier control
0
0
0
0
32
0
0
0
0
56
0
c
15
15
100
0
5
10
15
180
30
45
50
55
320
75
85
100
100
aHours.
215
-------
Appendix G-5. Test concentrations and mortality of spot
(Leiostonus xanthurus) exposed to EPN in static
acute toxicity tests using 20 antrals per concen-
tration. Natural seawater v/as adjusted to 20
°/oo; temperature, 25j^l°C.
Nominal concentration Mortality (%)
(ug/A) 2&P l8h 72 h %F
Contro:
0
in
10
10
Carrier control
0 .
0
2C
20
24
0
0
0
0
32
0
0
0
20
42
n
40
70
90
56
0
so
100
100
75
0
20
90
90
aHou^s.
216
-------
Appendix G-6. Test concentrations and mortality of mysic shrimp
(Mysidopsis bahia) exposed tc E3N in a flowing seawate"-
test. Mean total length of adult mysids used was 7.5 nm;
twenty animals were tested in each treatment. Test
temperature averaned 23°C (22 to 24°C); salinity
24 /oo (22 to 26 °/oo).
Concentration *(
uq/i.;
Mortality (%)
Nomi nal
Measured
24ha
48h
7 2 h
96 h
Cont >~o1
NPb
0
n
0
0
Carrier control
"ID
0
0
0
5
l.n
1.1
0
0
0
5
2.0
1.7
0
0
5
15
fi.n
3.8
5
10
20
50
8.0
B .6
15
65
85
100
j^Hours.
'P = f'ondetectablc (<0.02 ug/A).
217
-------
Appendix C-7. Test concentrations and mortality of pink shrimp
(Penaeus duorarun) exposed to EPN in a flowing seawater
test. Shrino s">ze averaged 69 nn, rostrum-to-tel son
length; twenty anina's were tested in each treatment.
Test temperature averaqed ?5°C (24 to 26°C); test
salinity 24 °/oo (22 to 26 °/oo).
Concentration (uq/z) Mortality (%)
Nomi nal Measured 24ha ' 48h 72h 36 h
Control
ndd
0
0
0
Carrier control
ND
0
0
0
5
0.032
NO
0
10
20
20
0.1
0 .042
0
5
10
10
0.32
0.*2
5
20
35
45
1.0
1.3
95
100
100
100
3.2
_
90
100
100
100
^Hours.
bND = Mondetectable (<0.02 ug/A).
218
-------
Appendix G-8. Test concentrations and mortality of sheepshead minnows
(Cyprinodon varienatus) exposed to EPM in a flowing
seawater test. Fish size averaged 14 vn standard
length; twenty animals were tested in each t^eatnent.
Test tennerature averaged 24.fi°C (24.0 to 25.3°C);
test salinity 18.?. °/oo (12.5 to 24.5 °/oo).
Concentration (uq/z) Mortality (%)
Noni na1
Measured
24 ha
48h
72h
96h
Control
n
0
0
0
Carrier control NO
0
0
0
0
75
58,
10
25
30
35
140
76
1 G
M sJ
25
40
45
2*0
150
65
90
°0
90
420
240
80
ion
100
100
7 50
-
100
100
100
:oo
^Hours.
NP = Nondetectahle (CO.n;? ug/i).
-------
Appendix G-9. Test concentrations and mortality of spot (Leiostomus
xanthurus) exposed to EPN in a flowing seawater test.
Fish size averaged 51 mm, standard, length; twenty
aninals were tested in each treatment. Test temperature
averaged ?4.0°C (23 to 25°C); test salinitv
23 °/oo (20 to 26 °/oo}.
Concentration {uq/i)
Mort
ality [%)
Nominal Measured
2dha
48 h
72h
96
Control ND^
0
0
0
0
Carrier control NO
0
•o
0
0
3.2 3.2
0
0
0
0
5.6 5.9
0
0
0
- E,
10 12
0
o
0
15
30
•XJ
00
o
15
20
45
32 a2
0
0
100
100
^Hours.
• ID = "londetectabl e (<0.D2 uo/£).
220
-------
Appendix G-10.
Test concentrations and mortality of pinfish (Lagod.cn
rhomboides) exposed to EnN in a flowing seawater test.
Fish size averaged oh nm, standard length; twenty ani-
mals were used in each treatment. Test temperature av-
eraged 25°C {?A to 26°C); test •salinity 21.1 °/co
(27 to 3? °/oo).
Concentration (u9/
'I)
Mortal 1 ty '.*•.)
¦ !omi nal
Measu rod
24 h3"
iVn
72h
96h
Cont rol
nnb
n
0
0
0
Carrier control
ND
r\
J
0
c
0
3.2
3.0
0
0
0
o
5.6
5.0
0
n
0
0
10
6.7
0
0
0
0
18
12
a
5
10
15
32
26
40
30
90
90
^Hours.
ND ¦= "londetectflhle {<0.D2 un/s.).
221
-------
Appendix G-11. Effect of EPN on the swimming performance of
Cypri nodon vari egatus
by
Ge>"aldine M. Cripe
Measurements of swinming ability of toxicant-exposed fish may
indicate changes
-------
by Srett (Brett, 1964):
CSS = LRV + VI (TE/TT)
where
CSS = critical swimming speed (cm/sac)
LRV « last recorded velocity which was maintained for the pre-
scribed time period (cm/sec)
TE = time endured at the final velocity (sec)
TT = total time increment (sec)
VI = velocity increment between final velocity and the oreceding
velocity (cm/sec)
The critical swimming speeds por the cish tested are shown in
Table a. Only CSS neasucements from animals exposed to 7.2 and -.1 yj
EP'l/i were significantly different from controls, therefore, the thres-
hold level of effect on swimming performance lies between 2.2 and 0.33
yg EPtl/i.
From these tests, one could speculate that a fish in the field
exposed to these compounds might also show a reduced swinning perform-
ance. This loss may a'fect its ability to remain in favorable environ-
mental conditions (light, oxygen levels, temperature and food supply)
against the influence of ebb and flood tides. It al*o may be impor-
tant in terms of spawning behavior such as territorial defense and in
predator avoidance of prey capture.
223
-------
Literature Cited
Brett, J.R. 1964. The respiratory metabol ism and swimming performance
of young sockeye salmon. J. Fis'n. Res. Ed. Canada 21:1184-1226.
Dodson, u.J. and C.I. Mayfield. 1979. Modification of tne rheotropic
response of rainbow trout (Sa• mo gairdneri) by sublethal doses of
the aquatic herbicides dicuat and simazine. Environ. Pollut.
18:14 7-
MacLeod, J.C. and L.L. Smith, Jr. 1966. Effect of ouipwood fibe*" on
oxygen consumption and swimming endurance of the fathead minnow,
Pimephales orcmelas. Trans. Amer. Fish. Soc. 95: 71-84,
Deterson, R.H. 1974. Influence of fenitrothion on swimming velocities
of brood trout (Salve!inus fontinalis). J. Fish. Res. Bd. Canada
31:1757-1762.
224
-------
Table 'I. Critical swimnin;] speed (cn/sec) of individual sheepshead minnows continuously exposed to measured concentrations
of FPU for 256 days.
Fxposure
Concentration Fish Number
1
?
3
4
5
6
7
8
9
10
.. ^ ...
SD
nna
:v). 3
4b.4
52.0
52.0
25.6
52.0
42.4
52.0
52.0
52.0
46.6
8.7
n.?5
19.4
52.0
46.3
51.4
52.0
52.0
41.9
47.7
21.1
52.0
43.6
12.7
0.5
52.0
42.3
51.6
52.0
33.9
52.0
27.2
15.9
52.0
16.0
39.5
15.2
n.M
11 .3
52.0
44.')
33.0
52.0
52.0
52.0
35.4
37.2
3.0
40.3
15.1
2.?
0.3
1/.2
33.2
14.6
45. 8
28.3
7.4
39.4
26.0
52.0
26.4b
16.7
4 .1
3ft.a
0.3
52.0
1ft.3
9.6
14.7
17.8
4.3
40.3
21 .6h
17.6
^Control-v/ith-carrier, NO = nondetectable (<0.05 vig/Jt).
Significantly different from control (u = 0.05).
-------
Anpendix H-la. Number of normal eastern oyster (Crassostrea
virginica) embryos per milliliter counted following
48 h of exposure to ethoprop in static, unaerated
seawater. If all embryos in the initial inoculum had
developed, the expected count would have been 1,059
embryos per milliliter. Salinity was 20 °/oo and
temperature, 25_+l°C.
Nominal concentration
Number of normal embryos
(ug/*)
RepaA Rep B Rep C
Mean
Control
Carrier control
714
758
731
724
395
217
56
827
821
773
653
438
793
809
805
682
335
178
34
778
793
759
686
402
225
48
+ 57
+ 30
+37
+ 35
+ 32
j+51
+ 16
3,200
5,600
10,000
32,000
56,000
280
46
aReplicate.
^Standard deviation
2j?6
-------
a r
Appendix H-Ib- Toxicity of ethoprop to embryos of eastern oysters
(Crasscstrea virgi nica) exposed for 48 h in static,
unaerated seawater. Criterion of effect was reduction
of the number of normal enbryos in test concentrations
compared to the number cf norma- control enbryos. Sal'nity
was ?S\ °/oo and temperature 2r>+l0C.
Nominal concentration Percentage reduction9
(uG/A)
Control
Carrier control
3,200 1
5,600 12
10,000 48
3?,non 71
56,nno 94
Number. of nornal 4R-h control embryos ninus number of
Percentage _ nornal 4P-h embryos in each test concentration ^ ^
reduction ~ Number of normal 48-h control embryos
0O7
t,
-------
Appendix H-2. Test concentrations and mortality of mysid shrimp
(Mysidopsis bahia) exposed to ethoprop in static acute
toxicity tests using 20 animals.per concentration.
Natural seawater was adjusted to 20 °/oo; tempera-
ture 25+l°C.
Nominal concentration Mortality [%)
(ug/A)
24 ha
48h
72h
96n
Control
0
0
0
0
Carrier control
0
5
5
5
10
C
5
1C
10
18
a
0
5
15
32
0
15
60
80
56
45
95
100
100
100
95
100
100
100
aHours.
228
-------
Appendix H-3. Test concentrations and mortality of postla^val penaeid
shrimp (penaeus stylirostris) exposed to ethoprop in
static icute toxicity tests using 20 annals pe* con-
centration. Natural seawater was adjusted to 20
°/oo; temperature 25+2°C.
Nominal concentration
Mortali ty
<%)
(yg/A)
24 hd
4R'o
72h
96
Control
3
3
5
8
Carrier control
•J
13
20
23
1.8
0
0
5
15
3.2
n
0
20
25
5.6
n
0
25
35
10
8
45
60
90
18
95
ino
100
100
32
100
100
100
100
^Hours.
Excessive control nortality
229
-------
Appendix H-4. Test concentrations and norta^ity of sheepshead minnows
(Cyprinodon variegatus) exposed to ethoprop in static
acute toxicity tests using 20 aninals per concentration.
Natural seawater was adjusted to 20 °/oo; temperature
25+1°C.
Morninal concentration Mortality (")
(ug/£) 24 ha 48h 72h 96h"
Control
0
0
0
0
Carrier control
0
0
0
0
100
0
0
0
0
180
0
0
0
0
320
0
5
15
15
560
5
30
35
35
1,000
30
55
60
65
aHours.
230
-------
Appendix H-5. Test concentrations and mortality of spot (Leiostorous
xanthurus).exposed to ethoprop in static acute toxicity
tests using 20 aninals oer concentration. Natural sea-
water was adjusted to 20 °/oo; temperature 25+l°C.
Nominal concentration . Mortality (%)
(yg/£) ?4'nd 48h * 72h 96h
Control
0
0
0
0
Carrie"- control
0
0
17
17
32
0
0
33
50
56
50
50
83
83
100
83
83
100
10C
180
100
100
100
100
320
100
100
100
100
j^Hours.
Excessive control mortality (>10%).
231
-------
Appendix H-6. Test concentration and mortality of nysid shrinp
(Mysidopsis bahia) exposed to ethoprcp in a flowing
seawater test. Shrinp size averaged 5 nrr, total
length; twenty aninals were tested in each treatment.
Test temperature was 22+l°C; salinity 28+1 °/oo.
Concentrat i cn
(ug/O
Mortality (fO
Nomi nal
Measured
24 ha
48h
72h
96 h
Control
NDb
0
0
0
0
Carrier contro
1 ND
0
o ¦
o
n
v
4
2.2
5
5
5
10
6
4.9
0
5
10
20
11
6.8
5
10
20
35
18
7.7
0
10
25
55
30
14. C
20
60
75
100
^Hours.
ND = Nondetectable (<0.1 ug/fc).
232
-------
Apoendix H-7. Test concentration and mortality of pink shrimp
(Penaeus duorarum) exposed to ethoprop in a flowing
seawater test. Shrinp size ranged from 36-64 mm
(rostrum to telson length); twenty animals were used
in each treatment. Test temperature was 22^1°C; salinity
26 to 30 °/oo
Concentration (ug/a) Mortality (%)
Nominal Measured 24ha 48h 72h 96h
Control
NDb
0
0
0
0
Carrier control
PID
0
0
0
0
4
2
0
0
0
Q
6
2.6
0
0
0
0
11
5.6
0
0
0
5
18
8.2
0
0
15
20
30
19
0
0
45
75
^Hours.
d\'D = rlondetectable (<0.5 ug/z).
233
-------
Appendix H-8. Test concentrations and mortality of sheepshead minnows
(Cyprinodon variecatus) exposed to ethoorop in a flowing
seawater test. Fish size ranged from 10 to 15 mm,
standard length; twenty aninals were used in each treat-
ment. Test temperature was 3.0+l°C; salinity 23+1 °/oo.
Concentration (gq/jp Mortality (%)
Nominal
Measured
?Ah&
48 h
72h
96 h
Control
NDb
0
5
10
10
Carrier control
MD
0
20
2C
20 c
130
14C
0
15
30
40
216
220
c
15
30
50
360
280
0
35
70
90
600
600
20
80
°5
95
1,000
1,100
25
90
100
. 100
*Hours.
ND = Mondetectable (<0.5 ug/Jt).
cL'nacceptable control mortality (>10%).
234
-------
Appendix H-9. Test concentrations and mortality of pinfish (Lagodon
rhonboides) exposed to sthoprop in a ^lowing seawater test.
Fish size ranged from 53 to 90 Tin, standard length; twenty
aninals were tested in each treatment. Test temperature
was 22_^1°C; salinity 28 to 30 °/oo.
Concentration (ug/£) "ortalitv (%)
Nominal Measured 24F3 48h 7?h 96h
Control
NDb
0
0
0
0
Carrier control
PID
0
o
0
0
4
2.1
0
0
0
0
6
4.9
0
5
5
5
11
6.7
n
15
25
30
13
7.7
15
65
70
75
30
u
20
100
100
100
j^Hours.
ID = Nondetectable; (<0.5 ug/£).
235
-------
Appendix I-la. Number of normal eastern oyster (Crassostrea
vi rni ni ca) embryos per milliliter counted following 48
of exposure to methyl paratfrion in static, unaerated
seawater. If all embryos i-n the initial inoculum had
developed, the expected count would have been 1,059
embryos per milliliter,
temperature, 25+l°C.
Salinity was 20 /oo and
Nominal concentration
Number of noma! embryos
(ugfi)
RepaA
Rep B
Reo C
Mean
SDU
Contro1
714
827
793
778
+ 57
3,200
598
523
509
576
+46
5,600
518
552
523
531
+ 18
10,000
335
312
271
¦ 306
+ 32
32,000
301
264
298
287
+ 20
56,000
198
253
219
223
+27
^Peplicate.
"Standard deviation.
-------
Appendix I-1b. Toxicity of methyl parathion to embryos of eastern
oysters (Crassostrea virqinica) exposed for ^8 h
in static, unaerated seawater. Criterion of effect
was reduction of the number of normal embryos in
test concentrations as compared to the number of
normal control embryos. Salinity was 20 °/oo and
temperature 25+l°C.
Nominal concentration
(uG/a)
Percentage reduction'
Control
3,200
5,600
•10,000
32,000
56,000
26
71
61
63
71
Number of norma1 48-h control embryos minus the number of
Percentage _ normal 4B-h embryos in each test concentration „
reduction ~ "lumber of normal 48-h control embryos
237
-------
Appendix 1-2. Test concentrations and mortality of mysid shrimp
(M.ysidopsis bahia) exposed to methyl parathion in static
acute toxicity tests using 20 animals per concentration.
Natural seawater was adjusted to 20 °/oo; temperature,
25+l°C.
Nominal concentration Mortality (%)
(¦ug/z) m? 48h 72 h 96h
Cont ro"!
C
0
0
0
Carrier control
. 0
0
0
0
0.18
0
0
0
0
0.32
0
0
0
0
0.56
10
10
20
20
1.0
5
2°
40
<15
1.8
70
90
90
90
aHcurs.
238
-------
Appendix 1-3, Test concentrations and mortality of postlarval penaeid
shrimp (Penaeus stylirostris) exposed to methyl para-
thion in static acute toxicity test using 20 animals per
concent rati on. Natural seawater was adjusted to
20 °/oo; temperature, 25+2°C.
Nominal concentration Mortality (%)
(wg/t)
24ha
48 h
72 h
96 h
Control
7
15
32
40
Carrier control
7
18
23
33
0.32
8
25
33 .
33
0.56
10
?.?.
30
42
1.0
IB
40
53
57
1.3
38
55
65
70
3.2
84
90
93
98
5.6
ino
100
100
100
aHours.
239
-------
Appendix 1-4. Test concentration? and mortality of calanoid copepod
(Acartia tonsa) exposed for 96 h to methyl Darathion
in static, unaerated seawater. Salinity was 22 °/oo
and temperature, 22+1 °C.
Nominal concentration Mortality {%)
(yg/£)
24h°
48h
72h
96h
Control
0
0
3
0
Carrier control
0
0
0
3
13
7
10
13
20
22
3
3
10
27
36
3
13
27
43
60
17
37
53
67
100
43
63
30
100
aHours.
240
-------
Appendix 1-5. Test concentrations and mortality of sheepshead minnov/s
(Cyprincdon variegatus) exposed to nethyl parathion in
static acute toxicity tests using 20 aninals per concen-
tration. Natural seawater was adjusted to 20 °/oo;
temperature 25+l°C.
Nominal concentration Mortality (%)
(ugfi) ?4h* 48h 72h %h
Control
0
0
0
0
Carrier control
0
0 '
0
0
10,000
0
0
0
0
12,000
5
15
25
35
14,000
85
95
100
100
16,000
70
100
100
100
18,000
100
100
100
100
aHours.
2*1
-------
Appendix 1-6. Test concentrations and mortality of spot (Leiostonus
xanthurus) exposed to methyl parathion in static acute
toxicity tests using 20 aninals per concentration.
Natural seawater was adjusted to 20 °/oo; temperature,
25+l°C.
Nominal concentration Mortality (%)
(yg/£) 24F® ^8h 72 h 96h
Control
C
0
0
C
Carrier control
0
0
0
0
56
o'
0
0
0
100
Q
20
50
60
180
0
20
40
70
320
30
70
90
100
560
50
70
80
90
aHours.
242
-------
Appendix 1-7. Test concentrations and mortality of mysid shrimp
(Hysidopsis bahia) exposed to methyl parathion in a flowing
seawater test. Early juveniles (<48h; total length <2 mm)
were used. Twenty animals
Test temperature averaqed
14 °/oo (12 to 16 °/oo).
were
19.5°C
tested in each
(18-21°C); tes
treatment.
t salinity
Concentration (pn/l)
Mortality (%)
nominal Measured
2
-------
ADnendix 1-3. Test concentrations and mortality of pink shrimp (Penaeus
duorarun) exnoseri to methyl oarat'nicn in flowing seawater tests.
Shrimp size averaged 38 mm, rostrum to telson length; twenty
animals were tested in each treatment. Test temperature
averaned 24.8°C (24.5 to. 25.0°C); test salinity 21.6 °/oo
(17.0"to 27.0 °/oo).
Concentration (uq/&) Mortality {%}
Nominal . Measured 24ha 48h ~ 72h 96h
C
n
15
25
an
100
95
^Hours.
b,ID = 'londetectabl e (<0.02 yg/ji).
Control
o
0
0
Carrier control
rm
0
0
0
0.56
0.57
0
•o.
0
0.75
0.79
20
20
20
1.35
1 7
X • /
80
' 80
SO
1.8 .
1.8
• 95
05
100
2.4
2.3
90
90
OP
244
-------
Appendix 1-9. Test concentrations and mortality of soot (Leiostonus
xanthurus) exposed to methyl parathion in flowing,
natural seawater. Twenty anina^ wore tested in each
treatment. Salinitv was 10-15 °/oo and temperature
22-23 °C.
Concentrati on
(yq/o
Mort
alitv (%)
Momi nal
Measu red
24ha
*8 h
72h
96h
Control
MDb
0
0
r,
. 0
iarrier control
ND
c
0
0
e
65 '
25
5
5
10
10
108
56
0
15
40
40
180
77
30
55
65
75
300
130
60
70
75
85
500
250
100
100
100
100
^Hours.
MC = 'londetectable; (<0.5 uQ/i).
-------
Appendix J-l'a. Number of normal eastern oyster (Crassostrea virqinica)
embryos per nilli.liter counted fol lowing 48 h of
exposure to ohorate in static, unaerated seawater. If
all embryos in the initial inoculum had developed, the
expected count would have been 739 embryos per milli-
liter. Salinity was 20 °/oo and temperature 25+l°C.
Nominal concentration ' Number of normal embryos
(ug/i.) RepaA Rep B Rep C Mean SD
Control
611
563
592
588
*24
Carrier control
548
587
579
574
-20
320
513
504
545
522
.*20
560
400
455'
378
411
+ 39
1,000
3^8
376
410
37R
±31
3,200
83
165
130
126
±41
5 ,600
0
0
0
0
0
^Rep1i cate.
^Standard deviation.
246
-------
Appendix J-lh. Toxicity of nhorate to embryos of eastern oysters
(Crassostrea virqi nica) exposed for 4R h in static,
unaerated seawater. Criterion of effect was '-eduction of
the nunber of normal embryos in test concentrations as
compared to the nunber of control embryos. Salinity was
20 /oo and temperature, 25+l°C.
"loninal concentration
(ug/*)
Control
Carrier control
320
sen
1,000
3,200
5 ,600
Percentage reduction8
2
11
30
36
78
ino
Number of normal -8-h control embryos minus the rumber
Percentage _ of normal 48-h embryos in each test concentration y
reduction " Number of normal 48-h control embryos
247
-------
Appendix J-2. Test concentrations and nortality of postlarval penaeid
shrinp (Penaeus stylirostris) exposed to phorate in stat"
acute toxicity tests using 20 anima 1s per concentration.
Seav/ater was adjusted to 20 °/oo; temperature, 25-*-1 °C -
iloninal concentration
Mortality (%)
C-iig/i)
24 hd
48h
72h
96h
Control
0
0
5
3
Carrier control
10
15
23
28
0.18
8
13
18
18
0.32
20
63
80
85
0.56
58
¦ .83
88 ¦
90
1.0
mo
100
100
100
1.8
95
100
. 10C
100
aHours.
248
-------
Appendix J-3. Test concentrations and mortality of'mysid shrimp (H.ysidopsis
bahia) exposed to phorate in static acute toxicity tests using 20
animals per concentration. Seawater was adjusted to 20 °/oo;
temperature, 25+2°C.
Nominal concentration
Mortality
(ug/O
2^hi
48 h
. 72h
96 h
Control
0
0
0
0
Carrier control
n
C
0
0
.0.32
5
5
5
5
0.56
10
10
10
30
i.C
5
1C
15
20
1.8
. 5
15
35
45
3.2
20
40
90
95
aHours.
249
-------
Appendix J-4.. Test concentrations and mortality of sheepshead min-
nows (Cynrinodon variegatus) exposed to phorate in
¦static acute toxicity tests using 20 animals per
-concentration. Seawater was adjusted to 20 G/oo;
temperature, 25+l°C.
nominal concentration Mortality {%)
(ug/£)
.24 h"
48h
72 h
96h
Control
0
0
0
0
Carrier control
0
0
0
o
2.4
5
5
5
5
3.2
20
40
45
50
4.2
30
40
45
50
5.6
60
70
yo-
70
7.5
95
95
gs
95
aHours.
250
-------
Appendix J-5. Test concentrations and mortality of spot (Leiostomus
xanthurus) exposed to phorate static acute toxicity
tests using 20 an-inals per concentration. Seawater was
adjusted to 20 °/oo; temperature, 25+l°C.
Nominal concentration Mortality {%)
(wg/£) 24h® ^8h 72h 96h
Control
0
0
C
5
Carrier control
10
10
15
15
3.2
0
0
0
0
4.2
0
10
10
10
5.6
45
90
90
90
7.5
95
:00
100
100
10.0
100
100
100
100
aHours.
251
-------
Appendix J-6. Test concentrations and mortality of pink shrimp
.(Penaeus duorarun) exposed to phorate in a.flowing
seawater test. Shrinp size averaged 88 rn, rostrurr1-
to-telson length; twenty animals v/ere tested in each
' treatment, "est tenperature averaged 25.3°C (25
to 26°C); test salinity 24.9 °/oo (22 to 28.5 °/oo).
Concentration (yq/f,)
¦Mortality (%}
Nonina1
Measured
?Ah6
CD
T
72h
96 h
Control
NDb
0
0
0
0
Carrier Control
MD
0
0
0
5
0.056.
fi.OR
5
5
5
10
O.lfi
o.:2
15
O
CO
60
70
0.32
0.26
50
100
100
100
0.56
0.49
85
100
' 100
100
1.8
; 1.7
100
100
100
100
^Hours.
N'D = Nondetectable (<0.02 yq/1).
252
-------
Appendix J-7. Test concentrations and .mortality of mysid shrinp (flysidopsis
bahia) exposed to phorate in a flowing seawater test.
Twenty early juveniles (<^8h; total length ~ 2 mn- were
tested in
2^.7°C (24
27 °/oo).
each treatnent. Test
to 25°C); test sal in1
temperature averaged
ty 1H.4 °/co (16 to
Concentration
(un/O
Mortali ty
(%)
floni nal
Pleasure
d 24ha
48 h
72 h
9fih
Control
NDb
0
10
10
10
Carrier contro
i ' fin
0
5
5
5
n .32
0.31
5
20
30
30
0.56
0.37
0
' 35
60
75
1.0
0.75
5
80
85
10Q
1.8
1.5
60
100
100
100
3.2
1.7
95
100
100
100
^Hours.
flD = nondetectable (<0.02 ug/ji;.
253
-------
Appendix J-8. Test concentrations and mortality of sheepshoad minnows.
(Cyprinodon varieqatus) exDosed to phorate in flowing
seawater test. Fish size averaged 6.6 nn, standard length
twenty animals were tested in each treatment. Test
temperature averaged 25°C (24 to 25°C); test salinity
16.9 °/oo (13 to 21 °/oo).
Concentration (ug/£)
Mortality (?,)
Nominal
Measured
24ha
43 h
72h
96 n
Control
\'Db .
0
0
O
u
¦ 0
Carrier control
•ID
0
0
0
0
0.14
0.12
0
0
o
0
0.24
0.22
0
. 0
' 0
0
0.42
0.50
0
o.
0
0
0.75
0.50
0
0
0
5
1.40
: .50
20
20
25
70
^Hours.
flD = nondetectable (<0.02 ug/i).
254
-------
Aopendix J-9. Test concentrations and mortality of spot (Lei ostomus
xanthurus) exposed to ohorate in fowing seawater test.
Fish size averaged 2~ mm, standard length; twenty an'nals
were tested in each treatment. Test temperature averaged
24.8°C (2* to 26°C); test salinity 16.9 "/bo (10.0 to
25 °/cc).
Concentration (yg/Q Mortality (%)
Nomi nal
Measured
24h
43 h
72h
96 h
Control
NDb
0
0
0
0
Carrier control
NO
0
0
0
0
0.42
0.39
0
0
0
0
0.75
0.47
0
0
0
0
1.4
1.1 .
0
0
0
10
2.4
¦ 2.8
0
25
25
25
i.2
4.2
0
45
50
55
^Hours.
ND = nondetectable (<0.02 pg/z).
255
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing;
1. REPORT NO.
EPA-600/4-81-041
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE . , _ u f u • _ L •
Acephate, nldicarb, Carbopnenothion,
DEF, iPN, Ethoprop, Methyl Parachion, and Phorate: Theic
Acute and Chronic Toxicity, 3iocon".entration Potential,
and Persistence as Related to Marine Environments
5 REPORT DATE
May 1931
6. PERFORMING ORGANIZATION CODE
7, AUTHOR(S)
Experimental Environments Branch
Environmental Research Laboratory, Gulf Breeze
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
10. PROGRAM ELEMENT NO.
A87E1A
11. CONTRACT/GRANT NC.
12. SPONSORING AGENCY NAME AND ADDRESS
Gulf Breeze Environmental Research Laboratory
13.-type of report and period COVERED
Office of Research and Development
Environmental Protection Agency
Gulf Breeze, Florida 32561
14. SPONSORING AGENCY CODE
EPA/60C/4
15. SUPPLEMENTARY NOTES
16. ABSTRACT
-•ie toxicity, bioconcentration, and persistence of the pesticides acephate, altii-
carb, carbophenothion, DEF, EPN, ethoprop, methyl parathion, and ohorate were deter-
mined for esiuarine environments. Static acute toxicity tests were conducted to deter-
mine -ne 96-h EC50 values for algae, 48-h EC5C values' for oyster larvae, and 96-h LC50
va—les -.or at least two crustacean and fish species.'1 Flow—through acute toxicity tests,
based on measured concentrations, were conducted to determine the 96-h LC50 values of
the pesticides for at least two crustacean and fish species. In addition, Maximum
Acceptable Toxicant Concentrations (MATC) were determined in life-cycle toxicitv tests
with mysid shrimp (Mysidopsis ba'nia) and sheepshead minnows (Cyprinodor. variega'tus) or
in partial lire-cycie tests with grass shrimp (Palaeraonetes pugio). MATCs were esti-
mated from embryo-juvenile toxicity tests with sheepshead minnows. Persistence, studies
on carbophenothion, DEE, EPN, methyl parathion,•and phorate investigated processes in
rcarj.ne sys.ens tnat contribute to those pesticides' disappearance. The relative impor-
tance or biological and nonbiological processes (including biodegradation, photolysis,
hydrolysis, sediment/water partitioning, and volatility) were examined. Bioconcentra-
tion -actors i.or fisn or moxlusks exposed to carbophenothion, EPN, ethoprop, and phorate
were determined at steady state or after >28-day exposures.
17.
KEY WORDS AND DOCUMENT ANALYSIS
a DESCRIPTORS
b. :DENT;F 5RS/OPEN ENDED TERMS
c. cosat; Field/Croup
Aquatic toxicology
Pesticides
Acephate, Aldicarb,
Carbophenothion, DEF,
EPN, Ethoprop, Methyl
parathion, and Phorate
06/F
18. DISTRIBUTION STATEMENT
19. SECURITY c_ASS 'This Report)
21 OF PAGES
Unclassified
255
Release to public
20 SECURITY ClASS i Tins page,
Unclassified
22. PRICE .
:PA Form 2220—' (R«v, 4-77^
S OGSOwc"
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